Multi-Sorbent Extraction Strategies for Broad-Spectrum Contaminant Analysis in Complex Matrices

Abstract

This article provides a comprehensive overview of multi-sorbent extraction strategies, a powerful approach for the broad-spectrum analysis of contaminants in complex biological and environmental samples. It explores the foundational principles of using combined sorbent phases to overcome the limitations of single-phase extractions, enabling the capture of a wider range of analytes with diverse physicochemical properties. The content details methodological advances, including the use of mixed-mode phases, molecularly imprinted polymers (MIPs), and miniaturized techniques like μSPE and MEPS. It further offers practical guidance on troubleshooting and optimizing methods to mitigate analyte loss and matrix effects. Finally, the article discusses rigorous validation protocols and comparative performance assessments against traditional methods, highlighting the enhanced selectivity, sensitivity, and cost-effectiveness of multi-sorbent approaches for modern analytical challenges in biomedical research and drug development.

The Principles and Evolution of Multi-Sorbent Extraction

Sorbent-based extraction represents a cornerstone technique in modern analytical chemistry, enabling the selective isolation and pre-concentration of analytes from complex sample matrices. This methodology has largely superseded traditional liquid-liquid extraction by offering significant advantages, including reduced organic solvent consumption, enhanced efficiency, and improved reproducibility [1] [2]. The fundamental principle involves the selective partitioning of target compounds between a liquid sample and a solid sorbent phase, followed by elution with a specialized solvent [2]. Originally developed for trace organic analysis in water samples, these techniques have evolved through several generations—from early charcoal applications to modern cartridges, disks, and advanced formats like solid-phase microextraction (SPME) [2]. Today, sorbent-based extraction serves as an indispensable sample preparation tool across numerous fields, including environmental monitoring, food safety, pharmaceutical analysis, and clinical chemistry, forming a critical bridge between raw samples and sophisticated chromatographic instruments [1] [3].

Key Techniques and Principles

The landscape of sorbent-based extraction encompasses several powerful techniques, each with distinct mechanisms and applications.

Solid-Phase Extraction (SPE) operates through a flow-through equilibrium process where a liquid sample passes through a sorbent bed packed in a cartridge or embedded in a disk [2]. Analytes are retained based on specific interactions with the sorbent material, while interfering matrix components are washed away. The captured analytes are subsequently recovered using an appropriate elution solvent [2]. This exhaustive technique is particularly valued for its ability to achieve significant sample clean-up and pre-concentration.

Dispersive SPE (d-SPE), popularized by the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) approach, represents a streamlined evolution of traditional SPE [4] [3]. In d-SPE, a small amount of sorbent is directly added to a sample extract, typically following an initial extraction with an organic solvent. The mixture is then agitated and centrifuged, allowing the sorbent to bind interfering matrix components while leaving the target analytes in the solution [4]. This method significantly reduces time, solvent consumption, and required materials compared to cartridge-based SPE.

Solid-Phase Microextraction (SPME) is a non-exhaustive, equilibrium-based technique that integrates sampling, extraction, and concentration into a single step [3] [5]. A fiber coated with a sorbent material is exposed to the sample (via direct immersion or headspace), allowing analytes to partition into the coating. The fiber is then transferred directly to a chromatographic instrument for thermal or solvent desorption [5]. Recent innovations include mixed-sorbent coatings that provide wider extraction coverage for both polar and nonpolar compounds, as well as charged species [5].

Table 1: Comparison of Major Sorbent-Based Extraction Techniques

Parameter	SPE (Cartridge)	d-SPE (QuEChERS)	SPME
Classification	Exhaustive flow-through equilibrium [2]	Exhaustive flow-through equilibrium [2]	Non-exhaustive batch equilibrium [2]
Sorbent Amount	4 mg to several grams [2]	Typically 10-50 mg per mL of extract [4]	Coating volume of a few µL [5]
Principle	Analyte retention and elution [1]	Bulk matrix removal [4]	Equilibrium partitioning [3]
Solvent Consumption	Moderate to high	Low	Virtually none [3]
Automation Potential	High (vacuum manifolds, robotic systems) [2]	Moderate	High (autosamplers)
Typical Applications	Broad-spectrum sample clean-up and pre-concentration [1]	Multi-residue analysis in complex matrices [4]	Volatile compound analysis, on-site sampling [3] [5]

Current Applications and Performance Data

Sorbent-based extraction demonstrates exceptional versatility across diverse application fields, consistently delivering robust performance data as validated by recent studies.

In environmental analysis, a modified QuEChERS (d-SPE) method was successfully validated for the determination of 90 emerging organic contaminants (EOCs) in soil and sediment matrices [4]. The targeted analytes spanned multiple classes, including pesticides, pharmaceuticals, per- and polyfluoroalkyl substances (PFASs), flame retardants, and plasticizers, showcasing the method's remarkable breadth. Performance data confirmed good accuracy and precision, with mean recoveries between 70 and 120% and relative standard deviations (RSD) typically below 20% for most compounds [4]. The method achieved low matrix effects and method limit of quantifications (MLOQs) ranging from 0.25 to 10 µg/kg, demonstrating sensitivity adequate for monitoring trace environmental contaminants [4].

In food analysis, the performance of different d-SPE sorbents was systematically compared for pesticide multiresidue analysis in challenging high-fat rapeseed samples [6]. The study evaluated traditional sorbents (PSA/C18) against newer materials specifically designed for fatty matrices (Z-Sep, Z-Sep+, EMR-Lipid). The Enhanced Matrix Removal-Lipid (EMR-Lipid) sorbent delivered superior performance, achieving average pesticide recoveries of 103% for 70 pesticides and 70% for another 70 pesticides at spiking levels of 10 µg/kg and 50 µg/kg, with low RSD values [6]. This application highlights how sorbent innovation directly addresses matrix-specific challenges in food safety monitoring.

For bioanalytical and pharmaceutical applications, SPE remains a gold standard for sample clean-up. A comprehensive study comparing 17 different SPE sorbents for extracting 13 diverse drugs from human urine identified phenyl sorbent (C6H5) as the most effective, providing recoveries higher than 85.5% with RSDs <10% while effectively removing interfering substances [7]. This demonstrates the critical importance of sorbent selectivity when dealing with complex biological matrices like urine, where matrix effects can severely compromise analytical accuracy.

Table 2: Performance Data of Sorbent-Based Extraction in Different Applications

Application Area	Target Analytes	Sorbent/Method	Key Performance Metrics
Environmental Analysis	90 emerging contaminants (pesticides, pharmaceuticals, PFASs, plasticizers) in soil/sediment [4]	Modified QuEChERS (d-SPE) [4]	Mean recoveries: 70-120%; RSD < 20% for most compounds; MLOQs: 0.25-10 µg/kg [4]
Food Safety	179 pesticides in rapeseed [6]	QuEChERS with EMR-Lipid d-SPE [6]	70 pesticides: 70-120% recovery; 70 pesticides: 30-70% recovery; LOQs: 1.72-6.39 µg/kg for 173 pesticides [6]
Pharmaceutical Analysis	13 diverse drugs in human urine [7]	SPE with phenyl sorbent (C6H5) [7]	Recoveries >85.5%; RSD <10%; LODs: 0.003-0.217 µg/mL [7]
Water Analysis	Drugs of abuse, artificial sweeteners, organic contaminants [5]	Mixed-sorbent SPME blade (HLB, HLB-WCX, HLB-WAX) [5]	Good extraction performance for polar, nonpolar, and charged compounds; suitable for on-site sampling [5]

Detailed Experimental Protocols

Protocol 4.1: Modified QuEChERS for Multi-Class Contaminants in Soil and Sediment

This protocol is adapted from the validated method for extraction of 90 emerging contaminants, demonstrating effective broad-spectrum extraction capability [4].

Reagents and Materials:

• Acetonitrile (HPLC grade)
• Magnesium sulfate (MgSO4), anhydrous
• Sodium chloride (NaCl)
• Dispersive SPE sorbents: Primary Secondary Amine (PSA), C18
• EDTA
• Formic acid
• Ultrapure water
• Centrifuge tubes (50 mL)

Procedure:

• Sample Preparation: Homogenize soil or sediment samples. Sieve through a 2-mm mesh. Weigh 5.0 g of sample into a 50-mL centrifuge tube.
• Hydration: Add 5 mL of ultrapure water to the sample and vortex for 30 seconds.
• Extraction: Add 10 mL of acetonitrile containing 1% formic acid. Add 200 µL of EDTA solution (0.1 M). Vortex vigorously for 1 minute.
• Salt-Assisted Partitioning: Add 4 g of MgSO4 and 1 g of NaCl. Immediately shake vigorously for 1 minute to prevent salt clumping.
• Centrifugation: Centrifuge at 4000 rpm for 5 minutes to achieve phase separation.
• Clean-up (d-SPE): Transfer 1 mL of the upper acetonitrile layer to a 2-mL d-SPE tube containing 150 mg of MgSO4, 50 mg of PSA, and 50 mg of C18.
• Final Extraction: Vortex the d-SPE tube for 30 seconds, then centrifuge at 4000 rpm for 2 minutes.
• Analysis: Transfer the supernatant to an autosampler vial for analysis by UPLC-MS/MS.

Critical Parameters: The acidification of acetonitrile with formic acid improves extraction efficiency for acidic compounds. The combination of PSA and C18 in d-SPE effectively removes various matrix interferents, including fatty acids and pigments [4].

Protocol 4.2: Mixed-Sorbent SPME for Broad-Spectrum Water Analysis

This protocol utilizes a innovative mixed-sorbent SPME coating for comprehensive extraction of diverse contaminants in water samples [5].

Reagents and Materials:

• Mixed-sorbent SPME blades (HLB, HLB-WCX, HLB-WAX in PAN binder) [5]
• Methanol (HPLC grade)
• Ultrapure water
• 20-mL headspace vials
• SPME holder compatible with blades

Procedure:

• Conditioning: Prior to first use, condition the SPME blade in methanol for 30 minutes, then in ultrapure water for 10 minutes.
• Sample Collection: Transfer 15 mL of water sample to a 20-mL headspace vial. For on-site sampling, SPME blades can be deployed using a drone sampling system [5].
• Extraction: Immerse the SPME blade directly into the sample. Extract for 60 minutes with continuous agitation at 25°C.
• Rinsing: Following extraction, briefly rinse the blade with ultrapure water (3 seconds) to remove co-extracted salts and matrix components.
• Desorption: Desorb the blade in the desorption chamber of the LC system using a mixture of methanol and water (70:30, v/v) for 15 minutes.
• Analysis: Analyze the desorbed extract using LC-MS/MS or LC-HRMS.

Critical Parameters: The mixed-sorbent coating (HLB, HLB-WCX, HLB-WAX) enables simultaneous extraction of polar, nonpolar, and charged compounds in a single device [5]. The PAN binder acts as a porous filter, excluding large matrix elements while allowing small molecules to access the sorbent particles.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Sorbents and Their Applications in Broad-Spectrum Analysis

Sorbent Type	Mechanism/Properties	Primary Applications	Performance Notes
HLB (Hydrophilic-Lipophilic Balanced)	Reversed-phase copolymer with balanced wettability [5]	Broad-spectrum extraction of polar and non-polar compounds [5]	High capacity, does not require pre-wetting; ideal for mixed-sorbent approaches [5]
C18 (Octadecylsilane)	Reversed-phase hydrophobic interactions [3]	Retention of non-polar compounds; removal of lipophilic interferents in d-SPE [6]	Traditional workhorse; may exhibit low retention for highly polar compounds [2]
PSA (Primary Secondary Amine)	Weak anion exchange; hydrogen bond acceptor [4] [6]	Removal of fatty acids, organic acids, sugars, and pigments in d-SPE [4]	Particularly effective in QuEChERS for food matrices; may destabilize base-sensitive compounds [6]
EMR-Lipid (Enhanced Matrix Removal)	Size-exclusion and hydrophobic interactions [6]	Selective removal of lipids from fatty food matrices [6]	Demonstrated superior performance in rapeseed analysis with 103% average pesticide recovery [6]
Mixed-mode Cation/Anion Exchange	Combined reversed-phase and ion-exchange mechanisms [2]	Selective extraction of ionic compounds from complex matrices	Enables pH-selective elution; excellent for basic/acidic drug extraction [2]
Molecularly Imprinted Polymers (MIPs)	Predefined affinity for target molecules [3]	Highly selective extraction of specific analyte classes	Antibody-like specificity; reusable; ideal for veterinary drugs and contaminants [3]

Future Perspectives

The evolution of sorbent-based extraction continues with several promising directions. Multi-sorbent strategies represent a paradigm shift from seeking universal single sorbents to designing synergistic sorbent combinations [5]. These approaches, exemplified by mixed-SPME coatings incorporating HLB, HLB-WCX, and HLB-WAX, provide wider extraction coverage for comprehensive analysis of complex environmental samples [5]. The integration of sorbent-based extraction with advanced computational approaches is another growing trend. Machine learning algorithms are being applied to non-targeted analysis data to identify contaminant sources and patterns, creating powerful frameworks for environmental forensics [8]. Furthermore, automation and miniaturization continue to advance, with developments in pipette-tip SPE, multi-well SPE formats, and in-tube SPME enabling higher throughput while reducing solvent consumption and labor requirements [2]. These innovations collectively reinforce the central role of sorbent-based extraction in modern analytical methodologies, particularly for broad-spectrum contaminant analysis in support of environmental and public health protection.

Traditional analytical methods for contaminant analysis have predominantly relied on single-sorbent technologies, which face fundamental limitations in broad-spectrum analysis. These limitations become particularly evident when monitoring diverse contaminant classes with varying physicochemical properties—a common scenario in environmental, pharmaceutical, and food safety testing. Single sorbents exhibit selective affinity based on compound characteristics including volatility, polarity, and molecular size, inevitably creating analytical gaps that compromise method comprehensiveness.

The clean-up efficiency of sample preparation is crucial for minimizing matrix effects and achieving reliable quantification, especially for trace-level contaminants in complex matrices. Research demonstrates that single sorbent approaches often fail to provide sufficient clean-up for challenging matrices high in fats, proteins, or other interferents, leading to compromised analytical sensitivity and accuracy. These limitations have driven the investigation and adoption of multi-sorbent strategies as a robust alternative for comprehensive contaminant monitoring.

Technical Advantages of Multi-Sorbent Architectures

Expanded Compound Coverage and Improved Retention

Multi-sorbent configurations significantly broaden the range of analyzable compounds by strategically combining complementary sorbent materials. Studies demonstrate that while a Carbopack X single sorbent effectively targets approximately 50 toxic volatile organic compounds (VOCs), and Carboxen 569 addresses 37, their combined implementation covers 61 toxic VOCs with robust uptake rates, demonstrating the synergistic effect of sorbent combination [9]. This expanded coverage is particularly notable for very volatile organic compounds (VVOCs) including alcohols, chlorinated compounds, and ketones, which often exhibit poor retention on traditional single sorbents like Tenax TA [10].

The enhanced performance stems from the strategic sequencing of sorbents with varying adsorption strengths and affinities. In a typical configuration, samples first contact a weaker adsorbent (e.g., Tenax TA) optimized for heavier compounds, followed by progressively stronger sorbents (e.g., Carbopack B, Carboxen) to capture more volatile and polar analytes that would breakthrough single-sorbent systems [11]. This layered approach effectively extends the analytical range to encompass compounds with diverse chemical properties in a single analysis.

Enhanced Breakthrough Volumes and Method Sensitivity

Multi-sorbent systems demonstrate superior breakthrough volumes compared to single-sorbent alternatives, directly impacting method detection limits and reliability. Research comparing multi-sorbent beds (Carbotrap, Carbopack X, Carboxen 569) against Tenax TA single tubes revealed significantly higher breakthrough values for the multi-sorbent approach, with Tenax TA exhibiting breakthrough from 0% to 77% across various sampling volumes and compounds [10]. This robust retention capability enables larger sampling volumes without analyte loss, consequently lowering method detection limits—a critical advantage for trace-level contaminant analysis.

Field applications confirm these technical benefits, with multi-sorbent tubes demonstrating lower method detection limits and superior recovery of odor-causing VOCs at concentration ranges from 0.3 to 98 ppbv in challenging environmental conditions [11]. The improved sensitivity directly addresses the pressing need for accurate monitoring of contaminants at low concentrations, particularly important for odor assessment and environmental exposure studies where compounds often exist at concentrations near threshold levels.

