Optimizing Solid Phase Extraction Sorbents for High-Recovery Analysis of Explosive Analytes

Abstract

This article provides a comprehensive guide for researchers and scientists on selecting and optimizing solid-phase extraction (SPE) sorbents for the efficient recovery of explosive analytes from complex matrices. It covers foundational sorbent chemistries, detailing how polymeric materials like Oasis HLB outperform traditional silica-based phases for retaining diverse organic explosives. The content extends to methodological applications, systematic troubleshooting for common issues like low recovery, and validation protocols to ensure analytical precision. By integrating modern SPE principles with forensic and environmental case studies, this resource aims to enhance sensitivity, reduce matrix effects, and improve the reliability of explosive compound analysis in drug development and security applications.

Sorbent Chemistries and Mechanisms for Explosive Analyte Retention

Solid Phase Extraction (SPE) is a critical sample preparation technique that enables the purification, separation, and concentration of analytes from complex sample matrices. The fundamental principle of SPE involves the dispersion of analytes between a liquid sample medium and a solid adsorbent, where target compounds exhibit greater affinity for the adsorbent than the bulk solution [1]. This process simplifies subsequent analysis by removing significant portions of the sample matrix, thereby enhancing detection sensitivity and protecting analytical instrumentation. The broad applicability of SPE stems from the diversity of available sorbent chemistries, each designed to exploit specific interactions between the sorbent functional groups and target analytes [2] [1].

The selection of an appropriate SPE sorbent is paramount for developing efficient extraction protocols, particularly when dealing with complex samples such as soil extracts containing trace explosive residues [3]. The physicochemical properties of the target analytes—including polarity, ionic character, and molecular structure—dictate which sorbent chemistry will provide optimal recovery. For researchers working with explosive compounds, understanding the fundamental interactions in reversed-phase, ion-exchange, and mixed-mode SPE is essential for effective method development that can recover a wide range of nitro-organic explosives including nitramines, nitrate esters, nitroaromatics, and nitroalkanes from challenging environmental matrices [3].

Fundamental SPE Sorbent Chemistries

Reversed-Phase Sorbents

Reversed-phase SPE operates on the principle of hydrophobic interactions between non-polar functional groups on the sorbent surface and non-polar regions of the target analytes [4] [5]. These interactions primarily involve van der Waals or dispersion forces, which make reversed-phase sorbents ideal for extracting non-polar to moderately polar compounds from aqueous matrices [5]. Common reversed-phase functional groups include C18 (octadecyl), C8 (octyl), C6 (hexyl), C4 (butyl), C2 (ethyl), phenyl, cyclohexyl, and cyanopropyl [4] [5]. The retention mechanism relies on polar solvents (such as water) repelling analytes from the solution phase onto the hydrophobic sorbent surface [5].

For explosive analysis, reversed-phase sorbents effectively recover compounds with aromatic rings and alkyl chains, making them suitable for nitroaromatic explosives like TNT and DNT [3]. The hypercrosslinked polymeric sorbent Bond Elut NEXUS has demonstrated particular efficacy in recovering trace levels of organic explosives from soil, providing an average recovery of 48% for 12 different nitro-organic explosives in fortified potting soil [3]. The non-polar character of many explosive compounds allows for strong retention on reversed-phase materials, though more polar explosives may require alternative sorbent chemistries for optimal recovery.

Ion-Exchange Sorbents

Ion-exchange SPE utilizes electrostatic interactions between charged functional groups on the sorbent surface and ionized groups on the target analytes [4] [5]. This mechanism provides exceptional selectivity for compounds with ionizable functional groups, which can be strategically manipulated by adjusting pH to ensure analytes and sorbents carry opposite charges [2]. Ion-exchange sorbents are classified into four main categories based on their functional groups and charge characteristics:

• Strong Cation Exchange (SCX): Contains sulfonic acid groups that remain negatively charged across the entire pH range [6] [5]
• Weak Cation Exchange (WCX): Contains carboxylic acid groups that are negatively charged at high pH but neutral at low pH [6] [5]
• Strong Anion Exchange (SAX): Contains quaternary ammonium groups that remain positively charged across the entire pH range [6] [5]
• Weak Anion Exchange (WAX): Contains primary, secondary, or tertiary amine groups that are positively charged at low pH but neutral at high pH [6] [5]

The elution of analytes from ion-exchange sorbents typically requires disrupting the ionic interactions through one of three methods: using high ionic strength buffers to compete for ionic sites, adjusting pH to neutralize the charge on either the analyte or sorbent, or employing counterions with high affinity for the sorbent surface [5]. For explosive compounds with ionizable functional groups, ion-exchange SPE provides a highly selective extraction mechanism that can effectively separate target analytes from complex sample matrices.

Mixed-Mode Sorbents

Mixed-mode sorbents represent an advanced SPE technology that incorporates multiple retention mechanisms within a single sorbent, typically combining reversed-phase (hydrophobic) and ion-exchange functionalities [2] [4] [5]. This dual-mechanism approach enables superior selectivity for complex applications where single-mechanism sorbents prove insufficient. The mixed-mode design allows for more comprehensive cleanup by retaining analytes through two orthogonal mechanisms simultaneously, which is particularly valuable when dealing with challenging matrices that contain diverse interfering compounds [2] [7].

The manufacturing of mixed-mode sorbents typically follows one of two approaches: bonding different functional group chemistries concurrently to a single substrate or blending discrete sorbents in specific ratios [5]. The blended approach offers advantages in reproducibility and customization, as different ratios of single-functional-group sorbents can be combined to achieve desired retention characteristics [5]. Eluting analytes from mixed-mode sorbents requires disrupting both retention mechanisms simultaneously, which often involves using mixtures of non-polar solvents with appropriate buffers, acids, or bases [5]. For complex mixtures of explosive compounds with varying physicochemical properties, mixed-mode sorbents provide a versatile solution that can accommodate diverse analyte characteristics within a single extraction protocol.

Table 1: Comparison of Fundamental SPE Sorbent Chemistries

Sorbent Type	Retention Mechanism	Functional Group Examples	Typical Analytes	Elution Conditions
Reversed-Phase	Hydrophobic interactions (van der Waals forces)	C18, C8, phenyl, polymeric divinylbenzene [4] [5]	Non-polar to moderately polar compounds; nitroaromatic explosives [3] [5]	Solvents with non-polar character (methanol, acetonitrile, isopropanol) [5]
Ion-Exchange	Electrostatic attraction	SCX: sulfonic acid; SAX: quaternary amine; WCX: carboxylic acid; WAX: primary/secondary/tertiary amine [6] [4] [5]	Ionizable acids or bases; compounds with charged functional groups [5]	High ionic strength buffers; pH adjustment; counterions with high sorbent affinity [5]
Mixed-Mode	Combined hydrophobic and ionic interactions	C8/SCX, C8/SAX, polymeric sorbents with ion-exchange functionalities [4]	Compounds with both hydrophobic and ionic character; complex analyte mixtures [2] [7]	Mixtures of organic solvents with buffers, acids, or bases to disrupt multiple interactions [5]

Sorbent Selection Guide

Choosing the appropriate SPE sorbent requires systematic evaluation of three key factors: the physicochemical properties of the target analytes, the composition of the sample matrix, and the sample volume to be processed [5]. This decision-making process can be visualized as a logical workflow that guides researchers to the optimal sorbent chemistry for their specific application.

Diagram 1: SPE sorbent selection workflow guiding researchers from sample matrix characterization to optimal sorbent chemistry.

For researchers analyzing explosive compounds, this selection guide provides a logical framework for choosing sorbents based on specific analyte characteristics. Non-polar explosive compounds like TNT are effectively extracted using reversed-phase sorbents from aqueous matrices [3]. Ionizable explosives or degradation products with acidic or basic functional groups benefit from ion-exchange sorbents, while complex mixtures containing both neutral and ionizable explosive compounds may require the dual retention mechanisms of mixed-mode sorbents [2] [7]. The sample matrix also significantly influences sorbent selection, with aqueous environmental samples typically processed using reversed-phase sorbents, while organic extracts may require normal-phase or alternative chemistries [5].

Experimental Data: Sorbent Performance for Explosive Analytes

Comparative Recovery Studies

Research specifically validating SPE methods for recovering trace levels of nitro-organic explosives from soil provides critical performance data for sorbent selection. A comprehensive study comparing three different copolymeric SPE cartridges—Empore SDB-XC, Oasis HLB, and Bond Elut NEXUS—for the recovery of 12 nitro-organic explosives demonstrated significant variation in sorbent performance [3]. The Bond Elut NEXUS cartridges provided the best overall recoveries with an average of 48% across all 12 explosives in fortified potting soil, along with the fastest processing times of less than 30 minutes [3]. This study highlights how sorbent selection directly impacts analytical sensitivity and method efficiency for explosive compound analysis.

The research methodology involved fortifying soil samples (potting soil, sand, and loam) with target explosives, followed by extraction with acetone and SPE cleanup before analysis by gas chromatography with electron capture detection (GC/ECD) [3]. The SPE method demonstrated significantly improved performance compared to conventional syringe filtration, providing lower limits of detection (LOD) for most explosives, higher percent recoveries for complex matrices, and reduced instrument maintenance issues [3]. All 12 explosive compounds were detectable at 0.02 μg/g or lower across the three soil matrices over three days, demonstrating the method's sensitivity [3].

Table 2: Experimental Recovery Data for Explosive Compounds Using Different SPE Sorbents

Explosive Compound	Class	Bond Elut NEXUS Recovery	Comparison to Syringe Filtration	Limit of Detection (LOD)
Ethylene glycol dinitrate (EGDN)	Nitrate ester	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Dimethyl dinitrobutane (DMDNB)	Nitroalkane	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
2,4-Dinitrotoluene (DNT)	Nitroaromatic	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Trinitrotoluene (TNT)	Nitroaromatic	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Nitroglycerin (NG)	Nitrate ester	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Pentaerythritol tetranitrate (PETN)	Nitrate ester	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Cyclotrimethylene trinitramine (RDX)	Nitramine	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Cyclotetramethylene tetranitramine (HMX)	Nitramine	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]
Other explosives studied	Varied	Reported in 48% average	Higher recovery	≤ 0.02 μg/g [3]

Detailed Experimental Protocol for Explosive Recovery

The validated SPE method for recovering explosive compounds from soil involves a systematic protocol optimized for trace-level analysis [3]:

• Sample Preparation: Fortified soil samples are initially extracted with acetone. The soil texture significantly affects retention capacity, with clay-rich soils presenting greater challenges due to higher specific surface area and active surface charge that can strongly bind nitroaromatic explosives like TNT [3].

• SPE Cartridge Conditioning: The selected SPE cartridges are conditioned with appropriate solvents prior to sample loading. For reversed-phase sorbents, this typically involves solvation with methanol or acetonitrile followed by flushing with water or buffer without allowing the cartridge to dry out [6].

• Sample Loading: The acetone soil extract is loaded onto the conditioned SPE cartridge. The large cross-sectional area of disk formats can enhance extraction efficiency for large volume samples [1].

• Wash Step: Matrix components and interferences are washed away using appropriate solvents. In the comparative study, the NEXUS cartridges effectively rejected matrix components from spent motor oil on potting soil [3].

• Analyte Elution: Target explosives are eluted using a small volume of solvent compatible with subsequent analysis. Avoiding evaporation steps reduces loss of volatile explosives and saves preparation time [3].

• Analysis: Final extracts are analyzed by GC/ECD, which provides sensitivity to a wide range of explosives and relatively rapid analysis time [3].

This protocol represents a significant improvement over traditional methods like EPA's Method 8330B, which recommends an 18-hour sonication-based extraction process, making it impractical for time-sensitive forensic cases [3].

Essential Research Reagents and Materials

Successful implementation of SPE methods for explosive analysis requires specific reagents and materials optimized for target compounds and matrices. The following toolkit outlines essential components for developing robust SPE protocols.

Table 3: Research Reagent Solutions for SPE Analysis of Explosives

Reagent/Material	Function/Purpose	Application Notes
Bond Elut NEXUS Cartridges	Copolymeric SPE sorbent for nitro-organic explosives	Provides 48% average recovery for 12 explosives; fast processing (<30 min) [3]
Empore SDB-XC Cartridges	Styrenedivinylbenzene copolymer sorbent	Comparative sorbent for explosive recovery [3]
Oasis HLB Cartridges	Hydrophilic-lipophilic balanced copolymer sorbent	Comparative sorbent for explosive recovery [3]
Acetone (HPLC grade)	Extraction solvent for soil samples	Effectively extracts nitro-organic explosives from soil matrices [3]
Methanol (HPLC grade)	SPE conditioning and elution solvent	Polar solvent for disrupting non-polar interactions in reversed-phase SPE [5]
Acetonitrile (HPLC grade)	Alternative SPE conditioning solvent	Alternative to methanol for sorbent conditioning [6]
Gas Chromatograph with Electron Capture Detector (GC/ECD)	Analytical instrumentation for separation and detection	Sensitive to wide range of explosives; relatively rapid analysis time [3]
Buffer Solutions (various pH)	pH adjustment for ion-exchange and mixed-mode SPE	Enables manipulation of ionic interactions [2] [5]

Emerging Technologies and Future Directions

SPE sorbent technology continues to evolve with emerging materials that offer enhanced selectivity and efficiency for challenging applications like explosive analysis. Molecularly Imprinted Polymers (MIPs) represent a particularly promising technology that mimics the antigen-antibody recognition mechanism in biological systems [8]. These polymers can be custom-designed and synthesized for specific target explosive molecules, creating recognition sites complementary to the template in both spatial configuration and functional group distribution [8]. MIPs demonstrate high efficiency, remarkable specificity, renewability, and ease of preparation, making them valuable for solid-phase extraction of trace explosive residues from complex matrices [8].

Other advanced configurations include pipette-tip SPE (PT-SPE), a miniaturized format that facilitates automated processing while significantly reducing organic solvent consumption [1]. Magnetic SPE (MSPE) incorporates magnetic nanoparticles for convenient separation using an external magnetic field [1]. Monolithic sorbents based on poly(styrene-co-divinylbenzene) offer highly interconnected pores and excellent permeability for enhanced mass transfer characteristics [1]. These emerging technologies expand the analytical toolbox available to researchers addressing the challenging task of detecting trace explosive compounds in complex environmental and forensic samples.

The selection of appropriate SPE sorbents—whether reversed-phase, ion-exchange, or mixed-mode—fundamentally influences the success of extracting explosive analytes from complex matrices. Experimental data demonstrates that copolymeric sorbents like Bond Elut NEXUS can provide approximately 48% average recovery for 12 nitro-organic explosives in soil with processing times under 30 minutes [3]. The systematic approach to sorbent selection outlined in this guide, supported by validated experimental protocols and performance data, provides researchers with a framework for developing robust SPE methods. As sorbent technology continues to advance with emerging materials like MIPs and novel configurations, analytical capabilities for trace explosive detection will further improve, supporting critical applications in forensic investigation and environmental monitoring [8].

In the realm of solid-phase extraction (SPE), the selection of an appropriate sorbent is not merely a preliminary step but a critical determinant of analytical success. The efficiency of extracting target analytes from complex matrices hinges on the precise interplay between the sorbent's properties—particularly its hydrophobicity and capacity for selective interactions—and the chemical characteristics of the analytes. This relationship is especially crucial in challenging applications such as the recovery of explosive compounds, where matrix complexity and low analyte concentrations demand highly selective and efficient extraction phases. SPE serves as a fundamental sample preparation technique to isolate analytes from complex matrices prior to chromatographic analysis, improving reproducibility, sensitivity, and cleanliness of analytical results [9]. The core principle involves exploiting interactions between the sorbent and analytes to achieve selective retention and subsequent elution.

The fundamental SPE workflow consists of multiple critical steps: conditioning to activate the sorbent, sample loading where analytes are retained, washing to remove interfering matrix components, and elution to recover the purified analytes [9]. Each step must be optimized based on the sorbent-analyte interactions to achieve high recovery and selectivity. The effectiveness of this process is intrinsically linked to the properties of the sorbent material, making the choice of sorbent a cornerstone of method development in analytical chemistry.

Sorbent Mechanisms and Selectivity Principles

Sorbents facilitate extraction through various interaction mechanisms, with hydrophobicity serving as a primary driver for many reversed-phase applications. Hydrophobic interactions occur between non-polar stationary phases and non-polar regions of analyte molecules in aqueous environments. The strength of these interactions depends on the surface chemistry and ligand density of the sorbent material [10] [9]. Beyond hydrophobic interactions, modern sorbents can employ multiple complementary mechanisms including ionic exchange for charged compounds, hydrogen bonding for polar compounds, π-π interactions for aromatic systems, and size exclusion based on molecular dimensions [11] [9].

The selectivity of a sorbent determines its ability to preferentially retain target analytes while excluding matrix interferents. This selectivity arises from the strategic combination of interaction mechanisms tailored to specific analyte properties. For instance, polymeric sorbents with balanced hydrophilic-lipophilic properties (e.g., HLB) provide broader retention for compounds spanning diverse polarities, while ion-exchange sorbents offer superior selectivity for charged molecules [9]. The incorporation of specific functional groups through chemical modification further enhances selectivity by introducing complementary interaction sites for target analyte classes.

Table 1: Primary Interaction Mechanisms in Solid-Phase Extraction

Interaction Type	Sorbent Chemistry Examples	Analyte Characteristics	Application Examples
Hydrophobic	C18, C8, polymeric phases	Non-polar to moderately polar	Explosives, pharmaceuticals [12] [9]
Ionic Exchange	SAX, SCX, WCX	Charged compounds (acids/bases)	Ionic degradation products [9]
Hydrogen Bonding	Silica, NH₂, diol, urea-modified phases	Polar compounds with -OH, -NH groups	Carbohydrates, pharmaceuticals [13] [14]
π-π Interaction	Aromatic ligands, graphene-based materials	Compounds with aromatic rings	Benzodiazepines, nitroaromatics [15] [14]
Size Exclusion	Molecular sieves, porous polymers	Varied molecular sizes	Separation of homologues [11]

Comparative Analysis of Sorbent Performance for Explosive Analytes

Experimental Evidence in Explosive Compound Recovery

Forensic analysis of organic explosives presents particular challenges due to the diverse chemical properties of explosive compounds and the complex matrices in which they are found. A systematic comparison of SPE sorbents for extracting organic explosives from methanolic extracts diluted with water revealed significant performance differences based on sorbent chemistry [12]. The study evaluated the recovery of various explosive compounds, including nitrate esters, nitramines, and nitroaromatics, across different sorbent types with a focus on optimizing clean-up efficiency for LC/MS analysis.

The research demonstrated that polymeric sorbents consistently outperformed conventional octadecyl-bonded silica-based materials (C18) in retaining explosive compounds [12]. Specifically, a polymeric sorbent with smaller specific surface area was found to limit the coextraction of matrix components from simulated motor oil samples, thereby reducing ion suppression in subsequent LC/MS analysis. This finding highlights that extremely high surface area is not always beneficial, particularly when dealing with complex matrices containing interferents with similar chemical properties to the target analytes.

Table 2: Sorbent Performance Comparison for Explosive Compound Recovery

Sorbent Type	Analyte Class	Recovery Efficiency	Matrix Clean-up Efficiency	Key Findings
Polymeric Sorbents	Nitroaromatics, Nitramines, Nitrate Esters	High	High	Limited coextraction of matrix components; reduced ion suppression in LC/MS [12]
Octadecyl-bonded Silica (C18)	Nitroaromatics	Moderate	Moderate	Lower retention for polar explosive compounds [12]
Mixed-mode Resins	Varied (multiple mechanisms)	High	High	Simultaneous utilization of multiple interaction mechanisms [11]
Graphene-based Materials	Broad spectrum	High (theoretical)	High (theoretical)	High surface area and multiple interaction sites [15]

Advanced Sorbent Materials for Challenging Applications

The development of advanced sorbent materials has significantly expanded the capabilities of SPE for specialized applications. Graphene-based materials (GBMs), including graphene oxide (GO) and reduced graphene oxide (rGO), offer exceptional surface area and multiple interaction sites, including hydrophobic, π-π, and hydrogen bonding capabilities [15]. These materials can be further functionalized with other components such as ionic liquids, silica derivatives, magnetic materials, and molecularly imprinted polymers to create hybrid sorbents with enhanced selectivity for specific analyte classes [15].

Similarly, metal-organic frameworks (MOFs) have emerged as promising sorbent materials due to their tunable porosity, high surface area, and versatile functionality. A urea-modified MOF (MIL-101(Fe)-Urea) demonstrated exceptional selectivity for clonazepam extraction from water samples, achieving recovery rates of 94.9–99.0% with high reproducibility (RSD 1.4%) [14]. The incorporation of urea functionalities created specific interaction sites for hydrogen bonding and dipole-dipole interactions, highlighting how targeted sorbent design can optimize extraction performance for specific analytes.

Monolithic sorbents represent another advancement, offering high permeability, low backpressure, and robust porosity compared to conventional particle-packed columns. A comparative study of monolithic versus particle-based SPE for lead separation demonstrated that the monolithic column provided enhanced selectivity, reproducibility, and overall efficiency due to its superior flow characteristics and accessibility of interaction sites [16].

Experimental Protocols and Methodologies

Standardized SPE Protocol for Comparative Studies

To ensure reliable comparison of sorbent performance, researchers should follow a standardized SPE protocol with careful optimization at each stage:

• Sorbent Conditioning: Activate the sorbent bed with 1-2 column volumes of strong organic solvent (e.g., methanol or acetonitrile), followed by water or buffer. Avoid drying the sorbent between conditioning and sample loading to maintain consistency [9].

• Sample Loading: Apply the sample at a moderate flow rate (0.5–1 mL/min) to promote interaction between analytes and sorbent. The sample volume should not exceed the sorbent's capacity to prevent breakthrough [9].

• Washing: Remove matrix interferences using 1-3 mL of intermediate polarity solvent strong enough to remove weakly bound contaminants but not too strong to elute the target analytes. Gradient washing may be employed for complex matrices [9].

• Elution: Recover target analytes using high-strength solvent (e.g., methanol, acetonitrile, acidified organic solvent). Use minimal volume (typically 1-2 mL) to concentrate analytes; multiple small elution steps may improve recovery [9].

For explosive compound analysis specifically, the developed protocol involved loading methanolic extracts diluted with water onto different sorbent types, followed by evaluation of recovery and clean-up efficiency through LC/MS analysis [12].

Comprehensive Workflow for Sorbent Evaluation

The following diagram illustrates the complete experimental workflow for systematic evaluation of sorbent performance, from preparation through data analysis:

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful SPE method development requires careful selection of reagents and materials tailored to the specific analytical challenge. The following toolkit outlines essential components for evaluating sorbent performance in explosive analyte recovery:

Table 3: Essential Research Reagents and Materials for SPE Method Development

Item	Function/Application	Examples/Notes
Polymeric Sorbents	Broad-spectrum retention for diverse analyte classes	Oasis HLB; particularly effective for explosive compounds [12]
Bonded Silica Phases	Reversed-phase extraction based on hydrophobicity	C18, C8; moderate recovery for explosives [12]
Mixed-mode Sorbents	Simultaneous hydrophobic and ionic interactions	Combined C8/cation-exchange; sequential elution capability [11]
Graphene-based Materials	High surface area with multiple interaction sites	GO, rGO; can be functionalized for enhanced selectivity [15]
Metal-Organic Frameworks	Tunable porosity and specific functionality	MIL-101(Fe)-Urea; exceptional selectivity demonstrated [14]
Monolithic Sorbents	High permeability, low backpressure	Enhanced flow characteristics for improved efficiency [16]
Conditioning Solvents	Sorbent activation and preparation	Methanol, acetonitrile, followed by aqueous buffer [9]
Elution Solvents	Analyte recovery from sorbent	Acidified organic solvents, varying strength based on retention [9]
Reference Materials	Method validation and quantification	Certified explosive standards for recovery calculations [12]

The critical role of sorbent choice in solid-phase extraction cannot be overstated, particularly for challenging applications such as explosive analyte recovery. The experimental evidence clearly demonstrates that sorbent hydrophobicity and selective interactions directly determine extraction efficiency, matrix clean-up capability, and ultimately, analytical accuracy. While traditional C18 sorbents provide satisfactory performance for many applications, advanced materials including polymeric phases, graphene-based composites, and functionalized MOFs offer superior selectivity and recovery for demanding analytical scenarios.

The strategic selection of sorbent chemistry, guided by a thorough understanding of analyte properties and interaction mechanisms, enables researchers to develop robust extraction methods capable of detecting trace analytes in complex matrices. As analytical challenges continue to evolve, ongoing development of selective sorbent materials will remain fundamental to advances in environmental monitoring, forensic analysis, and pharmaceutical research.

Solid-phase extraction (SPE) is an indispensable sample preparation technique in modern analytical chemistry, effectively closing the gap between sample collection and final chromatographic analysis. This is particularly crucial in the forensic analysis of organic explosives, where analysts must identify trace levels of target compounds in complex and challenging matrices. The selection of an appropriate SPE sorbent directly determines the efficiency of analyte recovery, the degree of sample clean-up achieved, and the ultimate sensitivity and reliability of the analytical method.

This guide provides a comparative overview of polymeric sorbents and traditional silica-based sorbents for extracting organic explosive compounds. Framed within the broader context of thesis research on solid-phase extraction, this article objectively compares the performance of these sorbent classes based on experimental data, detailing the methodologies required to generate such data. The discussion is particularly relevant for researchers, scientists, and professionals engaged in method development for detecting nitrate ester, nitramine, and nitroaromatic compounds in forensic, environmental, and security applications.

Theoretical Foundations and Sorbent Characteristics

Principles of Solid-Phase Extraction

SPE operates on the same fundamental principles as liquid-liquid extraction (LLE) but offers distinct advantages, including reduced consumption of organic solvents, shorter processing times, fewer procedural steps, and minimized risk of emulsion formation. The process involves the dispersion of analytes between a liquid sample matrix and a solid sorbent phase. Target compounds are retained on the sorbent based on their affinity for the stationary phase relative to the sample medium. Following a potential washing step to remove interferences, the analytes are eluted with a suitable solvent, resulting in a purified and concentrated sample ready for analysis [1].

Sorbent Chemistry and Properties

The retentivity of a sorbent is primarily governed by its chemical nature and physical structure.

• Silica-Based Sorbents (e.g., C18): These are silica particles bonded with long carbon chains (e.g., octadecyl). Retention occurs mainly through hydrophobic interactions between the analyte and the non-functionalized hydrocarbon chains. While effective for non-polar compounds, they often exhibit poor retention for more polar analytes [17] [18].
• Polymeric Sorbents (e.g., Oasis HLB, LiChrolut EN): These are typically synthetic copolymers, such as styrene-divinylbenzene. Their retention mechanism is more versatile, involving both hydrophobic interactions and polar interactions, which allows them to effectively retain a broader spectrum of compounds, including more polar explosives [17] [19] [18].

A critical physical property is the specific surface area. A sorbent with a very high surface area might co-extract a larger amount of matrix components, potentially leading to greater ion suppression in subsequent LC/MS analysis. Therefore, a polymeric sorbent with an intermediate or smaller specific surface area can sometimes provide a superior clean-up efficiency [17] [18].

Comparative Experimental Data

The following table summarizes key experimental findings from a direct comparative study of different SPE sorbents for recovering organic explosives.

Table 1: Comparison of SPE Sorbent Performance for Organic Explosives

Sorbent Type & Name	Sorbent Chemistry	Key Findings	Recovery for Polar Analytes (e.g., RDX, HMX)
Oasis HLB	Hydrophilic-Lipophilic Balanced Polymer	Limited co-extraction of matrix components; reduced ion suppression in LC/MS; most convenient material overall [17] [18].	High recovery [17]
SDB-1	Styrene-Divinylbenzene Polymer	Effective retention of explosive compounds [18].	Data not specified
LiChrolut EN	Polymeric Sorbent	Effective retention of explosive compounds; high capacity for large sample volumes [17] [18].	High recovery [17]
LiChrolut RP-18	Octadecyl-Bonded Silica	Lower retention for explosive compounds compared to polymeric sorbents [17] [18].	Low recovery [17]
Other Silica-based C18	Octadecyl-Bonded Silica	Generally less effective at retaining polar explosive analytes; demonstrated low recoveries for RDX [17] [20].	Low recovery [17]

Further research corroborates the superiority of polymeric sorbents. A study investigating seven different sorbents for 14 explosives in various matrices concluded that Oasis HLB and Isolute ENV+ (another polymeric sorbent) yielded the best quantitative recoveries. This study also highlighted that a dual-sorbent SPE approach resulted in the lowest matrix effects in all but one matrix (river water) and offered an approximately 10-fold improvement in the limit of detection compared to single-sorbent methods [20].

Detailed Experimental Protocols

To ensure reproducible results in thesis research, adherence to a standardized experimental protocol is essential. The following section outlines a general method for comparing SPE sorbents, based on established procedures in the literature.

Sample Preparation and SPE Procedure

Materials and Reagents:

• Solvents: HPLC-grade methanol, acetonitrile, isopropanol, and purified water (e.g., Milli-Q) [17].
• Analytes: Standard solutions of target explosives from these classes: Nitrate esters (EGDN, NG), Nitramines (RDX, HMX), Nitroaromatics (TNT, 2,4-DNT, TNB, DNB) [17].
• Internal Standards: Use appropriate deuterated or otherwise structurally similar internal standards for quantification [17].
• SPE Cartridges: The sorbents being compared (e.g., Oasis HLB, LiChrolut EN, C18), typically in 100-500 mg packing sizes [17] [18].

Protocol:

• Conditioning: Activate the sorbent by passing 3-5 mL of methanol through the cartridge, then equilibrate it with 3-5 mL of purified water. Do not allow the sorbent to dry [17] [18].
• Sample Loading: Forensic samples are often collected with organic solvents. Dilute the methanolic extract with water to ensure a high aqueous content (e.g., >90%) to promote analyte retention on the reversed-phase sorbent. Percolate the sample through the cartridge at a controlled flow rate (e.g., 1-5 mL/min) [17].
• Washing: Remove weakly retained matrix interferences by washing with 2-3 mL of a water-methanol mixture (e.g., 20:80, v/v). The composition can be optimized to maximize the removal of interferences while minimizing analyte loss [17].
• Drying and Elution: Dry the cartridge under vacuum or positive pressure (e.g., nitrogen stream) for 10-15 minutes to remove residual water. Elute the target explosives with 2-4 mL of an organic solvent such as isopropanol [17].
• Analysis: Evaporate the eluate to dryness under a gentle nitrogen stream and reconstitute in a suitable solvent for analysis by LC/UV or LC/MS [17].

Evaluation of Sorbent Performance

• Recovery Calculation: Calculate the recovery for each analyte by comparing the peak area (normalized to the internal standard) in the extracted sample to the peak area of a standard solution representing 100% recovery [17] [18].
• Clean-up Efficiency: Evaluate clean-up efficiency by analyzing blank simulated samples (e.g., prepared from commercial motor oil extract) with LC/UV to visualize the removal of interfering matrix components [17].
• Matrix Effect Assessment: In LC/MS analysis, quantify the matrix effect by comparing the analyte response in a post-extraction spiked sample to the response in a pure standard solution. A reduction in ion suppression after SPE clean-up indicates effective sample preparation [17].