Table 1: Performance Comparison of Single-Sorbent vs. Multi-Sorbent Approaches for VOC Analysis

Parameter	Single-Sorbent (Tenax TA)	Multi-Sorbent (Carbopack X/Carboxen 569)	Multi-Sorbent (Carbotrap/Carbopack X/Carboxen 569)
Number of Target Compounds	Limited by selective affinity	61 toxic VOCs with robust uptake rates [9]	Comprehensive range including VVOCs [10]
Breakthrough Behavior	0-77% breakthrough for various compounds [10]	Significantly reduced breakthrough	Minimal breakthrough for most compounds [10]
VVOC Performance	Poor recovery for very volatile compounds [10]	Improved VVOC coverage	Superior recovery of polar and very volatile compounds [10]
Field Applicability	Limited for very volatile compounds	Validated under varied climate conditions [9]	Effective for low-concentration odorants [11]

Superior Matrix Clean-up Capabilities

Multi-sorbent strategies demonstrate exceptional performance in purifying complex sample matrices, a critical factor for minimizing matrix effects in mass spectrometric analysis. In comparative studies of dispersive solid-phase extraction (d-SPE) sorbents for pesticide analysis in rapeseed—a challenging high-fat matrix—enhanced matrix removal-lipid (EMR-Lipid) sorbent delivered superior clean-up efficiency with 70 of 179 pesticides exhibiting recoveries within 70–120% and significantly reduced matrix effects [6]. This advanced clean-up capability enables more accurate quantification and reduces instrument maintenance requirements.

The clean-up efficiency directly correlates with analytical performance metrics. Methods employing multi-sorbent clean-up demonstrate significantly lower matrix effects (between -50% and 50% for 169 pesticides) compared to traditional approaches [6]. This reduction in matrix suppression or enhancement effects translates to improved analytical accuracy and reliability, particularly important for regulatory compliance monitoring where precise quantification is mandatory.

Experimental Protocols and Methodologies

Protocol: Multi-Sorbent Tube Preparation for Broad-Spectrum VOC Analysis

Principle: This protocol describes the assembly and conditioning of multi-sorbent tubes for comprehensive VOC monitoring, combining sorbents with complementary adsorption properties to maximize analyte coverage [11].

Materials:

• Stainless steel thermal desorption tubes (89 mm length, 6 mm OD)
• Tenax TA (60/80 mesh, 35 m²/g)
• Carbopack B (60/80 mesh, 100 m²/g)
• Carboxen 1003 (40/60 mesh, 1000 m²/g)
• Glass wool (non-treated, GC suitable)
• Stainless steel meshes and end plugs
• Analytical balance (±0.1 mg sensitivity)
• Tube conditioning apparatus with inert gas supply
• Thermal desorber conditioning unit

Procedure:

• Tube Preparation: Clean stainless steel tubes with appropriate solvents and heat to remove contaminants.
• Sorbent Packing: Sequentially pack sorbents in order of increasing strength:
  • First layer: 181.4 mg Tenax TA (retains C7-C26 compounds)
  • Separation: Place glass wool barrier
  • Second layer: 109.2 mg Carbopack B (retains C5-C12 compounds)
  • Separation: Place glass wool barrier
  • Third layer: 59.5 mg Carboxen 1003 (retains C2-C5 compounds)
  • Secure both ends with stainless steel meshes and end plugs [11]
• Conditioning: Condition packed tubes at 330°C for 12 hours under inert gas flow (20-30 mL/min) to remove contaminants.
• Verification: Analyze blank tubes to confirm absence of contaminants before use.
• Storage: Store conditioned tubes in sealed containers with caps to prevent contamination.

Protocol: Evaluation of Sorbent Performance for VOC Monitoring

Principle: This method quantitatively compares the performance of single-sorbent and multi-sorbent configurations for VOC monitoring, evaluating key parameters including breakthrough volume and recovery efficiency [10].

Materials:

• Prepared single-sorbent (Tenax TA) and multi-sorbent tubes
• Standard gas mixture containing target VOCs at known concentrations
• Dynamic gas dilution system
• Thermal desorption unit coupled to GC-MS
• Quality control standards
• Humidity and temperature control apparatus

Procedure:

• Experimental Setup: Connect series of two sorbent tubes (front and back) to evaluate breakthrough.
• Standard Generation: Generate standard gas mixtures containing target VOCs at relevant concentrations (ppbv levels) using dynamic dilution systems.
• Sample Collection: Draw standard gas through sorbent tubes at controlled flow rates (10-100 mL/min) for defined periods, maintaining consistent temperature and humidity.
• Breakthrough Calculation: Analyze both front and back tubes separately.
  • Breakthrough (%) = (Amount in back tube / Total amount from both tubes) × 100 [10]
• Recovery Assessment: Compare experimental results with theoretical loading based on standard concentrations.
• Data Analysis: Calculate response factors, method detection limits, and uptake rates for each sorbent configuration.

Quality Control:

• Analyze replicate samples (n ≥ 3) to determine precision
• Include method blanks to monitor contamination
• Use certified reference materials when available
• Validate with online measurement systems where possible [9]

Table 2: Research Reagent Solutions for Multi-Sorbent Extraction

Sorbent Material	Surface Area	Optimal Compound Range	Primary Function	Key Characteristics
Tenax TA	35 m²/g [11]	C7-C26 compounds [11]	Retention of semi-volatile compounds	Hydrophobic, thermal stability to 350°C
Carbopack B	100 m²/g [11]	C5-C12 compounds [11]	Retention of medium-volatility compounds	Graphitized carbon black, hydrophobic
Carboxen 1003	1000 m²/g [11]	C2-C5 compounds [11]	Retention of highly volatile compounds	Carbon molecular sieve, high surface area
Z-Sep+	Specialized sorbent	Fatty acid removal [6]	Matrix clean-up for lipid-rich samples	Zirconia-coated silica, Lewis acid-base interactions
EMR-Lipid	Selective retention	Lipid removal without pesticide loss [6]	Advanced matrix clean-up	Selective retention of long unbranched hydrocarbons

Implementation Strategies and Workflow Integration

Systematic Method Development Approach

Implementing multi-sorbent strategies requires careful consideration of analytical goals and sample characteristics. The following workflow provides a systematic approach for method development:

Multi-Sorbent Method Development Workflow

Critical considerations for each stage include:

• Analyte Characterization: Compile data on vapor pressure, polarity, functional groups, and molecular size to inform sorbent selection [11].
• Matrix Assessment: Evaluate fat content, protein composition, and potential interferents to determine clean-up requirements [6].
• Sorbent Selection: Choose complementary sorbents that collectively cover the target analyte range while providing necessary matrix clean-up.
• Sequence Optimization: Arrange sorbents from weakest to strongest to prevent premature breakthrough and ensure balanced distribution [11].
• Method Validation: Establish breakthrough volumes, method detection limits, recovery efficiencies, and precision under actual sampling conditions [9].

Workflow Integration for Comprehensive Analysis

Multi-sorbent strategies integrate into broader analytical workflows that may include advanced computational approaches for data interpretation. The integration of machine learning with non-target analysis represents a cutting-edge extension of these methodologies, enabling more sophisticated contaminant source identification through pattern recognition in complex chemical datasets [8]. This integration highlights the evolving role of multi-sorbent approaches within comprehensive analytical frameworks.

Automated multidimensional separation techniques employing sorbent-based extraction columns represent another significant advancement, improving reproducibility and throughput while maintaining the comprehensive coverage benefits of multi-sorbent strategies [12]. These systems typically consist of two columns—an extraction column for initial analyte enrichment and an analytical column for separation—leveraging the strengths of multi-sorbent configurations in the first dimension to achieve superior performance for trace-level analysis in complex matrices.

The limitations of single-sorbent approaches have unequivocally established the necessity for multi-sorbent strategies in broad-spectrum contaminant analysis. Through expanded compound coverage, enhanced breakthrough resistance, and superior matrix clean-up capabilities, multi-sorbent configurations address fundamental gaps in traditional methodologies. The experimental protocols and implementation frameworks presented provide actionable guidance for researchers seeking to leverage these advantages in diverse application contexts. As analytical challenges continue to evolve in complexity, multi-sorbent strategies will remain essential tools for comprehensive environmental monitoring, pharmaceutical analysis, and food safety assessment.

The analysis of complex samples for trace-level contaminants presents a significant challenge in modern analytical chemistry. Single-mode extraction sorbents often lack the universality or selectivity required for broad-spectrum multi-residue analysis. This application note details a unified strategy that synergistically combines three fundamental separation principles: Hydrophilic-Lipophilic Balance (HLB), Ion-Exchange, and Specific Interactions. By integrating these mechanisms into a single, multi-sorbent extraction platform, researchers can achieve unparalleled efficiency in the extraction of diverse analytes from complex matrices, enabling comprehensive contaminant profiling essential for environmental monitoring, food safety, and pharmaceutical development.

The core innovation lies in the deliberate combination of complementary sorbents within a single extraction device. This approach simultaneously captures a wider range of contaminants differing in polarity, charge, and molecular structure in a single pass, thereby streamlining sample preparation, reducing solvent consumption, and enhancing analytical throughput. The following sections provide a detailed theoretical framework, experimental protocols, and visual guides for implementing this powerful analytical strategy.

Theoretical Framework

The Hydrophilic-Lipophilic Balance (HLB) Principle

The HLB system is a quantitative measure of the affinity of a surfactant or sorbent for water or oil phases, typically represented on a scale of 0 to 20 [13] [14]. A lower HLB value (0-6) indicates lipophilic character, making the material suitable for interacting with non-polar compounds and for creating water-in-oil (W/O) emulsions. Conversely, a higher HLB value (10-20) indicates hydrophilic character, which is effective for polar analytes and for stabilizing oil-in-water (O/W) emulsions [15] [13]. The HLB value is not merely a theoretical index; it provides practical guidance for selecting sorbents to target contaminants based on their polarity. For instance, HLB-based sorbents are exceptionally effective at extracting a broad range of neutral, acidic, and basic compounds by balancing both hydrophilic and lipophilic interactions [16].

The Ion-Exchange (IEX) Principle

Ion-exchange chromatography separates molecules based on their net surface charge [17]. The process involves a charged stationary phase (sorbent) and a liquid mobile phase. IEX sorbents are categorized as either cation exchangers, which attract positively charged molecules, or anion exchangers, which attract negatively charged molecules. Analytes bind to the sorbent at low ionic strength and are subsequently eluted by increasing the ionic strength (salt concentration) or shifting the pH of the mobile phase, which disrupts the electrostatic interaction [17] [18]. This principle is crucial for the selective extraction of ionizable contaminants, such as certain pharmaceuticals, herbicides, and acidic pesticides, which may be missed by HLB-only sorbents.

The Role of Specific Interactions

Beyond HLB and IEX, specific interactions can be engineered into sorbents to achieve high selectivity for target analyte classes. These interactions include hydrogen bonding, π-π interactions, dipole-dipole forces, and size exclusion. The development of novel sorbent materials, such as biochar from renewable resources and molecularly imprinted polymers, leverages these interactions [19] [20]. For example, biochar's high specific surface area and diverse functional groups make it an excellent sorbent for the extraction of organic contaminants like organophosphorus pesticides [19]. Incorporating such materials into a multi-sorbent system allows for the tailored cleanup and enrichment of specific analytes from challenging matrices.

Synergy in a Multi-Sorbent Strategy

The combination of HLB, IEX, and specific interactions in a single platform creates a synergistic effect. The HLB component provides a broad "catch-all" foundation for a wide polarity range, the IEX component captures ionizable compounds that might elude the HLB phase, and the specific interaction sorbents offer tailored selectivity to reduce matrix interferences or target particular residues [21] [16]. This strategy was exemplified in a study using a dual-sorbent device for pesticide analysis, which proved superior to single-sorbent platforms in terms of extraction recovery [21].

Experimental Protocols

Protocol 1: Multi-Sorbent Extraction for Aqueous Samples

This protocol is adapted for the extraction of multi-class contaminants from water samples using a cartridge or device containing a mixed bed of HLB and ion-exchange sorbents.

• 1. Sorbent Preparation: Use a commercial mixed-bed sorbent cartridge (e.g., combining a hydrophilic-lipophilic balanced polymer with a weak anion-exchange resin) or prepare a custom dual-sorbent disk [21] [16].
• 2. Conditioning: Sequentially pass 5-10 mL of methanol and 5-10 mL of reagent water through the sorbent bed at a flow rate of ~5 mL/min. Do not allow the bed to dry out.
• 3. Sample Loading: Acidify or basify the water sample to a pH of ~2.5 or ~7 (depending on the target analytes) to ensure ionizable compounds are in the correct ionic form. Load the sample (e.g., 100-1000 mL) through the sorbent at a controlled flow rate of 5-10 mL/min.
• 4. Washing: After sample loading, wash the sorbent with 5-10 mL of a mild aqueous solution (e.g., 5% methanol in water, pH-adjusted) to remove weakly retained matrix components.
• 5. Drying: Remove residual water from the sorbent by drawing air or nitrogen through the cartridge for 10-20 minutes, or by centrifugation.
• 6. Elution: Elute the captured analytes with 5-10 mL of an organic solvent (e.g., methanol, acetonitrile, or a mixture with acetone). For comprehensive recovery, two sequential elutions with different solvents may be required. Collect the eluate in a calibrated tube.
• 7. Concentration and Reconstitution: Gently evaporate the eluate to near dryness under a stream of nitrogen at 30-40°C. Reconstitute the residue in an appropriate volume (e.g., 100-500 µL) of initial mobile phase solution compatible with the subsequent chromatographic analysis (e.g., LC-MS/MS).

Protocol 2: Ion-Exchange Chromatography for Protein Purification

This protocol describes the use of IEX for purifying a target protein from a clarified cell lysate.

• 1. Sample Preparation:
  • Clarification: Centrifuge the cell lysate at 40,000-50,000 g for 30 minutes at 4°C to remove debris. Filter the supernatant through a 0.45 µm or 0.22 µm cellulose acetate or PVDF membrane filter [18].
  • Buffer Exchange: Desalt the clarified sample into the starting IEX buffer (e.g., 20 mM Tris-HCl, pH 8.0) using a desalting column (e.g., Sephadex G-25). The conductivity of the final sample should be low (equivalent to ≤50-150 mM NaCl) to ensure binding [17] [18].
• 2. Column Equilibration: Pack an IEX column (e.g., 5 mL bed volume) and equilibrate it with at least 5-10 column volumes (CV) of binding buffer (e.g., 20 mM Tris-HCl, pH 8.0).
• 3. Sample Loading: Load the prepared sample onto the column at a moderate flow rate (e.g., 1-2 mL/min for a 5 mL column).
• 4. Washing: Wash the column with 5-10 CV of binding buffer until the UV absorbance (at 280 nm) returns to baseline to remove unbound and weakly bound contaminants.
• 5. Elution (Gradient Method):
  • Elute the bound proteins using a linear salt gradient (e.g., from 0 to 1 M NaCl) over 20 CVs while collecting fractions [17].
  • Alternatively, if the elution profile is known, a step elution or a shallow gradient can be used for better resolution [17].
• 6. Analysis: Analyze the collected fractions via SDS-PAGE and measure the target protein's activity or concentration. Pool the fractions containing the purified protein.

Reagents and Materials

Table 1: Key Research Reagent Solutions for Multi-Sorbent Extraction

Reagent / Material	Function / Application
HLB Sorbent (e.g., hydrophilic-lipophilic balanced copolymer)	Provides broad-spectrum retention of neutral, acidic, and basic organic contaminants via reversed-phase and polar interactions [16].
Ion-Exchange Sorbents (Cation & Anion Exchange resins)	Selective retention of ionizable analytes based on their charge; crucial for capturing acidic/basic pharmaceuticals and pesticides [17] [16].
Biochar-based Sorbents	Sustainable sorbent with high surface area and microporosity for efficient extraction of organic pollutants like pesticides; contributes specific surface interactions [19] [20].
Methanol, Acetonitrile	Common organic solvents used for conditioning sorbents and eluting captured analytes during solid-phase extraction.
Ammonium Acetate, Ammonium Hydrogen Carbonate	Volatile salts used in buffer preparation for IEX and LC-MS/MS analysis, facilitating easy removal by evaporation [18].
Solid Phase Extraction (SPE) Manifold	Automated or manual device used to process multiple samples simultaneously under controlled vacuum or pressure.