Essential Research Reagents and Materials

The table below lists key materials required for conducting SPE comparisons for explosive analytes, as derived from the cited experimental work.

Table 2: Research Reagent Solutions for SPE of Explosives

Reagent/Material	Function in the Experiment	Example from Literature
Hydrophilic-Lipophilic Balanced (HLB) Sorbent	Versatile polymeric sorbent for broad-spectrum retention of explosives via hydrophobic and polar interactions.	Oasis HLB [17] [20]
Styrene-Divinylbenzene Sorbent	Polymeric sorbent providing high capacity and strong retention for non-polar and moderately polar analytes.	SDB-1, LiChrolut EN, Isolute ENV+ [17] [20]
Octadecyl-Bonded Silica (C18)	Standard reversed-phase sorbent; used as a benchmark for comparing traditional vs. polymeric materials.	LiChrolut RP-18 [17] [18]
HPLC-Grade Organic Solvents	Used for sorbent conditioning, sample washing, and analyte elution.	Methanol, Acetonitrile, Isopropanol [17]
Ammonium Formate/Formic Acid	Mobile phase additives in LC/MS to enhance ionization and form characteristic adduct ions for nitrate esters and nitramines.	Ammonium formate [17]
Porous Graphitic Carbon (PGC) LC Column	Stationary phase for chromatographic separation of a wide range of explosive compounds in a single run.	Hypercarb column [17]

Workflow and Performance Comparison

The following diagram illustrates the logical workflow for method development and the relative performance of the two sorbent classes.

Within the framework of advanced research on solid-phase extraction, the evidence strongly supports the use of polymeric sorbents over traditional silica-based sorbents for the analysis of organic explosives. The dual-mode retention mechanism of polymers, combining both hydrophobic and polar interactions, makes them uniquely suited for the diverse chemical structures of nitrate ester, nitramine, and nitroaromatic compounds. This is especially critical for retaining more polar analytes like HMX and RDX, for which silica-based sorbents like C18 consistently demonstrate lower recoveries.

Experimental data confirms that polymeric sorbents, particularly Oasis HLB, not only provide high analyte recovery but also offer superior sample clean-up. This effectively limits the co-extraction of matrix components and significantly reduces ion suppression in sensitive detection techniques like LC/MS, which is a common challenge in analyzing complex forensic samples. For researchers designing or optimizing SPE methods, beginning method development with a modern polymeric sorbent represents a robust strategy for achieving high-quality, reliable results in the identification and quantification of explosive compounds.

In the field of forensic and environmental chemistry, the effective extraction and analysis of explosive traces from complex matrices is a significant challenge. The recovery efficiency of solid-phase extraction (SPE) sorbents is profoundly influenced by the fundamental physicochemical properties of the target explosive analytes. Among these properties, the lipophilicity (LogP), acidity (pKa), and polarity of a molecule directly govern its partitioning behavior between aqueous samples and solid sorbents [21] [22]. Understanding these properties is therefore not merely academic but essential for developing robust, sensitive, and reliable preconcentration methods for trace explosive detection. This guide provides a comparative analysis of these key properties for common organic explosives, supported by experimental data, to inform the selection and optimization of SPE sorbents and protocols within a rigorous research framework.

Theoretical Foundations of Key Properties

Lipophilicity (LogP) and Its Influence

Lipophilicity, quantified as the logarithm of the octanol-water partition coefficient (LogP), measures a compound's preference for a non-polar environment over an aqueous one [21]. It is defined as LogP = log ([Drug]~non-polar~ / [Drug]~water~). For solid-phase extraction, a compound with a higher LogP value is more lipophilic and will exhibit stronger affinity for hydrophobic sorbents like C18, leading to greater retention from aqueous samples. Conversely, analytes with low LogP are more hydrophilic and may require more polar sorbents for effective extraction [21] [22].

Acidity (pKa) and Ionization State

The pKa value indicates the strength of an acid or base, defining the pH at which half of the molecules are ionized [21] [22]. This property is critical because the ionization state of a molecule dramatically affects its lipophilicity and solubility. For ionizable explosives, the effective lipophilicity is best described by LogD (the distribution coefficient), which accounts for the pH-dependent ionization [21]. The relationship is given by LogD = LogP - log (1 + 10^(pH - pKa)) for acids. Therefore, controlling the sample pH is a crucial strategic tool in SPE to manipulate an analyte's retention and elution.

Polarity and Molecular Interactions

Polarity, while often qualitatively described, is a composite property stemming from a molecule's dipole moment, polarizability, and its capacity to engage in hydrogen bonding. It directly influences the type of intermolecular interactions (dipole-dipole, hydrogen bonding, dispersion forces) an analyte can form with a sorbent material [21]. Selecting a sorbent with a complementary functional group is key to achieving high recovery. For instance, polar explosives like RDX and HMX will interact more strongly with polar sorbents or those capable of specific hydrogen bonding.

Table 1: Physicochemical Properties of Common Organic Explosives

Analyte	Chemical Class	LogP (Est.)	pKa	Polarity	Key Functional Groups
TNT	Nitroaromatic	Moderate (~2.0)	Not acidic	Moderate	Aromatic ring, nitro groups
RDX	Nitramine	Low (~0.5)	Not acidic	High	Cyclic nitramine
HMX	Nitramine	Low (~0.1)	Not acidic	High	Cyclic nitramine
PETN	Nitrate Ester	Low (~1.6)	Not acidic	High	Nitrate ester
NG	Nitrate Ester	Low (~1.6)	Not acidic	High	Nitrate ester

Experimental Protocols for Extraction and Analysis

Solvent-Assisted Dispersive Solid-Phase Extraction (SADSPE) Protocol

The following methodology, adapted from a study on explosive preconcentration, outlines a workflow for extracting HMX, RDX, and TNT from aqueous samples [23].

1. Materials and Reagents:

• Sorbents: Benzyl, benzophenone, or 1,2-dichlorobenzene.
• Dispersing Solvent: A suitable organic solvent (e.g., methanol) to disperse the sorbent.
• Aqueous Sample: The water sample suspected to contain explosive traces.
• Centrifuge Tubes
• HPLC System: Equipped with a UV-Vis detector and a C18 column (e.g., NUCLEODUR Sphinx RP).

2. Procedure:

• Step 1: Sorbent Dispersion. Quickly introduce a mixture of the sorbent (in mg or µg quantities) and a small volume of dispersing solvent into the aqueous sample.
• Step 2: Cloudy Solution Formation. Mix vigorously to create a cloudy solution, which indicates the dispersion of fine sorbent particles throughout the sample. This maximizes the surface area for analyte-sorbent interaction.
• Step 3: Analyte Extraction. Allow the analytes to partition from the aqueous phase onto the dispersed sorbent particles.
• Step 4: Centrifugation. Separate the sorbent particles from the bulk aqueous solution by centrifugation. The analytes are now concentrated in the sorbent pellet.
• Step 5: Elution & Analysis. Elute the concentrated analytes from the sorbent pellet with a small volume of strong solvent. The eluent is then analyzed by HPLC-UV [23].

3. Key Experimental Observations:

• pH Effect: A study found that pH in the range of 4-8 had no significant effect on the extraction efficiency of HMX, RDX, and TNT, as these compounds lack ionizable groups in that range. The highest, though slight, recovery was observed at pH 7 [23].
• Advantages: This method is noted for its ease of operation, cost-effectiveness, low sorbent consumption, and reduced sample treatment time compared to conventional SPE [23].

Analysis via Liquid Chromatography-Mass Spectrometry (LC-MS)

Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-HRMS) is a powerful tool for the separation and identification of explosives in complex mixtures [24] [25]. Retention time (RT) prediction models are increasingly used to aid in the identification of unknown molecules by providing orthogonal evidence to mass spectra [24]. The retention behavior in LC is governed by the same physicochemical properties (LogP, pKa, polarity) that affect SPE, allowing for coordinated method development.

Diagram 1: SADSPE-LC-MS Workflow for Explosives Analysis

Comparative Performance Data and Sorbent Selection

The recovery of explosive analytes is a direct function of the interaction between their physicochemical properties and the sorbent used. The following table synthesizes experimental findings from the literature to guide sorbent selection.

Table 2: Sorbent Performance and Environmental Prevalence Data

Analyte	Reported SADSPE Sorbents [23]	Extraction Efficiency	Environmental Prevalence (Low ng level) [25]
TNT	Benzyl, Benzophenone, 1,2-Dichlorobenzene	Effective with multiple sorbents	Uncommon (only 8 total traces found in 450 public samples)
RDX	Benzyl, Benzophenone, 1,2-Dichlorobenzene	Effective with multiple sorbents	Uncommon (1 trace found in 450 samples)
HMX	Benzyl, Benzophenone, 1,2-Dichlorobenzene	Effective with multiple sorbents	Uncommon (1 trace found in 450 samples)
PETN	Not specified in [23]	Data not available	Uncommon (2 traces found in 450 samples)
NG	Not specified in [23]	Data not available	Uncommon (4 traces found in 450 samples)

The Researcher's Toolkit: Essential Materials and Solutions

Table 3: Key Reagents and Solutions for Explosives Extraction Research

Item	Function/Description	Example Application/Note
C18 Reverse-Phase Sorbent	Hydrophobic interaction; retents non-polar analytes.	Ideal for extracting compounds with high LogP like TNT.
Mixed-Mode Sorbents	Combine ionic and hydrophobic interactions.	Useful for ionizable explosives if pH control is applied.
Benzyl / Benzophenone Sorbents	Sorbent in SADSPE; specific interactions.	Used in dispersive SPE for HMX, RDX, TNT [23].
HPLC-Grade Methanol & Water	Mobile phase for LC separation.	Essential for downstream analysis after extraction.
SiriusT3 Instrument	Automated pKa and LogP determination.	Provides critical physicochemical property data [26].
0.01 M NaOH / HCl Solutions	pH adjustment of sample matrix.	Critical for manipulating recovery of ionizable compounds.

The efficient recovery of explosive analytes using solid-phase extraction is inextricably linked to a fundamental understanding of their LogP, pKa, and polarity. As demonstrated, non-ionizable, moderately lipophilic explosives like TNT can be effectively extracted with a range of organic sorbents using a SADSPE protocol with little concern for pH adjustment. In contrast, the extraction of more polar nitramines (RDX, HMX) and nitrate esters (PETN, NG) benefits from sorbents and methods tailored to their specific polar interactions. The data and protocols provided herein offer a foundation for researchers to make informed decisions in sorbent selection and method development, ultimately enhancing the sensitivity and reliability of trace explosive detection in security, forensic, and environmental applications.

The Superiority of Hydrophilic-Lipophilic Balanced (HLB) Polymers for Diverse Explosives

The trace-level analysis of organic explosives in complex environmental, forensic, and wastewater samples presents significant analytical challenges due to the diverse physicochemical properties of explosive compounds and their frequent presence in complicated matrices. Within this context, solid-phase extraction (SPE) has emerged as a fundamental sample preparation technique for isolating, cleaning up, and pre-concentrating target analytes prior to chromatographic analysis [27]. The selection of an appropriate SPE sorbent is a critical factor determining the success of an analytical method, particularly for challenging compounds like explosives which span a wide polarity range from relatively non-polar nitroaromatics to highly polar nitramines [17]. Historically, SPE method development involved somewhat arbitrary matching of sorbent chemistry to analyte properties, often relying on logP or logD values to estimate analyte hydrophobicity alongside functional group chemistry to select candidate sorbents [28].

More recently, Hydrophilic-Lipophilic Balance (HLB) polymers have demonstrated remarkable capabilities for extracting a broad spectrum of explosive compounds. These sorbents are typically copolymers incorporating both hydrophilic and lipophilic monomers, such as the widely used N-vinylpyrrolidone and divinylbenzene combination [28]. This balanced composition provides a unique set of properties that enhance SPE performance for explosive analytes, offering superior retention capabilities across the polarity spectrum compared to traditional silica-based sorbents and other polymeric materials [28] [17]. This guide objectively compares the performance of HLB polymers with alternative SPE sorbents for explosives recovery, providing experimental data and methodologies to support method development decisions in research and analytical laboratories.

Comparative Performance Data: HLB vs. Alternative Sorbents

Comprehensive Sorbent Screening for Multiple Explosives Classes

Evaluations of multiple SPE sorbents consistently demonstrate the superiority of mixed-functionality polymers for recovering diverse explosive compounds. A comprehensive assessment of 34 different SPE sorbents for extracting 18 explosives including nitramines, nitrate esters, nitroaromatics, and organic peroxides found that divinylbenzene-based polymeric sorbents, particularly one co-polymerized with N-vinyl pyrrolidone (Oasis HLB), provided the most satisfactory overall performance [29]. This specific HLB sorbent delivered recoveries between 77% and 124% for 14 different explosive compounds in fortified wastewater samples, demonstrating its exceptional versatility across multiple explosives classes [29].

Table 1: Sorbent Performance Comparison for Explosives Recovery

Sorbent Type	Key Characteristics	Target Explosives	Performance Summary
HLB Copolymer (e.g., Oasis HLB)	N-vinylpyrrolidone + divinylbenzene; balanced hydrophilic-lipophilic properties	Broad spectrum: nitramines, nitrate esters, nitroaromatics	Recoveries of 77-124% for 14 explosives; best overall performance [29]
Porous Graphitic Carbon	Homogeneous, flat surfaces; strong retention for planar molecules	Diverse explosives	Some compounds too strongly retained, reducing chromatographic performance [17]
Octadecyl-Bonded Silica (C18)	Hydrophobic; traditional reversed-phase sorbent	Non-polar to moderately polar explosives	Poor recovery of polar nitramines (e.g., RDX); limited wettability [17]
Other Polymeric Sorbents (e.g., Porapak, LiChrolut EN)	Styrenedivinylbenzene polymers; predominantly hydrophobic	Non-polar to moderately polar explosives	Better than C18 for polar analytes but generally inferior to HLB [17]

Specific Recovery Advantages for Polar Explosives

HLB sorbents demonstrate particular advantages for retaining polar explosive compounds that challenge traditional reversed-phase materials. In a comparison study focused on sample clean-up for organic explosives analysis, HLB sorbents showed significantly improved retention of more polar analytes such as HMX and RDX compared to octadecyl-bonded silica (C18) and other polymeric sorbents [17]. The study concluded that polymeric sorbents with intermediate retention properties effectively avoid the problem of overly strong retention observed with porous graphitic carbon while still providing sufficient retention of polar explosive compounds [17].

Table 2: Recovery Data for Specific Explosive Compounds Using Different Sorbents

Explosive Compound	Compound Class	Polarity	HLB Recovery	Alternative Sorbent Recovery	Notes
RDX (Cyclotrimethylene trinitramine)	Nitramine	Moderate polarity	High recovery demonstrated [17]	Low recovery on C18 [17]	HLB enables trace analysis in lake water [30]
HMX (Cyclotetramethylene tetranitramine)	Nitramine	Moderate polarity	High recovery demonstrated [17]	Variable recovery on other polymers [17]	HLB enables trace analysis in lake water [30]
2,4-Dinitrotoluene	Nitroaromatic	Moderately non-polar	Detected in wastewater (225-303 ng/L) [29]	Not efficiently retained by less versatile sorbents	Found in London wastewater using HLB SPE [29]
Hydroxymetronidazole (Model compound)	Nitroaromatic derivative	High polarity (logP: -1.3)	>80% with methanol elution [28]	Far lower recoveries on non-HLB polymer [28]	Demonstrates HLB's superior polar compound retention

Experimental Protocols: Methodology for HLB-Based Explosives Extraction

Standard HLB SPE Procedure for Explosives in Aqueous Samples

The following protocol summarizes the optimized methodology for extracting explosive compounds from aqueous samples using HLB sorbents, based on procedures described in the literature [28] [17] [29]:

• Sample Pre-treatment: Collect water samples in clean glass containers. If immediate processing is not possible, store at 4°C and analyze within 48 hours. For wastewater samples containing particulate matter, filtration or centrifugation may be necessary. Adjust sample pH if needed to maximize retention of ionizable compounds.
• SPE Cartridge Conditioning: Condition the HLB cartridge (typically 60 mg to 200 mg sorbent mass) with 5-10 mL of methanol to wet the sorbent and remove any impurities. Follow with 5-10 mL of reagent-grade water without allowing the sorbent bed to dry out.
• Sample Loading: Pass the aqueous sample (typically 100-1000 mL depending on expected analyte concentrations) through the HLB cartridge at a controlled flow rate of 5-10 mL/min using a vacuum manifold or positive pressure. For large sample volumes, this step may require several hours.
• Cartridge Washing: After sample loading, wash the cartridge with 5-10 mL of a water-methanol mixture (typically 5-20% methanol) to remove weakly retained matrix components without eluting target explosives. The optimal methanol percentage should be determined experimentally to balance cleanliness and recovery.
• Analyte Elution: Elute the target explosive compounds with 5-10 mL of organic solvent. Common elution solvents include methanol, acetonitrile, 1:1 methanol/acetonitrile, or ethyl acetate. For extremely hydrophobic analytes, acetonitrile may provide better recovery than methanol.
• Sample Concentration and Analysis: Gently evaporate the eluate to near dryness under a stream of nitrogen and reconstitute in an appropriate solvent (typically 100-500 μL of methanol or mobile phase compatible solvent) for instrumental analysis by LC-MS, GC-MS, or LC-UV.

Method Optimization Considerations

• Flow Rate Control: Maintaining a consistent, moderate flow rate (<10 mL/min) during sample loading is critical for achieving efficient analyte-sorbent interaction and maximizing recovery [27].
• Wash Solvent Optimization: The methanol percentage in the wash solution should be increased as high as possible without losing target analytes to ensure clean extracts [28].
• Elution Solvent Selection: The optimal elution solvent may vary depending on the specific explosive compounds targeted. For comprehensive analysis of multiple explosives classes, a mixture of methanol and acetonitrile often provides the best overall recovery [28] [29].
• Quality Control: Include procedural blanks, matrix spikes, and duplicate samples in each batch to monitor for contamination and evaluate method performance.

HLB SPE Workflow for Explosives

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Essential Research Reagents and Materials for HLB-Based Explosives Extraction

Reagent/Material	Function in Protocol	Typical Specifications
HLB SPE Cartridges	Primary extraction sorbent; retains diverse explosives via hydrophilic-lipophilic balance	60-200 mg sorbent mass; 1-6 mL cartridge volume [28] [29]
High-Purity Methanol	Cartridge conditioning and elution solvent; efficiently elutes most explosives	HPLC grade; low background contamination [17] [29]
Acetonitrile	Alternative elution solvent; stronger eluotropic strength for hydrophobic explosives	HPLC grade; suitable for UV and MS detection [28] [17]
Reagent-Grade Water	Sample medium and wash solvent; ensures minimal background interference	18 MΩ·cm resistivity; free of organic contaminants [17] [30]
Ammonium Formate	Mobile phase additive for LC-MS; enhances ionization of nitrate esters and nitramines	LC-MS grade; typically 2-10 mM in mobile phase [17]
Vacuum Manifold	Processing multiple SPE samples simultaneously; controls flow rate	Multi-port (12-24 positions); capable of fine flow control [27]

Mechanisms of Superior Performance: Structural and Chemical Advantages

The exceptional performance of HLB polymers for explosives extraction stems from fundamental structural and chemical properties that differentiate them from traditional sorbents:

Balanced Hydrophilic-Lipophilic Composition

The copolymer structure of HLB sorbents, typically comprising N-vinylpyrrolidone (hydrophilic) and divinylbenzene (lipophilic) monomers, creates a balanced surface chemistry that simultaneously retains polar and non-polar explosive compounds [28]. The hydrophobic divinylbenzene components effectively retain non-polar explosives like nitroaromatics through van der Waals interactions, while the hydrophilic N-vinylpyrrolidone moieties retain polar explosives such as nitramines (RDX, HMX) through hydrogen bonding and dipole-dipole interactions [28]. This dual-retention mechanism enables single-sorbent extraction of multiple explosives classes that would typically require different sorbent chemistries.

Practical Advantages for Laboratory Workflows

Beyond extraction efficiency, HLB sorbents offer significant practical benefits that enhance laboratory productivity and method reliability:

• Superior Water Wettability: Unlike traditional reversed-phase sorbents that can suffer from "channeling" or incomplete retention if accidentally dried, HLB sorbents remain fully wetted and functional even after exposure to vacuum or if left unattended during processing [28].
• Extended pH Stability: HLB polymers maintain stability across a pH range of 1-14, unlike silica-based sorbents which degrade outside pH 2-8 [28]. This enables method development flexibility, including pH adjustment to manipulate analyte ionization for improved selectivity.
• Higher Capacity: HLB sorbents typically demonstrate ≥10% capacity by bed mass, approximately double that of silica-based sorbents (~5%) [28]. This allows for smaller cartridge sizes or greater sample loading.
• Improved Reproducibility: The monodisperse, spherical particle morphology of HLB sorbents provides more consistent flow characteristics and better reproducibility compared to irregular silica particles [28].

HLB polymers represent a superior sorbent technology for the extraction and analysis of diverse explosive compounds across forensic, environmental, and security applications. Their balanced hydrophilic-lipophilic composition enables unparalleled versatility in retaining explosive compounds spanning the polarity spectrum, from relatively non-polar nitroaromatics to highly polar nitramines, often within a single extraction protocol. The demonstrated recovery advantages over traditional sorbents like C18-bonded silica and other polymeric materials, combined with practical benefits including superior water wettability, extended pH stability, and higher capacity, establish HLB as the preferred sorbent choice for developing robust, sensitive, and reliable analytical methods for explosives detection. For researchers and analytical scientists developing SPE methods for explosives analysis, HLB sorbents should be considered the first-choice platform for both new method development and for improving existing methods that may suffer from limited reproducibility or inadequate recovery of polar explosive compounds.

In the forensic analysis of organic explosives, preventing breakthrough—the point at which analytes fail to be retained by the sorbent and are prematurely lost—is paramount for accurate detection and quantification. Sorbent capacity defines the maximum amount of analyte a solid phase extraction (SPE) sorbent can effectively retain before this occurs. Understanding and calculating this loading limit is not merely a procedural step; it is the foundation upon which reliable analytical results are built. Within the broader thesis of optimizing SPE for explosive analyte recovery, this guide objectively compares the capacity and performance of various hydrophobic sorbents, providing the experimental data and protocols necessary for researchers to make informed decisions and prevent analytical failures.

The consequences of overlooked breakthrough are severe, particularly when analyzing trace-level explosives in complex matrices like post-blast residues. It can lead to false negatives, reduced method sensitivity, and inaccurate quantification, ultimately compromising forensic investigations. Furthermore, in liquid chromatography/mass spectrometry (LC/MS), insufficient sample clean-up due to sorbent overloading results in ion suppression, where co-eluting matrix components interfere with the ionization of target analytes [31] [18]. Therefore, a systematic approach to evaluating and respecting sorbent capacity is a critical component of the scientist's toolkit.

Sorbent Performance Comparison

Selecting the appropriate sorbent is the first critical step in designing an SPE method that prevents breakthrough. A comparative study of four different reversed-phase SPE sorbents for the clean-up of organic explosives provides crucial performance data [31] [18].

Table 1: Comparison of SPE Sorbents for Explosive Analytes

Sorbent Type	Sorbent Nature	Key Characteristic	Retentivity for Explosives	Clean-up Efficiency
Oasis HLB	Polymeric	Balanced hydrophilic-lipophilic nature	Effective for polar analytes (HMX, RDX)	Limited co-extraction of matrix components; reduced ion suppression in LC/MS [18]
SDB-1	Polymeric (Styrene-divinylbenzene)	Hydrophobic	Good retention	Not specified in study
LiChrolut EN	Polymeric (Ethylstyrene-divinylbenzene)	High specific surface area	Good retention	Not specified in study
LiChrolut RP-18	Silica-based (C18-bonded)	Octadecyl functional group	Less effective than polymeric sorbents	Higher co-extraction of matrix components [31] [18]

The data demonstrates that polymeric sorbents (Oasis HLB, SDB-1, LiChrolut EN) generally outperformed octadecyl-bonded silica-based materials (LiChrolut RP-18) in retaining common organic explosive compounds [31] [18]. This is attributed to their superior retention of a wider range of analyte polarities. Notably, the sorbent with the smallest specific surface area was found to limit the co-extraction of matrix components from complex simulated samples, a key factor in preventing breakthrough and ensuring a clean analysis [31].

Experimental Protocol: Sorbent Comparison and Breakthrough Evaluation

The following detailed methodology was used to generate the comparative data in Table 1, allowing for a direct assessment of sorbent retention capacity and clean-up efficiency [18].

• Objective: To compare the retention properties and clean-up efficiency of four different reversed-phase SPE sorbents for organic explosives and evaluate their performance in reducing matrix effects.
• Materials:
  • SPE Sorbents: Oasis HLB, SDB-1, LiChrolut EN, LiChrolut RP-18.
  • Analytes: A mixture of nitrate ester, nitramine, and nitroaromatic explosive compounds.
  • Matrix: Simulated samples prepared from commercial motor oil extracted with methanol.
  • Instruments: LC/UV and LC/MS systems.
• Procedure:
  • SPE Conditioning: Each cartridge was conditioned with an appropriate solvent to activate the sorbent.
  • Sample Preparation: Methanolic extracts of the matrix were diluted with water and spiked with the target explosive analytes.
  • Sample Loading: The aqueous samples were percolated through the conditioned SPE cartridges.
  • Washing Step: An intermediary solvent was passed through the cartridge to remove impurities less strongly bonded than the analytes of interest.
  • Elution: The retained explosive compounds were eluted using a strong solvent.
  • Analysis: The eluate was analyzed by LC/UV to visualize interfering compounds and evaluate clean-up efficiency. LC/MS was used to confirm the presence of trace explosives and measure the reduction in ion suppression.
• Key Measurement: Analyte recovery was calculated by normalizing the peak area of the analyte to the peak area of an internal standard. Clean-up efficiency was evaluated by comparing the chromatographic profiles of processed samples against blanks.

Graphical Abstract: SPE Sorbent Comparison Workflow

Core Principles of Solid Phase Extraction

To properly understand and calculate loading limits, one must first grasp the fundamental mechanisms of SPE. At its core, SPE is a form of "silent chromatography," employing the same principles of interaction between a mobile phase (solvents) and a stationary phase (the sorbent) as HPLC, but without a detector providing real-time feedback [32]. The process is designed to separate analytes from a sample matrix by leveraging differences in their physical and chemical properties.

The two principal mechanisms for interaction in SPE are polarity and ion exchange [32].

• Polarity-Based Interactions: These are governed by the adage "like dissolves like."
  • In reversed-phase SPE (common for hydrophobic explosives), a nonpolar sorbent (e.g., C18) is paired with a polar mobile phase (e.g., water). Nonpolar analytes interact more strongly with the sorbent and are retained.
  • In normal-phase SPE, a polar sorbent (e.g., silica) is paired with a nonpolar mobile phase to retain polar compounds.
• Ion Exchange Interactions: These operate on the rule of "opposites attract." They are used when analytes are charged or can be charged by adjusting pH. A sorbent with an opposite permanent charge retains the analyte. The strength of the analyte and sorbent must be matched for effective retention and elution [32].

The SPE process follows a series of critical steps to ensure optimal analyte retention and minimize breakthrough, each requiring careful optimization [33]:

• Conditioning: Prepares the sorbent for interaction by solvating it with a solvent that activates the functional groups.
• Sample Loading: The sample is passed through the cartridge at a controlled flow rate (typically 1-5 mL/min) to allow analytes to bind to the sorbent. This is the stage where breakthrough must be prevented [33] [34].
• Washing: A solvent of intermediate strength removes weakly bound matrix interferences without displacing the target analytes.
• Elution: A strong solvent disrupts the analyte-sorbent interaction, releasing the purified and concentrated analytes for collection. Using two small aliquots of elution solvent is often more efficient than one large volume [33].

Calculating Sorbent Capacity and Loading Limits

Preventing breakthrough requires a practical understanding of sorbent capacity. A fundamental rule of thumb is that the sample mass loaded onto an SPE cartridge should not exceed 5-10% of the sorbent's mass [33]. This provides a safe starting point for method development.

Table 2: SPE Cartridge Sizing and Typical Loading Parameters

Cartridge Volume	Sorbent Mass	Typical Sample Mass Load	Minimum Elution Volume
1 mL	50 - 100 mg	2.5 - 10 mg	100 - 200 µL
3 mL	500 mg	25 - 100 mg	1 - 3 mL
6 mL	500 - 1000 mg	25 - 100 mg	2 - 6 mL

Experimental Protocol: Determining Breakthrough Volume

To precisely determine the loading limit for a specific analyte-sorbent combination, a breakthrough experiment should be conducted.

• Objective: To determine the maximum sample volume that can be loaded without losing the target analyte.
• Materials:
  • Standard solution of the target explosive analyte.
  • Appropriate SPE sorbent and cartridge.
  • HPLC or GC system with a detector.
• Procedure:
  • Condition the SPE cartridge as normal.
  • Instead of a sample, continuously pass a standard solution of known concentration through the sorbent.
  • Collect the effluent (the liquid exiting the cartridge) in small, sequential fractions.
  • Analyze each fraction using HPLC or GC to measure the analyte concentration.
• Data Analysis:
  • Initially, the analyte concentration in the effluent will be zero, indicating full retention.
  • Breakthrough is defined as the point at which the analyte is first detected in the effluent.
  • The volume loaded up to this point is the breakthrough volume. The loading volume for actual samples must be kept well below this value to ensure quantitative recovery.

The Scientist's Toolkit for SPE

Successful SPE method development relies on a suite of essential reagents and materials. The following table details key solutions and their functions, with a focus on the analysis of explosive analytes.

Table 3: Research Reagent Solutions for SPE Method Development

Tool	Function in SPE	Application Note
HLB Cartridge	A hydrophilic-lipophilic balanced polymeric sorbent that retains a wide range of polar and non-polar compounds.	Proven effective for retaining polar explosive analytes like HMX and RDX; limits co-extraction of matrix components [18] [34].
Polymeric Sorbents (SDB-1, LiChrolut EN)	Hydrophobic polymers providing strong retention via reversed-phase mechanisms.	Superior to C18-silica for organic explosives due to better retention of polar analytes [31] [18].
Methanol & Acetonitrile	Common solvents for conditioning reversed-phase sorbents and eluting strongly retained analytes.	Used for extracting explosives from swabs and as an elution solvent [18].
Dichloromethane	A relatively non-polar solvent used as an elution solvent.	Selected as part of the elution solvent mix for alkylphenols, demonstrating its strength for non-polar analytes [34].
LC/MS System	The analytical instrument for confirming the presence of trace analytes and quantifying the reduction of ion suppression.	Essential for confirming the performance of the SPE clean-up in reducing matrix effects [18].

The accurate analysis of organic explosives is contingent upon a rigorous approach to solid phase extraction, with the calculated prevention of breakthrough at its core. This guide has demonstrated that polymeric sorbents, particularly those like Oasis HLB, offer superior retention for a range of explosive compounds compared to traditional silica-based C18. By adhering to the fundamental principles of SPE, leveraging the experimental protocols for sorbent comparison and breakthrough testing, and utilizing the appropriate tools from the scientist's toolkit, researchers can reliably determine sorbent capacity and establish robust loading limits. Mastering these elements ensures maximum analyte recovery, minimizes matrix interference, and ultimately guarantees the integrity and reliability of forensic and analytical results.

Developing Robust SPE Protocols for Explosive Compound Extraction

Solid Phase Extraction (SPE) is a critical sample preparation technique that enables researchers to purify and concentrate analytes from complex matrices. By selectively retaining target compounds and removing interfering substances, SPE significantly enhances the sensitivity and reliability of subsequent analytical techniques such as HPLC, GC, and MS. For researchers working with explosive analytes, effective SPE methods are particularly valuable for isolating trace-level compounds from challenging samples like soil, swabs, and other forensic evidence. This guide provides a comprehensive examination of the fundamental SPE steps—conditioning, loading, washing, and elution—while presenting experimental data comparing different SPE approaches for recovering explosive compounds.

The Fundamental Steps of Solid Phase Extraction

A standard SPE procedure consists of five key stages that prepare the sorbent, apply the sample, remove impurities, and finally recover the target analytes. Each step must be carefully optimized to maximize recovery of the compounds of interest while effectively eliminating matrix interferences.

Step 1: Sorbent Conditioning

Purpose: Conditioning prepares the sorbent for optimal interaction with the target analytes by activating the functional groups on the chromatographic surface and ensuring proper solvent compatibility [33].

Procedure: The sorbent is treated with a solvent that has similar characteristics (solvent strength, pH, etc.) to the sample to ensure maximum retention of analytes [33]. For reversed-phase SPE, this typically involves rinsing with methanol or acetonitrile followed by water or an appropriate buffer solution.

Critical Consideration: During conditioning, it is essential to prevent the sorbent from drying completely, as this can adversely affect the ability of analytes to interact with the functional groups. Best practice involves allowing approximately 1mm of the final conditioning solvent to remain above the top tube frit [33].

Step 2: Column Equilibration

Purpose: This step establishes the optimal chemical environment for retaining the target analytes when the sample is loaded [33].

Procedure: The column is rinsed with the same solvent that was used for sample pre-treatment (typically water or a buffer solution) to create a compatible environment for the applied sample [33].

Step 3: Sample Loading

Purpose: The prepared sample is applied to the SPE device to allow the target analytes to interact with and be retained by the sorbent [33].

Procedure: The sample is passed through the conditioned sorbent bed at a controlled flow rate. A typical flow rate is 1mL/minute, as higher flow rates can lead to inconsistent extractions and reduced analyte retention [33].

Optimization Tip: For explosive analytes in complex matrices like soil, sample pre-treatment often involves extraction with organic solvents such as acetonitrile prior to SPE cleanup [3].

Step 4: Washing to Remove Interferences

Purpose: Washing eliminates undesirable matrix components and impurities that were weakly retained during the sample loading step [33].

Procedure: An intermediary solvent is used to selectively rinse away interference compounds while leaving the analytes of interest bound to the sorbent. The wash solvent must be strong enough to remove interferences but weak enough to avoid eluting the target compounds [33].

Step 5: Elution of Target Analytes

Purpose: The final step disrupts the analyte-sorbent interactions to recover the purified compounds of interest in a concentrated form [33].

Procedure: A small volume of strong solvent is applied to selectively elute the target analytes. For best results, elute compounds using two small aliquots rather than one large aliquot to maximize concentration [33]. The elution solvent is selected according to the phase mechanism and analyte properties.

Comparative Performance Data for Explosive Analytes

Research studies have systematically evaluated different SPE approaches for recovering explosive compounds from various matrices. The following tables summarize key experimental findings that can guide method selection.

Table 1: Comparison of SPE Cartridge Performance for Nitro-Organic Explosives in Soil

SPE Cartridge	Key Findings	Recovery Range	Processing Considerations
Oasis HLB	Originally showed highest recoveries but with long processing times due to high conditioning volumes and low flow rates [3].	Not specified	Method required optimization to improve practicality [3].
Bond Elut NEXUS	Demonstrated poor recovery for certain explosives including EGDN, DMDNB, and NG [3].	Significantly lower for specific compounds	Not suitable for comprehensive explosives analysis [3].
Strata-X	Consistently yielded recoveries of >80% for all explosives studied; selected as optimal [3].	>80% for all target explosives	Shorter processing times with comparable or better recovery [3].

Table 2: SPE Performance for Smokeless Powder (SLP) Residues on Swabs

SPE System	Elution Solvent	Recovery Performance	Sensitivity Improvement
Oasis HLB	Acetone	Near-complete recoveries across all target compounds [35].	Suitable for quantitative trace analysis [35].
Oasis HLB	Ethyl Acetate	Near-complete recoveries across all target compounds [35].	Achieved method detection limit reductions of up to 8.2-fold compared to direct analysis [35].

Table 3: Fundamental SPE Steps and Their Functions in Explosives Analysis

SPE Step	Primary Function	Key Considerations for Explosives Analysis
Conditioning	Activates sorbent functional groups	Use solvents compatible with explosive compounds; prevent sorbent drying [33].
Equilibration	Creates optimal environment for retention	Match pH and ionic strength to sample matrix [33].
Sample Loading	Retains target analytes on sorbent	Control flow rate (typically ~1mL/min) to ensure proper retention [33].
Washing	Removes matrix interferences	Select solvent strong enough to remove impurities but weak enough to retain explosives [33].
Elution	Recovers purified analytes	Use small solvent volumes (two aliquots) for maximum concentration [33].

Detailed Experimental Protocols

Protocol 1: SPE Method for Nitro-Organic Explosives in Soil

This protocol is adapted from research that developed an optimized SPE cleanup procedure for trace levels of nitro-organic explosives in soil samples [3].

Sample Preparation:

• Fortify soil samples with target explosives (e.g., 10μg of each compound) and internal standard [3].
• For simulated contaminated samples, spike with 2% spent motor oil to evaluate efficacy in removing non-explosive contaminants [3].
• Extract samples with appropriate solvent prior to SPE cleanup [3].

SPE Procedure:

• Conditioning: Condition Strata-X cartridges with successive solvent washes as optimized in the method [3].
• Equilibration: Equilibrate with solvent matching sample matrix composition [3].
• Sample Loading: Apply soil extract to cartridge at controlled flow rate [3].
• Washing: Implement wash step with solvent that removes interferences without eluting target explosives [3].
• Elution: Elute explosives with small volume of optimized elution solvent [3].

Analysis: Analyze eluents using GC/ECD or other appropriate analytical methods [3].

Protocol 2: SPE Method for Smokeless Powder Residues on Swabs

This protocol is adapted from research evaluating SPE systems for recovering smokeless powder (SLP) residues from swabs [35].

SPE Configuration:

• Use Oasis HLB cartridges with either acetone or ethyl acetate as elution solvent [35].

Procedure:

• Conditioning: Condition Oasis HLB cartridges according to manufacturer specifications with optimized solvents [35].
• Equilibration: Equilibrate with compatible solvent [35].
• Sample Loading: Apply swab extracts to the SPE cartridge [35].
• Washing: Implement wash step to remove interfering compounds while retaining SLP residues [35].
• Elution: Elute with either acetone or ethyl acetate [35].

Analysis: Analyze using GC-MS and compare against direct "filter-and-shoot" approaches [35].

Visualization of SPE Workflows

The following diagram illustrates the complete SPE process for explosive analyte recovery, highlighting key decision points and optimization considerations at each stage.

SPE Workflow for Explosive Analytes

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 4: Key Materials and Reagents for SPE of Explosive Analytes

Item	Function	Application Notes
Strata-X Cartridges	Copolymeric sorbent for mixed-mode interactions	Optimal for nitro-organic explosives in soil; provides >80% recovery [3].
Oasis HLB Cartridges	Hydrophilic-lipophilic balanced sorbent	Effective for smokeless powder residues; compatible with acetone or ethyl acetate elution [35].
Acetonitrile	Extraction and elution solvent	Used for soil extraction prior to SPE; compatible with nitro-aromatic explosives [3].
Acetone	Elution solvent	Effective for eluting smokeless powder compounds from Oasis HLB [35].
Ethyl Acetate	Elution solvent	Alternative to acetone for Oasis HLB; provides similar recovery with potential background reduction [35].
GC-ECD Instrumentation	Analytical detection	Sensitive for nitro-organic explosives; used for method validation [3].
GC-MS Instrumentation	Analytical detection and confirmation	Provides identification capability for smokeless powder compounds [35].

The step-by-step SPE methodology—conditioning, loading, washing, and elution—provides a robust framework for purifying and concentrating explosive analytes from complex matrices. Experimental comparisons demonstrate that sorbent selection critically impacts recovery efficiency, with Strata-X cartridges achieving >80% recovery for nitro-organic explosives in soil and Oasis HLB with ethyl acetate elution offering up to 8.2-fold detection limit improvements for smokeless powder residues. By following optimized protocols and understanding the mechanistic basis for each SPE step, researchers can develop highly effective sample preparation methods that enhance analytical sensitivity and reliability for explosive compound analysis.

The effectiveness of solid phase extraction (SPE) for isolating explosive analytes is critically dependent on the careful manipulation of the sample matrix. Parameters such as pH, ionic strength, and solvent composition directly control the efficiency of the interaction between the target analyte and the sophisticated sorbents designed for their recovery. In the context of explosive compounds, which often contain nitro-functional groups and exist in complex environmental matrices, optimizing these parameters is not merely beneficial but essential for achieving high recovery, superior selectivity, and reproducible results. This guide objectively compares the performance of various SPE sorbents and formats, providing a detailed examination of the experimental data and methodologies that underpin optimal protocol design for researchers and scientists in the field.

Fundamental Principles of SPE Optimization

Solid phase extraction is a powerful sample preparation technique that purifies and concentrates analytes by adsorbing them onto a solid sorbent, followed by a cleanup and elution process using appropriate solvents [36]. Its advantages over liquid-liquid extraction include improved throughput, significantly decreased organic solvent usage and waste, higher and more reproducible recoveries, and the avoidance of emulsions [37].

The recovery of explosive analytes is governed by the precise control of chemical interactions within the sample matrix. Three key principles form the foundation of effective optimization:

• pH Control: The sample pH profoundly influences the ionization state of both the analyte and the functional groups on the sorbent material. For ionizable analytes, adjusting the pH to suppress ionization is crucial for effective retention on reversed-phase sorbents. A general guideline is to adjust the sample to 2 pH units above the pKa for basic compounds and 2 pH units below the pKa for acidic compounds to neutralize their charge, making them more hydrophobic and enhancing retention [38].

• Ionic Strength Adjustment: The addition of salts can alter the ionic strength of the sample solution, affecting analyte solubility and its interaction with the sorbent through the "salting-out" effect. Increased ionic strength can enhance the retention of hydrophobic analytes by reducing their solubility in the aqueous phase. However, excessively high ionic strength may also promote the retention of interfering compounds, necessitating a careful balance [39].

• Solvent Composition: The selectivity of the extraction process is heavily influenced by the polarity and composition of the loading, washing, and elution solvents. The "like dissolves like" principle applies here; understanding the hydrophobicity (logP) of the target analyte is essential for selecting solvents with appropriate elution strength [36] [38].

Table 1: Key Compound Properties and Their Impact on SPE Optimization

Property	Description	Optimization Guidance
pKa	The pH at which an analyte is half-ionized and half-un-ionized	Adjust sample pH to suppress ionization for improved retention on reversed-phase sorbents [38]
logP	Measure of a compound's hydrophobicity	Guides selection of wash and elution solvent strength; low logP analytes require weaker elution solvents [36] [38]
logD	Distribution coefficient at a specific pH	Accounts for both ionization and hydrophobicity, providing a more complete picture for method development [36]

Comparative Performance of SPE Sorbents and Formats

The selection of an appropriate sorbent and SPE format is a critical decision that significantly impacts the efficiency of explosive analyte recovery. The following section compares the performance of various SPE materials and configurations, supported by experimental data.

Molecularly Imprinted Polymers (MIPs) vs. Traditional Sorbents

Molecularly Imprinted Polymers (MIPs) represent a significant advancement in sorbent technology. These are synthetic polymers containing tailor-made binding sites complementary to the target molecule in size, shape, and the position of functional groups [39]. When used as sorbents in SPE (MISPE), they offer superior selectivity for the pre-concentration of analytes and removal of disruptive compounds from complex sample matrices compared to non-imprinted polymers [39].

The optimization of MISPE is complex due to numerous interdependent variables. Key parameters that require optimization include contact time, sample pH, sample ionic strength, sorbent mass, sample flow rate, and the selection of loading, washing, and elution solvents [39]. The formation of appropriate bonds between the sorbent and analyte, influenced by these factors, directly impacts the percentage recovery [39].

Monolithic vs. Particle-Packed SPE Columns

A recent comparative study on the selective separation of trace lead from aqueous matrices provides a valuable model for evaluating SPE format performance, with implications for metallic markers or catalysts in explosive residues.

Table 2: Performance Comparison of Monolithic vs. Particle-Packed SPE Columns

Parameter	Particle-Based SPE (p-SPE)	Monolithic SPE (m-SPE)
Column Architecture	Sorbent particles packed into a column [40]	Single, porous polymer rod [40]
Permeability & Backpressure	Lower permeability, higher backpressure [40]	High permeability, low backpressure [40]
Porosity	Dependent on particle size and packing [40]	Inherently robust porosity [40]
Key Performance Advantages	Satisfactory retention; diverse sorbent chemistries [40]	Enhanced selectivity, reproducibility, and overall efficiency [40]
Optimal Flow Rate Regime	Requires controlled, slower flow (~1 mL/min) for proper binding [38]	Accommodates higher flow rates without performance loss due to its structure [40]

The m-SPE column demonstrated enhanced performance due to its physical characteristics, which resulted in superior selectivity and reproducibility for trace metal separation [40]. Both column types showed minimal interference from common matrix ions and were reusable over multiple cycles without significant efficiency loss [40].

Magnetic Dispersive Solid-Phase Extraction (MDSPE)

Magnetic DSPE is a streamlined variation of conventional SPE that simplifies the extraction process. In MDSPE, magnetic nanoparticles (MNPs) are directly dispersed into the sample solution, allowing for immediate and effective contact with the analytes. The sorbent is then easily separated using an external magnet, eliminating the need for centrifugation or filtration steps [41]. This format is particularly advantageous for complex samples, as it avoids common problems like cartridge clogging [41].

The synthesis of magnetic composites, such as the magnetic Ni-MOF-I used for extracting organochlorine pesticides, often involves combining MNPs with metal-organic frameworks (MOFs). MOFs are hybrid porous materials known for their high surface area, adjustable porosity, and structural flexibility [41]. The resulting composite benefits from easy magnetic separation and high adsorption capacity due to interactions like hydrophobic interactions, hydrogen bonding, and π–π stacking between the MOF and the analytes [41].

Experimental Protocols for Optimization

This section details specific methodologies for optimizing critical parameters, providing a reproducible framework for researchers.

Protocol for Optimizing MISPE Conditions

The following protocol, adapted from research on Molecular Imprinted Solid-Phase Extraction, outlines a systematic approach to optimizing an SPE method [39]:

• Column Packing: Pack MIP particles (15–500 mg) into an empty SPE cartridge, placing frits at the top and bottom.
• Pre-treatment: Wash the column consecutively with the selected washing solvent until no compounds are detected, then condition it with the loading solvent.
• Sample Loading: Load a standard solution of the target analyte dissolved in the optimal loading solvent onto the column at a controlled flow rate.
• Washing: Flush the column with the washing solvent to remove non-specifically bound molecules while retaining the target analytes.
• Elution: Elute the specifically bound analytes using an optimized elution solvent.
• Analysis: Collect all fractions (loading, washing, and elution) and analyze them using a suitable analytical technique (e.g., HPLC, LC-MS).

To achieve maximum recovery, multiple factors must be investigated: contact time, ion strength, sample pH, amount of sorbent, sample flow rate, and the nature of the loading, washing, and elution solvents [39].

Protocol for Investigating pH and Ionic Strength Effects

A study on the aqueous photooxidation of green leaf volatiles provides a robust model for systematically investigating the impact of pH and ionic strength, which is directly applicable to optimizing SPE methods for polar explosive residues [42].

• Solution Preparation: Prepare sample solutions with the same analyte-to-matrix molar ratio but different total concentrations to simulate dilute (e.g., cloud/fog) and concentrated (e.g., aerosol) conditions relevant to the sample matrix.
• Parameter Adjustment:
  • pH: Adjust using standard buffer solutions (e.g., acetate for pH 3–5, MES for pH 6, HEPES for pH 7–8, TAPS for pH 9–10) [40].
  • Ionic Strength: Modify by adding salts such as ammonium sulfate or sodium nitrate [42].
  • Sulfate/Anion Effects: Investigate the role of specific anions (e.g., sulfate, perchlorate, acetate) by adding their sodium salts [42] [40].
• Extraction and Analysis: Perform the SPE procedure under each set of conditions and analyze the eluents to determine reaction kinetics, degradation rates, or analyte recovery yields.

This study highlighted that the effects of pH, ionic strength, and sulfate are governed by the aqueous reaction medium conditions, with more pronounced effects often observed in concentrated solutions versus dilute ones [42].

The Scientist's Toolkit: Essential Research Reagents

Successful optimization of SPE for explosive analytes requires a suite of specialized reagents and materials. The table below details key solutions and their functions in method development.

Table 3: Essential Reagents for SPE Method Optimization

Reagent / Material	Function in SPE Optimization
Buffer Solutions (0.1 M)	Controls sample pH to manipulate analyte charge and optimize retention. Common buffers: Acetate (pH 3-5), MES (pH 6), HEPES (pH 7-8), TAPS (pH 9-10) [40].
Salt Solutions (e.g., NH₄NO₃, (NH₄)₂SO₄)	Modifies ionic strength to influence analyte solubility (salting-out effect) and sorbent-analyte interactions [42].
Conditioning Solvent (e.g., Methanol)	Activates the sorbent bed, causing bonded functionalities to "stand up" for maximum interaction with analytes [38].
Wash Solvents (varying organic strength)	Removes interfering compounds from the sorbent without eluting the target analyte; strength is tuned based on analyte logP [36] [38].
Elution Solvents (with additives)	Displaces the target analyte from the sorbent; strength and composition (e.g., with acid, base, or ion-pairing agents) are critical for high recovery [39].
Supramolecule-equipped Sorbents	Provides high selectivity for specific ions (e.g., Pb²⁺) via host-guest interactions, ideal for complex matrices [40].
Magnetic Nanocomposites (e.g., Ni-MOF-I)	Serves as a dispersible sorbent for MDSPE, enabling easy separation with a magnet and efficient extraction via multiple interaction mechanisms [41].

The optimization of pH, ionic strength, and solvent composition is a foundational aspect of developing robust and efficient SPE methods for the recovery of explosive analytes. As demonstrated, the performance of different sorbent formats—from highly selective MIPs and efficient monolithic columns to convenient magnetic dispersive sorbents—is profoundly influenced by these matrix conditions. The experimental data and protocols presented provide a clear roadmap for researchers to systematically optimize their SPE procedures. By understanding and controlling these critical parameters, scientists can achieve the high levels of recovery, reproducibility, and selectivity required for advanced analytical applications in security and environmental monitoring. The continuous development of novel sorbent materials promises even greater capabilities for the sensitive and selective detection of explosive compounds in the future.

In solid-phase extraction (SPE), the elution step is not merely the final stage but is often the determining factor for the success of the entire analytical method. This is particularly true in the demanding context of explosive analyte research, where complex matrices and trace-level concentrations necessitate exceptional recovery and selectivity. The fundamental challenge lies in disrupting the specific chemical interactions retaining target analytes on the sorbent without co-eluting excessive matrix interferences. Achieving this requires a deliberate balance—the elution solvent must be strong enough to quantitatively recover the analyte, yet selective enough to yield a clean extract [43].

The principles governing this process are universal across application fields, from pharmaceutical development to environmental monitoring and security screening. For researchers isolating explosive compounds, the stakes are high: inadequate elution strength leads to low recovery and poor quantification, while excessive strength compromises selectivity and can lead to ion suppression in subsequent mass spectrometric analysis [44]. This guide systematically compares elution solvent strategies, providing experimental data and protocols to empower scientists in making informed decisions that maximize recovery while maintaining specificity.

Fundamental Principles of Elution in SPE

Primary Interaction Mechanisms and Their Disruption

Successful elution requires understanding the primary mechanisms through which analytes interact with the sorbent. These interactions fall into two principal categories, each with distinct strategies for disruption [45].

• Polarity-Based Interactions: These include van der Waals forces, hydrogen bonding, and dipole-dipole interactions. In reversed-phase SPE (using C18, C8, or polymeric sorbents), retention occurs via hydrophobic interactions, which are effectively disrupted by organic solvents like methanol or acetonitrile. The "like dissolves like" principle applies—nonpolar analytes require nonpolar organic solvents for elution. Conversely, normal-phase SPE (using silica, cyano, or diol sorbents) retains polar analytes through polar interactions, which are disrupted by introducing more polar solvents like alcohols, acetone, or ethyl acetate into the nonpolar mobile phase [45] [5].

• Ion Exchange Interactions: These involve electrostatic attractions between charged analytes and oppositely charged sorbent functional groups. Disruption requires neutralizing the charge on either the analyte or the sorbent. This is typically achieved by adjusting the pH of the elution solvent—for instance, lowering the pH to protonate (and thus neutralize) a basic analyte for elution from a cation-exchange sorbent, or raising the pH to deprotonate an acidic analyte for elution from an anion-exchange sorbent. Alternatively, using a high ionic strength buffer introduces competing ions that displace the analyte through mass action [45] [5]. Ion-exchange interactions are significantly stronger than hydrophobic interactions, often requiring more strategic elution conditions [44].

The Concept of Elution Strength and Selectivity

Elution strength refers to a solvent's ability to desorb an analyte from a given sorbent. However, this is not an absolute property; it is relative to the specific analyte-sorbent interaction. In reversed-phase SPE, elution strength generally increases with the solvent's hydrophobicity (e.g., water < methanol < acetonitrile < isopropanol). In normal-phase SPE, strength increases with polarity (e.g., hexane < ethyl acetate < acetone < methanol) [5].

Selectivity, on the other hand, is the ability of an elution solvent to recover the target analyte while leaving interfering matrix components behind. Achieving selectivity often involves exploiting subtle differences in analyte properties, such as pKa, log P, or hydrogen-bonding capacity. For example, a solvent with a carefully tuned pH can neutralize and elute a specific basic analyte while leaving other basic compounds with different pKa values retained on an ion-exchange sorbent [43] [44].

Systematic Optimization of Elution Conditions

Optimizing elution is a multivariate challenge. A structured approach that systematically adjusts key parameters is far more efficient than one-factor-at-a-time experimentation.

Key Parameters for Optimization

The following parameters act as independent "control levers" that can be adjusted to fine-tune elution performance [43]:

• Solvent Strength and Composition: The type and percentage of organic modifier are the primary controls in reversed-phase SPE. Acetonitrile often provides sharper subsequent LC-MS peaks, while methanol can be more effective for polar compounds.
• pH Control: For ionizable analytes, this is the most powerful parameter. The goal is to elute the analyte in its neutral form. A general rule is to set the elution pH at least 2 units above the pKa for basic analytes or at least 2 units below the pKa for acidic analytes [43].
• Ionic Strength and Additives: In ion-exchange SPE, volatile salts (e.g., ammonium acetate, ammonium formate) are used as counter-ions to displace analytes. Additives like formic acid or ammonium hydroxide can modify pH and ionic interactions.
• Elution Volume: Expressed in bed volumes (BV), where 1 BV equals the volume of the sorbent bed itself. Typical elution volumes range from 2–5 BV. Stepwise elution with multiple fractions can help identify the optimal volume and minimize dilution [43] [9].
• Temperature: Moderate heating (30–40 °C) can reduce solvent viscosity and enhance the desorption of strongly retained analytes, particularly those with significant hydrogen-bonding capacity [43].

Experimental Workflow for Method Development

The following workflow provides a robust protocol for developing and optimizing an elution method [43].

Step 1: Select a Starting Recipe Choose an initial solvent composition based on the sorbent type and known analyte properties. The table below provides science-based starting points for common sorbents [43].

Table 1: Initial Elution Strategies by Sorbent Type

Sorbent Type	Initial Elution Strategy
Reversed Phase (C18, C8)	80–100% MeOH or ACN; add 0.5–2% formic acid for acidic analytes or 0.5–2% NH₄OH for basic analytes.
Strong Cation Exchange (SCX)	MeOH:water (80:20) + 2% NH₄OH or triethylamine (TEA); 2–4 BV.
Weak Cation Exchange (WCX)	ACN:water (90:10) + 1% NH₄OH; 2–3 BV.
Strong Anion Exchange (SAX)	ACN:water (80:20) + 1–2 M ammonium formate or 2% formic acid; 2–4 BV.
Weak Anion Exchange (WAX)	MeOH:water (90:10) + 2% formic acid or 1–2 M ammonium formate; 2–3 BV.
HILIC / Polar Interaction	ACN:water (90:10) with 50–200 mM volatile salt; increase water to 30–50% if retention is strong.

Step 2: Small-Scale Screening Prepare multiple identical SPE cartridges loaded with the target analyte. Using a design-of-experiments (DoE) approach, test a matrix of conditions. For example, for a reversed-phase extraction, you might test a combination of 3 organic solvent ratios and 3 pH levels [43].

Step 3: Fractional Elution and Analysis Elute the analyte using the test conditions, collecting the eluate in small fractions (e.g., 1 BV per fraction). Analyze each fraction to determine the recovery profile and identify the condition and volume that yield the highest recovery of the target analyte with the lowest matrix interference [9].

Step 4: Refinement and Validation Narrow the experimental conditions to the most promising range identified in the screening. Conduct final validation experiments to confirm robustness, testing the finalized protocol across different sample matrices and loading amounts to ensure consistent performance [43].

Comparative Elution Performance Data

The effectiveness of an elution strategy is ultimately quantified by recovery rates and selectivity. The following table synthesizes experimental data and best practices for achieving maximum recovery from various sorbent chemistries, with a focus on strategies relevant to challenging analytes.

Table 2: Elution Solvent Comparison for Maximum Recovery

Sorbent Chemistry	Optimal Elution Solvent Formulations	Expected Recovery Range	Key Considerations for Explosive Analytes
C18 / C8 (Reversed-Phase)	ACN:MeOH (90:10) + 1% Acetic Acid [43]	90-98%	High organic content is critical for non-polar nitroaromatics.
	MeOH:Water (95:5) + 0.5% NH₄OH [43]	85-95%	Basic modifiers can aid recovery of certain nitramines.
Polymeric Reversed-Phase (e.g., HLB)	ACN with 5-10% Dichloromethane [5]	92-99%	Added dichloromethane enhances elution of very hydrophobic compounds.
	MeOH:ACN (50:50) [5]	88-96%	Balanced polarity useful for complex analyte mixtures.
Strong Cation Exchange (SCX)	MeOH:Water (80:20) + 5% NH₄OH [43]	90-98%	Essential for eluting basic compounds; high pH neutralizes analyte.
	Pyridine Buffer (0.1M, pH 6.0) [5]	85-92%	Competing ion mechanism; useful for stubborn retention.
Strong Anion Exchange (SAX)	ACN:Water (80:20) + 2% Formic Acid [43]	90-98%	High ionic strength and low pH neutralize acidic analytes.
	ACN:20mM Ammonium Acetate (90:10) [46]	85-95%	MS-compatible volatile buffer.
Mixed-Mode (C18/SAX)	ACN:MeOH (80:20) + 2% Formic Acid [5]	85-95%	Disrupts both hydrophobic and ionic interactions simultaneously.

Troubleshooting Common Elution Problems

Even with a structured method, challenges can arise. The table below diagnoses common elution problems and provides targeted solutions.

Table 3: Troubleshooting Guide for Elution Problems

Observed Problem	Root Cause	Corrective Actions
Low Recovery	Inadequate elution strength or volume; analyte still charged [44].	Increase organic solvent percentage; adjust pH to neutralize analyte; increase elution volume; add a solvent "soak" step (stop flow for 30-60 sec) [43] [44].
High Background/Matrix Co-elution	Wash step was too weak; elution solvent is too strong and non-selective [44].	Optimize wash solvent strength to remove interferences before elution; consider a weaker, more selective elution solvent or stepwise elution [43] [9].
Poor Reproducibility	Inconsistent flow rates; variable drying of sorbent bed between steps [44].	Standardize and control flow rates; ensure sorbent does not run dry after conditioning and before sample loading [44] [9].
Analyte Breakdown	Overly aggressive elution conditions (extreme pH or reactive solvents).	Use milder solvents and buffers; avoid prolonged exposure to strong acids/bases; consider temperature-controlled elution.

The Scientist's Toolkit: Essential Research Reagents

Selecting the right materials is fundamental to developing a robust SPE elution method. The following table details key reagents and their functions in the context of this research.

Table 4: Essential Research Reagents for SPE Elution Optimization

Reagent / Material	Primary Function in Elution	Application Notes
Methanol (MeOH)	Polar protic elution solvent for reversed-phase and normal-phase SPE.	Effective for a wide polarity range; can cause higher backpressure [46].
Acetonitrile (ACN)	Polar aprotic elution solvent for reversed-phase SPE.	Often provides better LC-MS compatibility and sharper peaks than MeOH [43] [46].
Ammonium Hydroxide	pH modifier to deprotonate acidic analytes and elute from anion-exchange sorbents.	Typically used at 0.5-5% v/v; highly volatile for easy evaporation [43].
Formic Acid	pH modifier to protonate basic analytes and elute from cation-exchange sorbents; also a volatile ion-pairing agent.	Commonly used at 0.1-2% v/v; excellent MS compatibility [46].
Ammonium Acetate/Formate	Volatile buffers to control ionic strength and act as counter-ions in ion-exchange elution.	Concentrations of 10-200 mM are common; essential for MS-compatible methods [46].
Isopropanol (IPA)	Stronger, less polar organic solvent for eluting very hydrophobic analytes.	Can be mixed with ACN or MeOH to increase elution strength without increasing volatility excessively [43].

The selection and optimization of elution solvents in SPE is a critical analytical exercise in balancing opposing forces—strength versus specificity. For researchers working with explosive analytes, where recovery and data integrity are paramount, a systematic approach is non-negotiable. By understanding the fundamental chemical interactions, leveraging structured optimization workflows like DoE, and utilizing the comparative data and troubleshooting guides presented here, scientists can develop robust, reliable, and reproducible SPE methods. This ensures maximum recovery of target compounds, minimizes matrix effects in downstream analysis, and ultimately strengthens the validity of analytical results in critical research and development.

The forensic analysis of organic explosives in post-blast debris is often complicated by the presence of complex, interfering matrices such as motor oil. These matrices can cause significant ion suppression in Liquid Chromatography-Mass Spectrometry (LC/MS), reducing the sensitivity and reliability of the analysis [31] [18]. Solid-phase extraction (SPE) serves as a critical sample clean-up technique to mitigate these effects by isolating explosive analytes from co-extracted matrix components.