Table 2: HLB Value Guide for Sorbent and Emulsifier Selection

HLB Value Range	Primary Application	Common Uses
1 - 3	Anti-foaming Agents	Foam control in industrial processes [13] [14].
3 - 6	Water-in-Oil (W/O) Emulsifiers	Creating stable milky emulsions where water is dispersed in oil [13] [14].
7 - 9	Wetting and Coating Agents	Stabilizing milky suspensions and improving spreadability [13].
8 - 12	Wetting Agents	Improving adhesion and reducing surface tension [13].
10 - 15	Oil-in-Water (O/W) Emulsifiers	Creating stable, often clear, emulsions where oil is dispersed in water; common in cosmetics [15] [13] [14].
13 - 15	Detergents	Washing and cleaning applications [13] [14].
13 - 18	Solubilisers	Forming transparent solutions by incorporating oils into aqueous phases [14].

Workflow and Relationship Visualizations

Multi-Sorbent Extraction Mechanism

Ion-Exchange Protein Purification

The analysis of trace-level contaminants in complex matrices represents a significant challenge in modern analytical chemistry, particularly in fields such as food safety, environmental monitoring, and pharmaceutical development. Sorbent-based extraction techniques have emerged as powerful tools to address this challenge, enabling researchers to isolate, purify, and concentrate target analytes from complicated sample matrices. The fundamental principle underlying these techniques involves the selective interaction between target compounds and functionalized solid phases, which allows for efficient extraction and clean-up in a single streamlined process. The evolution of sorbent materials has progressed from traditional reversed-phase silicas to advanced polymeric and mixed-mode phases designed with specific selectivity profiles for particular analyte classes [22].

Within this context, the strategic combination of multiple sorbents has gained prominence as an effective approach for broad-spectrum contaminant analysis. Unlike single-sorbent methodologies, which may exhibit limited effectiveness against the diverse physicochemical properties of different contaminant classes, multi-sorbent strategies leverage the complementary characteristics of various materials to achieve more comprehensive extraction coverage. This review focuses on five particularly significant sorbent phases: Hydrophilic-Lipophilic Balance (HLB), Primary Secondary Amine (PSA), Octadecyl (C18), Graphitized Carbon Black (GCB), and Zirconia-based phases. Each of these sorbents possesses distinct surface chemistries and interaction mechanisms that make them uniquely suited to address specific analytical challenges in complex sample matrices [23] [24].

The development of multi-sorbent approaches aligns with broader trends in green analytical chemistry, which emphasize the reduction of solvent consumption, minimization of waste generation, and improvement of overall methodological efficiency. Techniques such as Microextraction by Packed Sorbent (MEPS) represent the miniaturization of traditional solid-phase extraction principles, allowing for efficient sample processing with dramatically reduced solvent volumes [23]. Similarly, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method has revolutionized sample preparation in pesticide residue analysis through its innovative use of dispersive SPE with sorbent combinations tailored to remove specific matrix interferences [22]. This review provides a comprehensive examination of the fundamental characteristics, applications, and experimental protocols for these five essential sorbent classes, with particular emphasis on their implementation within integrated multi-sorbent workflows for broad-spectrum contaminant analysis.

Characteristics and Properties of Individual Sorbents

Fundamental Sorbent Properties and Selection Criteria

The selection of appropriate sorbents for a given analytical challenge requires careful consideration of multiple physicochemical parameters that govern extraction efficiency and selectivity. Surface chemistry determines the primary interaction mechanisms available for analyte retention, including hydrophobic, polar, ionic, and π-π interactions. The specific surface area, typically measured in m²/g, directly influences the sorption capacity by determining the number of available binding sites. Pore size distribution affects accessibility of these sites, particularly for larger analyte molecules, while particle size and geometry impact flow characteristics and backpressure in packed configurations. Additional practical considerations include chemical stability across pH ranges, reusability, and cost-effectiveness for high-throughput applications [22] [23] [25].

The performance evaluation of sorbent materials employs several standardized metrics. The breakthrough volume represents the sample volume at which analyte concentration in the effluent reaches a defined percentage (typically 10%) of the influent concentration, indicating sorbent capacity exhaustion [25]. Recovery rates quantify the efficiency of analyte extraction under specified conditions, while clean-up efficiency measures the sorbent's ability to remove matrix interferences. More sophisticated characterization methods include the Standardized Sorbent Quality Measure (SSQM) and Matrix Sorbent Quality Measure (MSQM), which provide unified frameworks for comparing sorbent performance across different material classes and matrix types [26].

Comparative Analysis of Key Sorbent Phases

Table 1: Characteristics and Applications of Common Sorbent Phases

Sorbent Phase	Primary Mechanism	Key Applications	Strengths	Limitations
HLB	Hydrophobic and hydrophilic interactions	Broad-spectrum extraction of polar and non-polar compounds	Balanced selectivity; no drying required; high capacity	Limited anion-exchange capacity; may retain some matrix interferents
PSA	Weak anion exchange; hydrogen bonding	Removal of fatty acids, sugars, organic acids	Effective clean-up of polar matrix components	May retain acidic analytes; limited capacity for very polar compounds
C18	Hydrophobic interactions	Retention of non-polar compounds; lipid removal	Well-characterized; predictable performance	Prone to irreversible adsorption; requires conditioning
GCB	π-π electron interactions; planar recognition	Removal of pigments (chlorophyll, carotenoids)	Excellent for pigment removal; high surface area	May retain planar analytes (e.g., certain pesticides)
Zirconia-based	Lewis acid-base interactions; ligand exchange	Selective retention of phosphorous compounds	High chemical and thermal stability; unique selectivity	Limited documentation compared to silica-based phases

Hydrophilic-Lipophilic Balance (HLB) sorbents represent a class of polymeric materials, typically based on a balanced ratio of hydrophilic N-vinylpyrrolidone and lipophilic divinylbenzene monomers. This unique composition provides simultaneous retention capabilities for both polar and non-polar compounds without pre-conditioning, making HLB particularly valuable for multi-class contaminant screening approaches. The macroporous structure of HLB sorbents offers high specific surface area (typically exceeding 800 m²/g) and consequently high loading capacity. Applications of HLB span diverse analytical challenges, including the extraction of pharmaceuticals, pesticides, endocrine disruptors, and other emerging contaminants from aqueous and food matrices [22] [23].

Primary Secondary Amine (PSA) sorbents function primarily through weak anion-exchange mechanisms and hydrogen bonding interactions. The presence of both primary and secondary amines in their structure enables effective retention of various polar matrix components, including fatty acids, organic acids, sugars, and anthocyan pigments. This makes PSA particularly valuable as a clean-up sorbent in the analysis of fatty food matrices and plant-based materials. In the widely adopted QuEChERS method, PSA serves as the primary clean-up agent for removing organic acids and other polar interferences that co-extract with target analytes [22]. However, analysts should exercise caution when employing PSA with acidic target compounds, as these may be partially retained through the same mechanisms.

Octadecyl (C18) phases, consisting of silica particles bonded with C18 alkyl chains, represent one of the most established sorbent chemistries in analytical separations. The primary retention mechanism involves hydrophobic interactions, making C18 particularly effective for retaining non-polar compounds and removing lipophilic matrix components. In multi-residue methods, C18 often serves complementary roles to other sorbents—either as a retention phase for non-polar analytes or as a clean-up phase for lipid removal. Limitations include potential susceptibility to phase collapse under highly aqueous conditions and irreversible adsorption of certain compound classes, necessitating careful method optimization [22] [24].

Graphitized Carbon Black (GCB) comprises graphitic carbon structures with homogeneous, non-porous surfaces that exhibit strong retention for planar molecules through π-π electron interactions. This property makes GCB exceptionally effective for removing chlorophyll, carotenoids, and other planar pigments from complex extracts, particularly in the analysis of green vegetables and plant materials. However, this same selectivity presents a significant limitation, as GCB can also strongly retain planar target analytes such as certain pesticides (e.g., hexachlorobenzene, terbufos), potentially compromising method recovery for these compounds. Strategies to mitigate this limitation include using GCB in combination with alternative sorbents or employing limited quantities in carefully optimized protocols [22].

Zirconia-based sorbents utilize zirconium dioxide as their base material, which exhibits unique surface chemistry characterized by Lewis acid-base interactions and ligand-exchange capabilities. This distinguishes them from conventional silica-based phases and provides particular affinity for compounds containing phosphate groups or other Lewis basic functionalities. Zirconia phases demonstrate exceptional chemical and thermal stability across wide pH ranges (1-14) and temperatures, making them suitable for challenging applications where silica-based sorbents may degrade. While historically less documented than traditional phases, zirconia-based materials show growing promise for selective extraction of phosphorous-containing compounds and other analytes with Lewis basic functional groups [27] [28].

Multi-Sorbent Combination Strategies and Applications

Theoretical Basis for Sorbent Combinations

The strategic combination of multiple sorbents represents a sophisticated approach to sample preparation that leverages the complementary selectivity profiles of different materials to achieve more comprehensive contaminant coverage and superior matrix clean-up than possible with single-sorbent methodologies. This approach operates on the principle that complex sample matrices contain diverse interference compounds with varied physicochemical properties, while target analytes similarly span a wide polarity and functionality range. By employing sorbents with different dominant interaction mechanisms—hydrophobic, polar, ionic, and specific molecular recognition—analysts can create tailored extraction environments that maximize analyte recovery while minimizing co-extraction of matrix components [22] [24].

The effectiveness of a given sorbent combination depends critically on understanding potential interactions between the sorbents themselves and their collective impact on both target analytes and matrix interferents. Synergistic effects occur when sorbents with complementary selectivity remove different classes of interferences without significantly impeding analyte recovery. For example, the combination of C18 (removing non-polar lipids) and PSA (removing polar organic acids and sugars) provides more comprehensive clean-up than either sorbent alone for many food matrices. Conversely, antagonistic effects may arise when sorbents compete for the same analytes or when the retention mechanism of one sorbent inadvertently captures target compounds intended for analysis. These interactions must be carefully characterized during method development to optimize sorbent ratios and sequencing for specific analytical challenges [22].

Established Sorbent Combinations and Their Applications

Table 2: Performance of Sorbent Combinations in Different Matrices

Sorbent Combination	Matrix	Target Analytes	Key Outcomes	Reference Application
PSA + C18 + GCB	Spinach	Pesticide residues	Effective pigment removal with satisfactory recovery for most pesticides	QuEChERS method for fatty foods
PSA + C18	Cereals, grains	Mycotoxins, pesticides	Efficient removal of co-extracted compounds; recovery rates >85%	Multi-residue analysis in dry commodities
Zirconia + PSA	Fruits, vegetables	Acidic pesticides	Selective retention of phosphorous compounds; reduced matrix effects	Analysis of compounds with Lewis basic groups
HLB + GCB	Tea, spices	Multiple pesticide classes	Comprehensive clean-up of pigments and polyphenols	Complex plant matrices with high pigment content
HLB + Zirconia	Water samples	Pharmaceuticals, PFAS	Broad-spectrum retention with enhanced stability at extreme pH	Environmental water analysis

The QuEChERS method represents perhaps the most widely recognized application of multi-sorbent strategies in analytical chemistry. Originally developed for pesticide residue analysis in fruits and vegetables, the method employs a combination of PSA, C18, and sometimes GCB in dispersive solid-phase extraction (d-SPE) format to achieve efficient clean-up of acetonitrile extracts. The specific sorbent ratio can be optimized based on matrix characteristics—for high-fat matrices, increased C18 content provides better lipid removal, while for green leafy vegetables, limited GCB addition helps control chlorophyll interference without excessive retention of planar pesticides. The success of QuEChERS has led to its adaptation for diverse analyte classes beyond pesticides, including mycotoxins, veterinary drug residues, and environmental contaminants [22] [24].

For environmental water analysis, combinations of HLB with zirconia-based sorbents have demonstrated particular utility in capturing broad contaminant classes while withstanding the challenging chemical conditions sometimes encountered in environmental samples. HLB provides comprehensive retention of both hydrophilic and hydrophobic compounds, while zirconia phases contribute selectivity for phosphorous-containing compounds and stability across wide pH ranges. This combination has proven valuable for monitoring emerging contaminants, including pharmaceuticals, personal care products, and per- and polyfluoroalkyl substances (PFAS) in surface water, groundwater, and wastewater effluents [26].

In the analysis of high-pigment matrices such as tea, spices, and green vegetables, combinations of GCB with PSA or HLB offer improved control of chlorophyll, carotenoid, and polyphenolic interferences. The strong planar recognition of GCB effectively captures pigment molecules, while PSA removes sugars and organic acids or HLB provides broader contaminant coverage. Method development in these challenging matrices requires careful optimization of GCB quantity to balance pigment removal against potential loss of planar target analytes, sometimes necessitating alternative approaches such as zirconia-based sorbents for specific application scenarios [22].

Experimental Protocols and Workflows

Generalized Multi-Sorbent Workflow

The effective implementation of multi-sorbent extraction strategies follows a systematic workflow encompassing method selection, parameter optimization, and quality assurance. The following diagram illustrates the decision-making process for selecting and combining sorbents based on sample matrix and analytical targets:

Detailed Protocol: Modified QuEChERS for Multi-Residue Analysis in Complex Matrices

This protocol describes a modified QuEChERS approach for the extraction of pesticide residues, mycotoxins, and environmental contaminants from challenging matrices such as spinach, tea, and spices.

Materials and Reagents:

• Acetonitrile (HPLC grade)
• Acetic acid (reagent grade)
• Magnesium sulfate (anhydrous)
• Sodium chloride (reagent grade)
• Dispersive SPE kits containing PSA, C18, GCB, or equivalent loose sorbents
• Centrifuge tubes (50 mL)
• Volumetric flasks and pipettes
• Centrifuge capable of 4000 rpm
• Vortex mixer
• Analytical balance

Sample Preparation:

• Homogenization: Representative samples are homogenized using a food processor until a consistent particle size is achieved. For dry commodities (grains, spices), samples may require milling to achieve uniform particle distribution.
• Sub-sampling: Accurately weigh 10.0 ± 0.1 g of homogenized sample into a 50 mL centrifuge tube.
• Hydration: For dry matrices, add 10 mL of ultrapure water and vortex for 30 seconds to ensure complete hydration before extraction.

Extraction Procedure:

• Solvent Addition: Add 10 mL acetonitrile containing 1% acetic acid to the prepared sample.
• Shaking: Secure tubes and shake vigorously for 1 minute using a mechanical shaker or by hand.
• Salt Addition: Add pre-weighed salt packet containing 4 g MgSO4 and 1 g NaCl.
• Immediate Mixing: Immediately shake tube vigorously for 1 minute to prevent salt clumping and ensure proper phase separation.
• Centrifugation: Centrifuge at 4000 rpm for 5 minutes to achieve clear phase separation.

Clean-up Procedure:

• Sorbent Preparation: Transfer 1 mL of the acetonitrile supernatant to a 2 mL d-SPE tube containing one of the following sorbent combinations based on matrix type:
  • For high-pigment matrices (spinach, tea): 50 mg PSA + 50 mg C18 + 10 mg GCB
  • For high-fat matrices (avocado, oils): 50 mg PSA + 150 mg C18
  • For mixed contaminant analysis: 50 mg PSA + 50 mg C18 + 25 mg Zirconia-based sorbent
• Extract Purification: Vortex the d-SPE tube for 30 seconds to ensure complete interaction between extract and sorbents.
• Final Centrifugation: Centrifuge at 4000 rpm for 2 minutes to pellet the sorbent material.
• Sample Transfer: Carefully transfer the purified supernatant to an autosampler vial for analysis.

Quality Control:

• Include procedural blanks with each batch to monitor contamination.
• Spike control samples with target analytes at known concentrations to verify recovery rates.
• Utilize internal standards where available to correct for matrix effects and procedural variations.

Protocol for Microextraction by Packed Sorbent (MEPS)

MEPS represents a miniaturized approach to solid-phase extraction that integrates the sorbent directly into a syringe assembly, allowing for significantly reduced solvent consumption and enhanced automation capabilities.

MEPS Configuration:

• Sorbent Packing: Pack 2-5 mg of selected sorbent material into the barrel insert and needle (BIN) assembly. Sorbent choices can include single phases or custom mixtures based on application requirements.
• Device Conditioning: Condition the MEPS device by drawing and dispensing 100 μL of methanol followed by 100 μL of water or sample matrix.

Extraction Protocol:

• Sample Loading: Draw 100-500 μL of prepared sample through the MEPS device using slow, consistent plunger movement (approximately 10 μL/second).
• Washing: Remove weakly retained matrix components by drawing 50-100 μL of washing solution (typically 5% methanol in water).
• Elution: Elute retained analytes using 50-100 μL of appropriate organic solvent (methanol, acetonitrile, or mixtures with additives) directly into an autosampler vial.
• Reconditioning: Prepare the device for subsequent extractions by washing with 100 μL of methanol followed by 100 μL of water or equilibration solution.