This case study objectively compares the performance of different hydrophobic SPE sorbents for the clean-up of organic explosives from methanolic extracts diluted with water, with a specific focus on a simulated sample prepared from commercial motor oil [31]. The findings are positioned within the broader thesis of optimizing SPE protocols to enhance analytical accuracy in forensic explosives research, a field that continuously adapts to the evolving nature of homemade explosives and complex crime scene samples [20].

Experimental Protocol

Materials and Reagents

• Explosive Standards: The study analyzed common organic explosives spanning different classes, including nitrate esters, nitramine, and nitroaromatic compounds [18].
• Internal Standards: Appropriate internal standards were used. Analyte peak areas were normalized to the internal standard peak area for quantitative analysis [18].
• SPE Sorbents: Four different reversed-phase SPE sorbents were compared [18]:
  • Oasis HLB: A hydrophilic-lipophilic balanced polymeric sorbent.
  • SDB-1: A styrene-divinylbenzene polymeric sorbent.
  • LiChrolut EN: A polymeric sorbent with a high specific surface area.
  • LiChrolut RP-18: An octadecyl-bonded silica-based sorbent.
• Solvents: High-purity solvents were used for extraction, dilution, and elution. Methanol was used to prepare the initial extracts from samples, which were then diluted with water before SPE [18].
• Simulated Sample: A challenging matrix was simulated by extracting commercial motor oil with methanol, spiking it with the target analytes, and concentrating it [31] [18].

Sample Preparation and SPE Procedure

The developed SPE procedure was performed manually as follows [18]:

• Conditioning: Each SPE cartridge was conditioned with an appropriate solvent.
• Sample Loading: The aqueous methanolic sample extract was percolated through the conditioned sorbent.
• Washing: A washing step with a water-methanol mixture was introduced to remove weakly retained matrix components without compromising analyte recovery.
• Drying: The sorbent bed was dried.
• Elution: The retained explosive compounds were eluted with a suitable organic solvent.

Instrumental Analysis

• LC/UV Analysis: LC with Ultraviolet (UV) detection was primarily used to evaluate the clean-up efficiency, as it allowed for the visualization of interfering compounds from the matrix [18].
• LC/MS Analysis: LC/MS was essential for confirming the presence of trace explosive compounds in complex samples and for verifying the reduction of ion suppression after SPE clean-up [31] [18].

Comparison of SPE Sorbent Performance

Retention of Explosive Analytes

The retention of various explosive compounds was studied by percolating different volumes of water samples containing methanol through each sorbent. A key finding was that polymeric sorbents (Oasis HLB, SDB-1, LiChrolut EN) demonstrated superior retention of explosive compounds compared to the octadecyl-bonded silica-based material (LiChrolut RP-18) [31] [18]. Polymeric sorbents were particularly effective at retaining more polar analytes like HMX and RDX [18].

Table 1: Retention Efficiency of Different SPE Sorbents for Explosive Analytes

Sorbent Type	Sorbent Name	Base Material	Retention of Explosives	Key Characteristics
Polymeric	Oasis HLB	Hydrophilic-Lipophilic Balanced Polymer	Excellent	Effective for polar analytes; limited co-extraction of matrix
Polymeric	SDB-1	Styrene-Divinylbenzene Polymer	Excellent	Good retentivity
Polymeric	LiChrolut EN	Polymeric	Excellent	High specific surface area
Silica-Based	LiChrolut RP-18	Octadecyl-Bonded Silica (C18)	Good, but inferior to polymers	Less effective for polar analytes

Clean-up Efficiency and Matrix Effects

The clean-up efficiency was evaluated using the simulated motor oil samples. The sorbent's specific surface area was identified as a critical factor. The polymeric sorbent with the smallest specific surface area was found to limit the co-extraction of non-volatile matrix components most effectively [31]. Among the tested sorbents, Oasis HLB was highlighted as the most convenient material, striking an optimal balance between high analyte recovery and efficient matrix removal [18].

The performance of the SPE method was quantitatively confirmed by a demonstrable reduction of ion suppression in subsequent LC/MS analysis, leading to improved detection limits and analytical accuracy [31] [18].

Table 2: Clean-up Performance and Analytical Outcomes for SPE Sorbents

Sorbent Name	Clean-up Efficiency (Motor Oil Matrix)	Effect on LC/MS Ion Suppression	Overall Assessment
Oasis HLB	Limited co-extraction of matrix components	Significant reduction	Most convenient and effective
SDB-1	Good clean-up	Data not specified in sources	Effective for retention
LiChrolut EN	Good clean-up	Data not specified in sources	Effective for retention
LiChrolut RP-18	Inferior clean-up compared to polymers	Less effective reduction	Less suitable for this application

The Scientist's Toolkit: Essential Research Reagents

The following table details key reagents and materials essential for conducting this SPE clean-up research.

Table 3: Key Research Reagents and Materials for SPE of Explosives

Item Name	Function/Brief Explanation
Oasis HLB SPE Cartridge	Hydrophilic-lipophilic balanced copolymer for broad-spectrum retention of explosive analytes.
SDB-1 SPE Cartridge	Styrene-divinylbenzene polymer sorbent for efficient extraction of organic compounds from water.
LiChrolut EN SPE Cartridge	A polymeric sorbent with a high surface area for high analyte retention.
C18 (RP-18) SPE Cartridge	Octadecyl-silica bonded phase; a common reversed-phase sorbent used as a comparison.
Organic Explosive Standards	Certified reference materials for nitrate esters, nitramines, and nitroaromatics for method development and calibration.
Deuterated Internal Standards	Isotopically labeled analogs of target analytes to correct for losses during sample preparation and matrix effects in LC/MS.
LC/MS System	Analytical instrument for separating, detecting, and confirming target explosives with high sensitivity.
LC/UV System	Used for monitoring interfering compounds and evaluating SPE clean-up efficiency visually.

Workflow and Logical Diagrams

The following diagram illustrates the logical sequence of the experimental and decision-making process for selecting an optimal SPE sorbent, as derived from the case study.

Diagram 1: SPE Sorbent Selection Logic

The experimental workflow for comparing sorbent performance, from sample preparation to final analysis, is outlined below.

Diagram 2: Experimental Workflow for SPE Comparison

This comparative study demonstrates that the choice of SPE sorbent is paramount for the effective clean-up of organic explosives from complex matrices like motor oil. The evidence shows that polymeric sorbents, particularly Oasis HLB, are markedly more effective than traditional octadecyl-bonded silica for this application [31] [18]. The key to its performance lies in its strong retention of a wide range of explosive compounds, coupled with its ability to limit the co-extraction of interfering matrix components due to its specific surface area properties.

The successful implementation of the developed SPE procedure with a washing step significantly reduces matrix effects, as confirmed by the observed reduction in ion suppression during LC/MS analysis [31] [18]. This translates directly to more reliable and sensitive identification of trace-level explosives in forensic casework.

For researchers and laboratory managers, especially those working under ISO 17025 accreditation, this case study provides a validated pathway for method improvement. While adopting new techniques requires thorough validation, the clear advantages demonstrated here underscore the value of updating sample preparation protocols to include advanced polymeric SPE sorbents for explosives analysis in challenging matrices [20].

Solid-phase extraction (SPE) remains a cornerstone technique in analytical chemistry, particularly for the clean-up and pre-concentration of complex samples in forensic and environmental analysis. Within the specialized field of explosives residue analysis, the choice of SPE configuration directly impacts method sensitivity, selectivity, and operational efficiency. While traditional cartridge and disk formats have long dominated laboratory practice, a significant trend toward miniaturization has emerged, with pipette-tip SPE (PT-SPE) representing a paradigm shift in sample preparation technology. This evolution responds to the growing need for analyzing limited-volume samples while maintaining stringent sensitivity requirements for trace-level analytes.

The analysis of organic explosives presents unique challenges due to the diverse chemical properties of target compounds—including nitrate esters, nitramines, and nitroaromatics—and the complex matrices in which they are found. Effective sample preparation must isolate these analytes from interfering substances while achieving high recovery rates for compounds with varying polarities. The selection of appropriate sorbent materials and device formats constitutes a critical methodological decision that can determine analytical success or failure. This guide systematically compares the performance characteristics of disk formats and pipette-tip SPE configurations, providing researchers with experimental data and protocols to inform their method development decisions for specific analytical scenarios in explosives recovery.

Sorbent Materials: Performance Comparison for Explosives Analysis

The efficacy of SPE depends fundamentally on the selective interactions between target analytes and the sorbent material. Research has extensively evaluated various sorbents for their retention capabilities toward organic explosives, with significant differences observed in recovery rates and clean-up efficiency.

Table 1: Comparison of SPE Sorbent Performance for Organic Explosives Recovery

Sorbent Material	Sorbent Type	Mean Recovery (%)	Key Strengths	Optimal Application
Oasis HLB	Hydrophilic-lipophilic balance polymer	>80% [17] [47]	Excellent for polar analytes (HMX, RDX); limits co-extraction of matrix components	Broad-spectrum explosives analysis in complex matrices
Isolute ENV+	Hydrophilic divinylbenzene polymer	>80% [47]	High retention of polar explosives	Aqueous samples; polar analyte pre-concentration
Porous Graphitic Carbon	Carbon-based	Variable [17]	Strong retention of large, polar molecules; efficient at extreme pH	Challenging separations of closely related structures
Octadecyl-bonded silica (C18)	Reversed-phase silica	Lower than polymeric [17]	Widely available; familiar chemistry	Less complex matrices; non-polar explosives
LiChrolut EN	Polymeric styrene-divinylbenzene	Moderate [17]	Good capacity; balanced retention	General purpose clean-up
Dual-sorbent combinations	Mixed modes	Improved vs single sorbent [47]	Complementary selectivity; reduced matrix effects	Complex samples (dried blood, oily residues)

Polymeric sorbents, particularly Oasis HLB, have demonstrated superior performance for retaining polar explosive analytes like HMX and RDX compared to traditional octadecyl-bonded silica materials [17]. Their balanced hydrophilic-lipophilic properties enable effective extraction of explosives spanning a wide polarity range from aqueous samples. The development of dual-sorbent SPE approaches represents a significant advancement, with research showing that combining sorbents in series can reduce matrix effects by approximately 10-fold while improving detection limits to femtogram levels in multiple matrices including wastewater, cooking oil residues, and dried blood [47].

Disk Format SPE: Configurations and Applications

Traditional Disk SPE Platforms

Disk format SPE configurations feature sorbent material embedded in a membrane or enclosed in a cartridge, providing a uniform, predictable flow path for samples and solvents. This format typically accommodates larger sample volumes than pipette-tip alternatives and is available in various diameters to suit different capacity requirements. In explosives analysis, disk SPE has proven valuable for processing environmental water samples and extracts from post-blast investigation materials.

A comprehensive study evaluating sorbents for organic explosives clean-up found that polymeric sorbents with smaller specific surface areas, particularly Oasis HLB, limited the co-extraction of matrix components from simulated samples prepared from commercial motor oil [17]. The inclusion of an optimized washing step in the disk SPE procedure significantly reduced ion suppression effects in subsequent LC/MS analysis, demonstrating the importance of method optimization alongside sorbent selection.

Experimental Protocol: Disk SPE for Organic Explosives in Complex Matrices

Objective: Extract and clean-up organic explosives from complex matrices using disk SPE prior to LC-MS analysis [17] [47].

Materials and Reagents:

• SPE disks: Oasis HLB (60 mg, 3 mL cartridge) or equivalent polymeric sorbent
• Explosives standards: Nitroglycerin (NG), ethylene glycol dinitrate (EGDN), pentaerythritol tetranitrate (PETN), cyclotrimethylenetrinitramine (RDX), cyclotetramethylenetetranitramine (HMX), 2,4,6-trinitrotoluene (TNT)
• Internal standards: [^{15}N]-TNT, [^{13}C]-RDX for quantification
• Solvents: HPLC-grade methanol, acetonitrile, acetone, reagent water
• Sample: Methanolic extracts of post-blast swabs diluted with water (final methanol concentration <10%)

Procedure:

• Conditioning: Sequentially pass 2 mL of methanol and 2 mL of reagent water through the disk under gentle vacuum (≈1-2 mL/min).
• Sample Loading: Dilute methanolic sample extract with water to reduce organic content (<10% methanol). Load 100-500 mL sample at controlled flow rate (3-5 mL/min).
• Washing: Pass 2 mL of 20:80 (v/v) methanol:water solution to remove interfering compounds.
• Drying: Apply full vacuum for 10-15 minutes to remove residual water.
• Elution: Pass 2 × 1 mL aliquots of ethyl acetate:acetone (90:10, v/v) without vacuum, letting each aliquot soak for 1 minute before applying gentle vacuum.
• Concentration: Evaporate eluate to dryness under gentle nitrogen stream at 30°C.
• Reconstitution: Redissolve in 100 μL methanol for LC-MS analysis.

Performance Data: This protocol achieved recoveries >80% for most organic explosives, with limits of detection improved approximately 10-fold compared to single-sorbent approaches in complex matrices [47].

Diagram 1: Disk SPE Workflow for Explosives Analysis

Pipette-Tip SPE: Miniaturized Configurations for Specific Scenarios

Principles and Advantages of Pipette-Tip SPE

Pipette-tip SPE (PT-SPE) represents the miniaturization of conventional solid-phase extraction, with sorbent material packed between two frits within a standard pipette tip. This configuration enables efficient extraction using microliter volumes of samples and solvents, significantly reducing consumption while maintaining analytical performance. The technique operates on dispersive SPE principles, where loose sorbent mixes with the sample solution during aspirating and dispensing cycles, facilitating optimal analyte recovery and reduced matrix effects [48].

PT-SPE offers several distinct advantages for specific scenarios in explosives analysis: minimal sample volume requirements (as low as 25 μL elution volume), compatibility with high-throughput automation, dramatically reduced solvent consumption, and elimination of evaporation steps through concentration effects [49]. These characteristics make pipette-tip SPE particularly valuable when sample quantity is limited—common in forensic applications—or when processing large sample batches for environmental monitoring.

Experimental Protocol: Pipette-Tip SPE for Trace Explosives Analysis

Objective: Extract trace-level organic explosives from small-volume samples using pipette-tip SPE with concentration for enhanced sensitivity [49] [50].

Materials and Reagents:

• PT-SPE tips: All carbon reverse phase, 50 mg bed weight, 1 mL capacity [48] or equivalent
• Explosives standards: Target analytes based on application (e.g., NG, EGDN, TNT, RDX)
• Internal standards: Deuterated or (^{13})C-labeled explosives for quantification
• Solvents: LC-MS grade methanol, acetonitrile, ethyl acetate, reagent water
• Sample: Aqueous extracts from forensic swabs or environmental water samples

Procedure:

• Tip Conditioning: Aspirate and dispense 2 × 1 mL methanol, then 2 × 1 mL reagent water (5 cycles each).
• Sample Loading: Aspirate and dispense 1 mL sample solution for 10-15 cycles at controlled speed.
• Washing: Aspirate and dispense 1 mL of 5:95 (v/v) methanol:water solution for 5 cycles.
• Elution: Aspirate and dispense 2 × 50 μL aliquots of ethyl acetate:methanol (80:20, v/v) for 10 cycles each.
• Direct Analysis: Transfer eluate to autosampler vial for LC-MS analysis without concentration.

Performance Data: The micro-elution capability of PT-SPE tips enables elution volumes as low as 25 μL, achieving up to 8-fold concentration factors without evaporation steps [49]. This approach has demonstrated recovery rates comparable to conventional SPE with significantly reduced solvent consumption and processing time.

Diagram 2: Pipette-Tip SPE Workflow for Trace Explosives

Comparative Performance Data: Disk vs. Pipette-Tip SPE

Direct comparison of disk and pipette-tip SPE formats reveals distinct performance characteristics that inform their application in specific analytical scenarios. The following data, synthesized from multiple experimental studies, provides quantitative metrics for evaluating each format's suitability for particular requirements in explosives analysis.

Table 2: Performance Comparison of Disk SPE and Pipette-Tip SPE Formats

Parameter	Disk SPE	Pipette-Tip SPE	Analytical Implications
Sample Volume Range	100-1000 mL [17]	0.1-1 mL [48] [49]	Disk preferred for large volume pre-concentration
Elution Volume	1-5 mL [17]	25-100 μL [49]	PT-SPE provides automatic concentration factor
Solvent Consumption	10-20 mL per extraction [17]	0.1-2 mL per extraction [50]	PT-SPE reduces solvent use >80%
Processing Time	30-60 minutes (manual) [17]	10-20 minutes [49]	PT-SPE offers faster throughput
Automation Compatibility	Limited without specialized systems	High compatibility with liquid handlers [49]	PT-SPE preferable for high-volume labs
Limit of Detection	Low femtogram range with concentration [47]	Comparable with concentration factor [49]	Both achieve ultra-trace sensitivity
Recovery for Polar Explosives (RDX, HMX)	>80% with polymeric sorbents [17]	Comparable with optimized cycles [50]	Sorbent selection more critical than format
Matrix Tolerance	Excellent with optimized washing [17]	Good with sufficient wash cycles [50]	Disk format slightly superior for very dirty samples

The data indicates that disk formats maintain advantages for processing large sample volumes and highly complex matrices, while pipette-tip configurations excel in scenarios requiring minimal solvent consumption, rapid processing, and automated workflows. For ultra-trace analysis, both formats can achieve comparable sensitivity when optimized, though through different concentration mechanisms—manual solvent evaporation for disk SPE versus minimal elution volumes for pipette-tip SPE.

Application Scenarios: Strategic Selection Guidance

Scenario-Based Format Recommendations

The optimal selection between disk and pipette-tip SPE formats depends on specific analytical requirements and constraints. Based on comparative performance data, the following scenarios illustrate strategic format selection:

Large Volume Environmental Sampling: For monitoring explosives in groundwater, surface water, or wastewater where sample volumes of 100-1000 mL require processing to achieve adequate method detection limits, disk SPE formats are unequivocally superior. Their higher capacity and compatibility with vacuum manifolds enable efficient extraction of large volumes, with demonstrated success in detecting new explosives traces in untreated wastewater at femtogram levels [47].

Forensic Samples with Limited Availability: When analyzing explosives residues from crime scene investigations where sample material is precious and limited—such as swabs from human skin or small surface areas—pipette-tip SPE provides distinct advantages. The ability to achieve satisfactory recovery from sample volumes as low as 100 μL makes PT-SPE ideal for these applications, with the additional concentration factor compensating for limited starting material [49].

High-Throughput Screening Laboratories: In facilities processing large batches of samples for security screening or environmental monitoring, the automation compatibility of pipette-tip SPE formats significantly enhances throughput. Compatibility with common liquid handling systems enables simultaneous processing of 96 samples with minimal manual intervention, reducing analyst time while maintaining data quality [49].

Complex or Dirty Matrices: For samples with high levels of interfering compounds—such as motor oil extracts, biological fluids, or soil extracts—the more extensive washing capabilities and larger sorbent bed mass of disk SPE formats provide superior clean-up efficiency. Research demonstrates that polymeric sorbents like Oasis HLB in disk format effectively limit co-extraction of matrix components from challenging samples like commercial motor oil [17].

Research Reagent Solutions: Essential Materials for Explosives Analysis

Successful implementation of SPE methods for explosives analysis requires specific materials and reagents optimized for target analytes and matrices. The following table details essential components for establishing robust SPE protocols in this specialized application area.

Table 3: Essential Research Reagents for Explosives Analysis SPE

Reagent/Sorbent	Function/Purpose	Example Applications
Oasis HLB Sorbent	Hydrophilic-lipophilic balanced copolymer; retains polar and non-polar explosives	Broad-spectrum extraction of nitrate esters, nitramines, nitroaromatics [17]
Porous Graphitic Carbon	Retains large molecules, highly polar compounds; stable at extreme pH	Separation of closely related structures; challenging polar explosives [48]
All Carbon Reverse Phase Tips	Pipette-tip format with graphite carbon source; efficient for large/polar molecules	High-throughput processing of limited samples before LC-MS or PCR [48]
Mixed-Mode Cation Exchange Sorbents	Combined reversed-phase and cation exchange mechanisms	Selective extraction of basic explosive compounds; cleaner extracts [50]
Deuterated Internal Standards	Compensation for matrix effects and variability; quantification accuracy	[^{15}N]-TNT, [^{13}C]-RDX for precise quantification in complex matrices [47]
Ethyl Acetate:Acetone (90:10)	Elution solvent for organic explosives; high elution strength	Efficient recovery of non-polar to moderately polar explosives from polymeric sorbents [17]

Emerging Trends and Future Perspectives

The continuing evolution of SPE technologies for explosives analysis points toward several promising developments. The integration of novel sorbent materials—including metal-organic frameworks (MOFs), molecularly imprinted polymers (MIPs), and functionalized nanomaterials—into both disk and pipette-tip formats shows potential for enhanced selectivity and capacity [51]. These advanced materials may address current limitations in extracting particularly challenging analyte classes, such as organic peroxide explosives.

Miniaturization trends continue to advance, with μ-SPE techniques gaining traction for their environmental benefits and compatibility with limited sample volumes. Recent research demonstrates successful determination of 40 drugs of abuse in urine using homemade pipette-tip extraction, highlighting the potential for similar approaches in explosives analysis [50]. The development of low-cost, cellulose-based composite sorbents offers sustainable alternatives to commercial materials while maintaining analytical performance, particularly valuable for resource-limited settings [52].

The combination of multiple sorbents in series represents another significant advancement, with dual-sorbent SPE demonstrating improved recoveries and reduced matrix effects across diverse sample types including wastewater, cooking oil residues, and dried blood [47]. This approach provides a versatile solution for robust and highly sensitive detection of explosives residues in forensic and environmental applications.

As analytical challenges continue to evolve—driven by the emergence of new explosive compounds and increasingly stringent detection requirements—both disk and pipette-tip SPE formats will maintain important roles in the analytical chemist's toolkit, with format selection increasingly dictated by specific scenario requirements rather than generic preferences.

In modern analytical laboratories, the demand for faster and more efficient sample analysis is ever-increasing. Liquid chromatography-mass spectrometry (LC-MS) has become a cornerstone technology across various fields, from clinical biochemistry to forensic explosives analysis. A significant challenge in these applications is the need for high-throughput analysis without compromising sensitivity or accuracy, particularly when dealing with complex samples and trace-level analytes. Online coupling techniques, which integrate sample preparation and concentration directly with the LC-MS system, have emerged as powerful solutions to this challenge. This guide objectively compares the performance of different online coupling approaches for improving analytical throughput, with specific application to the recovery of explosive analytes using solid phase extraction sorbents.

Key Online Coupling Approaches for Throughput Enhancement

Trapping and Micro-LC Systems

The trapping-micro-LC (T-µLC) system represents a significant advancement for ultra-sensitive, robust, and high-throughput quantification of challenging analytes. This approach utilizes two synchronized LC components: a high-flow LC for fast, large-capacity sample loading and a low-flow LC for sensitive analysis [53].

Experimental Protocol: The T-µLC method involves three critical steps: (I) selective, rapid trapping at a high-flow rate (1000 μL/min) on a large internal diameter trap that concentrates target analytes while removing hydrophilic matrix components; (II) selective delivery and peak compression where the trap is switched in line with the analytical µLC column and back-flushed with a microflow gradient (25 μL/min); and (III) analysis on the µLC-MS system which only processes the fraction containing concentrated analytes, preventing hydrophobic and hydrophilic matrix components from entering and potentially damaging the system [53].

Performance Data: When applied to vitamin D metabolites in serum samples, the T-µLC system with narrow-window isolation selected reaction monitoring (NWI-SRM) achieved remarkable lower limits of quantification (LOQs) within a 9-minute cycle: 1.0 pg/mL for 1,25(OH)₂D₃, 5.0 pg/mL for 24,25(OH)₂D₃, and 30 pg/mL for both 25(OH)D₂ and 25(OH)D₃. These LOQs were markedly lower than any existing LC-MS methods, demonstrating the sensitivity benefits of this approach [53].

LC Multiplexing Systems

LC multiplexing provides an alternative approach to throughput enhancement by operating multiple LC streams in parallel. This methodology involves running two independent LC channels simultaneously before introduction to a single mass spectrometer, effectively minimizing mass spectrometer downtime during column equilibration and dead volume periods [54].

Experimental Protocol: A typical multiplexing setup utilizes a dual-channel UHPLC system with identical columns in each channel. The system is programmed to stagger injections between the two channels, allowing one channel to perform sample loading and separation while the other is actively eluting compounds into the MS. Synchronization between LC components and the mass spectrometer is controlled by specialized software that manages valve switching and timing [54].

Performance Data: In a comparative study analyzing 55 drugs of abuse, the use of dual LC channels reduced acquisition time from 7:07 minutes per sample (single channel) to 4:25 minutes per sample, representing a 1.7× improvement in sample throughput. The precision (%CV) for both single and dual channel modes was below 10% for all analytes, demonstrating that multiplexing maintained data quality while significantly improving efficiency [54].

System Optimization Approaches

Beyond specialized instrumentation, substantial throughput gains can be achieved through systematic optimization of conventional LC-MS parameters. These approaches are particularly valuable for laboratories working with existing equipment and limited budgets.

Experimental Protocols:

• Dead Volume Reduction: Bypassing the mixing chamber and dampener in Agilent 1100/1200 series HPLC systems reduced retention times by almost a full minute, enabling 2.4-minute injection-to-injection cycles [55].
• Gradient Optimization: Modifying gradient profiles to quickly return to original conditions allows system re-equilibration to occur during instrumental "think time," reducing overall cycle times [55].
• Flow Rate Increase: Raising flow rates from 500 to 1000 μL/min, despite an average 34% reduction in signal intensity, enabled shorter run times with minimal impact on detection capabilities for most compounds in absorption, distribution, metabolism, and excretion (ADME) assays [55].
• Column Technology Utilization: Employing monolithic columns, which feature higher permeability than packed-bed columns, allows two- to three-fold increases in mobile phase flow rates without exceeding pressure limits [56].

Table 1: Comparison of Throughput Enhancement Approaches for LC-MS Analysis

Approach	Mechanism	Throughput Gain	Key Applications	Limitations
Trapping-Micro-LC	Online sample concentration and matrix removal	9-minute cycles for ultra-sensitive quantification	Low-abundance metabolites (e.g., vitamin D metabolites)	System complexity; method development intensity
LC Multiplexing	Parallel analysis with staggered injections	1.7× improvement (4:25 min vs. 7:07 min per sample)	High-volume targeted screening (e.g., drugs of abuse)	Requires specialized hardware and software
System Optimization	Parameter optimization on existing equipment	2× reduction in run time (10 to 5 minutes)	General laboratory applications	Sensitivity trade-offs at higher flow rates

Application to Explosive Analytes Recovery

The analysis of explosive traces presents particular challenges, including low environmental concentrations and complex sample matrices. Online coupling techniques offer significant advantages for this application by improving sensitivity while maintaining the high throughput necessary for security and forensic applications.

Magnetic Solid Phase Micro-Extraction

Magnetic solid phase micro-extraction (MSPME) with LC-MS/MS detection has been developed as an efficient online approach for trace explosive analysis in water samples.

Experimental Protocol: A method utilizing oxidized multiwalled carbon nanotube/Fe₃O₄ composite material as an adsorbent was developed for 2,4,6-trinitrotoluene (TNT) analysis. The optimized protocol involves: (1) adsorbent preparation through acid oxidation of MWCNT to enhance surface functional groups; (2) extraction with 25 mg of adsorbent in 10 mL sample for 60 seconds; (3) magnetic separation; (4) elution with 1 mL of methanol; and (5) LC-MS/MS analysis using a C18 column with mobile phase of 5 mM ammonium formate and methanol in gradient mode [57].

Performance Data: The MSPME method achieved an impressive limit of detection of 0.025 ng/mL for TNT with high precision (%RSD 2.3%). The method demonstrated excellent linearity (0.1-100 ng/mL) and high enrichment factor (83), enabling reliable detection of TNT at concentrations significantly below the US EPA maximum allowable limit of 2 μg/L for drinking water [57].

Environmental Prevalence and Significance

Understanding background levels of explosive residues is crucial for interpreting analytical results in forensic investigations. Recent surveys utilizing advanced LC-MS methodologies have provided updated data on environmental prevalence.

Experimental Protocol: A comprehensive study collected 450 swab and vacuum samples from public locations across Great Britain, including airports, public transportation, and stadiums. Analysis was performed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and ion chromatography-mass spectrometry (IC-MS) to screen for a wide range of explosives analytes with sub-nanogram detection limits [25].

Performance Data: The study detected only eight low nanogram-level traces of organic high explosives (HMX, NG, PETN, and RDX) across all samples, representing a mere 1.8% positivity rate. This demonstrates that high explosives traces remain uncommon in public environments, strengthening the association between detection and explosives-related activities. The low prevalence highlights the critical need for highly sensitive analytical methods capable of detecting these rare occurrences [25].

Table 2: Analytical Performance Comparison for Explosive Compound Analysis

Analytical Method	Target Analyte	Limit of Detection	Throughput	Application Context
MSPME-LC-MS/MS	TNT	0.025 ng/mL	High (simple extraction)	Water samples
LC-HRMS Screening	Multiple explosives (HMX, NG, PETN, RDX)	Sub-nanogram	High-throughput screening	Environmental prevalence
Traditional Methods	Varies	Varies (typically higher)	Moderate to low	General explosive analysis

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Online SPE-LC-MS Analysis

Item	Function	Application Example
Oxidized MWCNT/Fe₃O₄ composite	Magnetic adsorbent for explosive traces	Extraction of TNT from water samples [57]
C8 and C18 stationary phases	Trap and analytical column chemistries	T-µLC systems for selective trapping and separation [53]
Monolithic columns	High-permeability separation media	Fast separations at elevated flow rates without excessive backpressure [56]
Cookson-type reagents	Derivatization agents	Enhancement of ionization efficiency for vitamin D metabolites [53]
Multiple reaction monitoring (MRM)	Highly specific MS detection	Targeted analysis of explosive compounds in complex matrices [58]

Workflow Integration and Logical Relationships

The integration of online sample preparation with LC-MS analysis involves carefully orchestrated steps and decision points, as illustrated in the following workflow:

Diagram 1: Online SPE-LC-MS Workflow. NWI-SRM: Narrow-Window Isolation Selected Reaction Monitoring; MRM: Multiple Reaction Monitoring.

Online coupling techniques for LC-MS analysis provide powerful options for enhancing throughput while maintaining or improving sensitivity. The optimal approach depends on specific application requirements, available instrumentation, and throughput needs. Trapping-micro-LC systems offer exceptional sensitivity for challenging low-abundance analytes, LC multiplexing maximizes instrument utilization for high-volume screening, and systematic optimization extends the capabilities of existing equipment. For explosive analyte recovery, magnetic solid phase micro-extraction coupled with LC-MS/MS demonstrates excellent performance for trace-level detection. The continued advancement of these online coupling methodologies will further streamline analytical workflows across diverse fields, from clinical research to forensic investigation.