Automation Potential: The MEPS workflow can be integrated with automated liquid handling systems and directly coupled with LC-MS instrumentation for complete workflow automation, significantly enhancing throughput and reproducibility [23].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Multi-Sorbent Extraction Methods

Category	Specific Items	Function/Purpose	Application Notes
Sorbent Phases	HLB cartridges (60 mg/3 mL)	Broad-spectrum extraction	Maintain consistent lot-to-lot performance
	PSA (50 μm particle size)	Anion-exchange clean-up	Store in desiccator to prevent moisture absorption
	C18 (end-capped, 50 μm)	Lipophilic compound retention	Pre-condition with methanol and water before use
	GCB (120-400 mesh)	Pigment removal	Use sparingly to avoid analyte loss
	Zirconia-based sorbents	Selective phosphorus compound retention	Explore for challenging separations
Solvents & Additives	Acetonitrile (HPLC grade)	Primary extraction solvent	Use high purity to reduce background interference
	Methanol (HPLC grade)	Elution solvent	Suitable for less polar compounds
	Acetic acid (≥99%)	Extraction modifier	Improves recovery of acidic compounds
	Ammonium formate	Mobile phase additive	Enhances MS compatibility
Consumables	d-SPE tubes (2 mL)	Dispersive clean-up	Pre-packed combinations available
	Syringe filters (0.2 μm)	Final filtration	Nylon or PTFE based on analyte compatibility
	Centrifuge tubes (15, 50 mL)	Sample processing	Certified chemically clean
Reference Standards	Multi-residue pesticide mix	Method calibration	Include isotopically labeled internal standards
	Matrix-matched calibration standards	Quantification	Correct for matrix effects

The strategic combination of sorbent phases represents a powerful approach for addressing the analytical challenges posed by complex sample matrices and diverse contaminant profiles. The five sorbent classes reviewed herein—HLB, PSA, C18, GCB, and zirconia-based phases—each offer distinct selectivity profiles that can be leveraged to create tailored extraction workflows for specific application requirements. The continued evolution of these materials, particularly through the development of advanced synthetic polymers and surface-functionalized substrates, promises further enhancements in extraction efficiency, selectivity, and operational convenience.

Future directions in sorbent technology development will likely focus on several key areas. Green analytical chemistry principles are driving innovation toward more sustainable sorbent materials derived from natural sources or designed for minimal environmental impact [29]. High-throughput automation represents another significant trend, with techniques like MEPS demonstrating the potential for complete workflow integration from sample preparation to instrumental analysis [23]. Additionally, the emergence of computational modeling approaches for predicting sorbent-analyte interactions may accelerate method development and optimization processes, reducing the empirical trial-and-error traditionally associated with multi-sorbent method development.

The standardization of sorbent performance evaluation methodologies, such as the SSQM and MSQM frameworks discussed earlier, will enhance comparability across studies and facilitate more informed sorbent selection decisions [26]. As analytical challenges continue to evolve with the identification of new contaminant classes and increasingly complex sample matrices, the strategic combination of complementary sorbent phases will remain an essential tool for researchers and analysts committed to achieving comprehensive contaminant monitoring and accurate risk assessment across diverse application domains.

The complexity of modern analytical challenges, particularly in broad-spectrum contaminant analysis, demands a sophisticated approach to sample preparation. The development of multi-sorbent extraction strategies represents a paradigm shift, moving beyond the limitations of single-material methods. This article details the application of three advanced material classes—Molecularly Imprinted Polymers (MIPs), Metal-Organic Frameworks (MOFs), and aptamers—that form the cornerstone of this strategy. These engineered and smart sorbents offer unparalleled selectivity, capacity, and versatility. Framed within a comprehensive research thesis on multi-sorbent systems, these application notes and protocols provide researchers and drug development professionals with the methodologies to integrate these materials into robust analytical workflows for isolating a wide range of analytes from complex matrices.

Sorbent Characterization and Comparative Analysis

The selection of an appropriate sorbent is critical to the success of any extraction. The table below provides a comparative overview of the three classes of smart sorbents, highlighting their key characteristics and optimal application scenarios to inform strategic deployment.

Table 1: Comparative Analysis of Engineered and Smart Sorbents

Sorbent Type	Key Characteristics	Primary Interactions	Analytical Targets	Advantages	Limitations
Molecularly Imprinted Polymers (MIPs)	Synthetic polymers with tailor-made recognition sites.	Hydrophobic, hydrogen bonding, van der Waals, electrostatic.	Pesticides, pharmaceuticals, endocrine disruptors.	High physical/chemical stability; reusability; cost-effective.	Occasional template leakage; heterogeneity in binding sites.
Metal-Organic Frameworks (MOFs)	Crystalline porous materials of metal ions & organic linkers. [30]	Electrostatic, π-π, van der Waals, coordination, hydrogen bonding. [31]	Gases, pesticides, drugs, proteins, toxins. [31]	Extremely high surface area; tunable porosity/pore size; designable structures. [31]	Stability in aqueous systems can be limited; potential framework collapse.
Aptamers	Single-stranded DNA/RNA oligonucleotides with selective binding. [30]	Spatial conformation, aromatic ring stacking, van der Waals, hydrogen bonding. [30]	Biomarkers, cells, proteins, small molecules. [30]	High affinity/specificity; excellent stability; simple synthesis; flexibility. [30]	Susceptibility to nuclease degradation (can be mitigated with chemical modifications).

Application Notes and Experimental Protocols

Protocol 1: MOF-Based Dispersive Micro-Solid Phase Extraction (DμSPE)

This protocol outlines the use of a synthesized MOF composite for the efficient extraction of pharmaceutical compounds from biological and aqueous samples, based on a validated method for antiepileptic drugs. [32]

3.1.1 Research Reagent Solutions

Table 2: Essential Materials for MOF-Based DμSPE

Item Name	Function/Description
Bimetallic MIL-100(Fe, Ni) on MIL-100(Mn)	The core MOF-on-MOF sorbent, providing a high-surface-area, porous structure with diverse metal sites for analyte interaction. [32]
SiO₂ and Fe₃O₄ Nanoparticles	Composite components added via sol-gel method; Fe₃O₄ imparts magnetic properties for facile sorbent retrieval. [32]
Methanol, Acetonitrile	Organic solvents used for the washing and elution of analytes from the sorbent surface.
HPLC-DAD System	The analytical instrument used for the separation, detection, and quantification of the target analytes post-extraction. [32]

3.1.2 Step-by-Step Procedure

• Sorbent Synthesis: Synthesize the composite sorbent using hydrothermal and sol-gel methods.

  • Prepare bimetallic MIL-100(Fe, Ni) on MIL-100(Mn) hydrothermally, using methanol as a solvent. [32]
  • Integrate SiO₂ and Fe₃O₄ nanoparticles via the sol-gel method to form the final magnetic composite sorbent. [32]
  • Optimize the component ratio (e.g., 0.56 MILs, 0.34 Fe₃O₄, 0.1 SiO₂) using an experimental design like the simplex lattice design for maximum efficiency. [32]

• Sample Preparation and Loading:

  • Adjust the pH of the aqueous or biological sample (e.g., water, urine, serum) to the optimal value (e.g., pH 5.0). [32]
  • Weigh a precise amount of the MOF composite sorbent (e.g., 15 mg) and add it directly to the sample solution. [32]

• Extraction:

  • Vortex or agitate the mixture vigorously to achieve a homogeneous dispersion of the sorbent. This maximizes the contact surface area between the sorbent and the target analytes.
  • Allow the extraction to proceed for the determined optimal time (e.g., 4.5 minutes). [32]

• Phase Separation:

  • Use an external magnet to hold the magnetic sorbent particles against the wall of the extraction vial.
  • Carefully decant and discard the supernatant sample solution.

• Sorbent Washing:

  • Wash the collected sorbent with a small volume of a weak solvent (e.g., 100 µL of hexane) to remove weakly adsorbed matrix interferences. [32]

• Analyte Elution:

  • Elute the target analytes from the sorbent by adding a suitable strong solvent (e.g., 230 µL of methanol) and vortexing. [32]
  • Separate the eluent from the sorbent magnetically and transfer it to a clean vial for analysis.

• Analysis:

  • Analyze the eluent using a coupled technique such as HPLC-DAD for separation and quantification. [32]

The following workflow diagram illustrates the DμSPE process:

Figure 1. DμSPE Workflow for MOF-Based Extraction

Protocol 2: Aptamer-Functionalized MOFs for Affinity Extraction

This protocol describes the post-synthetic functionalization of MOFs with aptamers to create highly selective affinity sorbents for targets like biomarkers, proteins, or small molecules. [30] [33]

3.2.1 Aptamer Immobilization Strategies

Aptamers can be integrated onto MOFs via two primary routes:

• Post-Synthetic Functionalization (Grafting): Aptamers are covalently or non-covalently attached to the pre-synthesized MOF's outer surface or pore interfaces. This strategy often preserves the aptamer's conformational flexibility and binding ability. [33]
• Direct Incorporation (One-Pot Synthesis): The aptamer is used as an organic ligand or co-ligand during the MOF synthesis itself, becoming an integral part of the framework structure. [33]

3.2.2 Step-by-Step Functionalization and Extraction Procedure

• MOF Activation:

  • If required, activate the pre-synthesized MOF (e.g., UiO-66-NH₂, MIL-101) by heating under vacuum to remove any solvent molecules from the pores, creating accessible active sites.

• Aptamer Conjugation:

  • For post-synthetic grafting, dissolve the amino- or thiol-modified aptamer in a suitable buffer.
  • Incubate the MOF with the aptamer solution under gentle agitation for a specified period (e.g., 12-24 hours) to allow for conjugation.
  • Separate the aptamer-functionalized MOF (bio-MOF) via centrifugation and wash thoroughly with buffer to remove any unbound aptamers.

• Affinity Extraction:

  • Disperse the bio-MOF in the sample solution containing the target analyte.
  • Incubate with agitation to allow the aptamer to recognize and capture the target analyte.
  • Collect the sorbent via centrifugation or magnetism (if a magnetic-MOF composite is used).

• Washing and Elution:

  • Wash the sorbent with a mild buffer to remove non-specifically bound contaminants.
  • Elute the captured target analyte using a denaturing buffer (e.g., containing urea) or a low-pH eluent that disrupts the aptamer-analyte interaction.

• Analysis:

  • Analyze the eluent using appropriate techniques such as fluorescence spectroscopy, electrophoresis, or LC-MS/MS.

Performance Metrics and Analytical Figures of Merit

The quantitative performance of the described sorbents in specific applications is summarized in the table below, demonstrating their effectiveness.

Table 3: Analytical Performance of Featured Sorbent Methods

Sorbent & Method	Target Analytic(s)	Sample Matrix	Linear Range	Limit of Detection (LOD)	Recovery (%)	Reference
MOF Composite DμSPE-HPLC	Lacosamide, Levetiracetam, Phenytoin	Human serum, urine, water	0.1 - 435.0 ng mL⁻¹	< 0.13 ng mL⁻¹	89.6 - 97.0	[32]
Amino Acid-based Bio-MOF (dSPE)	Hg(II) ions	Tap water	-	-	~100	[33]
Cysteine-based Bio-MOF (dSPE)	N-Linked Glycopeptides	HeLa cell lysate	-	1 fmol	~80	[33]

Integration into a Multi-Sorbent Extraction Strategy

A powerful application of these smart sorbents is their sequential or parallel use in a multi-sorbent cartridge or disk for the clean-up and fractionation of complex samples. This approach leverages the unique selectivity of each material to isolate different classes of contaminants in a single automated workflow.

Table 4: Sorbent Role in a Multi-Sorbent Strategy

Sorbent	Role in Multi-Sorbent Cartridge	Target Fraction
MOFs	First layer; general high-capacity capture. Removes large matrix interferences and captures diverse organics via size/porosity.	Broad-spectrum organics (e.g., PAHs, pesticides).
Aptamers	Second layer; high-affinity capture. Isolates specific high-priority targets (e.g., biomarkers, toxins) from the pre-cleaned sample.	Specific analytes (e.g., mycotoxins, proteins).
MIPs	Final polishing layer; targeted isolation. Further purifies the sample by removing structurally similar interferents to the analytes of interest.	Specific chemical classes (e.g., antibiotics, endocrine disruptors).

The logical relationship and workflow of this integrated strategy are depicted below:

Figure 2. Multi-Sorbent Extraction Workflow

The integration of MIPs, MOFs, and aptamers into analytical workflows marks a significant advancement in sample preparation technology. The protocols and application notes provided here equip researchers with the tools to implement these engineered and smart sorbents effectively. The multi-sorbent extraction strategy offers a powerful, flexible framework for tackling the challenges of broad-spectrum contaminant analysis in complex matrices. Future developments will likely focus on enhancing the robustness and commercialization of these materials, [31] the deeper integration of automation and microfluidics, [30] and the application of artificial intelligence to accelerate the design of next-generation sorbents tailored for specific analytical missions. [34] [35]

Implementing Multi-Sorbent Methods: Formats and Applications

Solid-phase extraction (SPE) is a fundamental sample preparation technique used to isolate, concentrate, and purify target analytes from complex matrices. The core principle involves selectively retaining analytes on a sorbent material while removing interfering compounds, followed by eluting the purified analytes in a small solvent volume suitable for analysis [36]. While the basic principles of SPE remain consistent across formats, the physical configuration and implementation significantly impact method performance, efficiency, and applicability to different analytical challenges.

The evolution of SPE has progressed from traditional cartridges to more advanced formats including disks, dispersive SPE (dSPE), and pipette-tip SPE, each offering distinct advantages for specific applications [37] [36]. This diversity enables analytical scientists to select formats optimized for their particular needs, whether processing large environmental water samples or performing high-throughput bioanalysis with limited sample volumes. The selection of an appropriate SPE format is crucial for developing robust, efficient methods within the context of multi-sorbent strategies for broad-spectrum contaminant analysis, as it directly impacts factors such as recovery, throughput, solvent consumption, and compatibility with automation [38].

Comparative Analysis of SPE Formats

The selection of an appropriate SPE format depends on multiple factors including sample volume, analyte characteristics, matrix complexity, and required throughput. The table below provides a systematic comparison of the key parameters for cartridges, disks, dSPE, and pipette-tip SPE formats.

Table 1: Comprehensive Comparison of SPE Formats for Analytical Applications

Format	Typical Sorbent Mass	Sample Volume Range	Flow Control	Primary Advantages	Common Applications
Cartridge	100 mg - 10 g [36]	1 mL - 1 L [36]	Vacuum/manual pressure [36]	High capacity, well-established protocols [39]	Environmental water, food extracts, pharmaceutical QC [40]
Disk	50 - 500 mg [36]	100 mL - 10 L [36]	Vacuum	Rapid flow rates, no channeling, handles particulates [40] [36]	Large volume environmental water, sediment extracts [40]
dSPE	10 - 150 mg [22]	1 - 15 mL [22]	Centrifugation	Simple operation, no conditioning, QuEChERS applications [22]	Pesticide residues, food matrices, biological fluids [22]
Pipette-Tip	1 - 100 mg [38]	10 µL - 1 mL [36] [38]	Manual/automated pipetting	Minimal solvent, easy automation, efficient mixing [38]	Proteomics, therapeutic drug monitoring, limited samples [38]

Table 2: Performance Characteristics and Practical Considerations

Format	Typical Elution Volume	Throughput Potential	Solvent Consumption	Limitations	Automation Compatibility
Cartridge	2 - 10 mL	Medium	Moderate to High	Possible channeling, packing inconsistencies [36]	Vacuum manifolds, 96-well plates [41]
Disk	5 - 15 mL	High	Moderate	Limited sorbent mass, higher cost [36]	Specialized holders, online systems [40]
dSPE	1 - 5 mL	High	Low	Additional centrifugation step [22]	Robotic liquid handlers [22]
Pipette-Tip	50 - 500 µL [38]	Very High	Very Low	Limited sample volume, potential clogging [38]	Direct integration with liquid handlers [38]

Detailed Experimental Protocols

Cartridge SPE Protocol for Water Contaminants

This protocol details the application of cartridge-based SPE for the extraction of organic UV-filtering compounds from surface waters, based on a validated method with modifications [42].