Solving Common SPE Problems to Maximize Explosive Analyte Recovery

The forensic analysis of organic explosives presents a significant analytical challenge, particularly when trace-level residues are embedded in complex matrices such as motor oils, blood, or swabs from bombing scenes [17] [47]. Achieving high recovery rates in solid-phase extraction (SPE) is paramount, as low recovery compromises quantification accuracy, leads to poor reproducibility, and can ultimately invalidate method validation [59]. The core issues of sorbent mismatch, weak elution solvents, and insufficient sorbent volume or sample loading capacity are frequently interconnected problems that plague methods for explosive analytes, which range from relatively polar nitramines like RDX and HMX to less polar nitroaromatics [17]. This guide objectively compares the performance of different SPE sorbents and methodologies, providing experimental data to help researchers optimize recovery of explosive analytes.

Sorbent Mismatch: Selecting the Optimal Chemistry for Explosive Analytes

The selection of an appropriate sorbent is the foundational step in designing an efficient SPE protocol. A sorbent mismatch, where the chemistry of the stationary phase is ill-suited to the properties of the target explosive analytes, is a primary cause of irreversible analyte loss and low recovery [60] [59].

Comparative Performance of Sorbent Types

Experimental comparisons consistently demonstrate that polymeric sorbents outperform traditional silica-based materials for a broad spectrum of organic explosives. A systematic study comparing four different reversed-phase SPE sorbents for the clean-up of forensic samples found that polymeric sorbents such as Oasis HLB and Isolute ENV+ retained explosive compounds significantly better than octadecyl-bonded silica-based materials (e.g., LiChrolut RP18) [17] [18]. This is particularly true for more polar analytes like HMX and RDX, which showed poor retention on C18 silica [17]. The superior performance of Oasis HLB and Isolute ENV+ was confirmed in a larger study of 44 organic explosives, where they yielded average recoveries of over 80% [47].

Table 1: Recovery of Explosive Analytes on Different SPE Sorbents

Sorbent Type	Specific Example(s)	Key Characteristics	Recovery for Polar Explosives (e.g., RDX, HMX)	Overall Recovery for Explosive Mixtures
Hydrophilic-Lipophilic Balanced (HLB) Polymer	Oasis HLB	Retains a wide spectrum of analytes; good for polar compounds	High [17]	>80% [47]
Porous Styrene-Divinylbenzene Polymer	Isolute ENV+, SDB-1, LiChrolut EN	High surface area; strong retention for non-polar and some polar compounds	High [17]	>80% (Isolute ENV+) [47]
Octadecyl-Bonded Silica (C18)	LiChrolut RP18	Hydrophobic interactions; well-suited for non-polar compounds	Low [17]	Lower than polymeric sorbents [17]

Experimental Protocol: Sorbent Comparison

The protocol for comparing sorbent efficacy, as detailed in the study by [17], is as follows:

• Sorbent Conditioning: Cartridges (e.g., 60 mg Oasis HLB, 100 mg SDB-1, 200 mg LiChrolut EN, 200 mg LiChrolut RP18) are conditioned with 2 mL of methanol followed by 2 mL of water.
• Sample Loading: Aqueous samples (100 mL at pH 7) containing a mixture of explosive compounds (nitrate esters, nitramines, nitroaromatics) and a small percentage of methanol (e.g., 5%) are percolated through the sorbent at a flow rate of 5-10 mL/min.
• Washing: The sorbent bed is dried under vacuum for 10 minutes and then washed with 1 mL of a water/methanol mixture (e.g., 80/20, v/v).
• Elution: Analytes are eluted with 2 mL of isopropanol/dichloromethane (e.g., 10/90, v/v).
• Analysis: The eluate is evaporated to dryness under a gentle stream of nitrogen, reconstituted in methanol, and analyzed via LC/UV or LC/MS. Recovery is calculated by comparing the peak areas of extracted samples to those of standard solutions [17].

Weak Eluents: Ensuring Complete Analyte Desorption

The elution step must be strong enough to overcome the interactions retaining the analytes on the sorbent. Using an elution solvent that is too weak is a common, yet easily remedied, cause of low recovery [60] [61].

Elution Solvent Optimization

Experiments show that explosive compounds, which are often retained strongly on polymeric sorbents, require solvents of sufficient elution strength for efficient recovery. For reversed-phase SPE, relatively non-polar solvents like dichloromethane and isopropanol are effective [17]. The study on explosive analysis found that a mixture of isopropanol/dichloromethane (10/90, v/v) provided effective elution from Oasis HLB sorbent after a proper washing step [17]. It is critical to balance the solvent strength to ensure complete analyte elution while leaving highly hydrophobic matrix interferences behind, thereby achieving a cleaner extract [60] [61].

Protocol: Evaluating Elution Efficiency

To diagnose and solve poor elution, a systematic evaluation of the elution step is necessary [59] [61]:

• Fractional Analysis: Process a standard sample through the SPE protocol. Collect the eluate as multiple fractions (e.g., 2 x 1 mL instead of 1 x 2 mL) and analyze each separately.
• Identify Loss: If a significant amount of the analyte is found in the second fraction, the elution volume is insufficient. If analyte is found in both the wash and elution fractions, the wash solvent is too strong. If the analyte is not found in any fraction, it was never eluted, indicating the elution solvent is too weak.
• Optimize Solvent: If the eluent is too weak, increase its strength incrementally. For reversed-phase SPE, this means using a solvent with a higher eluotropic value (e.g., moving from methanol to acetonitrile to dichloromethane, or using mixtures with additives). For instance, a basic drug recovered poorly (~40%) with a methanol wash was successfully eluted with 5% NH4OH in methanol, improving recovery to over 85% [59].

Insufficient Volume: Addressing Capacity and Breakthrough

Insufficient volume in SPE can refer to two issues: an undersized sorbent bed that lacks the capacity to retain all analytes, or an excessive sample volume that leads to analyte "breakthrough" before loading is complete [60] [59].

The Dual-Sorbent Solution for Complex Matrices

A powerful approach to manage complex matrices and prevent column overloading is the use of dual-sorbent SPE [47]. This strategy employs two different sorbents in series to achieve superior clean-up. The first sorbent is chosen to remove matrix interferences, while the second, more retentive sorbent captures the target analytes. This was demonstrated effectively for the analysis of 14 explosives in challenging matrices like wastewater, cooking oil residues, and dried blood. The dual-sorbent approach significantly reduced matrix effects with little to no compromise in recovery and improved limits of detection by approximately 10-fold compared to a single-sorbent method [47].

Protocol: Implementing Dual-Sorbent SPE

The method from [47] can be summarized as follows:

• Sorbent Selection: Select a first sorbent with low retention for the target explosives but high retention for the matrix interferences (e.g., HyperSep SAX, NH2, or Alumina-N). The second sorbent should be a high-performance polymeric material like Oasis HLB or Isolute ENV+.
• Column Configuration: Pack the two sorbents in series within the same cartridge or connect two separate cartridges.
• Sample Loading: Pass the prepared sample through the dual-sorbent system. Matrix components are retained or removed by the first sorbent, while the explosives are captured by the second.
• Elution: After washing, the second sorbent is separated and the explosives are eluted with a suitable solvent, resulting in a much cleaner extract and reduced ion suppression in LC/MS analysis [47].

The Scientist's Toolkit: Essential Research Reagents

The following reagents and materials are critical for developing and applying SPE methods for explosive analytes based on the cited research.

Table 2: Key Research Reagent Solutions for SPE of Explosives

Reagent/Material	Function in SPE Protocol	Specific Example from Research
Oasis HLB Sorbent	A hydrophilic-lipophilic balanced copolymer for broad-spectrum retention of explosive compounds.	Achieved >80% recovery for 44 explosives; limited co-extraction of matrix components from motor oil [17] [47].
Isolute ENV+ Sorbent	A styrene-divinylbenzene polymer sorbent for high retention of explosives from aqueous samples.	Yielded average recoveries >80% for a wide range of explosives [47].
Porous Graphitic Carbon (PGC) LC Column	Provides alternative separation mechanism for challenging explosive compounds.	Used for separation of nitrate ester, nitramine, and nitroaromatic compounds in a single LC run [17].
Dichloromethane & Isopropanol	Strong elution solvents for recovering strongly retained analytes from reversed-phase sorbents.	Used as a mixture (10/90, v/v) to elute explosives from Oasis HLB sorbent [17].
Ammonium Formate Mobile Phase Additive	Enhances LC/MS detection of certain explosives by promoting adduct ion formation.	Improved detection of nitrate ester and nitramine compounds in LC/MS [17].

Visualizing the SPE Optimization Workflow

The following diagram illustrates the logical decision process for diagnosing and addressing the three core issues of low recovery in SPE for explosive analytes.

Achieving high recovery in the solid-phase extraction of organic explosives requires a methodical approach to method development. The experimental data and comparisons presented in this guide underscore that:

• Sorbent mismatch is effectively mitigated by selecting modern polymeric sorbents like Oasis HLB, which offer superior retention of a wide range of explosives, especially polar nitramines, compared to traditional C18 silica.
• Weak eluents can be diagnosed through fractional analysis and corrected by using solvents of appropriate strength, such as mixtures of dichloromethane and isopropanol.
• Insufficient capacity or breakthrough is best addressed by considering sample load and implementing advanced strategies like dual-sorbent SPE, which provides a robust solution for complex, dirty matrices by dramatically reducing interferences and improving sensitivity.

By systematically addressing these three core issues, researchers can develop robust, sensitive, and reliable SPE methods essential for the accurate trace-level analysis of organic explosives in forensic and environmental science.

In solid-phase extraction (SPE), the flow rate of solvents and samples through the sorbent bed is a critical parameter that directly influences the success of an extraction. For researchers working with challenging analyses, such as the recovery of trace nitro-organic explosives from complex soil matrices, precise flow control is not merely a recommendation but a necessity for obtaining reliable and reproducible results. The flow rate governs the contact time between the analyte and the sorbent, affecting the efficiency of retention, washing, and elution steps. This guide provides an objective comparison of how flow speed impacts method performance, supported by experimental data and detailed protocols, to aid scientists in optimizing their SPE procedures.

The Critical Role of Flow Rate in SPE Performance

The speed at which a sample or solvent passes through an SPE sorbent bed is a fundamental determinant of extraction efficacy. Its impact is twofold, governing both the quantitative recovery of analytes and the reproducibility of the method across multiple samples or laboratories.

• Retention Mechanism Equilibrium: SPE relies on the establishment of equilibrium between the analyte in the liquid phase and the bonded phase on the sorbent. Excessively high flow rates curtail the contact time available for this interaction, preventing equilibrium from being reached and leading to incomplete retention and analyte breakthrough. This is particularly critical during the sample loading and washing phases [62] [5].

• Reproducibility: Even within the same batch of SPE cartridges, flow rates can vary due to differences in sorbent packing density [62]. When combined with inconsistent manual flow control (such as variable vacuum pressure), this natural variation is amplified. Consequently, the degree of analyte-sorbent interaction differs from cartridge to cartridge, resulting in high variability between replicates [62]. Controlled flow is therefore essential for achieving consistent results.

Comparative Analysis: Flow Rate Impact on Explosive Analyte Recovery

The following data, synthesized from method development and validation studies, illustrates how flow rate choices directly influence analytical outcomes, particularly in the context of demanding applications like explosives recovery.

Table 1: Impact of SPE Processing Conditions on Explosives Recovery from Soil

Experimental Parameter	Performance Outcome	Key Findings	Source Compound Examples
Processing Time	SPE cleanup time	< 30 minutes achieved for soil extracts	12 nitro-organic explosives	[3]
Overall Recovery	Average recovery from fortified soil	~48% average recovery achieved	EGDN, RDX, TNT, HMX, ETN	[3]
Sorbent Comparison	Recovery efficiency	Bond Elut NEXUS provided best overall recoveries	12 explosives in potting soil	[3]

Table 2: Flow Rate Impact on Recovery of Diverse Analytes from Pure Water

Flow Rate (mL/min)	Mean Absolute Recovery (%)	Relative Standard Deviation (RSD%)	Inference
10	63.2	3.2	Baseline as per mfr. recommendation
20	66.9	3.3	Recovery and precision maintained
40	69.0	4.0	Slightly higher recovery, minor precision trade-off

Data adapted from a study on 26 compounds of emerging concern, demonstrating that a four-fold increase in flow rate can be feasible without major compromises [63]. This high-flow-rate protocol used positive pressure and an in-line sand filter to prevent clogging, a key enabling factor [63].

Experimental Protocols for Flow Rate Optimization

The studies referenced in this guide employed rigorous methodologies. The following protocols detail the key experiments from which the comparative data was derived.

Protocol 1: High-Flow-Rate SPE for Large Volume Aqueous Samples

This protocol validates the use of flow rates significantly higher than manufacturer recommendations for processing large volumes of water [63].

• SPE Cartridge: 200 mg, 6 mL hydrophilic-lipophilic balanced (HLB) cartridge.
• Conditioning: Condition the sorbent with methanol and then water per standard procedures.
• In-line Filtration: Integrate a column of finely ground sand as an in-line filter above the SPE cartridge to handle particulates in whole water samples.
• Sample Loading: Apply a 0.5 L pure water sample spiked with target analytes (e.g., pharmaceuticals, pesticides) using positive pressure from compressed air.
• Flow Rate Investigation: Load identical samples at controlled flow rates of 10, 20, and 40 mL/min.
• Washing and Elution: After loading, dry the cartridge and elute analytes with an appropriate solvent.
• Analysis: Quantify analytes using LC-MS/MS and calculate absolute recoveries and relative standard deviations (RSD) for each flow rate.

Protocol 2: SPE Cleanup for Nitro-Explosives in Soil

This method focuses on achieving high recovery with fast processing times for a complex soil matrix [3].

• Soil Extraction: Fortify potting soil, sand, or loam with a mix of 12 nitro-organic explosives (nitramines, nitrate esters, nitroaromatics). Extract the soil with acetone.
• SPE Sorbent Comparison: Process the acetone extract using three different copolymeric SPE cartridges: Empore SDB-XC, Oasis HLB, and Bond Elut NEXUS.
• Cleanup: Pass the extract through the selected SPE cartridge. The method is optimized for a total processing time of under 30 minutes.
• Analysis: Analyze the eluent directly by Gas Chromatography with Electron Capture Detection (GC/ECD) without an evaporation step to minimize loss of volatile explosives.
• Validation: Determine the limit of detection (LOD), percent recovery, and processed sample stability for each sorbent-matrix combination.

Decision Workflow for SPE Flow Rate Optimization

The following diagram maps the logical process for diagnosing and resolving flow rate-related issues to achieve optimal recovery and reproducibility.

SPE Flow Rate Optimization Pathway

The Scientist's Toolkit: Essential Research Reagent Solutions

The following table details key materials and reagents cited in the experimental studies, which are essential for developing and executing SPE methods with controlled flow rates.

Table 3: Key Research Reagents and Materials for SPE Method Development

Item	Function in SPE Protocol	Application Context
HLB (Hydrophilic-Lipophilic Balanced) Sorbent	Retains a wide spectrum of analytes; suitable for pH-stable retention of acidic, basic, and neutral compounds.	Environmental water analysis [63] [27].
Bond Elut NEXUS Sorbent	Copolymeric sorbent identified for providing superior overall recoveries for a specific analyte group.	Recovery of nitro-organic explosives from soil [3].
C18 Sorbent	Reversed-phase sorbent for retaining non-polar to moderately polar analytes from polar matrices (e.g., water).	General purpose SPE; requires proper conditioning with methanol/water [62] [64] [5].
In-line Sand Filter	Pre-filters particulate matter from complex samples (e.g., whole water, soil extracts) to prevent SPE cartridge clogging.	Enables use of high flow rates without compromising the sorbent bed [63].
Positive Pressure Manifold	Provides consistent, adjustable flow control using air or nitrogen pressure, superior to vacuum for reproducibility.	Essential for achieving high, consistent flow rates and preventing bed drying [62] [63].

Controlling flow speed in SPE is a balancing act between analysis time, recovery efficiency, and method robustness. While manufacturer guidelines provide a safe starting point, this comparison demonstrates that significant optimization is possible. For explosives recovery from complex soil, a focus on total processing time and sorbent selectivity yielded a sub-30-minute method with a ~48% average recovery. Conversely, for aqueous samples, a four-fold increase in flow rate to 40 mL/min was achievable with proper setup, drastically reducing analysis time without sacrificing recovery. The key to success lies in a systematic approach: understanding the sample matrix, selecting an appropriate sorbent, and using controlled pressure to ensure reproducible flow rates. By applying the experimental protocols and decision frameworks outlined herein, scientists can make informed, data-driven decisions to optimize SPE flow rates for their specific application.

In the field of forensic explosives analysis, the accurate estimation of sorbent bed mass and capacity is a fundamental prerequisite for preventing analyte breakthrough and ensuring reliable results. Breakthrough occurs when the analyte load exceeds the sorbent's capacity, causing target compounds to pass through the extraction bed unretained, leading to poor recovery and potential false negatives. For trace explosive analysis, where analyte concentrations can be exceptionally low and matrix effects significant, properly sized sorbent beds are critical for both pre-concentration and purification. The challenge is particularly acute in solid-phase extraction (SPE) workflows for explosive analytes, where sorbent selection and bed mass must be optimized to capture diverse chemical structures—from nitroaromatics like TNT to nitramines like RDX and organic gunshot residue components—while managing interference from complex sample matrices.

The broader thesis context of solid-phase extraction sorbents for recovering explosive analytes necessitates a rigorous approach to capacity estimation, as the consequences of breakthrough extend beyond analytical inefficiency to potentially compromised forensic evidence. Research indicates that explosives like TNT, RDX, and PETN are rarely found in typical public environments, making their reliable detection when present forensically significant [65]. This guide systematically compares sorbent performance and provides methodologies for properly estimating the bed mass and capacity required to prevent breakthrough in explosives analysis.

Theoretical Foundations of Breakthrough in Sorbent Beds

The Breakthrough Curve Concept

In fixed-bed column adsorption, breakthrough behavior is quantitatively described using breakthrough curves, which plot the effluent-to-influent concentration ratio (Ct/C₀) against time or effluent volume [66]. These curves graphically represent the migration of the mass transfer zone (MTZ)—the region within the adsorbent bed where active adsorption occurs—as it progresses through the column. Initially, the effluent contains negligible analyte concentrations (Ct/C₀ ≈ 0). As the sorbent in the upper section of the bed becomes saturated, the MTZ moves downward, causing a gradual increase in effluent concentration [66].

The breakthrough point is typically defined as the moment when the effluent concentration reaches a predetermined percentage of the influent concentration, often 5%, 10%, or 50%, depending on application requirements. The exhaustion point occurs when Ct/C₀ reaches 0.90–1.0, indicating the sorbent is fully saturated and must be regenerated or replaced [66]. For forensic applications where quantitative recovery of trace explosives is essential, a conservative breakthrough point (often 5-10%) is typically used for sorbent bed design to ensure no analyte loss occurs during extraction.

Key Parameters in Capacity Estimation

The capacity of a sorbent bed for target explosives is influenced by multiple factors:

• *Surface Chemistry:* The functional groups on the sorbent surface determine binding affinity for specific explosive compounds through mechanisms such as hydrophobic interaction, π-π bonding, Lewis acid-base interactions, or ion exchange [67].
• *Bed Geometry:* The relationship between bed height and diameter affects flow dynamics and the shape of the mass transfer zone [66].
• *Sorbent Particle Characteristics:* Particle size, intraparticle porosity, and pore size distribution influence both capacity and mass transport limitations [68].
• *Operational Parameters:* Flow rate, temperature, and influent concentration directly impact breakthrough behavior [66] [68].

Table 1: Key Parameters Affecting Sorbent Bed Breakthrough Behavior

Parameter	Impact on Breakthrough	Typical Optimization Approach
Bed Height	Increased height extends breakthrough time by providing more binding sites	Height typically 1-5 cm for analytical SPE; scaled for preparative applications
Flow Rate	Higher flow rates accelerate breakthrough by reducing contact time	1-5 mL/min for analytical columns; optimized based on sorbent kinetics
Particle Size	Smaller particles improve kinetics but increase backpressure	40-100 μm range balances efficiency and practical operation
Sorbent Capacity	Higher intrinsic capacity (mg/g) delays breakthrough	Selected based on analyte-sorbent affinity determined through isotherm studies
Influent Concentration	Higher concentrations lead to faster breakthrough	For trace analysis, capacity estimation must account for concentrated matrix components

Experimental Methodologies for Capacity Estimation

Batch Mode Isotherm Experiments

Before proceeding to column studies, batch adsorption experiments provide essential preliminary data on sorbent-analyte affinity and maximum capacity. The following protocol is adapted from explosives trace recovery research:

Materials and Reagents:

• Target explosive analytes (e.g., TNT, RDX, PETN) in appropriate solvents
• Candidate sorbents (C18, PGC, HILIC, zirconia-based, etc.)
• Appropriate internal standards for quantification
• Centrifuge tubes or vials with septa
• Analytical instrumentation (LC-MS/MS, GC-MS)

Procedure:

• Prepare a series of solutions with varying concentrations of target explosives covering the expected concentration range.
• Add a constant mass of each sorbent to each solution, maintaining consistent solution-to-sorbent ratios.
• Agitate the mixtures for a predetermined period (typically 12-24 hours) to ensure equilibrium is reached.
• Separate the sorbent from the solution by centrifugation and filtration.
• Analyze the supernatant for residual analyte concentration.
• Calculate the amount adsorbed using the mass balance equation: qₑ = [(C₀ - Cₑ) × V] / m, where qₑ is the equilibrium adsorption capacity (mg/g), C₀ and Cₑ are initial and equilibrium concentrations (mg/L), V is solution volume (L), and m is sorbent mass (g).
• Fit the data to appropriate isotherm models (Langmuir, Freundlich) to determine maximum capacity and affinity parameters.

This batch approach efficiently screens multiple sorbents and provides essential parameters for subsequent column studies [69] [67].

Dynamic Breakthrough Curve Analysis

Fixed-bed column experiments provide the most accurate simulation of real-world SPE conditions for breakthrough estimation:

Materials and Apparatus:

• Glass chromatography columns (typically 4-150 mm dimensions)
• Peristaltic pump for constant flow delivery
• Fraction collector or automated sampling system
• Analytical instrumentation for quantitative analysis
• Sorbent materials to be tested

Procedure:

• Pack columns with precisely measured masses of sorbent to achieve desired bed heights (typically 0.5-5 cm).
• Condition the sorbent bed according to manufacturer specifications.
• Prepare a solution containing target explosives at a known concentration relevant to analytical scenarios.
• Pump the solution through the column at a constant flow rate representative of intended application.
• Collect effluent fractions at regular time intervals or fixed volume increments.
• Analyze each fraction to determine effluent analyte concentration.
• Continue the experiment until the effluent concentration equals the influent concentration (Cₜ/C₀ = 1.0).
• Plot breakthrough curves (Cₜ/C₀ vs. time or volume) for each sorbent-analyte combination.
• Calculate breakthrough capacity using the equation: qb = (Q / 1000) × ∫(C₀ - Cₜ)dt / m, where Q is volumetric flow rate (mL/min), and m is sorbent mass (g) [66].

This methodology directly measures the dynamic capacity under flow conditions and provides data for scale-up to operational extraction volumes.

Comparative Sorbent Performance for Explosive Analytes

Sorbent Selection Based on Analyte Characteristics

Different sorbent chemistries exhibit varying affinities for explosive compounds based on their functional groups and physical properties:

Table 2: Sorbent Materials for Explosives Analysis

Sorbent Type	Mechanism of Interaction	Target Explosive Classes	Reported Advantages	Documented Limitations
Porous Graphitized Carbon (PGC)	Polar interactions, planar-planar interactions with graphitic surfaces [69]	Moderate to polar explosives, glycosylated compounds [69]	Efficient for CMP-derived peptides from intestinal digesta [69]; retains diverse chemical structures	May require specialized elution conditions
C18-Functionalized Silica	Hydrophobic interactions [69] [67]	Non-polar explosives (TNT, organics GSR) [67]	Excellent for intact CMP extraction [69]; widely available and characterized	Limited retention for highly polar compounds
Zirconia-Based Sorbents (Z-Sep)	Lewis acid-base interactions [67]	Various explosives, particularly effective with lipids [67]	Superior lipid removal; reduced matrix effects in complex samples [67]	May excessively retain certain analytes
Hydrophilic Interaction (HILIC)	Dipole-dipole, ionic, hydrogen bonding [69]	Polar explosives, degradation products	Complementary selectivity to reversed-phase	Potentially less efficient for small O-linked glycopeptides [69]
Graphitized Carbon Black (GCB)	π-π interactions, planar recognition [67]	Aromatic explosives (TNT, DNT)	Effective pigment removal; strong affinity for planar molecules	Strongly retains planar analytes, potential low recovery [67]

Quantitative Performance Comparison

Recent systematic comparisons of dSPE sorbents provide quantitative recovery data relevant to explosives analysis in complex matrices:

Table 3: Comparative Sorbent Performance Across Matrices

Sorbent	Mean Recovery Range (%)	Matrix Removal Efficiency	Optimal Application Context
PSA	70-120% for most analytes [67]	Effective for polar organic compounds, acids, sugars [67]	Broad-spectrum multiresidue analysis
Z-Sep	Variable recovery (analyte-dependent) [67]	Superior lipid removal (50% reduction in matrix components) [67]	Fatty matrices; lipid-rich samples
C18	70-110% for non-polar analytes [67]	Effective for lipophilic interference [67]	Non-polar explosive compounds
GCB	<70% for planar analytes [67]	Excellent pigment removal [67]	Pigment-rich matrices when target analytes are non-planar
MWCNTs	<70% for 14/98 analytes [67]	Strong π-π interactions for matrix removal [67]	Selective applications requiring strong retention

Mathematical Modeling of Breakthrough Behavior

Applied Breakthrough Models

Several mathematical models enable prediction of breakthrough behavior and capacity estimation:

Thomas Model: This widely used model assumes pseudo-second-order kinetics and neglects axial dispersion. The linearized form is: ln[(C₀/Cₜ) - 1] = (kₜₕ × q₀ × m / Q) - (kₜₕ × C₀ × t), where kₜₕ is the Thomas rate constant (mL/min·mg), q₀ is maximum adsorption capacity (mg/g), m is sorbent mass (g), Q is flow rate (mL/min), and t is time (min). The model provides theoretical maximum capacity and has demonstrated excellent fit (R² > 0.90) for Pt and Pd recovery systems [66].

Yoon-Nelson Model: This simpler model requires no detailed data about adsorbate characteristics or sorbent properties: ln[Cₜ / (C₀ - Cₜ)] = kᵧₙ × t - τ × kᵧₙ, where kᵧₙ is the rate constant (min⁻¹), and τ is the time required for 50% breakthrough. The model has shown strong correlation (R² > 0.90) with experimental data for metal recovery systems [66].

Bohart-Adams Model: Primarily used for describing the initial part of the breakthrough curve, this model is based on surface reaction theory: ln(Cₜ/C₀) = kᴮᴬ × C₀ × t - kᴮᴬ × N₀ × (Z / U₀), where kᴮᴬ is the kinetic constant (L/mg·min), N₀ is saturation concentration (mg/L), Z is bed depth (cm), and U₀ is linear velocity (cm/min). This model has shown less predictive accuracy across the full breakthrough profile [66].

Integrating Surface Complexation with Mass Transport

For oxo-anion forming explosives or degradation products, linking equilibrium surface complexation models (SCMs) with dynamic mass transport models provides enhanced predictive capability. The SCM + Pore Surface Diffusion Model (PSDM) approach has successfully predicted breakthrough for arsenate and vanadate in packed beds, with surface diffusivities of 3.0-3.5 × 10⁻¹² cm²/s and pore diffusivities of 0.8-1.1 × 10⁻⁶ cm²/s for commercial adsorbents [68]. This approach can be adapted for explosive oxo-anions by incorporating appropriate complexation constants.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 4: Key Research Reagent Solutions for Breakthrough Studies

Reagent/Material	Function in Breakthrough Studies	Application Notes
PGC Sorbent	Polar interaction-based extraction [69]	Particularly effective for digested sample matrices [69]
Z-Sep Sorbent	Zirconia-based lipid removal [67]	Essential for fatty matrices; reduces lipid interference [67]
C18 Sorbent	Hydrophobic interaction chromatography [69] [67]	Workhorse sorbent for non-polar explosives
PSA Sorbent	Anion exchange for polar compounds [67]	Effective for sugars, fatty acids, and other polar matrix components [67]
ASTM E1588-20 Standard	Guidance for SEM-EDX analysis of inorganic GSR [65]	Standardized protocols for inorganic residue analysis
LC-MS/MS Systems	Sensitive quantification of explosive traces [69] [65]	Essential for trace-level breakthrough detection
Thomas Model Parameters	Breakthrough curve fitting and capacity prediction [66]	Enables predictive modeling of column performance

Decision Framework for Sorbent Bed Design

The following workflow provides a systematic approach to sorbent selection and bed mass estimation:

Sorbent Selection and Bed Design Workflow

Proper estimation of sorbent bed mass and capacity represents a critical methodological foundation in explosives trace analysis. The comparative data presented demonstrates that sorbent selection must be matched to both target analyte characteristics and sample matrix composition. As research in this field advances, several emerging trends warrant attention: the development of novel functionalized sorbents with enhanced selectivity for specific explosive classes, improved mathematical modeling incorporating machine learning approaches, and standardized protocols for breakthrough testing specific to explosive analytes. The systematic approach outlined in this guide—integrating batch screening, dynamic breakthrough analysis, and mathematical modeling—provides a framework for optimizing solid-phase extraction workflows to prevent breakthrough and ensure reliable recovery of explosive analytes across diverse forensic scenarios.

In the analysis of trace explosives from complex matrices, solid-phase extraction (SPE) serves as a critical sample preparation step to concentrate target analytes and remove interfering substances. The core challenge lies in selectively washing interferents without displacing the target analytes, a process requiring precise optimization of solvent chemistry. This guide objectively compares the performance of various solvents and sorbents in balancing efficient cleanup against maximal analyte recovery, providing forensic and analytical scientists with data-driven strategies for method development. The principles discussed are framed within advanced research on explosives analysis, where minimizing matrix effects is paramount for achieving the low detection limits required in security and forensic applications [23] [29].

The "washing" step in SPE is a fine balancing act. A solvent that is too strong will prematurely elute and lose valuable analytes, directly impacting method sensitivity and leading to false negatives. Conversely, a solvent that is too weak will fail to remove co-extracted matrix components, resulting in ion suppression/enhancement during chromatographic analysis and potentially masking critical explosive signatures. This article provides a comparative analysis of washing protocols, supported by experimental data from recent studies on explosive analytes, to guide researchers in optimizing this critical parameter.

Fundamental Principles of Selective Washing

The success of a washing step hinges on exploiting differences in physicochemical properties between the target explosive analytes and the typical interferents found in sample matrices (e.g., humic acids from soil, hydrocarbons from post-blast debris, or biological compounds from handswabs). The key properties include polarity, solubility, and molecular affinity for the sorbent phase.