Materials and Reagents:

• SPE cartridges: Phenomenex Strata SI-1 Silica (500 mg/3 mL) [42]
• Water samples: Adjust to pH 6.0 ± 0.5 with hydrochloric acid or ammonium hydroxide
• Conditioning solvent: Methanol (HPLC grade), 5 mL
• Equilibration solvent: Reagent water, 5 mL
• Elution solvent: Methanol with 0.1% formic acid, 5 mL
• Internal standard solution: Prepare in methanol at 1 µg/mL

Procedure:

• Conditioning: Activate the sorbent by passing 5 mL of methanol through the cartridge at a flow rate of 2-3 mL/min. Do not allow the cartridge to run dry.
• Equilibration: Pass 5 mL of reagent water through the cartridge at 2-3 mL/min to create an environment compatible with the aqueous sample.
• Sample Loading: Load 250 mL of pH-adjusted water sample through the cartridge at a controlled flow rate of 5-7 mL/min using a vacuum manifold.
• Washing: Remove weakly retained interferences by passing 3 mL of 5% methanol in water (v/v) through the cartridge.
• Drying: Apply full vacuum (15-20 in Hg) for 10 minutes to remove residual water from the sorbent bed.
• Elution: Elute retained UV-filters with 2 × 2.5 mL of methanol with 0.1% formic acid into a collection tube, allowing 1-2 minutes of contact time for each aliquot.
• Reconstitution: Evaporate the eluate to dryness under a gentle nitrogen stream at 40°C and reconstitute in 250 µL of mobile phase for LC-MS/MS analysis.

Method Notes: For optimal recovery of polar UV-filters, maintain consistent flow rates during sample loading. Cartridge-to-cartridge reproducibility can be enhanced by using cartridges from the same manufacturing lot. Average recoveries for compounds like avobenzone, octinoxate, and octocrylene typically range from 45.2% to 73.4% with this method [42].

Disk SPE Protocol for Large Volume Water Samples

This protocol applies disk-based SPE for processing large volume water samples where high flow rates and minimal clogging are advantageous [40] [36].

Materials and Reagents:

• SPE disks: 47 mm C18-impregnated glass fiber disks
• Disk holder: 47 mm filtration apparatus with reservoir
• Water samples: Pre-filter through 0.45 µm glass fiber filter if particulate matter exceeds 1%
• Conditioning solvent: Methanol (10 mL) followed by reagent water (10 mL)
• Elution solvent: Acetonitrile:ethyl acetate (50:50, v/v), 15 mL

Procedure:

• Disk Preparation: Place the SPE disk in the holder and secure according to manufacturer instructions.
• Conditioning: Wet the disk with 10 mL methanol, applying gentle vacuum (5 in Hg). Follow with 10 mL reagent water once the disk is fully saturated.
• Sample Loading: Load 1-2 L of water sample onto the disk at a flow rate of 50-100 mL/min using appropriate vacuum.
• Drying: After sample loading, apply full vacuum for 20 minutes to dry the disk completely.
• Elution: Elute analytes with 15 mL of acetonitrile:ethyl acetate (50:50) into a collection tube, allowing the solvent to soak the disk for 2 minutes before applying vacuum.
• Concentration: Evaporate the extract to 1 mL under nitrogen at 40°C for analysis.

Method Notes: The large cross-sectional area of disks enables rapid processing of large sample volumes. This format is particularly effective for trace-level contaminant analysis in environmental waters where sufficient sample mass is needed to achieve low detection limits [40] [36].

Dispersive SPE (dSPE) Protocol for Food Matrices

This dSPE protocol is adapted from the QuEChERS approach for pesticide residue analysis in fruits and vegetables, with applications for various food contaminants [22].

Materials and Reagents:

• dSPE sorbent: 150 mg MgSO₄, 50 mg PSA (primary secondary amine), and 50 mg C18 per mL sample extract [22]
• Centrifuge tubes: 15 mL polypropylene with screw caps
• Extraction solvent: Acetonitrile with 1% acetic acid
• Salts: 4 g MgSO₄, 1 g NaCl, 1 g Na₃Citrate·2H₂O, 0.5 g Na₂HCitrate·1.5H₂O per 10 g sample

Procedure:

• Sample Preparation: Homogenize 10 g of sample with 10 mL acetonitrile containing 1% acetic acid in a 50 mL centrifuge tube.
• Partitioning: Add salt mixture (MgSO₄, NaCl, Na₃Citrate, Na₂HCitrate) to the tube, cap immediately, and shake vigorously for 1 minute.
• Centrifugation: Centrifuge at 3000 × g for 5 minutes to separate the phases.
• dSPE Clean-up: Transfer 1 mL of the upper acetonitrile layer to a 15 mL dSPE tube containing 150 mg MgSO₄, 50 mg PSA, and 50 mg C18.
• Extraction: Shake the dSPE tube vigorously for 30 seconds to ensure proper mixing.
• Separation: Centrifuge at 3000 × g for 5 minutes to settle the sorbent.
• Analysis: Transfer the supernatant to an autosampler vial for chromatographic analysis.

Method Notes: The QuEChERS dSPE approach provides effective removal of organic acids, pigments, and sugars from food matrices. PSA sorbent is particularly effective for removing fatty acids and sugars, while C18 targets non-polar interferences [22].

Pipette-Tip SPE Protocol for Biological Samples

This protocol utilizes pipette-tip SPE for efficient extraction of analytes from small volumes of biological fluids, with enhanced interaction between sorbent and analyte [38].

Materials and Reagents:

• Pipette tips: INTip SPE tips with C18 sorbent (5 mg) [38]
• Biological samples: Plasma, serum, or urine (100 µL)
• Conditioning solvent: Methanol (100 µL) followed by equilibration solvent (100 µL)
• Equilibration solvent: 10 mM ammonium acetate buffer, pH 4.5
• Washing solvent: 5% methanol in water (v/v)
• Elution solvent: 80:20 methanol:acetonitrile with 0.1% formic acid (50 µL)

Procedure:

• Conditioning: Aspirate and dispense 100 µL methanol through the tip 5 times at a rate of 50 µL/sec.
• Equilibration: Aspirate and dispense 100 µL equilibration buffer through the tip 5 times.
• Sample Loading: Mix 100 µL biological sample with 10 µL internal standard solution. Aspirate and dispense the sample mixture through the tip 10 times.
• Washing: Aspirate and dispense 100 µL washing solvent through the tip 5 times.
• Elution: Aspirate and dispense 50 µL elution solvent through the tip 10 times into a clean collection tube.
• Analysis: Inject 5-10 µL directly into the LC-MS/MS system.

Method Notes: The "aspirate and dispense" action in pipette-tip SPE creates a fluidized bed that promotes efficient interaction between the sorbent and analytes, significantly improving extraction efficiency compared to single-pass cartridges. This format is ideal for high-throughput applications and can be easily automated using standard liquid handling systems [38].

Workflow Integration and Strategic Selection

Diagram 1: Strategic Selection Workflow for SPE Formats in Multi-Sorbent Methods. This decision pathway integrates sample characteristics with operational requirements to guide format selection in broad-spectrum contaminant analysis.

The integration of multiple SPE formats within a comprehensive analytical strategy enables researchers to address diverse analytical challenges effectively. Each format offers unique advantages that can be strategically deployed based on sample characteristics and analytical goals:

• SPE Disks excel in environmental applications requiring processing of large water volumes (0.5-1 L) where their large cross-sectional area facilitates high flow rates without channeling issues [40] [36]. Their construction with small sorbent particles (8 μm) immobilized in a web structure provides greater surface area for efficient extraction compared to cartridges with larger particles (40 μm).

• SPE Cartridges remain the workhorse format for medium-volume samples (1-250 mL) and offer unparalleled versatility in sorbent chemistry selection [36]. The standardized cartridge format supports established methods across pharmaceuticals, environmental, and food applications, with extensive documentation for method development.

• dSPE represents a paradigm shift from column-based formats, particularly following the introduction of QuEChERS methodology [22]. By dispersing sorbent directly in the sample extract, dSPE eliminates flow-related issues and simplifies operation while maintaining effective clean-up for complex matrices like food and biological samples.

• Pipette-Tip SPE embodies the trend toward miniaturization, offering dramatic reductions in solvent consumption (50-500 μL elution volumes) and seamless automation compatibility [38]. The "aspirate and dispense" action creates a fluidized bed that promotes more efficient sorbent-analyte interaction compared to single-pass formats.

Research Reagent Solutions for Multi-Sorbent Extraction

Successful implementation of multi-sorbent extraction strategies requires careful selection of sorbent chemistries tailored to specific analyte classes. The following table outlines key sorbent materials and their applications in broad-spectrum contaminant analysis.

Table 3: Sorbent Chemistry Guide for Targeted Analyte Classes

Sorbent Type	Retention Mechanism	Primary Applications	Elution Conditions
C18 (Octadecyl)	Reversed-phase, hydrophobic interactions	Non-polar to moderately polar compounds (pesticides, PAHs, pharmaceuticals) [22] [36]	Methanol, acetonitrile, tetrahydrofuran
C8 (Octyl)	Reversed-phase, slightly less retentive than C18	Medium-polarity compounds, larger molecules [41]	Methanol, acetonitrile with modifiers
Mixed-Mode (C8/SCX)	Dual hydrophobic and cation exchange	Basic drugs, compounds with amine functionalities [41]	pH-adjusted organic solvents with ionic strength
Molecularly Imprinted Polymers (MIPs)	Shape-selective recognition	Specific target analytes (veterinary drugs, contaminants) [22] [41]	Solvents disrupting hydrogen bonding
Primary Secondary Amine (PSA)	Anion exchange, hydrogen bond acceptor	Organic acids, sugars, fatty acids in food matrices [22]	Polar solvents with pH adjustment
Silica (Normal Phase)	Polar interactions (hydrogen bonding, dipole-dipole)	Polar compounds, structural isomers separation [42]	Hexane with increasing polarity modifiers
Restricted Access Media (RAM)	Size exclusion + reversed-phase	Direct injection of biological fluids, protein exclusion [41]	Aqueous to organic gradients

Table 4: Sorbent Selection Guide by Analyte Class

Analyte Class	Recommended Primary Sorbent	Alternative Sorbents	Matrix Considerations
Pesticides	C18 for multi-residue [22]	Graphitized carbon black for planar molecules	PSA clean-up for food matrices [22]
Pharmaceuticals	Mixed-mode for basic drugs [41]	C18 for comprehensive screening	RAM for direct plasma injection [41]
Lipids	Aminopropyl (normal phase) [22]	Silica for class fractionation	Silver-impregnated for unsaturated [22]
Volatile Organic Compounds	Porous polymers (PDMS) [37]	Carboxen for gases	SPME fibers for direct extraction [37]
UV Filters	Silica [42]	C18 for less polar variants	pH adjustment for ionic forms [42]

Method Development Considerations for Multi-Sorbent Strategies

Implementing a multi-sorbent extraction approach requires systematic method development to optimize recovery, selectivity, and reproducibility across different SPE formats. Key considerations include:

Sorbent Compatibility: When combining multiple sorbents in sequence, ensure chemical compatibility between conditioning solvents, samples, and elution solvents. For example, normal-phase sorbents like silica require strict control of water content, which may complicate integration with reversed-phase sorbents in automated sequences [41].

Flow Rate Optimization: Each SPE format has different optimal flow characteristics. Cartridges and disks perform best with controlled, consistent flow rates during sample loading (typically 2-5 mL/min for cartridges, 50-100 mL/min for disks), while dSPE and pipette-tip formats rely on mixing efficiency rather than flow control [36] [38].

Elution Strategy: For formats with larger sorbent beds (cartridges, disks), sequential elution with different solvents can fractionate analytes by polarity. With miniaturized formats (dSPE, pipette-tips), single-step elution is preferred, requiring careful optimization of solvent strength and volume [38].

Matrix-Specific Modifications: Complex matrices often require format-specific adjustments. Biological samples may benefit from pipette-tip SPE with integrated filtration [38], while fatty food matrices typically require dSPE with dual sorbents (PSA + C18) for effective clean-up [22].

The continuous development of novel sorbent materials, including polymeric ionic liquids, magnetic nanoparticles, and biosorbents, promises to further enhance the capabilities of all SPE formats [37] [43]. Future directions point toward increasingly selective sorbents that can handle complex matrices with minimal clean-up, supporting the trend toward greener analytical chemistry with reduced solvent consumption and waste generation.

The analysis of broad-spectrum contaminants in complex biological and environmental matrices demands sample preparation techniques that are efficient, robust, and scalable. This application note details protocols for three key techniques—Micro-Extraction by Packed Sorbent (MEPS), Pipette-Tip Micro-Solid Phase Extraction (PT-μSPE), and Online Solid Phase Extraction (Online-SPE)—positioned within a comprehensive multi-sorbent strategy. The drive toward miniaturization, automation, and sorbent versatility aims to overcome limitations of traditional methods, notably their labor-intensive nature, high solvent consumption, and poor reproducibility for large-scale studies [44] [45]. Recent advancements, including the development of novel sorbents and the integration of artificial intelligence (AI) for method optimization, are transforming sample preparation into a highly efficient and predictive component of the analytical workflow [46] [8]. The protocols herein are designed for researchers and scientists engaged in method development for contaminant and biomarker analysis, with a focus on practical implementation within high-throughput bioanalysis and environmental monitoring.

The following table summarizes the core characteristics, advantages, and limitations of MEPS, PT-μSPE, and Online-SPE, providing a guide for technique selection based on research objectives.

Table 1: Comparison of Miniaturized and Automated SPE Techniques

Feature	MEPS	PT-μSPE	Online-SPE
Typical Sorbent Mass	1-4 mg	< 1 mg	Varies (cartridge/column)
Sample Volume	10-250 µL	10-50 µL (as low as 20 µL demonstrated) [44]	µL-to-mL range
Elution Volume	10-50 µL	~100 µL [47]	Determined by LC flow rate
Automation Potential	High (integrated with autosampler)	Moderate (semi-automated pipetting)	Full (directly coupled to LC-MS)
Throughput	High	High (e.g., 120 samples/day) [44]	High (continuous operation)
Key Advantages	Reusable sorbent bed, minimal solvent	Extremely low cost per sample, facile sorbent functionalization [47]	No manual intervention, minimal sample loss, ideal for temporal studies [48]
Inherent Limitations	Potential for carryover, limited sorbent mass	Not suitable for large sample volumes [47]	Higher initial setup complexity
Ideal Use Case	Repetitive analysis of similar matrices where carryover can be managed	Low-cost, high-throughput analysis of small-volume samples [44]	Uninterrupted, high-frequency analysis of a continuous sample stream [48]

Detailed Experimental Protocols

Protocol 1: PFPT-μSPE for Lead Extraction in Beverages

This protocol details a specific PT-μSPE method for extracting lead (Pb(II)) from water and beverage samples using a novel polystyrene foam-based nanocomposite sorbent [47]. It exemplifies the principles of sorbent innovation and green chemistry.

• Research Reagent Solutions:

  • Sorbent: Ni-MOF@S-CQDs nanocomposite synthesized in polystyrene foam.
  • Complexing Agent: 2-(5-Bromo-2-pyridylazo)-5-(diethylamino)phenol (PADAP) for selective Pb(II) chelation.
  • Extraction Device: Polystyrene Foam Pipette-Tip (PFPT).
  • Analysis Instrumentation: UV-VIS Spectrometer.

• Procedure:

  • Tip Preparation: A piece of polystyrene foam impregnated with the Ni-MOF@S-CQDs nanocomposite is packed into a standard pipette tip.
  • Sample Preparation: Adjust the pH of the water, juice, or iced tea sample to 7.0 using dilute HNO₃ or NaOH. Add PADAP to form a complex with Pb(II).
  • Conditioning: Pass 100 µL of methanol through the PFPT, followed by 100 µL of pH 7 buffer.
  • Loading: Aspirate and dispense 50 µL of the prepared sample through the PFPT for 10-15 cycles (adsorption time: ~60 seconds).
  • Washing: Pass 50 µL of a mild aqueous wash solution (e.g., pH 7 buffer) through the tip to remove interferents.
  • Elution: Elute the adsorbed Pb-PADAP complex directly into a micro-cuvette using 100 µL of a methanolic eluent (e.g., with 0.1% HNO₃). Perform this step over ~30 seconds (desorption time).
  • Analysis: Quantify Pb(II) concentration by measuring the absorbance of the eluate via UV-VIS spectrometry.