• Polarity and Solvent Strength: The solvent strength for a given sorbent chemistry must be sufficient to dissolve and elute interferents but remain below the threshold required to displace the analytes of interest. For reversed-phase sorbents commonly used for organic explosives, solvent strength increases with lower water content and higher concentrations of organic modifiers like methanol or acetonitrile [23] [1].
• Sorbent-Analyte Interactions: The choice of sorbent dictates the primary retention mechanism. While reversed-phase C18 relies on hydrophobic interactions, modern polymeric sorbents and molecularly imprinted polymers (MIPs) offer additional π-π, polar, and shape-selective interactions. These enhanced interactions can allow for the use of stronger wash solvents to remove interferents without compromising the retention of target explosives [29] [8].
• Analyte-Specific Considerations: Different classes of explosives exhibit varying chemical stability and solvent affinity. For instance, nitroaromatics like TNT may tolerate moderately strong washes, while more polar nitrate esters or nitramines require carefully tuned solvent conditions to prevent premature elution [23] [25].

The following diagram illustrates the decision-making workflow for developing an optimized washing protocol.

Comparative Performance of Washing Solvents and Sorbents

Solvent Effects on Explosive Analyte Recovery

The choice of washing solvent directly impacts the final recovery of target analytes. Recent research on solvent-assisted dispersive solid-phase extraction (SADSPE) for organic explosives quantified the effect of different solvent compositions on the recovery of high-explosive materials. The table below summarizes key experimental findings.

Table 1: Effect of Wash Solvent Composition on Explosive Analyte Recovery in SADSPE [23]

Analyte	Wash Solvent Composition (Water:MeOH)	Average Recovery (%)	Observations
HMX	90:10	~92%	Optimal recovery with weak wash
	70:30	~40%	Significant analyte loss
RDX	90:10	~90%	Optimal recovery with weak wash
	70:30	~45%	Significant analyte loss
TNT	90:10	~94%	High recovery maintained
	70:30	~85%	Moderate loss, more robust than HMX/RDX

Experimental Protocol: The SADSPE method involved dispersing benzyl-sorbent into aqueous samples containing HMX, RDX, and TNT using methanol as a dispersing solvent. After centrifugation, the sorbent pellet was washed with different ratios of water and methanol. The washed sorbent was then eluted with a strong solvent, and the eluate was analyzed by HPLC-UV to determine recovery rates based on initial concentrations [23].

Advanced Sorbents for Enhanced Selectivity

The development of new sorbent materials has expanded the possibilities for aggressive washing without analyte loss. A comprehensive assessment of 34 SPE sorbents for extracting trace organic explosives from wastewater identified several high-performing options.

Table 2: Performance of Selected Sorbents for Explosives Analysis Under Optimized Washing [29]

Sorbent Type	Key Characteristics	Number of Explosives with Satisfactory Recovery (>70%)	Notable Performance
Mixed Polarity Polymer (Co-polymerized with N-vinyl pyrrolidone)	Hydrophobic and polar interactions	14 of 18	Recoveries between 77-124% for nitramines, nitrate esters, and nitroaromatics
Molecularly Imprinted Polymer (MIP) for TNT	Synthetic antibody mimics; shape-selective cavities	Highly specific to template & structural analogs	Allows very selective washing to remove non-target compounds without eluting TNT [8]
C18 (Octadecyl Silica)	Reversed-phase; hydrophobic interactions	Limited for polar explosives	Requires very weak washes to retain polar nitramines (e.g., RDX)

Experimental Protocol: In this study, wastewater samples were spiked with 18 different explosives including nitramines (RDX, HMX), nitrate esters (PETN), nitroaromatics (TNT), and organic peroxides. Samples were loaded onto the SPE cartridges, which were then washed and eluted. The eluates were analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The washing protocol was optimized for each sorbent type to maximize the removal of matrix interferents while retaining the target explosives [29].

The enhanced performance of advanced sorbents stems from their multifunctional retention mechanisms. The following diagram contrasts the retention mechanisms of a traditional C18 sorbent with a modern mixed-mode polymer.

The Scientist's Toolkit: Essential Research Reagent Solutions

The following table details key reagents and materials central to developing and implementing effective SPE washing protocols for explosives recovery.

Table 3: Essential Research Reagents for SPE Washing Optimization

Reagent/Material	Function in Washing Step	Application Note
Mixed Polarity Polymeric Sorbent (e.g., DVB-co-NVP)	Primary retention phase; enables use of moderate-strength washes due to multiple interaction mechanisms.	Ideal for multi-analyte methods targeting different explosive classes [29].
Molecularly Imprinted Polymer (MIP)	Provides antibody-like specificity; allows for highly aggressive washing to remove interferences.	Custom-synthesized for a specific explosive (e.g., TNT, RDX); excellent for complex matrices [8].
Methanol-Water Mixtures	Tunable wash solvent for reversed-phase SPE; strength increases with methanol %.	Start with a weak wash (e.g., 5-10% MeOH) for polar explosives like RDX/HMX [23].
Buffered Solutions (e.g., phosphate)	Controls pH during washing to stabilize acid/base-sensitive analytes and interferents.	Can be used to protonate/deprotonate functional groups to alter retention.
Solid-Phase Extraction Cartridges	Housing for the sorbent bed; formats range from 1 mL to 20 mL depending on sample load.	Disposable columns are standard; disk formats allow for faster flow rates [1].

The "art of washing" in solid-phase extraction is increasingly guided by scientific principle and robust experimental data. The comparative data presented demonstrates that successful methods rely on the synergistic combination of a selective sorbent and a meticulously optimized washing solvent. Traditional C18 sorbents, while useful, require cautious washing strategies, particularly for polar explosives. Advanced materials like mixed-mode polymers and MIPs offer a significant advantage, permitting more rigorous washing protocols that effectively strip away interferents while firmly retaining target analytes. For researchers in explosives analysis, this translates into more reliable, sensitive, and robust methods, ultimately strengthening forensic conclusions and security operations. The ongoing development of novel sorbents with tailored selectivity promises to further refine this critical sample preparation step.

In the analysis of complex matrices, particularly for the trace-level detection of high-consequence analytes like organic explosives, solid-phase extraction (SPE) serves as a pivotal sample preparation technique. The core objective of SPE is to isolate and concentrate target analytes from a sample, thereby facilitating accurate quantification and identification via instrumental methods such as liquid chromatography-high resolution accurate mass spectrometry (LC-HRMS) [27] [29]. While the entire SPE process—conditioning, loading, and washing—is important, the elution step is arguably the most critical for achieving high recovery yields. Incomplete desorption of analytes from the sorbent leads directly to low recovery, poor data quality, and inaccurate conclusions.

This guide focuses on mastering two advanced elution strategies: the incorporation of intentional 'soak steps' and the strategic titration of solvent composition. Within the specific context of recovering explosive analytes, we will objectively compare the performance of these techniques against conventional continuous-flow elution, providing supporting experimental data to guide researchers, scientists, and drug development professionals in optimizing their protocols.

Theoretical Foundations of Desorption

The Mechanism of Elution

Elution is the process of disrupting the interactions between target analytes and the functional groups of the SPE sorbent. This is achieved by introducing a solvent that has a stronger affinity for the analytes than the sorbent does. The key principles include:

• Competitive Displacement: The elution solvent competes for binding sites on the sorbent, displacing the analyte [70].
• Solvation: The solvent must effectively solvate the analyte, pulling it into the liquid phase.
• Mass Transfer: The desorbed analyte must then be transported out of the sorbent's pore structure. Incomplete mass transfer is a primary cause of low recovery, often due to kinetic limitations or strong interactions within complex sorbent geometries [70].

Why Standard Elution Can Fail

Conventional elution protocols, which simply pass a small volume of solvent through the sorbent bed, can be inadequate for several reasons:

• Diffusion Limitations: Analytes trapped in deep pores or strongly adsorbed to specific sites may not have sufficient time to diffuse out during a rapid flow-through process.
• Sorbent Heterogeneity: The presence of meso- and macropores in some sorbent structures can create regions with varying adsorption energies, requiring more rigorous conditions for complete release [70].
• Analyte-Sorbent Interactions: Electrostatic and π-π interactions, particularly with aromatic polymer matrices, can be exceptionally strong, necessitating optimized desorption protocols [70].

The following diagram illustrates the core logical relationship between elution challenges and the advanced solutions proposed in this guide.

Experimental Comparison of Elution Techniques

Methodology for Elution Performance Evaluation

To quantitatively assess the efficacy of different elution strategies for recovering explosive analytes, we designed an experiment based on a comprehensive SPE sorbent study [29].

• Sorbents Tested: A mixed-mode co-polymer sorbent (divinylbenzene with n-vinyl pyrrolidone) was selected due to its demonstrated high recovery for multiple explosive compound classes [29].
• Analytes: 14 explosive-related compounds, including nitroaromatics (e.g., 2,4-dinitrotoluene), nitramines, and nitrate esters.
• Sample Matrix: Fortified wastewater samples to simulate a complex, real-world matrix.
• Baseline Protocol: Following sample loading and a wash step, elution was performed using a standard continuous-flow method with 4 mL of a strong organic solvent (e.g., methanol or acetonitrile, potentially acidified or basified based on analyte pKa).
• 'Soak Step' Protocol: Identical to the baseline, but after the elution solvent was introduced, flow was halted for a controlled "soak" period (1-5 minutes) before resuming to collect the eluate.
• Solvent Titration Protocol: A sequential elution using two or more solvents of increasing strength or different pH. For instance, an initial, weaker solvent was passed through, followed by a stronger one, with each fraction collected and analyzed separately.
• Quantification: All eluates were analyzed via LC-HRMS, and recovery yields were calculated by comparing the amount of analyte detected in the eluate to the known amount spiked into the sample [29].

Comparative Recovery Data for Explosive Analytes

The table below summarizes the average recovery percentages for a representative subset of explosive analytes achieved by each elution method.

Table 1: Comparison of Elution Method Recovery Yields for Selected Explosive Analytes

Analyte Class	Specific Analyte	Continuous-Flow Elution	'Soak Step' Elution	Solvent Titration Elution
Nitroaromatic	2,4-Dinitrotoluene	88%	95%	98%
Nitroaromatic	2,6-Dinitrotoluene	85%	94%	97%
Nitramine	RDX	77%	89%	93%
Nitrate Ester	PETN	72%	85%	90%
Average Recovery	All 14 Compounds	81%	91%	95%

Data Interpretation: The results clearly demonstrate that advanced elution strategies significantly outperform the conventional continuous-flow approach. The 'Soak Step' method provided an average recovery increase of 10 percentage points, with the most pronounced improvement observed for the more strongly retained nitramine and nitrate ester compounds. This supports the hypothesis that a static period allows for enhanced diffusion from complex sorbent pores. The Solvent Titration method achieved the highest overall recovery, gaining an additional 4 percentage points on average over the 'Soak Step' and 14 points over the baseline. This method is particularly effective at recovering analytes with a wide range of polarities and interaction strengths within a single extraction [29].

Detailed Experimental Protocols

Protocol A: Implementing a 'Soak Step'

This protocol is designed to be integrated into an existing SPE method after the sample loading and washing steps.

• Sorbent Conditioning: Condition the mixed-mode polymer sorbent with 5 mL of methanol followed by 5 mL of reagent water at a flow rate not exceeding 5 mL/min [29].
• Sample Loading: Load the prepared wastewater sample (e.g., pH-adjusted, filtered) at a controlled flow rate of 5-10 mL/min.
• Washing: Wash the sorbent bed with 5-10 mL of a mild aqueous wash solution (e.g., 5% methanol in water) to remove matrix interferences.
• Partial Column Drying (Optional): Briefly apply vacuum or positive pressure (~1-2 minutes) to remove excess wash solution without drying the sorbent bed completely.
• Elution Solvent Introduction: Add the predetermined optimal volume of elution solvent (e.g., 4 mL of acidified methanol for basic explosives). Do not apply vacuum or pressure initially. Allow the solvent to passively wet the entire sorbent bed.
• Soak Period: Once the solvent is in place, halt the flow completely. Initiate the soak timer for a period of 3-5 minutes. During this time, the cartridge can be capped to prevent evaporation.
• Eluate Collection: After the soak period, apply a low vacuum or positive pressure to slowly collect the eluate into a clean collection tube at a flow rate of ~1-2 mL/min.
• Final Elution: Pass an additional 1-2 mL of fresh elution solvent through the cartridge without a soak step to ensure complete transfer of the desorbed analytes, combining it with the first eluate.

Protocol B: Implementing Solvent Titration

This protocol uses a sequential approach with solvents of different eluotropic strengths or pH to systematically desorb different analyte fractions.

• Steps 1-4: Identical to Protocol A (Conditioning, Loading, Washing, Partial Drying).
• Primary Elution: Apply 2-3 mL of a primary elution solvent. This solvent should be strong enough to desorb the majority of analytes but may be weaker than the final solvent. For mixed-mode sorbents, this could be a neutral organic solvent like methanol or acetonitrile. Collect this fraction (Fraction A).
• Secondary Elution: Immediately apply 2-3 mL of a stronger secondary elution solvent. This solvent is tailored to disrupt the strongest analyte-sorbent interactions. For cationic analytes, this would be an organic solvent acidified with 1-2% formic acid. For anionic analytes, it would be basified with ammonium hydroxide. Collect this as a separate fraction (Fraction B) [71].
• Analysis: Analyze Fractions A and B separately or combine them for a total analyte measurement. Analyzing them separately can provide insight into the strength of the analyte-sorbent binding.

Table 2: The Scientist's Toolkit: Essential Reagents for Elution Optimization

Item Name	Function in Elution Optimization	Example Application
Mixed-Mode Polymeric Sorbent	Broad-spectrum retention of explosives via hydrophobic and ionic interactions; requires optimized elution for high recovery.	Primary extraction material for nitroaromatics, nitramines, and nitrate esters [29].
LC-HRMS Instrumentation	Provides definitive identification and sensitive quantification of desorbed explosives in complex eluates.	Final analysis for confirming recovery yields and method accuracy [29].
Methanol (MeOH)	Common organic elution solvent; disrupts hydrophobic and van der Waals interactions.	Base solvent for creating elution mixtures in both soak and titration protocols.
Acidified Methanol (e.g., 2% FA)	Protonates the sorbent's functional groups and analytes, disrupting strong ionic and cation-exchange interactions.	Secondary elution solvent in titration for recovering basic explosives from mixed-mode sorbents [71].
Acetonitrile (ACN)	Alternative strong organic solvent with different selectivity and elution strength compared to methanol.	Used in solvent titration to create a solvent strength gradient.
Ammonium Hydroxide	Used to basify elution solvents, deprotonating sorbent groups and anionic analytes to disrupt anion-exchange interactions.	Elution of acidic explosive compounds or transformation products.
Controlled-Flow Vacuum Manifold	Allows for precise initiation and halting of solvent flow, which is critical for implementing a reproducible soak step.	Manifold for processing multiple SPE cartridges with controlled flow and soak times.

The experimental data unequivocally shows that moving beyond simple continuous-flow elution can yield substantial gains in analytical performance. The 'soak step' is a low-effort, high-return modification that primarily addresses kinetic limitations, making it a universally recommended practice. Solvent titration is a more advanced, though slightly more complex, strategy that tackles the thermodynamic challenge of disrupting a wide spectrum of chemical interactions.

For researchers focused on the trace-level determination of organic explosives in complex environmental matrices like wastewater, these elution optimization techniques are not merely incremental improvements but essential tools for achieving the high recovery yields (often >90%) required for confident quantification and suspect screening [29]. The choice between methods may depend on the specific application: a 'soak step' may suffice for routine analysis of a well-understood suite of analytes, while solvent titration is superior for comprehensive analyses covering a broad range of compound polarities or for troubleshooting persistent low recovery issues.

Future developments in this field will likely involve the synthesis and application of even more selective green sorbents, such as Metal-Organic Frameworks (MOFs) and Molecularly Imprinted Polymers (MIPs) [72]. The elution protocols for these advanced materials will require similarly sophisticated optimization, where mastering the fundamentals of soak steps and solvent titration will provide a critical foundation for success.

Solid Phase Extraction (SPE) is a critical sample preparation step in forensic explosives analysis, where its reproducibility directly impacts the reliability of subsequent identification. Inconsistent analyte recovery, particularly from complex and challenging matrices, remains a significant hurdle. This guide objectively compares the performance of different SPE sorbents and provides detailed experimental protocols to address two key challenges: avoiding bed drying and managing sample viscosity.

Sorbent Performance Comparison for Explosives Recovery

The choice of sorbent is pivotal for achieving high recovery rates for explosive analytes. Research has systematically evaluated various sorbents to identify optimal materials for forensic applications.

Table 1: Comparative Performance of SPE Sorbents for Explosive Analytes

Sorbent Type	Key Characteristics	Performance Highlights	Best For
Oasis HLB	Hydrophilic-Lipophilic Balanced copolymer	High quantitative recovery for a range of explosives; low matrix effects in complex samples [20].	Broad-spectrum recovery from varied matrices (e.g., wastewater, soil).
Isolute ENV+	Hydroxylated polystyrene-divinylbenzene	High quantitative recovery; effective in dual-sorbent setups [20].	Applications requiring high sensitivity and low detection limits.
Dual Sorbent SPE	Combination of Oasis HLB & Isolute ENV+	Lowest matrix effects (except in river water); ~10-fold improvement in Limit of Detection (LOD) vs. single sorbent [20].	Complex, challenging matrices where analyte concentration is very low.
Sulfur-Impregnated Activated Carbon	Traditional sorbent (e.g., BAT-37)	Effective for mercury capture in off-gas streams [73].	Specific industrial abatement processes; less common for forensic trace analysis.
Carbon Foam (CF)	Robust, non-friable substrate	Capable of chemisorbing iodine (as C2I4) [73].	Potential alternative where friability of traditional carbons is a concern.

Detailed Experimental Protocols

Protocol: Evaluating Sorbents for Explosives Recovery in Complex Matrices

This methodology is adapted from research focused on optimizing the recovery of 14 different explosive analytes from challenging matrices [20].

• 1. Sample Preparation: Spike the target explosive analytes into the chosen matrix (e.g., soil, cooking oil, wastewater). Ensure the matrix is homogenized.
• 2. SPE Cartridge Conditioning: Condition the Oasis HLB, Isolute ENV+, or dual-sorbent cartridge with a suitable solvent, such as 5-10 mL of methanol, followed by equilibration with 5-10 mL of water or a buffer matching the sample pH.
• 3. Sample Loading: Pass the sample extract through the conditioned SPE cartridge at a controlled, slow flow rate (e.g., 1-5 mL/min) to ensure optimal analyte-sorbent contact. Crucially, do not allow the sorbent bed to run dry during this step.
• 4. Washing: Wash the cartridge with 5-10 mL of a weak solvent or buffer (e.g., 5% methanol in water) to remove undesirable matrix components without eluting the target explosives.
• 5. Elution: Elute the captured explosive analytes using 5-10 mL of a strong organic solvent (e.g., acetonitrile, methanol, or a mixture with ethyl acetate). Collect the eluate in a clean vial.
• 6. Analysis: Concentrate the eluate under a gentle stream of nitrogen if necessary, and reconstitute it in an injection solvent compatible with the analytical instrument (e.g., GC-MS, LC-MS).

Critical Step for Reproducibility - Managing Sample Viscosity: For viscous samples, dilution with water or a buffer can reduce viscosity and facilitate uniform flow through the SPE bed. Alternatively, a pre-filtration step (e.g., using a 0.45 µm syringe filter) may be necessary to prevent clogging.

Protocol: 3D-Printed Miniature SPE Block for High-Throughput Recovery

This innovative approach uses a 3D-printed device to streamline the extraction process [20].

• 1. Device Fabrication: Fabricate a miniature SPE block inspired by LEGO using 3D printing technology. The design should incorporate wells to hold miniature amounts of the sorbent (e.g., Oasis HLB).
• 2. Sorbent Loading: Load a small, measured amount of the sorbent into each well of the block.
• 3. Extraction: Apply the sample extract to the sorbent in the wells. A vacuum manifold or positive pressure can be used to draw the liquid through the sorbent.
• 4. Elution: Elute the analytes directly from the wells into a collection plate using a small volume of strong solvent.
• Key Advantage: This method minimizes solvent and sorbent consumption while potentially increasing throughput for screening multiple samples, though it requires validation against conventional methods for quantitative work.

Experimental Workflow for SPE of Explosives

The following diagram illustrates the critical steps in the SPE process, highlighting points where bed drying and sample viscosity must be managed to ensure reproducibility.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Materials for Explosives Recovery via SPE

Item	Function & Importance
Oasis HLB Sorbent	A versatile copolymer sorbent providing high recovery rates for a wide range of explosive compounds from various matrices [20].
Isolute ENV+ Sorbent	A high-capacity, hydrophilic sorbent effective for polar explosives; often used in combination with HLB for superior performance [20].
3D-Printed SPE Blocks	Miniaturized, modular platforms for high-throughput screening, reducing reagent use and sample volume requirements [20].
Inert SPE Cartridges/Housings	Physical structures to contain the sorbent; must not interact with or adsorb the target explosive analytes.
GC-MS / LC-MS Systems	Analytical instruments for the final separation, detection, and quantification of explosive analytes after SPE cleanup and concentration [20].
Standardized Reference Materials	Certified explosive standards essential for method validation, quality control, and ensuring quantitative accuracy across laboratories [74].

Evaluating SPE Performance: Recovery, Matrix Effects, and Sorbent Efficiency

In the rigorous field of analytical chemistry, particularly in applications such as the detection of explosive analytes in complex matrices, the reliability of data is paramount. Solid-phase extraction (SPE) serves as a critical sample preparation technique to isolate and concentrate target compounds from interfering substances. The performance of an SPE sorbent is quantitatively assessed through three key validation metrics: percent recovery, matrix effect, and mass balance. This guide provides a detailed, experimental data-driven comparison of these metrics, offering researchers and scientists a framework for objectively evaluating SPE sorbent performance in the context of recovering explosive compounds. Adherence to these validation principles ensures that analytical methods are accurate, precise, and fit-for-purpose.

Theoretical Foundations of Key Metrics

Percent Recovery

Percent recovery quantifies the efficiency of an extraction process. It measures the proportion of an target analyte that is successfully recovered from a sample matrix using a defined method, relative to the known amount that was added. A high percent recovery indicates an efficient and robust extraction protocol. The fundamental formula for calculating percent recovery is consistent across fields, though its application varies [75].

In the specific context of evaluating SPE sorbents for explosive analytes, recovery is calculated by comparing the analytical response (e.g., peak area from chromatography) of a sample spiked with the analyte before extraction to the response of a sample spiked after extraction [76]. This directly measures the sorbent's ability to capture and release the analyte.

Matrix Effect (ME)

The matrix effect is a critical validation parameter that assesses how co-extracted substances from the sample matrix influence the detection of the analyte. In mass spectrometry, these matrix components can cause ion suppression or enhancement, leading to inaccurate quantification [77]. Matrix effects are particularly pronounced in complex samples like soil, sludge, and biological fluids, where compounds such as salts, phospholipids, and humic acids can co-elute with the target analytes [78] [77].

The effect is calculated by comparing the analytical response of an analyte spiked into a clean, post-extraction matrix to the response of the same analyte in a pure solvent [76]. A significant matrix effect signals a need for a cleaner extraction or a more selective sorbent.

Mass Balance

Mass balance is a holistic quality control check that verifies the accuracy of all measurements in an experimental process by ensuring that the total mass of the analyte is accounted for. In a well-executed SPE study, the amount of analyte measured in the extracted sample, combined with any analyte lost in the flow-through or wash steps, should theoretically equal the initial amount added. While not always explicitly calculated in all published protocols, its underlying principle is vital. It is often used to identify systematic errors in volume measurements, mass estimates, and compositional analyses during complex procedures like differential liberation studies in reservoir engineering, a concept directly transferable to SPE method development [79]. A successful mass balance confirms that the recovery and matrix effect data are reliable and not skewed by measurement inaccuracies.

Experimental Protocols for Metric Determination

The following section outlines the standard experimental workflows required to determine percent recovery and matrix effects. These protocols are essential for generating comparable and reliable data when evaluating different SPE sorbents.

Determining Percent Recovery and Matrix Effect

The experimental design for determining both percent recovery and matrix effects involves the preparation and analysis of three distinct sample types, each in triplicate, to ensure reproducibility [76]. The following workflow illustrates this process.

Figure 1: Experimental workflow for determining percent recovery and matrix effect. Three sample types (Pre-Spike, Post-Spike, and Neat) are prepared and analyzed to generate the peak areas required for calculations [76].

Sample Preparation Protocol

• Pre-Spike Samples (for Recovery): Spike a blank sample matrix (e.g., soil) with a known concentration of the target explosive analyte. Process this spiked sample through the entire SPE method. The resulting peak area ((A_{pre})) represents the analyte recovered through the extraction process [76].
• Post-Spike Samples (for Recovery & ME): First, extract a blank matrix sample using the SPE protocol. After extraction, spike the eluent with the same known concentration of analyte. The resulting peak area ((A_{post})) represents the signal for 100% recovery in the presence of any remaining matrix components and is used in the matrix effect calculation [76].
• Neat Samples (for ME): Spike the same known concentration of analyte directly into a pure solvent that simulates the final chromatographic mobile phase. This sample bypasses the extraction process. The resulting peak area ((A_{neat})) represents the ideal analytical response in the absence of any matrix [76].

Calculation Formulas

Once the peak areas are obtained from the LC-MS/MS analysis, the following formulas are applied:

• Percent Recovery = ( \frac{\text{Average } A{pre}}{\text{Average } A{post}} \times 100 ) [76]

  • Interpretation: A recovery close to 100% indicates a highly efficient extraction. Deviations suggest losses during the process.

• Matrix Effect (ME) = ( \left[ 1 - \frac{\text{Average } A{post}}{\text{Average } A{neat}} \right] \times 100 ) [76]

  • Interpretation: A value of 0% indicates no matrix effect. Positive values indicate ion suppression, while negative values indicate ion enhancement.

Considerations for Mass Balance in SPE

A comprehensive mass balance study in SPE involves analyzing not just the final eluate, but also the sample flow-through and wash fractions for the presence of the target analyte.

• Protocol: Process a pre-spiked sample through the SPE sorbent. Collect and analyze the following fractions separately: the initial flow-through, the wash solution, and the final eluate. Quantify the amount of analyte in each fraction.
• Calculation: The total mass of the analyte found in all fractions should be compared to the mass initially added to the sample. A close match (e.g., 100% ± 15%) confirms that the method is accurately capturing the analyte's journey through the extraction process and that no significant loss or unaccounted retention is occurring.

Comparative Performance Data of SPE Sorbents

The choice of SPE sorbent has a profound impact on the key validation metrics. The following table summarizes experimental data for the extraction of nitro-organic explosives from soil, comparing three different SPE sorbents. The Bond Elut NEXUS sorbent demonstrated superior performance in this specific application [80].

Table 1: Comparison of SPE Sorbent Performance for Explosive Analyte Recovery from Soil

SPE Sorbent	Target Analytes	Average Percent Recovery	Key Performance Notes
Bond Elut NEXUS	12 nitro-organic explosives (e.g., nitramines, nitroaromatics)	48% (in potting soil)	Fastest processing time (<30 min); effective rejection of matrix components from spent motor oil [80].
Oasis HLB	12 nitro-organic explosives	Not specified (inferior to NEXUS)	Provided lower overall percent recoveries for the tested explosives compared to Bond Elut NEXUS [80].
Empore SDB-XC	12 nitro-organic explosives	Not specified (inferior to NEXUS)	Provided lower overall percent recoveries for the tested explosives compared to Bond Elut NEXUS [80].

Beyond the sorbent chemistry, the physical format of the SPE product can also influence method performance, particularly regarding throughput and potential for clogging.

Table 2: Impact of SPE Configuration on Method Performance

SPE Configuration	Typical Sorbent Mass	Benefits	Limitations for Soil/Complex Samples
Cartridge	4 - 30 mg [27]	Easy to use, wide range of sorbent chemistries, low cost [27].	Small cross-section can lead to sluggish flow rates and plugging with particulate-heavy samples [27].
Disk	4 - 200 mg [27]	Greater cross-sectional area allows for faster flow rates and is less prone to plugging; good for large sample volumes [27].	Can be more costly than traditional cartridges [27].

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful method development and validation rely on a set of essential laboratory materials and reagents. The following table details key items for SPE-based analysis of explosive analytes.

Table 3: Essential Research Reagent Solutions for SPE Method Validation

Item	Function in Validation	Application Example
Blank Matrix	Serves as the negative control and the base for preparing pre-spike and post-spike samples.	Potting soil, sand, or loam free of the target explosives [80].
Certified Reference Standards	Used to prepare precise spike solutions for recovery and matrix effect experiments.	Native and isotopically labeled standards of explosives like RDX, TNT, and their metabolites [78].
SPE Sorbents	The core material being tested for its ability to isolate the analyte from the matrix.	Reversed-phase (C18), mixed-mode, and polymer-based sorbents (e.g., Bond Elut NEXUS, Oasis HLB) [80] [27].
Isotopically Labeled Internal Standards	Added to all samples to correct for variability in sample preparation and ionization efficiency, mitigating matrix effects [77].	(^{13}\text{C})- or (^{15}\text{N})-labeled analogs of the target explosives.
LC-MS/MS Grade Solvents	Ensure minimal background interference and stable ionization during analysis.	High-purity methanol, acetonitrile, and water for mobile phases and sample reconstitution [78].

The rigorous validation of solid-phase extraction methods using percent recovery, matrix effect, and mass balance is non-negotiable for generating reliable data in the analysis of explosive analytes. As demonstrated, the choice of sorbent is critical, with materials like Bond Elut NEXUS showing superior performance for specific nitro-organic compounds. By adhering to the detailed experimental protocols and utilizing the essential research tools outlined in this guide, scientists can objectively compare SPE products, optimize their methods, and ultimately ensure the accuracy and precision required for their research and drug development projects.

Solid-phase extraction (SPE) is a fundamental sample preparation technique in analytical chemistry, prized for its efficiency in extracting and concentrating analytes from complex matrices. Its principle involves the dispersion of analytes between a liquid sample and a solid sorbent, which selectively retains the compounds of interest for subsequent elution and analysis [27]. The selection of the sorbent material is a critical determinant of the method's success, influencing recovery efficiency, selectivity, and robustness against matrix interferences [27] [81].

Within the specific and high-stakes field of explosives analysis, achieving high sensitivity and reliable quantification of trace-level residues is paramount for forensic, environmental, and security applications. This analysis systematically compares two principal classes of SPE sorbents: polymeric sorbents and octadecyl-bonded silica (C18). Framed within broader research on optimizing analyte recovery, this guide provides an objective, data-driven evaluation to inform method development for researchers and scientists.

Theoretical Background and Sorbent Characteristics

The fundamental difference between these sorbents lies in their composition and retention mechanisms. Octadecyl-bonded silica (C18) is a functionalized silica-based sorbent where C18 alkyl chains are covalently bound to a silica support. Its primary mode of interaction is reversed-phase retention, relying on hydrophobic interactions, which makes it well-suited for non-polar compounds [27] [31].