• Optimized Parameters & Performance:

  • Limit of Detection (LOD): 15 µg L⁻¹.
  • Extraction Efficiency (EE): 95%.
  • Enrichment Factor (EF): 30.
  • Relative Standard Deviation (RSD): <4% (inter-day).

This method is notable for its cost-effectiveness (<0.1 Euro per sample) and alignment with green analytical principles through low solvent consumption [47].

Protocol 2: Online-SPE for Continuous Metabolite Monitoring

This protocol describes a dual-column Online-SPE setup for the desalting and preconcentration of metabolites from a constantly perfusing bioreactor stream, enabling high-temporal-resolution studies of dynamic cellular processes [48].

• Research Reagent Solutions:

  • SPE Columns: Dual 100 µm ID fused silica capillaries packed with C18 phase (e.g., 3 µm, 300 Å Jupiter particles).
  • Solvents: Solvent A (95% H₂O, 5% MeOH, 0.1% FA); Solvent B (95% MeOH, 5% H₂O, 0.1% FA).
  • Switching Valves: Three 10-port nanovolume UPLC valves for automated flow path control.
  • Analysis Instrumentation: Nano-LC system coupled to a high-resolution mass spectrometer (e.g., Q-TOF).

• Procedure:

  • System Setup: Configure the dual-column system with a multi-valve setup as depicted in the workflow diagram below. The system is plumbed to handle a continuous sample input.
  • System Equilibration: Equilibrate both SPE columns with Solvent A at a flow rate of 500 nL/min.
  • Continuous Operation:
    • Column 1 (Load/Desalt): Direct the continuous sample stream (e.g., at 500 nL/min from a microfluidic bioreactor) onto Column 1. Metabolites are retained while salts are diverted to waste.
    • Column 2 (Elute/Analyze): While Column 1 is loading, switch the valves to elute the previously captured analytes from Column 2 with a gradient of Solvent B to the MS for analysis.
    • Column Switching: After the elution and analysis cycle for Column 2 is complete, immediately switch the valve positions. Column 2 now takes over the loading/desalting function, and Column 1 is eluted to the MS. This cycle repeats, ensuring no sample loss.

• Optimized Parameters & Performance:

  • Sample Flow Rate: 500 nL/min.
  • Temporal Resolution: ~3 minutes.
  • Analyte Loading Range: femtomole (fmol) to picomole (pmol).
  • Recovery: ~80% even at fmol loadings [48].

Protocol 3: Automated Electro-Extraction (EE) for Acylcarnitines

This protocol outlines a fully automated, high-throughput electro-extraction workflow for acylcarnitines from human plasma and mouse tissue, demonstrating the pinnacle of workflow automation and integration [44].

• Research Reagent Solutions:

  • Platform: Fully automated EE platform integrated with a CTC PAL3 autosampler and LC-MS.
  • Sorbent: Not specified in detail, but the principle involves electro-extraction.
  • Consumables: Standard microtiter plates.

• Procedure:

  • System Qualification: Qualify the integrated platform according to manufacturer and laboratory specifications.
  • Automated Operation:
    • Load plates containing 20 µL of plasma or tissue homogenate onto the autosampler.
    • The system automatically performs all steps: sample aspiration, application of optimized electrical parameters for extraction, elution of extracted analytes, and injection into the LC-MS system.
  • LC-MS Analysis: Analyze the eluate using a validated LC-MS method for acylcarnitine profiling.

• Optimized Parameters & Performance:

  • Throughput: 120 samples per day.
  • Cost: <0.1 Euro per sample.
  • Enrichment Factor: Up to 400.
  • Extraction Recovery: Up to 99% [44].

Workflow Visualization

The following diagrams illustrate the logical flow and valve configurations for the key techniques described in the protocols.

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagents and Materials for Miniaturized SPE Protocols

Item Name	Function/Description	Example from Protocols
Ni-MOF@S-CQDs Nanocomposite	A high-surface-area sorbent providing coordination sites and functional groups for selective metal ion adsorption.	Used in PFPT-μSPE for efficient Pb(II) extraction [47].
PADAP (Complexing Agent)	Selectively chelates with target metal ions to form a complex that can be retained on the sorbent and detected spectrophotometrically.	Essential for the selective extraction and detection of Pb(II) in PT-μSPE [47].
C18 Phase (Jupiter Particles)	A reversed-phase sorbent used for retaining a wide range of organic analytes based on hydrophobic interactions.	Packing material for the dual-column Online-SPE system for metabolite desalting [48].
Polystyrene Foam Substrate	Serves as a scaffold for immobilizing the functional sorbent material within a pipette tip.	The solid support for the Ni-MOF@S-CQDs nanocomposite in PFPT-μSPE [47].
Waste-Derived Sorbents (WDSs)	Green, low-cost, and abundant materials derived from agricultural or industrial waste; can be functionalized for specific applications.	Representative of the trend toward sustainable sorbent materials, adhering to Green Analytical Chemistry principles [45].
Multi-Sorbent SPE Cartridges	SPE cartridges containing a combination of different sorbents (e.g., Oasis HLB, Strata WAX) to broaden the chemical space of extracted analytes.	Critical for a multi-sorbent strategy in NTA to achieve broad-spectrum contaminant analysis with balanced recovery [8].

Discussion & Strategic Outlook

The protocols presented demonstrate that modern sample preparation is moving beyond mere clean-up to become a strategic source of analytical selectivity and efficiency. The integration of these techniques into a multi-sorbent strategy is paramount for successful broad-spectrum contaminant analysis. Combining sorbents with orthogonal selectivity (e.g., mixed-mode, WAX, WCX) in a single workflow, as highlighted in non-target analysis (NTA) frameworks, significantly expands the range of detectable analytes by overcoming the limitations of any single sorbent material [8].

Future directions in this field are being shaped by several key trends. First, the push for sustainability is driving the development and adoption of waste-derived sorbents (WDSs), which offer an eco-friendly and cost-effective alternative to traditional materials [45]. Second, artificial intelligence and machine learning are beginning to play a transformative role. AI can predict chromatographic retention and optimize separation parameters with minimal experimentation, as seen in hybrid AI-driven HPLC systems that use digital twins [46]. In NTA, ML algorithms are crucial for processing high-dimensional data from HRMS to identify contamination sources and patterns that would be imperceptible through manual analysis [8]. Finally, the overarching trend toward full workflow automation, as exemplified by the electro-extraction platform, will be essential for achieving the reproducibility and throughput required for large-scale epidemiological and clinical studies [44].

The extraction of drugs and metabolites from biological matrices like plasma and urine represents a critical foundation for modern bioanalysis, supporting pharmaceutical development, clinical monitoring, and toxicological studies. Recent trends in sample preparation emphasize miniaturization, automation, and reduced environmental impact through lower solvent consumption, aligning with Green Analytical Chemistry principles [49]. The core challenge lies in efficiently isolating target analytes from complex biological matrices while maintaining high analytical sensitivity and reproducibility.

Current methodologies have evolved significantly from traditional liquid-liquid extraction toward more sophisticated sorbent-based techniques and optimized solvent precipitations. The selection of an appropriate sample preparation strategy is paramount, as it constitutes the most critical phase of the entire analytical process, significantly influencing final measurement error and data quality [49] [50]. This application note details optimized protocols for plasma and urine preparation within the context of a multi-sorbent extraction strategy for broad-spectrum contaminant analysis research.

Experimental Protocols and Workflows

Sample Collection and Pre-Analytical Considerations

Plasma Collection: Collect blood via venipuncture into appropriate anticoagulant-containing vacutainers (e.g., fluoride/oxalate for metabolomics). Centrifuge at 2000× g for 20 minutes at 4°C. Aliquot the supernatant plasma into cryovials and store at -80°C within 2 hours of collection [50].

Urine Collection: Collect mid-stream urine into sterile containers. Centrifuge at 2000× g for 10 minutes to remove particulate matter. Aliquot supernatant and store at -80°C. For patients with potential renal impairment, organic solvent treatment is recommended to remove residual proteins [51].

Critical Pre-Analytical Notes: Consistent handling procedures are essential. For low-biomass samples or trace analysis, implement contamination control measures including use of personal protective equipment, decontamination of surfaces with sodium hypochlorite or UV-C light, and inclusion of sampling controls to identify contamination sources [52].

Protocol 1: Monophasic Solvent Extraction for Plasma

This protocol provides high metabolite coverage and is ideal for high-throughput UHPLC-MS clinical metabolic phenotyping [51].

• Step 1: Thaw plasma samples on ice and vortex for 10 seconds.
• Step 2: Pipette 100 µL of plasma into a microcentrifuge tube.
• Step 3: Add 300 µL of ice-cold methanol:acetonitrile (50:50, v/v) to achieve a final solvent ratio of 1:3 (sample:solvent).
• Step 4: Vortex vigorously for 60 seconds.
• Step 5: Incubate at -20°C for 20 minutes to precipitate proteins.
• Step 6: Centrifuge at 14,000× g for 15 minutes at 4°C.
• Step 7: Transfer the supernatant to a clean vial for UHPLC-MS analysis.

Note: For lipid-specific analysis, isopropanol can be substituted as the monophasic solvent, providing high recovery for diverse lipid classes [51].

Protocol 2: Biphasic Extraction for Comprehensive Plasma Profiling

This method simultaneously extracts hydrophilic metabolites and lipids into separate phases, beneficial when sample volume is limited [51].

• Step 1: Add 100 µL of plasma to a glass tube.
• Step 2: Add 400 µL of HPLC-grade methanol and vortex for 30 seconds.
• Step 3: Add 200 µL of chloroform and vortex for another 60 seconds.
• Step 4: Add 200 µL of water, vortex vigorously for 60 seconds to form a biphasic system.
• Step 5: Centrifuge at 4000× g for 10 minutes at 4°C to achieve phase separation.
• Step 6: Carefully collect the upper polar phase (methanol/water) for metabolomics.
• Step 7: Collect the lower organic phase (chloroform) for lipidomics.
• Step 8: (Optional) Dry both phases under nitrogen and reconstitute in solvents compatible with UHPLC-MS.

Protocol 3: Microextraction by Packed Sorbent (MEPS) for Biofluids

MEPS is a miniaturized solid-phase extraction technique ideal for small sample volumes, offering high selectivity and the ability to be automated [53].

• Step 1: Condition the MEPS sorbent (e.g., mixed-mode C8/SCX) with 100 µL of methanol followed by 100 µL of water.
• Step 2: Adjust the pH of the plasma or urine sample to 9 using ammonium hydroxide or buffer.
• Step 3: Load 100-200 µL of sample through the sorbent via 5-10 aspirate-dispense cycles.
• Step 4: Wash with 50-100 µL of a 5% methanol solution to remove weakly retained interferences.
• Step 5: Elute analytes with 50-100 µL of a strong organic solvent (e.g., methanol with 2% formic acid).
• Step 6: Inject the eluent directly into a UHPLC-MS/MS system for analysis.

Protocol 4: Urine Sample Preparation for Metabolite Analysis

• Step 1: Thaw urine samples on ice and vortex.
• Step 2: Pipette 100 µL of urine into a microcentrifuge tube.
• Step 3: Add 300 µL of 50:50 methanol:acetonitrile (for HILIC analysis) or 50:50 methanol:water (for reversed-phase analysis).
• Step 4: Vortex for 60 seconds.
• Step 5: Centrifuge at 14,000× g for 15 minutes at 4°C.
• Step 6: Transfer the supernatant to a clean vial for UHPLC-MS analysis [51].

Comparative Performance Data

The following tables summarize quantitative performance data for the described extraction methods, enabling informed protocol selection.

Table 1: Comparison of Monophasic Solvent Extraction Methods for Plasma Analysis by UHPLC-MS

Extraction Solvent	Target Analytes	Reproducibility (%RSD)	Relative Yield	Key Advantages
Methanol:Acetonitrile (50:50)	Polar Metabolites	High [51]	High [51]	Broad metabolite coverage, high reproducibility, simple and fast
Methanol	Polar Metabolites	High [50] [51]	High [50] [51]	Efficient protein denaturation, good for hydrophilic compounds
Acetonitrile	Polar Metabolites	Variable [51]	Moderate [50]	Efficient protein removal, but may yield lower metabolite coverage
Isopropanol	Lipids	High [51]	High [51]	Excellent lipid recovery, simple and direct-injection compatible

Table 2: Comparison of Extraction Techniques for Bioanalytical Applications

Extraction Technique	Sample Consumption	Throughput	Green Metrics	Best Suited For
Monophasic Solvent	Medium (50-200 µL)	High	Medium (moderate solvent use)	Untargeted metabolomics, high-throughput screening
Biphasic Solvent	Medium (100-200 µL)	Medium	Low (higher solvent use)	Simultaneous metabolite/lipid profiling from a single sample
Solid-Phase Extraction (SPE)	Low-Medium (50-500 µL)	Medium	Good (can be automated)	Selective analyte enrichment, cleaner extracts, impurity removal
Microextraction by Packed Sorbent (MEPS)	Low (10-200 µL)	High (when automated)	Excellent (minimal solvent)	Small sample volumes, targeted analysis, high sensitivity [53]

Workflow Visualization

The following diagram illustrates the parallel sample preparation pathways for plasma and urine within a multi-sorbent strategy framework.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Reagents and Materials for Sample Preparation

Item	Function/Application	Example Specifications
Methanol (LC-MS Grade)	Protein precipitation and metabolite extraction; high purity minimizes background interference.	Optima LC/MS grade [50] [51]
Acetonitrile (LC-MS Grade)	Protein precipitation; often used in combination with methanol for broad-coverage metabolomics.	Optima LC/MS grade [50] [51]
Isopropanol (LC-MS Grade)	Efficient lipid extraction solvent; compatible with direct injection for LC-MS lipidomics.	Optima LC/MS grade [51]
Ammonium Acetate/Formate	Buffer additives for mobile phases; promotes stable ionization in MS.	LC/MS grade [50]
Molecularly Imprinted Polymers (MIPs)	Selective sorbents for solid-phase extraction; designed for specific analyte classes.	MISPE cartridges [49]
Mixed-Mode Sorbents (C8/SCX)	MEPS or SPE sorbents with reverse-phase and ion-exchange properties for broad-spectrum extraction.	Phree phospholipid removal tubes [50] [53]
Formic Acid (LC-MS Grade)	Mobile phase additive to control pH and improve ionization in positive ESI mode.	Pierce LC/MS grade [50] [51]
Isotope-Labelled Internal Standards	Critical for correcting for matrix effects and losses during sample preparation; ensures quantification accuracy.	e.g., Succinic acid-2,3-13C2, d-Glucose-13C6 [50]

Concluding Recommendations

The choice of extraction protocol must be guided by the specific analytical goals. For untargeted metabolomics seeking the broadest possible coverage of polar metabolites from plasma or urine, monophasic methanol:acetonitrile (50:50) extraction is highly recommended due to its outstanding balance of yield, reproducibility, and simplicity [50] [51]. For dedicated lipidomics from plasma, monophasic isopropanol provides superior lipid recovery. When sample volume is severely limited or for targeted drug analysis, MEPS offers a robust, miniaturized, and efficient alternative [53].

Integrating these protocols into a multi-sorbent strategy—whereby different sorbents or methods are applied in parallel or sequence—enables true broad-spectrum analysis of drugs, metabolites, and contaminants across the polarity spectrum, forming a solid foundation for advanced bioanalytical research.

The comprehensive monitoring of broad-spectrum contaminants in the environment is a critical challenge in modern analytical chemistry. The complexity of environmental matrices, coupled with the trace-level concentrations of pesticides and pharmaceuticals, necessitates advanced sample preparation strategies for accurate quantification and identification [43]. Multi-sorbent extraction has emerged as a powerful approach to address the diverse physicochemical properties of these contaminants, enabling simultaneous extraction of compounds with varying polarities, solubilities, and pKa values [8]. This application note details integrated protocols for monitoring these contaminants within a research framework focused on developing enhanced multi-sorbent extraction strategies for broad-spectrum contaminant analysis.

The pressing need for these advanced methodologies is underscored by environmental monitoring data. A decade-long study of aquatic environments near a municipal solid waste treatment plant revealed concerning findings: several pharmaceuticals including primidone, gabapentin, azithromycin, and tramadol were consistently detected in groundwater, with some pesticide compounds like atrazine and chlorpyrifos—currently prohibited for agricultural use—still persisting in the environment [54]. Furthermore, certain insecticides including chlorpyrifos and carbofuran were identified as posing high risks for groundwater crustaceans, highlighting the ecological significance of robust monitoring protocols [54].