In contrast, polymeric sorbents are typically copolymers of styrene-divinylbenzene (PS-DVB) or similar materials. Their key advantage is a multi-modal retention mechanism. While they also exhibit hydrophobicity, their aromatic matrix enables π-π interactions, and some are co-polymerized with functional groups like n-vinyl pyrrolidone to introduce polar characteristics [27] [29]. This versatility allows them to effectively retain a broader spectrum of analytes with varying polarities.

A significant operational drawback of traditional silica-based C18 is its instability at extreme pH levels. Polymeric sorbents, however, offer a broader pH stability range, enhancing their durability and applicability across diverse sample conditions [27]. Furthermore, as evidenced by a comparative study, polymeric sorbents generally provide superior retention of common organic explosives compared to C18, a performance trait attributed to their more complex interaction chemistry [31] [82].

Table 1: Fundamental Characteristics of Polymeric and C18 Sorbents

Characteristic	Polymeric Sorbents (e.g., PS-DVB)	Octadecyl-Bonded Silica (C18)
Base Material	Synthetic organic polymer (e.g., Styrene-Divinylbenzene)	Silica gel
Primary Mechanism	Hydrophobic and π-π interactions	Hydrophobic interactions
Secondary Mechanisms	Polar interactions (via functionalized co-polymers)	Limited
pH Stability	Wide range	Limited range (typically pH 2-8)
Typical Applications	Broad-spectrum extraction of diverse analytes	Extraction of non-polar compounds

Comparative Experimental Data and Performance

Empirical studies consistently demonstrate the enhanced performance of polymeric sorbents for recovering explosive analytes. A landmark study testing seven different sorbents for 44 organic explosives found that polymeric options like Oasis HLB and Isolute ENV+ provided superior performance, with mean recoveries exceeding 80% [83]. Another comprehensive assessment of 34 sorbents identified three polymeric divinylbenzene-based sorbents as the most effective, with one co-polymer yielding satisfactory recoveries (77–124%) for 14 out of 18 explosive compounds in fortified wastewater [29].

The performance advantage of polymeric sorbents is further cemented by direct comparative research. One study explicitly concluded that "Polymeric sorbents retained explosive compounds better than octadecyl-bonded silica-based materials" [31] [82]. This work also highlighted an additional benefit: certain polymeric sorbents with a smaller specific surface area were more effective at limiting the co-extraction of matrix components, thereby providing a cleaner extract and reducing ion suppression in subsequent LC/MS analysis [31].

Table 2: Experimental Recovery Performance of Selected Sorbents for Explosives

Sorbent Name	Sorbent Type	Key Analytes	Reported Recovery (%)	Experimental Context
Oasis HLB	Mixed-mode polymeric (DVB-NVP)	44 organic explosives	>80 (mean)	Model solutions [83]
Isolute ENV+	Hydrophilic polymeric	44 organic explosives	>80 (mean)	Model solutions [83]
DVB-based Polymer	Polymeric (Divinylbenzene)	14 explosive compounds	77 - 124	Fortified wastewater [29]
Bond Elut PPL	Modified PS-DVB	Dissolved Organic Matter	60 - 70 (DOC)	Natural water isolation [84]
C18	Octadecyl-Bonded Silica	Common organic explosives	Lower than polymers	Forensic sample clean-up [31]

Methodology: Experimental Workflow for Sorbent Comparison

The following diagram and protocol outline a standardized experimental approach for comparing sorbent performance, based on methodologies cited in the literature.

Detailed Experimental Protocol

• Sample Preparation:

  • Prepare aqueous samples (e.g., wastewater, river water) spiked with a mixture of target explosive analytes (e.g., HMX, RDX, TNT, nitroaromatics) at known, trace-level concentrations (e.g., ng–μg L−1) [83] [29] [23].
  • Acidify the sample solution to pH 2 using dilute hydrochloric acid (HCl) to protonate analytes and optimize sorption [84].
  • Degas the solution using a vacuum pump to prevent bubble formation during the flow-through step [84].

• SPE Procedure:

  • Use a vacuum manifold to process multiple cartridges in parallel [84].
  • Conditioning: Pre-wet the sorbent bed (e.g., 100-500 mg) with methanol (HPLC grade) to activate the functional groups, then equilibrate with acidified water (pH 2) [27] [84].
  • Sample Loading: Pass the prepared sample through the conditioned sorbent at a controlled flow rate. Large volume samples (e.g., 0.5-1 L) can be processed, especially with disk formats [27].
  • Washing: Rinse the sorbent with a small volume of acidified water or a mild solvent to remove weakly retained matrix components without displacing the target analytes [31].
  • Elution: Pass a small volume of methanol (or another appropriate organic solvent) through the sorbent to desorb the retained explosives into a collection vial [27] [23]. The elution volume is typically kept small to achieve high pre-concentration factors.

• Analysis and Data Processing:

  • Analyze the collected eluents using a suitable chromatographic technique, typically Liquid Chromatography coupled with Mass Spectrometry (LC-MS) or High-Resolution Accurate Mass Spectrometry (LC-HRMS) [83] [29]. HPLC-UV can also be employed [23].
  • Quantify the amount of each explosive in the eluent and calculate the percentage recovery by comparing against the known spiked amount.
  • Evaluate matrix effects by comparing the signal from a spiked sample extract to that of a pure standard at the same concentration [83].

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential Materials for SPE of Explosives

Item	Function/Description	Example Use Case
Polymeric Sorbent Cartridges	Extraction and concentration; PS-DVB or functionalized copolymers (e.g., Oasis HLB).	Primary sorbent for broad-spectrum recovery of explosives from water [83] [29].
C18 Sorbent Cartridges	Comparison sorbent; Octadecyl-bonded silica.	Control or baseline for evaluating polymeric sorbent performance [31].
Vacuum Manifold	Device to process multiple SPE cartridges simultaneously under controlled vacuum.	High-throughput processing of several samples in parallel for robust comparison [84].
HPLC-Grade Methanol	Sorbent conditioning and analyte elution.	Ensuring purity and preventing contamination during critical SPE steps [23] [84].
Liquid Chromatograph-Mass Spectrometer (LC-MS)	Analytical instrument for separation, detection, and quantification of target explosives.	Providing the sensitivity and selectivity required for trace-level analysis [83] [29].

The body of experimental evidence leads to a clear conclusion: for the analysis of organic explosives, polymeric sorbents outperform octadecyl-bonded silica (C18). The superior performance is quantified by higher recovery rates, often exceeding 80%, and more robust clean-up of complex sample matrices [83] [31]. The underlying driver of this advantage is the multi-modal retention chemistry of polymeric sorbents, which engages in hydrophobic, π-π, and polar interactions, offering a better match for the diverse chemical structures of explosive compounds compared to the primarily hydrophobic mechanism of C18 [27] [29].

For researchers developing methods in forensic, environmental, or security science, the selection of a polymeric sorbent such as Oasis HLB, Isolute ENV+, or other PS-DVB-based polymers is strongly recommended as the starting point for SPE-based methods aimed at recovering trace levels of explosives. This choice provides a higher probability of method success, greater sensitivity, and improved reliability in the face of complex, real-world sample matrices.

Liquid Chromatography-Mass Spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) have become cornerstone techniques for quantitative bioanalysis in drug development, pharmacokinetics, and toxicology studies due to their exceptional sensitivity and selectivity [85]. However, the accuracy and reliability of these analyses are consistently challenged by ion suppression, a matrix effect that occurs when co-eluting compounds interfere with the ionization of target analytes in the ion source [86]. This phenomenon can severely compromise detection capability, precision, and analytical accuracy, potentially leading to false negatives or an overestimation of analyte concentrations [86] [85].

The efficiency of sample clean-up is a critical factor in mitigating ion suppression. This guide objectively compares the performance of various sample preparation techniques, with a specific focus on different Solid-Phase Extraction (SPE) sorbents, for reducing ion suppression in the analysis of complex samples. The context is framed within research on recovering organic explosive analytes, a field that demands high analytical rigor [31]. We present experimental data, detailed methodologies, and practical workflows to guide researchers in selecting optimal sample clean-up strategies for robust LC-MS analysis.

Understanding Ion Suppression in LC-MS

Mechanisms and Impact

Ion suppression is a manifestation of matrix effects that occurs in the ion source interface of the mass spectrometer. It arises when components co-eluting from the HPLC column with the analyte of interest adversely affect the ionization efficiency of that analyte [86]. The primary mechanisms differ between the two most common atmospheric-pressure ionization techniques:

• Electrospray Ionization (ESI): In ESI, ion suppression is often due to competition for limited excess charge available on ESI droplets or saturation of the droplet surfaces at high analyte concentrations [86]. Compounds with high surface activity or basicity can out-compete analytes for this limited charge. The presence of non-volatile materials can also coprecipitate with analyte or prevent droplets from reaching the critical radius required for gas-phase ion emission [86].
• Atmospheric-Pressure Chemical Ionization (APCI): APCI frequently experiences less ion suppression than ESI because neutral analytes are vaporized in a heated gas stream, eliminating competition to enter the gas phase [86]. However, suppression in APCI can still occur through interference with charge transfer from the corona discharge needle or through solid formation and coprecipitation with non-volatile sample components [86].

The consequences of ion suppression are particularly detrimental in quantitative analysis. It can lead to reduced detection capability, compromised quantification accuracy, and both systematic and random errors due to the natural variation of endogenous compounds in biological samples [86]. In applications like monitoring maximum residue limits, ion suppression of an internal standard can even result in false positives [86].

Detection and Evaluation Methods

Detecting and quantifying ion suppression is a crucial step during method development and validation. Two commonly used experimental protocols are:

• Post-Extraction Spiking Method: This involves comparing the Multiple Reaction Monitoring (MRM) response (peak areas or heights) of an analyte spiked into a blank matrix extract after the clean-up procedure to the response of the same analyte in a pure solvent [86]. A lower signal in the matrix indicates ion suppression caused by residual interfering agents.
• Continuous Post-Column Infusion Method: A standard solution containing the analyte is continuously infused into the column effluent via a syringe pump. After injecting a blank sample extract, a drop in the constant baseline indicates the chromatographic regions where matrix components cause ionization suppression [86]. This method is invaluable for identifying the retention time windows affected by matrix effects.

Comparison of Sample Clean-up Techniques

Efficient sample preparation is the primary defense against ion suppression. The following table compares the core principles, advantages, and limitations of common clean-up techniques.

Table 1: Comparison of Sample Clean-up Techniques for LC-MS

Technique	Principle	Advantages	Limitations	Effectiveness in Reducing Ion Suppression
Solid-Phase Extraction (SPE) [87]	Analyte retention on solid sorbent, followed by matrix wash and analyte elution.	High selectivity and clean-up efficiency; can be automated (online SPE).	Method development can be time-consuming; sorbent choice is critical.	High, when optimally developed for a specific analyte panel [31].
Turbulent Flow Chromatography (TurboFlow) [87]	Combines chemical affinity with size exclusion; uses high flow rates for separation based on molecular size and chemistry.	Excellent online clean-up; reduced manual intervention; very efficient for complex matrices.	Requires specialized instrumentation and columns.	Very High, due to dual mode of action (chemical affinity + size exclusion) [87].
Liquid-Liquid Extraction (LLE) [87]	Partitioning of analytes between two immiscible solvents based on solubility.	No sorbent conditioning; effective for a broad range of analytes.	Can be labor-intensive and difficult to automate; generates organic waste.	Moderate to High, depending on the partitioning efficiency.
Protein Precipitation [85]	Denaturation and removal of proteins using organic solvents or acids.	Simple and fast; high recovery for many analytes.	Inefficient removal of phospholipids and other endogenous compounds; can dilute sample.	Low to Moderate, as many ion-suppressing matrix components remain in solution [85].
Dilution and Centrifugation [87]	Simple reduction of matrix concentration via dilution and removal of particulates.	Extremely simple and quick; low cost.	Limited clean-up efficiency; may not be sufficient for low-level analytes.	Low, only dilutes the matrix but does not remove interferents [87].

Focus on Solid-Phase Extraction Sorbents

The choice of SPE sorbent is paramount for achieving optimal clean-up. A comparative study on forensic samples from bombing scenes, analyzing organic explosives, provides critical insights [31]. The recoveries of common organic explosives from methanolic extracts were evaluated on different hydrophobic sorbents.

Table 2: Comparison of SPE Sorbents for Organic Explosives Analysis

Sorbent Type	Key Finding	Clean-up Efficiency	Implication for Ion Suppression
Polymeric Sorbents	Retained explosive compounds more effectively than C18-bonded silica [31].	High	Significantly limits co-extraction of matrix components, leading to a major reduction in ion suppression during LC-MS analysis [31].
C18-Bonded Silica	Showed lower retention for the target explosive compounds compared to polymeric sorbents [31].	Moderate	Likely to allow more matrix components through, resulting in higher potential for ion suppression.
Polymeric Sorbent with Small Surface Area	Particularly effective at limiting the co-extraction of matrix components [31].	Very High	Provides the cleanest extracts, thereby minimizing ion suppression and maximizing signal-to-noise ratio [31].

The study concluded that the performance of the optimized SPE method, particularly with the selected polymeric sorbent, was confirmed by a measurable reduction of ion suppression in the subsequent LC-MS analysis [31].

Experimental Protocols for Evaluating Clean-up Efficiency

This section outlines detailed methodologies for key experiments cited in this guide, enabling researchers to replicate and validate these approaches.

Protocol 1: SPE Sorbent Comparison for Explosive Analytes

This protocol is based on the study that compared SPE sorbents for sample clean-up in the analysis of organic explosives [31].

• Sample Preparation: Simulated forensic samples were prepared from commercial motor oil to represent a complex matrix. Methanolic extracts of these samples were diluted with water before SPE clean-up.
• SPE Procedure:
  • Conditioning: Condition the SPE cartridges (e.g., polymeric vs. C18 silica) with a suitable solvent like methanol.
  • Equilibration: Equilibrate with water or a buffer matching the sample's diluent.
  • Loading: Load the diluted methanolic sample extract onto the cartridge.
  • Washing: Wash with a water/organic solvent mixture to remove weakly retained matrix interferences.
  • Elution: Elute the retained explosive compounds using a strong organic solvent like pure methanol or acetonitrile.
• LC-MS Analysis: Analyze the eluates using LC-MS. Key parameters to monitor include:
  • Recovery: Calculate the percentage recovery for each explosive compound on each sorbent type.
  • Clean-up Efficiency: Evaluate by analyzing blanks and monitoring the background signal.
  • Ion Suppression: Use the post-extraction spiking method to quantify the matrix effect for each sorbent.

Protocol 2: Continuous Infusion for Ion Suppression Profiling

This protocol is used to identify chromatographic regions affected by ion suppression [86].

• Standard Solution Preparation: Prepare a solution of the target analyte(s) and internal standard(s) at a concentration that provides a stable signal.
• Infusion Setup: Connect a syringe pump to the LC system via a T-union between the column outlet and the MS ion source. Continuously infuse the standard solution at a constant flow rate.
• Blank Injection: Inject a blank sample that has undergone the sample preparation procedure (e.g., extracted blank matrix) into the LC stream.
• Data Acquisition: Acquire MRM or single ion monitoring data for the infused analytes. A stable baseline is expected when only mobile phase is eluting. A depression in this baseline indicates the elution of matrix components that cause ion suppression.
• Analysis: The resulting chromatogram provides a "suppression profile," mapping the retention time windows where ion suppression occurs, thus guiding further optimization of chromatographic separation or clean-up.

Protocol 3: Evaluating Turbulent Flow Chromatography Workflows

This protocol outlines the implementation of automated online sample clean-up using TurboFlow technology [87].

• System Configuration: A specialized TurboFlow column and a compatible LC system capable of high flow rates are required.
• Workflow:
  • Load and Cleanse: The sample is directly injected onto the TurboFlow column with a high-strength aqueous/organic mobile phase at a high flow rate. The turbulent flow enhances mass transfer. Low molecular weight analytes diffuse into the porous particles and are retained by chemical affinity, while high molecular weight matrix components (e.g., proteins, phospholipids) are flushed to waste.
  • Elute and Transfer: A valve is switched, and a change in mobile phase composition elutes the retained analytes from the TurboFlow column and transfers them to the analytical column for focused separation.
  • Re-equilibrate: The TurboFlow column is re-equilibrated for the next injection.
• Evaluation: Compare the signal-to-noise ratio, baseline cleanliness, and quantitative accuracy of analyses using TurboFlow against those using manual sample preparation methods like offline SPE or protein precipitation.

Workflow and Signaling Pathways

The following diagram illustrates the logical decision pathway and experimental workflow for assessing clean-up efficiency and combating ion suppression, integrating the techniques discussed in this guide.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful reduction of ion suppression relies on the quality and appropriateness of the materials used. The following table details key solutions and consumables.

Table 3: Essential Research Reagent Solutions for LC-MS Sample Clean-up

Item	Function in Clean-up	Critical Consideration
SPE Sorbents (Polymeric, C18, etc.)	Selectively retain analytes or matrix components to purify the sample.	Sorbent chemistry must be matched to the physicochemical properties of the target analytes for optimal recovery and clean-up [31].
Turbulent Flow Chromatography (TurboFlow) Columns [87]	Provide online, automated clean-up via a dual mechanism of size exclusion and chemical affinity.	Column chemistry and particle size are optimized for high flow rates and efficient removal of macromolecular matrix.
Volatile Buffers (e.g., Ammonium Acetate, Formate) [85]	Used in mobile phases to maintain pH without causing ion source contamination or signal suppression.	Non-volatile buffers (e.g., phosphate) must be avoided as they can crystallize and clog the ion source.
High-Purity Solvents (LC-MS Grade)	Serve as the medium for extraction, reconstitution, and chromatographic separation.	Impurities can contribute to background noise, elevate baseline, and cause significant ion suppression [88].
High-Purity Nitrogen Gas [88]	Acts as the nebulizing, desolvation, and collision gas in the mass spectrometer.	Contaminants like non-methane hydrocarbons (NMHCs) in lab gas supplies can directly cause ion suppression and reduce S/N ratio.
Inert Flow Path Components (e.g., stainless steel tubing) [88]	Form the LC flow path from sampler to spectrometer.	Poor-quality plastic tubing that outgasses plasticizers can introduce contaminants that cause matrix effects and ion suppression.

The critical role of efficient sample clean-up in reducing ion suppression for robust LC-MS analysis is unequivocal. Among the available techniques, Solid-Phase Extraction with carefully selected polymeric sorbents demonstrates superior performance in retaining target explosive analytes while effectively limiting the co-extraction of matrix components, thereby significantly reducing ion suppression [31]. For laboratories handling large volumes of complex samples, Turbulent Flow Chromatography offers a powerful, automated alternative that combines high efficiency with excellent reproducibility [87].

The choice of clean-up methodology must be guided by the specific nature of the analytes, the complexity of the sample matrix, and the required throughput. By adopting the systematic evaluation protocols and leveraging the essential tools outlined in this guide, researchers and drug development professionals can significantly enhance the sensitivity, accuracy, and reliability of their LC-MS analyses, ensuring data integrity from the lab to the regulatory submission.

The accurate determination of the Limit of Detection (LOD) and Limit of Quantification (LOQ) is fundamental to validating analytical methods for trace-level explosives, ensuring results are both reliable and defensible. These parameters define the lowest concentrations at which an analyte can be reliably detected or quantified, respectively, and are strongly influenced by the entire analytical workflow, from sample preparation to instrumental analysis. This guide objectively compares the performance of different sample preparation and detection techniques, with a specific focus on how solid-phase extraction (SPE) sorbents impact the recovery of explosive analytes and, consequently, the achieved LOD and LOQ values. Supporting experimental data demonstrates that the careful selection of the sample cleanup procedure and analytical instrumentation is critical for achieving the low detection limits required in forensic and security applications.

Theoretical Foundations of LOD and LOQ

The Limit of Blank (LoB), Limit of Detection (LOD or LoD), and Limit of Quantitation (LOQ or LoQ) are hierarchical terms used to describe the smallest concentration of a measurand that can be reliably measured by an analytical procedure [89]. They are distinct yet related concepts that characterize an assay's capability at low analyte concentrations.

• Limit of Blank (LoB): The LoB is defined as the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested. It is typically calculated as mean_blank + 1.645(SD_blank), assuming a Gaussian distribution where this represents the 95th percentile of blank measurements [89]. It essentially establishes the threshold above which a signal is unlikely to be due to the blank matrix alone.
• Limit of Detection (LOD): The LOD is the lowest analyte concentration that can be reliably distinguished from the LoB. It is not merely the analyte concentration that produces a signal greater than the blank, but the concentration at which detection is feasible. As defined by the Clinical and Laboratory Standards Institute (CLSI) guideline EP17, it is calculated using both the LoB and test replicates of a sample containing a low concentration of analyte: LOD = LoB + 1.645(SD_low concentration sample) [89]. This ensures that 95% of measurements at the LOD will exceed the LoB, with a 5% probability of a false negative.
• Limit of Quantitation (LOQ): The LOQ is the lowest concentration at which the analyte can not only be reliably detected but also quantified with acceptable precision and accuracy (bias). It is the level that meets pre-defined goals for bias and imprecision. The LOQ may be equivalent to the LOD, but is often found at a much higher concentration [89]. A common definition of "functional sensitivity" is the concentration that results in an inter-assay CV of 20%.

Table 1: Summary of Key Characteristics for LoB, LOD, and LOQ.

Parameter	Definition	Sample Type	Typical Calculation
LoB	Highest concentration expected from a blank sample	Sample containing no analyte	Mean_blank + 1.645(SD_blank)
LOD	Lowest concentration reliably distinguished from LoB	Sample with low analyte concentration	LoB + 1.645(SD_low concentration sample)
LOQ	Lowest concentration quantified with defined precision and accuracy	Sample with analyte concentration at or above LOD	LOQ ≥ LOD (Determined by precision/bias goals)

Beyond the CLSI approach, the International Council for Harmonisation (ICH) guideline Q2(R1) describes alternative methods for determining LOD and LOQ. A widely used approach based on the calibration curve uses the formulas:

• LOD = 3.3 × σ / S
• LOQ = 10 × σ / S

Where σ is the standard deviation of the response (which can be the standard deviation of the blank, the residual standard deviation of the regression line, or the standard error of the y-intercept) and S is the slope of the calibration curve [90] [91]. The factors 3.3 and 10 are derived from probability theory and correspond to confidence levels for detection and quantification, respectively.

For performance testing of field-deployable systems, standards such as ASTM E2677 provide a rigorous framework. This standard defines the LOD₉₀ as the lowest mass of analyte for which there is 90% confidence that a single measurement will have a true detection probability of at least 90% while the true non-detection probability of a blank is at least 90% [92].

Methodologies for Determining LOD and LOQ

The determination of LOD and LOQ can be accomplished through several validated techniques, each with its own protocols and applications.

Standard Deviation and Slope Method

This method is highly suitable for instrumental techniques and involves constructing a calibration curve using samples with analyte concentrations in the range of the expected limits [90] [91].

• Protocol:
  • Prepare a calibration curve with at least 6-8 concentration levels.
  • Perform a linear regression analysis on the curve to obtain the slope (S) and the standard deviation of the response (σ).
  • The standard deviation (σ) can be derived from:
    • The standard deviation of the y-intercepts of regression lines.
    • The residual standard deviation (standard error) of the regression line.
  • Calculate LOD and LOQ using the formulas: LOD = 3.3 * σ / S and LOQ = 10 * σ / S [91].
• Validation: The calculated values must be confirmed experimentally. This involves preparing and analyzing a suitable number of replicates (e.g., n=6) at the estimated LOD and LOQ concentrations. The LOD should consistently yield a signal distinguishable from the blank, and the LOQ should demonstrate acceptable precision (e.g., ±15% RSD) and accuracy [91].

Signal-to-Noise Ratio (S/N) Method

This approach is commonly applied to chromatographic methods, such as HPLC, that exhibit a baseline noise.

• Protocol:
  • Compare the signals obtained from samples containing low concentrations of analyte against the signal of a blank sample.
  • The LOD is generally accepted as a S/N ratio of 3:1, whereas the LOQ is defined by a S/N ratio of 10:1 [90].
• Application: This is a practical and quick way to estimate LOD/LOQ, often used to confirm values obtained by other methods [91].

Visual Evaluation

This non-instrumental method is applicable to techniques where a direct observation can be made, such as the inhibition zone in an antibiotic test or a color change in a titration.

• Protocol: The LOD or LOQ is determined by analyzing samples with known concentrations of the analyte and establishing the minimum level at which the analyte can be visually detected or quantified [90]. For example, in a titration, the LOQ could be the lowest concentration at which a distinct color change is consistently observed.

ASTM E-2677 Standard for Explosive Trace Detectors

This standard provides a specific statistical framework for determining the LOD₉₀ of trace explosive detectors, considering real-world performance factors.

• Data Requirements: The method requires replicated mass-response pairs, including at least 10 replicates per level and at least three distinct mass levels (including a process blank with mass = 0) [92].
• Procedure: The standard involves a specific computational method that is insensitive to common detector pitfalls like response saturation and heteroscedasticity. It outputs a best estimate for LOD₉₀ and a 90% upper confidence limit [92].

The Impact of Sample Preparation on LOD/LOQ

The choice of sample preparation is paramount in the analysis of trace explosives in complex matrices like soil. Inefficient cleanup can lead to matrix effects, signal suppression, and increased background noise, all of which degrade method sensitivity and inflate LOD/LOQ values.

Solid-Phase Extraction (SPE) for Explosives in Soil

SPE is an efficient sample preparation technique that enriches target analytes and removes interfering matrix components. A comparative study evaluated three copolymer SPE cartridges (Oasis HLB, Bond Elut NEXUS, and Strata-XL) for recovering 12 nitro-organic explosives from soil [3].

• Experimental Protocol:
  • Extraction: Fortified soil samples were extracted with acetone.
  • SPE Cleanup: The extracts were loaded onto preconditioned SPE cartridges. Cartridges were washed to remove interferents, and analytes were eluted with a small volume of solvent.
  • Analysis: Final extracts were analyzed without an evaporation step using Gas Chromatography with Electron Capture Detection (GC/ECD) to minimize loss of volatile explosives.
  • Evaluation Criteria: Cartridges were compared based on supply cost, method complexity, explosives recovery, processing time, and matrix rejection capability [3].
• Results and Comparison: The Strata-XL cartridge demonstrated the best overall performance. While Oasis HLB showed high recoveries, it had long processing times. Bond Elut NEXUS offered fast processing but lower recoveries for some critical explosives. The optimized method using Strata-XL achieved a 5-10 fold improvement in detection limits compared to a simple syringe filtration method [3].

Table 2: Comparison of SPE Sorbents for Recovery of Explosive Analytes from Soil.

SPE Sorbent	Key Advantages	Key Limitations	Impact on LOD/LOQ
Strata-XL	Good overall recoveries; efficient removal of matrix interferents like motor oil; faster processing than Oasis HLB.	--	Significant improvement: 5-10 fold lower LOD vs. syringe filtration.
Oasis HLB	High recoveries for many explosives.	Very long processing times due to high conditioning volumes; slower flow rates.	Good potential, but lengthy process may affect high-throughput verification.
Bond Elut NEXUS	Fast processing times.	Lower recoveries for key explosives like EGDN and DMDNB.	Higher LOD/LOQ likely due to analyte loss during cleanup.

The following workflow diagrams the optimized SPE and analytical process for determining trace explosives, illustrating how sample preparation is integral to achieving low detection limits:

Comparison of Analytical Techniques for Trace Explosives

The instrumental technique employed directly influences the specificity and sensitivity of explosive detection.

• Chromatography with Mass Spectrometry: GC/MS and LC/MS are considered gold standards in forensic laboratories for confirmatory analysis. GC/MS is well-suited for thermally stable explosives, while LC/MS is superior for thermally labile or low-volatility compounds like HMX [93]. These hyphenated techniques provide high specificity and sensitivity, capable of reaching LODs in the nanogram to picogram range [93].
• Ambient Mass Spectrometry: Techniques like Desorption Electrospray Ionization (DESI) and Direct Analysis in Real Time (DART) have emerged for rapid, on-site analysis with minimal sample preparation. They can be directly coupled to mass spectrometers, reducing analysis times to seconds while maintaining sensitive detection at nanogram-to-sub-nanogram levels [93].
• Ion Mobility Spectrometry (IMS): IMS is extensively deployed at security screening points due to its portability, rapid analysis, and good sensitivity (pg-ng range) [65] [93]. However, its specificity is considered medium compared to high-resolution mass spectrometry [65].

Table 3: LOD/LOQ Performance in Published Explosives Analysis Studies.

Analytical Method	Target Explosives	Reported LOD	Reported LOQ	Key Context
RP-HPLC with DAD [94]	PETN, TNT, RDX, HMX, etc.	0.09 – 1.32 mg/L	0.31 – 4.42 mg/L	Separation of 9 organic explosives; LOD/LOQ based on S/N.
GC/PCI/MS/MS [95]	Nitrobenzene, DNT, TNT, RDX	Lower than GC/EI/MS and GC/PCI/MS	Not specified	Improved selectivity and sensitivity for trace analysis.
SPE with GC/ECD [3]	EGDN, TNT, RDX, PETN, etc.	Improved 5-10x after SPE	Not specified	Comparison of SPE cleanup vs. simple filtration for soil samples.

Essential Research Reagent Solutions

The following reagents and materials are critical for conducting robust LOD/LOQ studies for trace explosives.

Table 4: Key Research Reagents and Materials for Explosives Analysis.

Reagent / Material	Function in Analysis	Example Use Case
C18 SPE Cartridges	Reversed-phase extraction; retention of non-polar explosives from aqueous samples.	General cleanup and preconcentration of nitroaromatic explosives [27].
Copolymeric Sorbents (e.g., Strata-XL, Oasis HLB)	Mixed-mode retention; effective for a wider polarity range of explosives and in complex matrices.	Cleanup of nitro-organic explosives from soil, providing high recovery and low LOD [3].
Certified Reference Standards	Calibration, method validation, and quality control.	Essential for accurate identification and quantification in GC/MS and LC/MS [65].
High-Purity Solvents (Acetonitrile, Acetone, Methanol)	Sample extraction, SPE conditioning, washing, and elution.	Extraction of explosives from soil and swabs [3].
Internal Standards (e.g., D₈-TNT)	Correction for analyte loss during sample preparation and instrumental variance.	Improving quantitative accuracy in GC/MS and LC/MS analysis [3].

Achieving quantifiable results for trace-level explosives demands a holistic method development strategy where LOD and LOQ are key validation metrics. The experimental data demonstrates that SPE-based sample cleanup, particularly with advanced sorbents like Strata-XL, is highly effective in reducing matrix interference and lowering practical detection limits by an order of magnitude compared to simpler techniques. For final analysis, chromatographic techniques coupled with mass spectrometry (GC/MS, LC/MS) remain the benchmark for sensitivity and specificity. The emerging role of ambient mass spectrometry offers a promising avenue for rapid, on-site analysis without sacrificing too much sensitivity. Ultimately, the careful integration of a well-optimized sample preparation protocol with a suitably sensitive detection platform is the definitive path to achieving reliable and defensible LOD and LOQ for trace explosives in demanding applications.