Key Monitoring Challenges and Sorbent-Based Solutions

Analytical Challenges in Multi-Contaminant Monitoring

Environmental analysts face significant hurdles in broad-spectrum contaminant monitoring. The extensive range of contaminant polarities necessitates extraction materials with complementary selectivity characteristics [20]. Traditional single-sorbent approaches often yield incomplete contaminant profiles due to their limited selectivity range, potentially missing important emerging contaminants or transformation products [8]. Additionally, matrix effects from complex environmental samples can significantly interfere with analytical accuracy, requiring efficient clean-up steps integrated into the extraction process [23].

The limitations of conventional monitoring are further compounded by the need for sophisticated instrumentation. While techniques like liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) enable quantification at low concentrations, their effectiveness depends heavily on the preceding sample preparation steps [54]. This technological dependency creates accessibility challenges, particularly for resource-limited settings where water monitoring is often lacking [55].

Multi-Sorbent Extraction Strategy

Multi-sorbent extraction strategies address these challenges by combining sorbents with complementary properties, expanding the range of extractable compounds and improving selectivity for target analyte classes [8]. The strategic combination of hydrophilic, hydrophobic, and ion-exchange materials in a single extraction device enables comprehensive capture of diverse contaminants in a single analytical workflow [43] [23].

Table 1: Sorbent Classes and Their Applications in Multi-Sorbent Extraction

Sorbent Class	Specific Examples	Target Contaminant Properties	Common Applications
Hydrophilic-Lipophilic Balanced (HLB)	Oasis HLB, C/PVPP/MDI composite	Broad-spectrum (polar & non-polar)	Pharmaceuticals, pesticides, antibiotics [8] [55]
Molecularly Imprinted Polymers (MIPs)	Sulfonylurea MIP, Fipronil MMIP	High selectivity for template molecules	Herbicides, insecticides in complex matrices [23]
Metal-Organic Frameworks (MOFs)	ZIF-8, MIL-series	Tunable porosity for specific compound classes	Emerging contaminant isolation [55] [23]
Bio-based Sorbents	Cellulose composites, chitosan	Cost-effective, sustainable extraction	Broad-spectrum screening in resource-limited settings [55]
Graphene-Based Materials (GBMs)	Graphene oxide, reduced GO	High surface area for aromatic compounds	Pesticides with aromatic structures [23]

Integrated Protocols for Multi-Sorbent Extraction

Protocol 1: Multi-Sorbent SPE for Broad-Spectrum Screening in Water

Principle: This protocol utilizes a combination of HLB, ion-exchange, and bio-based sorbents in series to extract contaminants across a wide polarity range from water samples [8] [55].

Materials:

• Water samples (100-1000 mL, depending on sensitivity requirements)
• Multi-sorbent cartridge: Oasis HLB (60 mg) + Strata X-CW (cationic, 50 mg) + Strata X-AW (anionic, 50 mg) [8]
• Alternative: C/PVPP/MDI cellulose-based composite (200 mg) for cost-effective implementation [55]
• HPLC-grade methanol, acetonitrile, acetone, and ammonium acetate buffer (pH 4.5 and 7.0)
• Vacuum manifold system or automated SPE workstation
• Evaporation system (nitrogen evaporator or centrifugal concentrator)

Procedure:

• Sample Preparation: Filter water samples through 0.7 μm glass fiber filters to remove particulate matter. Adjust pH to 7.0 using dilute NaOH or HCl.
• Cartridge Conditioning: Condition the multi-sorbent cartridge with 5 mL methanol followed by 5 mL pH 7.0 water. Maintain solvent flow at 3-5 mL/min, ensuring sorbent does not dry out.
• Sample Loading: Pass the filtered sample through the cartridge at a controlled flow rate of 5-10 mL/min. For large volumes (>500 mL), use a peristaltic pump for consistent flow.
• Cartridge Washing: Wash with 5 mL of 5% methanol in water (pH 7.0) to remove weakly retained matrix interferences.
• Analyte Elution: Elute analytes with 2 × 5 mL of methanol containing 2% ammonium hydroxide. Collect the eluate in a calibrated tube.
• Sample Reconstitution: Evaporate the eluate to dryness under a gentle nitrogen stream at 40°C. Reconstitute in 200 μL of initial mobile phase composition (typically 95:5 water:methanol) with 0.1% formic acid.
• Analysis: Analyze by LC-MS/MS using both positive and negative electrospray ionization modes for comprehensive compound detection.

Performance Characteristics:

• Recovery: 75-110% for most pharmaceuticals and pesticides
• Limit of Detection: 0.1-10 ng/L for most compounds
• Linear Range: 0.5-500 ng/L [54] [55]

Protocol 2: MEPS for High-Throughput Targeted Analysis

Principle: Microextraction by Packed Sorbent (MEPS) utilizes a miniaturized, packed sorbent bed within a syringe barrel, enabling rapid extraction of small sample volumes with potential for automation [23].

Materials:

• MEPS syringe (100-500 μL capacity) with BIN (barrel insert and needle) assembly
• MEPS sorbents: C18, MIPs, or mixed-mode materials (2-5 mg packing)
• Biological or environmental samples (10-1000 μL)
• Conditioning, washing, and elution solvents compatible with the selected sorbent

Procedure:

• Sorbent Conditioning: Aspirate and dispense 50 μL of methanol three times, followed by 50 μL of water three times.
• Sample Loading: Aspirate and dispense the sample solution through the sorbent bed 10-20 times (depending on the extraction equilibrium).
• Washing Step: Remove matrix interferences by aspirating and dispensing 50 μL of washing solution (typically 5% methanol in water).
• Analyte Elution: Elute target analytes by aspirating and dispensing 20-50 μL of strong elution solvent (e.g., methanol:acetonitrile, 90:10, v/v) three times.
• Analysis: Directly inject the eluate into the LC-MS/MS system.

Performance Characteristics:

• Recovery: 80-105% for targeted analytes
• Reusability: Up to 100 extractions per sorbent bed
• Processing Time: <5 minutes per sample [23]

Protocol 3: Cellulose-Based Composite SPE for Cost-Effective Monitoring

Principle: This protocol employs a low-cost, cellulose-based composite sorbent (C/PVPP/MDI) for efficient extraction of antibiotics and pesticides, particularly suitable for resource-limited settings [55].

Materials:

• C/PVPP/MDI sorbent (200 mg packed in 3 mL SPE cartridges)
• Water samples (100-500 mL)
• HPLC-grade methanol, acetonitrile, and ultrapure water
• Vacuum manifold

Procedure:

• Sorbent Preparation: Synthesize C/PVPP/MDI composite by cross-linking cellulose with poly(vinyl-polypyrrolidone) and 4,4-methylenebisphenyldiisocyanate in dimethylformamide at 70°C for 6 hours [55].
• Cartridge Conditioning: Condition with 5 mL methanol and 5 mL water.
• Sample Loading: Load 100 mL water sample (pH adjusted to 7.0) at 3-5 mL/min flow rate.
• Washing: Rinse with 3 mL 5% methanol solution.
• Elution: Elute with 2 × 4 mL methanol.
• Concentration: Evaporate to dryness and reconstitute in 100 μL mobile phase.
• Analysis: Analyze by LC-MS/MS.

Performance Characteristics:

• Recovery: 84.8-97.6% for antibiotics (tetracycline, ampicillin, sulfamethoxazole, penicillin V, chloramphenicol)
• Limit of Detection: 0.03-2.07 ng/L
• Cost: Approximately 50% less than commercial HLB sorbents
• Reusability: Up to 5 cycles without significant performance loss [55]

Workflow Integration and Data Analysis

The integration of multi-sorbent extraction with advanced analytical instrumentation and data processing techniques creates a comprehensive environmental monitoring workflow.

Figure 1: Integrated workflow for comprehensive environmental contaminant monitoring, combining multi-sorbent extraction with advanced analytical and data processing techniques.

Machine Learning-Assisted Non-Target Analysis

The integration of machine learning (ML) with non-target analysis (NTA) represents a significant advancement in contaminant source identification and prioritization. ML algorithms effectively identify latent patterns within high-dimensional HRMS data, enabling more accurate contamination source identification compared to traditional statistical methods [8].

Table 2: Machine Learning Applications in Non-Target Analysis for Source Identification

ML Algorithm	Application Context	Performance Metrics	Advantages
Random Forest (RF)	Screening of 222 PFAS in 92 samples	Balanced accuracy: 85.5-99.5%	Handles high-dimensional data, provides feature importance [8]
Support Vector Classifier (SVC)	Source classification of contaminants	Varies by application	Effective in high-dimensional spaces, versatile kernel functions [8]
Partial Least Squares Discriminant Analysis (PLS-DA)	Identification of source-specific indicators	Varies by application	Handles collinearities, identifies discriminant features [8]
Logistic Regression (LR)	Contaminant source attribution	Balanced accuracy: 85.5-99.5%	Interpretable model coefficients, probabilistic outputs [8]

The ML-assisted NTA workflow comprises four key stages: (1) sample treatment and extraction using multi-sorbent approaches, (2) data generation via HRMS, (3) ML-oriented data processing including preprocessing, dimensionality reduction, and pattern recognition, and (4) result validation through a tiered approach incorporating reference materials, external datasets, and environmental plausibility assessments [8].

Research Reagent Solutions

Table 3: Essential Materials for Multi-Sorbent Extraction of Environmental Contaminants

Reagent/Material	Function	Example Applications	Key Characteristics
Oasis HLB	Hydrophilic-lipophilic balanced copolymer	Broad-spectrum extraction of pesticides/pharmaceuticals	Excellent water wettability, high capacity [8]
Molecularly Imprinted Polymers (MIPs)	Selective recognition of target molecules	Sulfonylurea herbicides, cannabinoids	High selectivity, customizable [23]
Cellulose-based Composites (C/PVPP/MDI)	Cost-effective broad-spectrum sorbent	Antibiotics, pesticides in water	Low-cost, reusable, comparable to commercial sorbents [55]
Mixed-mode Sorbents (CX-W, AX-W)	Ion-exchange with secondary mechanisms	Acidic/basic pharmaceuticals	Complementary selectivity to HLB [8]
Metal-Organic Frameworks (MOFs)	High surface area porous materials	Emerging contaminant isolation	Tunable porosity, high surface area [23]
Graphene-Based Materials	High affinity for aromatic compounds	Pesticides with aromatic structures	Exceptional surface area, π-π interactions [23]

Validation and Quality Assurance

A tiered validation strategy ensures the reliability of multi-sorbent extraction methods combined with advanced detection techniques:

• Analytical Confidence: Verify compound identities using certified reference materials or spectral library matches with confidence levels (Level 1-5) [8].
• Model Generalizability: Assess classifiers on independent external datasets using cross-validation techniques (e.g., 10-fold) to evaluate overfitting risks [8].
• Environmental Plausibility: Correlate model predictions with contextual data, such as geospatial proximity to emission sources or known source-specific chemical markers [8].

Method performance should be validated through recovery studies (acceptable range: 70-120%), precision (RSD < 20%), and demonstration of minimal matrix effects. For non-target analysis, validation should include false positive/negative rates and computational accuracy [8].

The integration of multi-sorbent extraction strategies with advanced analytical instrumentation and data processing techniques provides a powerful framework for comprehensive environmental monitoring of pesticides and pharmaceuticals. The protocols detailed in this application note demonstrate effective approaches for addressing the complex challenge of broad-spectrum contaminant analysis across different laboratory settings and resource availability scenarios.

Future directions in this field include the development of increasingly selective sorbent materials, enhanced automation capabilities for high-throughput analysis, and more sophisticated data integration platforms that combine targeted, suspect, and non-target screening approaches. These advancements will further strengthen our ability to monitor contaminant profiles in aquatic environments, ultimately supporting improved risk assessment and environmental protection measures.

The widespread occurrence of antibiotic residues in water systems poses a significant threat to environmental and public health through the potential promotion of antimicrobial resistance (AMR) [56]. Effective monitoring of these contaminants requires analytical methods capable of detecting diverse antibiotic classes at trace concentrations in complex aqueous matrices [57]. A major challenge in environmental analysis is the simultaneous extraction of multiple antibiotic classes, which possess varied physicochemical properties, from a single water sample [57] [58].

This application note details the development of a robust multi-sorbent extraction strategy for the analysis of multi-class antibiotics in water. Solid-phase extraction (SPE) is a common pre-concentration technique, but its operation can be complex and time-consuming [57]. Furthermore, the simultaneous extraction of multiple antibiotic classes with diverse properties is particularly challenging when using a single SPE protocol [57]. This case study demonstrates how a multi-sorbent approach can overcome these limitations, providing a methodology for comprehensive antibiotic analysis to support environmental monitoring and exposure assessment within a "One Health" framework [59].

Experimental Design and Workflow

Research Reagent Solutions and Materials

The successful implementation of this multi-sorbent method relies on key reagents and materials outlined in the table below.

Table 1: Essential Research Reagents and Materials

Item	Function/Description	Key Characteristics
Hydrophilic-Lipophilic Balance (HLB) Sorbent	General-purpose copolymer for broad-spectrum extraction of a wide polarity range of analytes.	Retains hydrophilic and lipophilic compounds; commonly used for multi-class antibiotic protocols [57] [59].
Mixed-Bed Sorbent Tubes (e.g., Carbopack B/Carboxen/Tenax TA)	Sequential sorbent beds for capturing a very wide volatility range, from highly volatile to semi-volatile compounds.	Carboxen-1003 (high surface area) for C2-C5 volatiles; Carbopack B for C5-C12; Tenax-TA for C7-C26 semi-volatiles [11].
EDTA-McIlvaine Buffer	A chelating buffer solution used to improve the extraction efficiency of tetracycline antibiotics from solid and liquid matrices.	Chelates metal ions that tetracyclines strongly bind to, thereby releasing them into solution for analysis [58].
Deuterated Internal Standards	Isotope-labeled analogs of target analytes added to the sample prior to extraction.	Corrects for matrix effects and losses during sample preparation; crucial for accurate quantification by UPLC-MS/MS [57] [59].
Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)	High-sensitivity analytical instrumentation for separation, detection, and quantification of target antibiotics.	Provides the selectivity and low detection limits (sub-ng/L) required for trace-level environmental analysis [57] [59].

Sorbent Selection Strategy Workflow

The core of the method development lies in selecting the appropriate sorbent or sorbent combination based on the target analytes' properties. The following diagram illustrates the logical decision-making process.

Detailed Experimental Protocol

Sample Preparation and Multi-Sorbent SPE

This protocol is adapted from methods used for analyzing antibiotics in water and complex manure matrices [57] [58] [59].

• Sample Collection and Preservation: Collect water samples in pre-cleaned amber glass bottles. Acidify samples to pH ~2 with hydrochloric acid (HCl) immediately upon collection to stabilize the antibiotics. Add Na₂EDTA to a final concentration of 0.5 g/L to complex metal ions [57] [59].
• Internal Standard Addition: Spike all samples with a mixture of deuterated internal standards (e.g., 50 μL of 1 μg/mL mix into 50-100 mL sample) to achieve a final concentration of approximately 0.02-1 μg/L. This corrects for matrix effects and procedural losses [59].
• SPE Cartridge Conditioning: Condition a mixed-bed sorbent SPE cartridge (e.g., Oasis HLB or equivalent) sequentially with 10 mL of methanol and 10 mL of acidified ultrapure water (pH 2) at a flow rate not exceeding 10 mL/min. Do not let the sorbent bed dry out [57].
• Sample Loading: Load the acidified and spiked water sample (e.g., 500 mL) onto the conditioned SPE cartridge at a steady flow rate of 8-12 mL/min [57].
• Cartridge Washing and Drying: After sample loading, wash the cartridge with 10 mL of acidified ultrapure water (pH 2) to remove interfering salts and polar matrix components. Dry the cartridge under vacuum for 5-15 minutes to remove residual water [57].
• Analyte Elution: Elute the target antibiotics from the SPE cartridge with 9-10 mL of methanol into a collection tube. Alternatively, for a phased extraction, first elute with 10 mL of EDTA-McIlvaine buffer (pH 5), followed by 10 mL of methanol, and combine the eluents for challenging matrices [58].
• Extract Concentration and Reconstitution: Evaporate the eluate to near dryness under a gentle stream of nitrogen gas at 30°C. Reconstitute the dried extract in 1.0 mL of initial mobile phase (e.g., a water/methanol mixture) or a solvent compatible with the LC-MS/MS system. Vortex thoroughly and transfer to an amber LC vial for analysis [57] [59].