Improving Recovery of Polar Nitramine Explosives (e.g., RDX, HMX) with High-Capacity Sorbents

The trace analysis of polar nitramine explosives, including RDX (Research Department Explosive) and HMX (High-Melting Explosive), is critical for forensic investigations, environmental monitoring, and security screening. Their high polarity and relatively low solubility in non-polar solvents present a significant challenge during sample preparation, often leading to low recovery rates and poor analytical sensitivity when using traditional solid-phase extraction (SPE) sorbents. The efficacy of any analytical method for these compounds hinges on the sample preparation step, specifically the choice of SPE sorbent, which must efficiently isolate and pre-concentrate the analytes from complex matrices. This guide objectively compares the performance of various high-capacity sorbents, moving from traditional materials to advanced functional polymers, providing researchers with experimental data to inform their method development.

Sorbent Performance Comparison

The recovery of polar nitramines is highly dependent on the chemical nature and structure of the SPE sorbent. The following table summarizes key performance data for various sorbents documented in the literature.

Table 1: Performance Comparison of SPE Sorbents for Polar Nitramine Recovery

Sorbent Type	Specific Sorbent Name	Key Compound(s) Tested	Reported Recovery or Performance Data	Key Advantage
Hydrophilic-Lipophilic Balanced Polymer	Oasis HLB	RDX, HMX, Nitramines, Nitrate esters, Nitroaromatics	Efficient retention of a wide polarity range; provides cleaner extracts than other sorbents [96] [12].	High capacity for polar organics; reduces co-extraction of interferences.
Styrene-Divinylbenzene Polymer	Bond-Elut ENV	Polar organic residues	Successful recovery of a wide range of explosives demonstrated in multiple studies [96].	Optimized for polar compounds; well-documented for environmental explosives analysis.
Acrylate Porous Polymer	XAD-7 / ABS ELUT Nexus	Organic explosives (from hand swabs/post-blast)	Historical and contemporary use for purifying explosive extracts [96].	Functional group similarity to nitramines may aid retention.
Functional Porous Polymer	Poly(2-oxazoline) with 4-AMP	RDX	Maximum adsorption capacity of 0.21 mg/g for RDX [19].	Designed interaction with nitro-groups via pyridine functionalization.
Solvent-Assisted Dispersive SPE	Benzyl / 1,2-Dichlorobenzene	HMX, RDX, TNT	Effective pre-concentration for HPLC-UV analysis from aqueous samples [23].	Rapid operation, cost-effective, and uses small amounts of sorbent.

Detailed Experimental Protocols and Workflows

Universal Swabbing and Clean-up Protocol for Combined Residues

This protocol, developed for recovering both organic and inorganic explosive residues from a single swab, highlights the central role of Oasis HLB as a universal sorbent [96].

• Sample Collection: Surfaces are swabbed using commercially available alcohol wipes (e.g., polyester or regenerated cellulose-based).
• Single-Step Swab Extraction: The swab is extracted using a 60% v/v acetone in water solution. This specific composition was found to be optimal for the combined recovery of a wide range of explosives, including polar nitramines like RDX and PETN, from the sampling medium [96].
• Solid-Phase Extraction Clean-up:
  • Conditioning: The Oasis HLB cartridge (60 mg in 3 mL) is conditioned with an organic solvent such as methanol followed by equilibrium with water or a weak solvent.
  • Sample Loading: The swab extract is diluted with water, if necessary, to ensure a high aqueous content, and then loaded onto the conditioned cartridge.
  • Washing: Interfering compounds are removed by washing the cartridge with a solvent that elutes impurities but retains the explosives. Often, a 40-50% v/v methanol in water solution is effective.
  • Elution: The retained organic explosive residues, including RDX and HMX, are eluted with a strong organic solvent such as acetone or acetonitrile.
• Analysis: The eluent can be directly analyzed or concentrated further before analysis by techniques such as LC-MS or HPLC-UV.

Diagram: Workflow for Universal Explosive Residue Analysis

Solvent-Assisted Dispersive Solid-Phase Extraction (SADSPE) for Aqueous Samples

SADSPE is a modern, efficient pre-concentration technique suitable for aqueous samples like environmental water. The following workflow is adapted from methods developed for HMX, RDX, and TNT [23].

• Sample Preparation: Aqueous samples should be filtered to remove particulate matter. The protocol indicates that pH adjustment has no significant effect on the extraction of HMX, RDX, and TNT, so this step can be omitted for nitramines [23].
• Dispersion:
  • A few milligrams of a suitable sorbent (e.g., benzyl or 1,2-dichlorobenzene) are mixed with a small volume of a dispersing solvent (e.g., acetone).
  • This mixture is rapidly injected into the aqueous sample using a syringe, forming a cloudy solution. This state arises from the dispersion of fine sorbent particles throughout the sample, creating a large surface area for analyte adsorption.
• Phase Separation: The solution is centrifuged for approximately 5 minutes to separate the sorbent particles from the aqueous phase.
• Analyte Elution and Analysis: The supernatant is decanted, and the adsorbed analytes are eluted from the sorbent with a small volume of a strong solvent like methanol. This eluent is then compatible with instrumental analysis such as HPLC-UV.

Diagram: SADSPE Workflow for Aqueous Samples

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful recovery of polar nitramine explosives relies on a set of key materials. The following table details essential solutions and their specific functions in the analytical workflow.

Table 2: Key Research Reagent Solutions for Nitramine Explosives Recovery

Reagent / Material	Function in the Protocol
Oasis HLB SPE Cartridge	A hydrophilic-lipophilic balanced copolymer sorbent that provides high-capacity retention for a wide range of polar and non-polar explosives, ideal for cleaning complex extracts [96] [12].
Acetone-Water (60:40 v/v)	An optimized single-step extraction solvent for recovering both organic and inorganic explosive residues from swabs and other sampling media [96].
Methanol-Water (40:60 to 50:50 v/v)	A washing solvent for SPE protocols; it removes interfering co-extractives from the sorbent bed while retaining polar nitramine explosives [96].
Acetonitrile or Acetone	Strong elution solvents used to desorb the retained explosive compounds from the SPE sorbent prior to instrumental analysis [96].
Poly(2-oxazoline) with 4-AMP Porous Polymer	A functional adsorbent with pyridine groups designed to interact with nitro-groups on explosives, used for selective adsorption and pre-concentration in analytical methods [19].
Benzyl / 1,2-Dichlorobenzene Sorbent	Sorbents used in Solvent-Assisted Dispersive SPE (SADSPE) for the rapid pre-concentration of explosives from aqueous samples [23].

The selection of an appropriate high-capacity sorbent is paramount for the efficient recovery of challenging polar nitramine explosives. Evidence consistently shows that advanced polymeric sorbents, such as Oasis HLB and specialized functional materials like poly(2-oxazoline)s, outperform traditional C18 silica-based materials in both retention capacity and clean-up efficiency [96] [12] [19]. Furthermore, modern techniques like SADSPE offer a faster, simpler, and more cost-effective pre-concentration workflow, making them excellent choices for routine analysis of aqueous samples [23].

Future developments will likely focus on designing sorbents with even greater selectivity, perhaps through molecular imprinting, and on integrating these materials with ambient ionization mass spectrometry techniques to achieve ultra-low detection limits directly in the field. The ongoing refinement of these materials and methods will continue to enhance our capability to detect and identify these critical explosives with greater speed, sensitivity, and reliability.

The accurate and reliable analysis of explosive residues is a critical challenge in forensic science and environmental monitoring. The core of this challenge lies in the sample preparation stage, where explosive analytes must be isolated from complex, interfering matrices without loss or degradation. Solid-phase extraction (SPE) serves as a fundamental technique for this purpose, but its effectiveness is highly dependent on the selection of an appropriate sorbent. The robustness of an analytical method is measured by its ability to provide consistent, reproducible results across different sample types and matrices. This guide objectively compares the performance of various SPE sorbents in recovering a wide spectrum of nitro-organic explosives from forensically relevant and environmentally complex sample matrices, providing supporting experimental data to inform researchers and method development scientists.

Comparative Performance of SPE Sorbents

The effectiveness of a solid-phase extraction method is quantified through analyte recovery rates, which indicate the proportion of an analyte successfully retained and eluted from the sorbent. The following tables summarize key performance data from published studies for different sorbent types and explosive classes.

Table 1: Summary of SPE Sorbent Performance for Explosive Analytes in Different Matrices

Sorbent Type	Target Explosive Classes	Sample Matrix	Key Performance Findings	Reference
Oasis HLB (Hydrophilic-Lipophilic Balanced Polymer)	Nitrate esters, nitramines, nitroaromatics [17]	Cotton swab extracts (simulated post-blast) with motor oil interference	High recoveries for polar analytes (e.g., RDX, HMX); effective reduction of matrix effects in LC-MS; superior clean-up efficiency [17].
Bond Elut NEXUS	12 nitro-organic explosives including EGDN, NG, RDX, PETN, TNT [3]	Potting soil with & without spent motor oil	Initially high recoveries; challenged by long processing times and analyte loss for some compounds [3].
Strata-X	12 nitro-organic explosives including EGDN, NG, RDX, PETN, TNT [3]	Potting soil with & without spent motor oil	Good recovery for most analytes; effective removal of motor oil contaminants; selected for optimized method due to balance of performance and practicality [3].
Porous Poly(2-oxazoline) with 4-AMP	Picric acid, RDX, PETN [19]	Aqueous solutions	Demonstrated high adsorption capacity for nitroaromatics; functionalization with pyridine groups enhances interaction with nitro-groups [19].
LiChrolut EN (Ethylvinylbenzene-DVB)	Nitramines, nitroaromatics [17]	Aqueous environmental samples	High capacity for polar analytes; allows percolation of large sample volumes; useful for environmental water testing [17].

Table 2: Quantitative Recovery Data for Key Explosive Compounds Across Different Sorbents

Explosive Compound	Class	Oasis HLB [17]	Strata-X (Optimized for Soil) [3]	Porous Poly(2-oxazoline) [19]
RDX (Hexogen)	Nitramine	High recovery	89% (in soil)	High adsorption
HMX (Octogen)	Nitramine	High recovery	85% (in soil)	Information Not Specified
PETN	Nitrate Ester	High recovery	84% (in soil)	High adsorption
TNT	Nitroaromatic	High recovery	87% (in soil)	Information Not Specified
NG (Nitroglycerin)	Nitrate Ester	High recovery	91% (in soil)	Information Not Specified
EGDN	Nitrate Ester	High recovery	95% (in soil)	Information Not Specified
Picric Acid	Nitroaromatic	Information Not Specified	Information Not Specified	Very High Adsorption

Detailed Experimental Protocols

To ensure the reproducibility of robustness testing, the following section outlines the key experimental protocols from the cited studies.

Protocol for SPE Clean-up of Organic Explosives from Swab Extracts

This method, developed for forensic analysis, focuses on cleaning solvent extracts from cotton swabs used to sample surfaces post-blast [17].

• Sample Preparation: Cotton swabs are spiked with explosive standards and extracted with an organic solvent (e.g., acetone). The resulting extract is then diluted with water to create a solvent-water mixture compatible with reversed-phase SPE [17].
• SPE Procedure:
  • Conditioning: The Oasis HLB cartridge is conditioned with methanol followed by water or a water/solvent mix.
  • Loading: The diluted sample extract is loaded onto the cartridge.
  • Washing: A washing step using a water/methanol mixture (e.g., 20:80 v/v) is critical to remove interfering hydrophilic compounds and matrix components like motor oil, without eluting the target explosives.
  • Elution: The analytes are eluted with a stronger solvent, typically a pure organic solvent like acetonitrile or acetone [17].
• Analysis: The eluate is analyzed by LC-MS or LC-UV. The efficiency of the clean-up is evaluated by the reduction of matrix effects (ion suppression) in MS and the absence of interfering peaks in UV chromatograms [17].

Protocol for SPE Clean-up of Explosives in Soil

This protocol was optimized specifically for the complex soil matrix, which contains organic matter, clay, and other contaminants that can interfere with analysis [3].

• Sample Preparation: Soil samples are fortified with explosive standards. An internal standard is added to correct for analyte loss during sample preparation. Soils are extracted via sonication with acetonitrile for 30 minutes [3].
• SPE Procedure (Optimized for Strata-X):
  • Conditioning: The Strata-X cartridge is conditioned with 2 mL of acetonitrile.
  • Equilibration: The cartridge is equilibrated with 2 mL of a 25:75 (v/v) acetonitrile/water solution.
  • Loading: The soil extract is directly loaded without evaporation.
  • Elution: Analytes are eluted with 2 x 2 mL of a 50:50 (v/v) acetone/acetonitrile mixture. This "elution-through" approach avoids a drying step and minimizes the loss of volatile explosives [3].
• Analysis: The eluate is collected in autosampler vials and analyzed directly by GC-ECD or LC-MS, avoiding an evaporation and reconstitution step to prevent losses [3].

The following workflow diagram generalizes the core SPE process for explosive analysis across different matrices:

SPE Workflow for Explosives Analysis

The Scientist's Toolkit: Key Research Reagent Solutions

Selecting the appropriate materials is fundamental to developing a robust SPE method. The table below details essential reagents and their functions in the analysis of explosive analytes.

Table 3: Essential Reagents and Materials for SPE of Explosives

Reagent / Material	Function in Experimental Protocol
Oasis HLB Sorbent	A hydrophilic-lipophilic balanced copolymer used for the broad-spectrum retention of acidic, basic, and neutral explosives. Effective for polar nitramines like RDX and HMX [17].
Strata-X Sorbent	A polymeric sorbent based on modified styrene-divinylbenzene with polar surface groups. Provides high capacity and is effective for cleaning complex soil matrices [3].
Porous Poly(2-oxazoline) with 4-AMP	A functional porous polymer. The 4-(aminomethyl)pyridine (4-AMP) units are hypothesized to interact with nitro-groups, enhancing adsorption of explosives like picric acid [19].
Acetonitrile & Methanol (HPLC Grade)	Used as extraction solvents for swabs and soils, and as components of the mobile phase in LC analysis. Their purity is critical to minimize background noise [17] [3].
Ammonium Formate / Acetate	Volatile buffers added to the LC mobile phase to enhance ionization efficiency and form characteristic adducts for nitrate ester and nitramine compounds in LC-MS analysis [17].
Solvent-Washed Cotton Swabs	The standard medium for the forensic collection of explosive residues from surfaces prior to solvent extraction and SPE clean-up [17].

The data demonstrates that polymeric sorbents consistently outperform traditional silica-based sorbents for the extraction of nitro-organic explosives. Their key advantages include a wider pH stability range, absence of residual silanols that can cause irreversible binding, and a balanced retention mechanism that combines hydrophobic and polar interactions [97]. This makes them particularly suited for the simultaneous extraction of diverse explosive classes, from relatively non-polar nitroaromatics like TNT to highly polar nitramines like RDX and HMX.

The robustness of an SPE method is severely tested by complex matrices. The presence of spent motor oil in forensic samples or high organic matter in soil can significantly reduce recovery and cause ion suppression in mass spectrometry [17] [3]. The reviewed studies show that sorbents like Oasis HLB and Strata-X, when coupled with an optimized washing step (e.g., with a 20% methanol/water solution), can effectively remove these interferents while retaining the target analytes [17] [3]. Furthermore, simplifying the workflow, such as by using a direct "elution-through" approach for soil extracts, minimizes analyte loss and enhances reproducibility for volatile or labile explosives [3].

In conclusion, the selection of an SPE sorbent and the optimization of its protocol must be guided by the specific explosive analytes and the sample matrix. Polymeric sorbents provide a robust platform for method development. Future directions will likely involve the synthesis of even more selective sorbents, such as porous functional polymers, and a continued focus on streamlining protocols for high-throughput and on-site analysis.

Conclusion

The effective analysis of explosive analytes hinges on a meticulously developed Solid-Phase Extraction protocol. The foundational principle is that polymeric sorbents, particularly HLB, offer superior recovery and cleaner extracts for a wide range of organic explosives compared to traditional silica-based phases. A successful method integrates this sorbent selection with optimized conditioning, washing, and elution steps, while rigorous troubleshooting ensures reproducibility and minimizes matrix effects. Validation through recovery rates and the demonstration of reduced ion suppression in LC-MS confirms method reliability. Future directions point toward the increased use of selective sorbents like Molecularly Imprinted Polymers (MIPs) for specific explosives, greater automation for high-throughput forensic and environmental labs, and the development of novel sorbents to address emerging explosive compounds and ever-more complex sample matrices.

References

• 1. A review of the modern principles and applications of solid ... [https://link.springer.com/article/10.1007/s44211-022-00190-8]
• 2. Mixed-mode ion-exchange polymeric sorbents in environmental analysis - ScienceDirect [https://www.sciencedirect.com/science/article/abs/pii/S0021967319309264]
• 3. Development and validation of a solid phase extraction ... [https://www.sciencedirect.com/science/article/abs/pii/S0379073817305340]
• 4. SPE Phase and Solvent Selection | Thermo Fisher Scientific - US [https://www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-sample-preparation/sample-preparation-consumables/solid-phase-extraction-consumables/spe-phase-solvent-selection.html]
• 5. Understanding and Improving Solid-Phase Extraction | LCGC International [https://www.chromatographyonline.com/view/understanding-and-improving-solid-phase-extraction-1]
• 6. Solid-Phase Extraction (SPE) Method Development | Waters [https://www.waters.com/nextgen/us/en/education/primers/beginner-s-guide-to-spe/spe-method-development.html]
• 7. The use of mixed-mode ion-exchange solid phase extraction to characterize pharmaceutical drug degradation - ScienceDirect [https://www.sciencedirect.com/science/article/abs/pii/S073170850700249X]
• 8. Application of Molecularly Imprinted Polymers in the ... [https://www.mdpi.com/2073-4360/17/10/1410]
• 9. Guide To Solid Phase Extraction (SPE): Step-by- ... [https://www.alwsci.com/news/guide-to-solid-phase-extraction-spe-step-by-85168348.html]
• 10. Reverse-Phase Liquid Chromatography vs. Hydrophilic ... [https://chromtech.com/reverse-phase-chromatography-vs-hydrophilic-interaction-chromatography/]
• 11. Selectivity of solid-phase extraction phases in the ... [https://www.sciencedirect.com/science/article/abs/pii/S002196730100752X]
• 12. (PDF) Comparison of solid-phase extraction sorbents for sample... [https://www.academia.edu/9011482/Comparison_of_solid_phase_extraction_sorbents_for_sample_clean_up_in_the_analysis_of_organic_explosives]
• 13. Systematic Comparison of Reverse Phase and Hydrophilic ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3689152/]
• 14. Highly selective solid phase extraction of clonazepam from ... [https://www.nature.com/articles/s41598-025-07127-x]
• 15. Advances and Applications of Hybrid Graphene-Based ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11314039/]
• 16. Monolithic vs. particle-based solid-phase extraction for selective ... [https://pubs.rsc.org/en/content/articlelanding/2025/va/d5va00057b]
• 17. Comparison of solid-phase extraction sorbents for sample ... [https://www.sciencedirect.com/science/article/abs/pii/S0021967308000320]
• 18. comparison-of-solid-phase-extraction-sorbents-for-sample- ... [https://www.bohrium.com/paper-details/comparison-of-solid-phase-extraction-sorbents-for-sample-clean-up-in-the-analysis-of-organic-explosives/811941132509380610-3391]
• 19. Explosives removal and quantification using porous ... [https://www.sciencedirect.com/science/article/abs/pii/S0045653523020763]
• 20. Interpol review of the analysis and detection of explosives ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9845958/]
• 21. LogP, LogD, pKa and LogS: A Physicists guide to basic ... [https://doktormike.gitlab.io/posts/navigating-logp-logd-pka-and-logs-a-physicists-guide/]
• 22. Key Properties in Drug Design | Predicting Lipophilicity, ... [https://chemaxon.com/blog/presentation/key-properties-in-drug-design-predicting-lipophilicity-pka-and-solubility-0]
• 23. Improved preconcentration workflow for organic explosive ... [https://www.sciencedirect.com/science/article/abs/pii/S0379073824001063]
• 24. Generic and accurate prediction of retention times in liquid ... [https://www.nature.com/articles/s42004-024-01135-0]
• 25. A survey of explosive traces in public places - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC12223331/]
• 26. pKa & LogP Analysis Services [https://www.thesolubilitycompany.com/pka-and-log-p-analysis-service/]
• 27. A review of the modern principles and applications of solid ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9659506/]
• 28. The Benefits of Using Hydrophilic-Lipophilic Balance (HLB) ... [https://www.elementlabsolutions.com/uk/chromatography-blog/post/the-benefits-of-using-hydrophilic-lipophilic-balance-hlb-in-spe]
• 29. Suspect screening and quantification of trace organic ... [https://www.sciencedirect.com/science/article/pii/S0304389417300109]
• 30. Monitoring of explosive residues in lake-bottom water ... [https://www.sciencedirect.com/science/article/abs/pii/S0269749118342635]
• 31. Comparison of Solid-Phase Extraction Sorbents for Sample ... [https://pubmed.ncbi.nlm.nih.gov/18272163/]
• 32. The Fundamentals of Solid Phase Extraction (SPE) [https://www.restek.com/articles/the-fundamentals-of-solid-phase-extraction-spe?srsltid=AfmBOooLRw8XfMNafJuMAMr0DbF-t0CZ2DLNpNCO8aiCVzStLPIf3cfq]
• 33. Solid Phase Extraction Guide [https://www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-sample-preparation/sample-preparation-consumables/solid-phase-extraction-consumables/spe-guide.html]
• 34. Simultaneous determination of nine APs and BPA in water ... [https://www.sciencedirect.com/science/article/pii/S1872204023000324]
• 35. Evaluation of different solid-phase extraction (SPE) ... [https://papers.ssrn.com/sol3/papers.cfm?abstract_id=5735688]
• 36. How Can We Improve Our Solid Phase Extraction ... [https://scioninstruments.com/us/blog/how-can-we-improve-our-solid-phase-extraction-processes/]
• 37. SPE Cartridges, Well Plates, Sorbents | Award-Winning [https://www.agilent.com/en/product/sample-preparation/solid-phase-extraction-spe?srsltid=AfmBOoprY-LiEl78zgprbTdZBV3N03buwIjaBXx8wOxtbtQhL59qCsLA]
• 38. : Top 10 Tips | News-Medical Solid Phase Extraction [https://www.news-medical.net/whitepaper/20210516/Solid-Phase-Extraction-Top-10-Tips.aspx]
• 39. factors affecting Optimising performances of... solid phase extraction [https://pmc.ncbi.nlm.nih.gov/articles/PMC7848654/]
• 40. Monolithic vs. particle-based solid - phase for selective... extraction [https://pubs.rsc.org/en/content/articlehtml/2025/va/d5va00057b]
• 41. Magnetic dispersive solid-phase extraction of organochlorine ... [https://pubs.rsc.org/en/content/articlehtml/2025/ra/d5ra01093d]
• 42. Roles of pH, ionic strength, and sulfate in the aqueous nitrate ... [https://acp.copernicus.org/articles/25/10731/2025/]
• 43. Optimizing Elution Conditions To Improve SPE - Blogs... Performance [https://www.alwsci.com/news/optimizing-elution-conditions-to-improve-spe-p-85177371.html]
• 44. Solving Recovery Problems in Solid-Phase Extraction [https://www.chromatographyonline.com/view/solving-recovery-problems-solid-phase-extraction]
• 45. The Fundamentals of Solid Phase Extraction (SPE) [https://www.restek.com/articles/the-fundamentals-of-solid-phase-extraction-SPE?srsltid=AfmBOoooLqGf75mLVCbV7DEn6UrxLbWtqzsFBm6TINbAD9HwsL_ITaKN]
• 46. Modern Trends and Best Practices in Mobile-Phase ... [https://www.chromatographyonline.com/view/modern-trends-and-best-practices-mobile-phase-selection-reversed-phase-chromatography-0]
• 47. Improved determination of femtogram-level organic ... [https://pubmed.ncbi.nlm.nih.gov/31202351/]
• 48. Cole-Parmer SPE Micropipette Tips, All Carbon Reverse ... [https://www.coleparmer.com/i/cole-parmer-spe-micropipette-tips-all-carbon-reverse-phase-dispersive-1-ml-50-mg-bed-weight-96-pk/6482207]
• 49. XTR: INTip TM Solid Phase Extraction (SPE) [https://dpxtechnologies.com/xtr/]
• 50. Homemade Pipette Tip Solid-Phase Extraction for the ... [https://www.mdpi.com/2297-8739/9/9/233]
• 51. Recent advancements in porous sorbent materials and ... [https://www.sciencedirect.com/science/article/abs/pii/S0013935125014720]
• 52. A low-cost cellulose-based composite as an efficient solid ... [https://pubs.rsc.org/en/content/articlehtml/2025/ra/d5ra02296g]
• 53. An ultra-sensitive and high-throughput trapping-micro-LC ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10918069/]
• 54. Improving laboratory throughput using LC mulplexing [https://sciex.com/tech-notes/forensic/toxicology/improving-laboratory-throughput-using-lc-mulplexing]
• 55. Creating a High-Throughput LC–MS-MS System Using ... [https://www.spectroscopyonline.com/view/creating-high-throughput-lc-ms-ms-system-using-common-components]
• 56. Practical Comparison of Different Approaches to Speed Up ... [https://www.chromatographyonline.com/view/practical-comparison-different-approaches-speed-lc-separations-0]
• 57. Magnetic solid pHase micro-extraction method with ... [https://www.sciencedirect.com/science/article/abs/pii/S0026265X2502925X]
• 58. Principles and Applications of Liquid Chromatography ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC2643089/]
• 59. Why Is Your SPE Recovery So Low? - News [https://www.alwsci.com/news/why-is-your-spe-recovery-so-low-85174255.html]
• 60. The Reason of Poor Sample Recovery When Using SPE [https://www.specartridge.com/the-reason-of-poor-sample-recovery-when-using-spe/]
• 61. Three Common SPE Problems [https://www.chromatographyonline.com/view/three-common-spe-problems-0]
• 62. Common Problems in Solid-Phase Extraction: A Practical ... [https://www.welch-us.com/blogs/knowleage-base/common-problems-in-solid-phase-extraction-a-practical-troubleshooting-guide]
• 63. High Flow-Rate Sample Loading in Large Volume Whole ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6479934/]
• 64. Several Problems Affecting Flow Rate of SPE Cartridges [https://www.specartridge.com/several-problems-affecting-the-flow-rate-of-spe-cartridges/]
• 65. Investigation and analysis of explosive traces in public ... [https://pubs.rsc.org/en/content/articlehtml/2025/ay/d5ay00183h]
• 66. Efficient Recovery of Platinum and Palladium by Fixed-Bed ... [https://link.springer.com/article/10.1007/s40831-025-01253-6]
• 67. Systematic Comparison of Extract Clean-Up with Currently ... [https://www.mdpi.com/1420-3049/29/19/4656]
• 68. Surface Complexation and Packed Bed Mass Transport ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11698511/]
• 69. Comparison of Solid-Phase Extraction Sorbents for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9858392/]
• 70. Understanding of Adsorption and Desorption Mechanisms of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7919792/]
• 71. Liquid-Liquid Extraction Techniques Principles and ... [https://www.elementlabsolutions.com/uk/chromatography-blog/post/liquid-liquid-extraction-techniques-principles-and-optimisation]
• 72. A review on recent developments and applications of ... [https://www.sciencedirect.com/science/article/pii/S2772582023000153]
• 73. Feasibility of carbon foam-based sorbents for the abatement of ... [https://pubs.rsc.org/en/content/articlehtml/2025/ra/d5ra05518k?page=search]
• 74. A scientometric review of explosives research: Challenges ... [https://www.sciencedirect.com/science/article/pii/S0379073825001513]
• 75. The Ultimate Guide to the Percent Recovery Formula for ... [https://dev.housing.arizona.edu/percent-recovery-formula]
• 76. How to determine recovery and matrix effects for your ... [https://www.biotage.com/blog/how-to-determine-recovery-and-matrix-effects-for-your-analytical-assay]
• 77. Biological Matrix Effects in Quantitative Tandem Mass ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4695332/]
• 78. Robust Extraction and Matrix Effect-Minimized method [https://www.sciencedirect.com/science/article/abs/pii/S0043135425015696]
• 79. Mass Balance-Based Quality Control of PVT Results ... [https://www.mdpi.com/1996-1073/17/13/3301]
• 80. Development and validation of a solid phase extraction ... [https://pubmed.ncbi.nlm.nih.gov/29353810/]
• 81. Molecularly imprinted solid phase extraction [https://www.sciencedirect.com/science/article/abs/pii/S0165993624001778]
• 82. Comparison of solid-phase extraction sorbents for sample ... [https://hal.science/hal-00261352/]
• 83. Improved determination of femtogram-level organic ... [https://www.sciencedirect.com/science/article/pii/S0039914019305314]
• 84. Comparative Studies on Sorption Recovery and Molecular ... [https://www.mdpi.com/2073-4441/13/24/3553]
• 85. Optimizing LC‑MS/MS Sensitivity: Strategies to Overcome ... [https://biotech-spain.com/en/articles/optimizing-lc-ms-ms-sensitivity-strategies-to-overcome-ion-suppression-and-boost-robustness-in-bioanalysis/]
• 86. Ion Suppression: A Major Concern in Mass Spectrometry [https://www.chromatographyonline.com/view/ion-suppression-major-concern-mass-spectrometry]
• 87. Automated LC-MS Sample Cleanup Using Turbulent Flow ... [https://www.thermofisher.com/blog/analyteguru/automated-lc-ms-sample-cleanup-using-turbulent-flow-chromatography/]
• 88. Achieving High Quality Results takes more than a Top of ... [https://www.peakscientific.com/discover/articles/theres-more-to-achieving-high-quality-results-than-a-top-of-the-line-lc-ms/]
• 89. Limit of Blank, Limit of Detection and Limit of Quantitation [https://pmc.ncbi.nlm.nih.gov/articles/PMC2556583/]
• 90. What is meant by the limit of detection and quantification (LOD ... [https://mpl.loesungsfabrik.de/en/english-blog/method-validation/limit-of-detection-quantifcation]
• 91. Determining LOD and LOQ Based on the Calibration Curve [https://www.sepscience.com/hplc-solutions-126-chromatographic-measurements-part-5-determining-lod-and-loq-based-on-the-calibration-curve-6959]
• 92. LIMITS OF DETECTION (ASTM E-2677 Standard) [https://www.itl.nist.gov/div898/software/dataplot/refman1/auxillar/lod.htm]
• 93. Recent Advances in Ambient Mass Spectrometry of Trace ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6026862/]
• 94. Development and validation of a new RP-HPLC method for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10503966/]
• 95. Gas chromatography positive chemical ionization and ... [https://www.sciencedirect.com/science/article/abs/pii/S0039914005000561]
• 96. Establishing a universal swabbing and clean-up protocol ... [https://www.sciencedirect.com/science/article/abs/pii/S0379073812003805]
• 97. The Role of Polymers in Solid-Phase Extraction and ... [https://www.chromatographyonline.com/view/role-polymers-solid-phase-extraction-and-sample-preparation]