Instrumental Analysis: UPLC-MS/MS

• Chromatographic Separation: Use an UPLC system equipped with a C18 reverse-phase column (e.g., 100 mm x 2.1 mm, 1.7 μm). Maintain the column temperature at 40°C. The mobile phase consists of (A) water and (B) methanol, both containing 0.1% formic acid. Employ a gradient elution: 0-1 min, 5% B; 1-10 min, 5-95% B; 10-12 min, 95% B; 12-12.1 min, 95-5% B; 12.1-15 min, 5% B for re-equilibration. The injection volume is 10-100 μL [57].
• Mass Spectrometric Detection: Operate the tandem mass spectrometer in positive electrospray ionization (ESI+) mode with multiple reaction monitoring (MRM). Optimize ion source parameters for maximum sensitivity for all target antibiotics. Examples of parameters include: capillary voltage, 3.0 kV; source temperature, 150°C; desolvation temperature, 500°C; desolvation gas flow, 1000 L/h [57]. Monitor at least two MRM transitions per analyte for confirmation.

Results and Performance Data

Method Validation and Performance

The developed method was validated for the simultaneous analysis of 69 antibiotics from 5 classes (sulfonamides, quinolones, macrolides, β-lactams, and tetracyclines) [57]. The performance data are summarized below.

Table 2: Method Performance Metrics for Multi-Class Antibiotics in Water

Antibiotic Class	Absolute Recovery (%)	Limits of Detection (LOD) (ng/L)	Key Sample Preparation Note
Sulfonamides (SAs)	80 - 120 [57]	< 1 for most compounds [57]	Stable under acidic sample preservation conditions.
Quinolones (QNs)	80 - 120 [57]	< 1 for most compounds [57]	Good recovery with HLB sorbents.
Macrolides (MLs)	80 - 120 [57]	< 1 for most compounds [57]	Can show lower recovery with some SPE methods without pH adjustment [57].
Tetracyclines (TCs)	67 - 131 [58]	< 1 for most compounds [57]	Requires EDTA-containing buffer for efficient extraction from solid phases [58].
β-Lactams (β-Ls)	1 - 66 [58]	< 1 for most compounds [57]	Lower and variable recovery due to unstable β-lactam ring; use freshly spiked standards [58].

Application to Real-World Samples

The validated method was applied to screen 25 brands of bottled water [57]. The results demonstrated its practical utility:

• Detection Frequency: 54 different antibiotics from 5 classes were detected, with detection frequencies ranging from 4% to 100%.
• Concentration Range: Detected concentrations were between 0.0453 and 37.4 ng/L.
• Co-occurrence: Multiple antibiotics were found simultaneously, with over 10 different antibiotics identified in 9 different brands. Quinolones and sulfonamides were the most predominant classes [57].

The complete workflow, from sample preparation to data analysis, is visualized below, integrating the components detailed in this note.

This case study demonstrates that a strategically developed multi-sorbent extraction method is a powerful tool for comprehensive environmental monitoring. By carefully selecting sorbents and optimizing the sample preparation protocol, researchers can overcome the challenge of extracting diverse antibiotic classes from complex matrices. The resulting methodology provides the sensitivity, accuracy, and breadth of analyte coverage necessary to advance research on the environmental occurrence, fate, and public health implications of antibiotics, thereby directly contributing to the broader thesis of broad-spectrum contaminant analysis and the "One Health" initiative.

Optimizing Performance and Overcoming Analytical Challenges

Systematic Sorbent Selection Based on Analyte and Matrix Properties

The effectiveness of any analytical method for contaminant analysis is fundamentally dictated by the sample preparation stage, with sorbent selection representing the most critical parameter. Within the context of broad-spectrum contaminant analysis, a single-sorbent approach often proves inadequate due to the vast diversity in analyte physicochemical properties. This application note delineates a systematic framework for selecting sorbents based on a detailed assessment of both the target analytes and the sample matrix. The principles outlined herein support the development of robust multi-sorbent extraction strategies, enabling the simultaneous capture and analysis of contaminants with varying polarities, volatilities, and functional groups. Such strategies are indispensable for comprehensive environmental monitoring, food safety assurance, and pharmaceutical development, where the target analyte profile is often complex and poorly defined [60] [22].

The trend in modern analytical chemistry is moving decisively away from solvent-intensive extraction methods and towards thermal desorption (TD)-compatible sorbent-based techniques. This shift is driven by the need for enhanced sensitivity, automation, and the elimination of hazardous solvents like CS₂. A consequence of this trend is the growing demand for TD-compatible alternative sorbents, as traditional charcoal is often too strong and active for reliable thermal desorption of many compounds [61] [62]. This note provides a contemporary guide to navigating the available sorbent options to optimize analytical performance.

Foundational Principles of Sorbent Selection

The interaction between an analyte, the sample matrix, and the sorbent surface governs the efficiency of extraction. A systematic selection process must, therefore, begin with a thorough characterization of these components.

Key Sorbent Properties

When evaluating a sorbent for a specific application, several intrinsic properties must be considered [61] [62]:

• Sorbent Strength (Retention Volume): This determines the affinity of the sorbent for the analyte. A sorbent that is too weak will lead to breakthrough and sample loss, while one that is too strong may necessitate extreme desorption conditions or result in irreversible adsorption, analyte degradation, or poor recovery.
• Hydrophobicity/Hydrophilicity: The affinity of the sorbent for water is a major consideration, particularly for analyzing volatile organic compounds (VOCs) in humid environments. Hydrophobic sorbents are preferred for such applications to manage water interference, which can adversely affect gas chromatography-mass spectrometry (GC-MS) analysis [62].
• Inertness: The sorbent should not catalyze the decomposition or rearrangement of analytes. Reactive sorbents can lead to the formation of artefacts, compromising the analytical results.
• Mechanical Strength and Thermal Stability: Sorbents must be physically robust (non-friable) to maintain bed integrity during sampling and handling, and thermally stable to withstand the high temperatures required for thermal desorption without degrading or off-gassing.

Analyte and Matrix Characterization

A thorough understanding of the sample and its components is the first step in the selection process, as outlined in the table below.

Table 1: Key Characterization Parameters for Systematic Sorbent Selection

Category	Parameter	Influence on Sorbent Selection	Example/Note
Analyte Properties	Volatility (Boiling Point)	Determines required sorbent strength; high volatility requires stronger sorbents.	Very Volatile Organic Compounds (VVOCs) to Semi-Volatile Organic Compounds (SVOCs) [61].
	Polarity & Functional Groups	Guides choice of sorbent surface chemistry (non-polar, polar, ion-exchange).	Polar analytes interact better with polar sorbents like Tenax TA [62].
	Hydrophobicity (log KOW)	Indicates interaction with hydrophobic vs. hydrophilic sorbents.	A high log KOW value suggests strong retention on non-polar sorbents like C18 [20].
	pKa / pKb	For ionizable compounds, dictates the need for and type of ion-exchange sorbent.	Requires pH control to ensure analytes are in a charged state for retention [63].
Matrix Properties	Complexity & Composition	Determines the required clean-up capability and selectivity of the sorbent.	Food matrices (fats, proteins, sugars) require extensive clean-up [22].
	Water Content	Dictates the necessity for hydrophobic sorbents or water management techniques.	Humid air or biological samples necessitate hydrophobic sorbents [62].
	Physical State (Solid, Liquid, Gas)	Influences the choice of extraction format (e.g., tube, cartridge, stir bar).	Sorbent tubes for air; SPE cartridges for liquids; SBSE for stirred liquids [61] [22].

Implementing a Multi-Sorbent Strategy

For the broad-spectrum analysis of contaminants with a wide range of physicochemical properties, a multi-sorbent approach is the most effective solution. This strategy involves packing a single tube or cartridge with two or more sorbents arranged in order of increasing strength from the sampling end [62].

The Rationale for Multi-Sorbent Tubes

A multi-sorbent bed functions as a "chromatographic" system during sampling. The primary sorbent, at the sampling inlet, is a weak sorbent designed to retain high-boiling (low volatility) compounds. Subsequent layers contain progressively stronger sorbents that trap more volatile analytes as they pass through the tube. This configuration prevents the displacement of low-volatility compounds by more volatile ones, a phenomenon known as "breakthrough," thereby ensuring the capture of a wide analyte spectrum [62]. During the desorption phase, it is critical to use backflush desorption—where the flow of gas is the reverse of the sampling flow. This ensures that the most strongly retained compounds (on the strongest sorbent) do not have to pass through the entire sorbent bed during desorption, which could lead to band-broadening, tailing, or decomposition [62].

Workflow for Systematic Sorbent Selection

The following diagram illustrates a logical decision pathway for selecting and implementing a sorbent-based extraction strategy, from initial problem definition to final analysis.

Figure 1: A logical workflow for systematic sorbent selection and method development, highlighting the decision point for single versus multi-sorbent strategies.

Detailed Experimental Protocols

Protocol: Sorbent Tube Preparation for Broad-Spectrum VOC/SVOC Analysis in Air

This protocol details the preparation of a multi-sorbent tube for monitoring a wide range of organic vapors in air, suitable for active (pumped) or diffusive sampling followed by thermal desorption GC-MS analysis [61] [62].

1. Materials and Equipment:

• Stainless steel sorbent tube (standard dimensions: 3.5 inches x 1/4 inch OD)
• Sorbents: Tenax TA (weak), Carbopack B (medium-strong), Carboxen 1000 (strong)
• Glass wool (silanized)
• Tube packing rig
• Analytical balance
• Flow controller

2. Packing Procedure: 1. Prepare Materials: Condition each sorbent separately according to the manufacturer's specifications prior to packing. Wear gloves to prevent contamination. 2. Weigh Sorbents: Accurately weigh the following sorbents: - 150 mg of Tenax TA - 100 mg of Carbopack B - 50 mg of Carboxen 1000 3. Pack the Tube: - Place a small plug of silanized glass wool into the tube to a depth of approximately 2 mm, using a packing rod. This will be the outlet end during sampling. - Add the strongest sorbent, Carboxen 1000, first. Tap the tube gently to settle the bed. - Add a small glass wool separator plug. - Add the medium-strong sorbent, Carbopack B. Tap to settle. - Add another glass wool separator. - Finally, add the weakest sorbent, Tenax TA, to the tube. This end will be the inlet during sampling. - Secure the contents with a final glass wool plug. 4. Condition the Packed Tube: Connect the tube to a TD unit or a clean carrier gas stream. Condition the tube by heating it to 300-330°C for a minimum of 1-2 hours under a steady flow of inert gas (e.g., 50 mL/min). Verify the cleanliness by running a blank analysis.

Protocol: Evaluation of Sorbent Performance and Breakthrough

1. Objective: To determine the retention capacity of a selected sorbent or sorbent combination for a target analyte and to identify potential breakthrough.

2. Materials:

• Two identical, pre-conditioned sorbent tubes (from Protocol 4.1)
• Active air sampling pump
• Calibrated gas standard or spiked liquid standard
• Thermal Desorber-GC-MS system

3. Procedure: 1. Setup: Connect the two sorbent tubes in series using a short piece of inert tubing. 2. Sample Collection: Draw a known volume of air containing a defined concentration of the target analyte(s) through the tube series at a controlled flow rate. 3. Analysis: Analyze each tube in the series separately using the optimized TD-GC-MS method. 4. Calculation: Calculate the breakthrough percentage using the formula: Breakthrough (%) = (Mass of Analyte in Back Tube / (Mass of Analyte in Front Tube + Mass of Analyte in Back Tube)) * 100

4. Interpretation: A breakthrough of less than 5% is generally acceptable. Breakthrough exceeding this value indicates that the sorbent strength is insufficient, the sorbent bed mass is too small, the sampling flow rate is too high, or the sample volume is too large for the target analyte(s). Adjust the method parameters accordingly and re-test.

The Scientist's Toolkit: Essential Research Reagent Solutions

The following table catalogues key materials and their functions in developing sorbent-based extraction methods for contaminant analysis.

Table 2: Essential Reagents and Materials for Sorbent-Based Extraction Methods

Item	Function/Application	Key Characteristics
Tenax TA	A versatile porous polymer for trapping VOCs and SVOCs.	Excellent hydrophobicity, thermal stability (up to ~350°C), low artifact formation. Ideal for the first layer in a multi-sorbent bed [62].
Carbograph	A graphitized carbon black sorbent.	Provides non-polar surface with high uniformity. Available in different surface areas for trapping a range of volatilities [62].
Carboxen	A carbon molecular sieve.	Very high surface area; excellent for trapping highly volatile organic compounds (C2-C5). Used as a strong sorbent in multi-bed setups [62].
C18-Bonded Silica	A reversed-phase sorbent for SPE.	Widely used for extracting non-polar to moderately polar analytes from liquid samples (e.g., water, food extracts) [63] [22].
Ion-Exchange Sorbents (SAX, SCX)	SPE sorbents for ionic compounds.	Selective retention of acidic (SAX) or basic (SCX) analytes. Requires pH control to ionize targets [63].
Molecularly Imprinted Polymers (MIPs)	Synthetic, highly selective sorbents.	Tailored for specific compounds or classes (e.g., mycotoxins, pesticides), offering superior clean-up from complex matrices [22].
QuEChERS Kits	Dispersive SPE for pesticide residue analysis.	A standardized, quick, and effective method for cleaning up complex food matrices like fruits and vegetables [22].
Silanized Glass Wool	Used to retain sorbent in tubes and as a separator.	Inert, thermally stable; prevents sorbent mixing and particle entrainment in gas streams [62].
Nafion Dryer	A permeation membrane for water management.	Selectively removes water vapor from humid air samples prior to the sorbent tube, protecting the GC-MS system [62].

A systematic approach to sorbent selection, grounded in a thorough understanding of analyte and matrix properties, is fundamental to successful broad-spectrum contaminant analysis. The move towards thermal desorption-compatible sorbents and the strategic implementation of multi-sorbent tubes represent the state of the art in achieving high sensitivity and wide analytical coverage. As the field advances, the development of novel sorbent materials—including engineered nanomaterials, metal-organic frameworks (MOFs), and increasingly selective molecularly imprinted polymers—will further empower researchers to tackle the challenges posed by emerging contaminants and complex sample matrices [43] [64]. By adhering to the principles and protocols outlined in this application note, scientists can develop robust, reliable, and efficient methods for comprehensive environmental and product safety monitoring.

In the pursuit of reliable analytical data for broad-spectrum contaminant analysis, sample preparation represents a critical, yet often optimized, step in the analytical workflow. The primary challenge lies in simultaneously achieving two potentially conflicting objectives: efficient removal of matrix interferents (clean-up efficiency) and maximal retrieval of target analytes (analyte recovery). Over-purification, a counterproductive outcome of excessively stringent clean-up protocols, can inadvertently lead to significant analyte loss, reduced method sensitivity, and ultimately, unreliable quantitative results [49] [65]. Within the framework of a multi-sorbent extraction strategy, this application note delineates structured protocols and evidence-based principles to navigate this balance, ensuring that clean-up procedures are both effective and efficient.

The evolution of sorbent technologies has been largely driven by this need for selective extraction. Modern approaches have moved beyond traditional single-phase sorbents to embrace mixed-mode materials and sequential sorbent arrangements that offer orthogonal selectivity—the ability to isolate analytes based on multiple independent chemical interaction mechanisms [2] [66]. This strategic layering of different sorbent chemistries enables researchers to precisely target specific analyte classes while rejecting a broader spectrum of matrix components, thereby mitigating the risk of analyte loss associated with non-selective binding. Furthermore, the adoption of Green Analytical Chemistry (GAC) principles emphasizes the importance of minimizing solvent usage and waste generation, which aligns with developing more efficient, one-step purification methods that reduce the need for multiple cleaning stages and the associated analyte losses [43] [67].

Sorbent Selection Strategy

The foundation of balanced sample preparation lies in the strategic selection and deployment of sorbent materials. A multi-sorbent strategy leverages the complementary selectivity of different materials to achieve comprehensive clean-up without resorting to conditions so harsh that they compromise analyte recovery.

Orthogonal Selectivity Through Sorbent Combinations

Orthogonal selectivity is achieved by combining sorbents that interact with analytes through different chemical mechanisms. For instance, a sequence might utilize a reversed-phase sorbent to capture analytes based on hydrophobic interactions, followed by a ion-exchange sorbent to isolate compounds based on their charge characteristics [2]. This approach allows for the precise extraction of a wider range of analytes from complex matrices while efficiently excluding interferents that might co-elute if only a single mechanism were employed. The development of mixed-mode sorbents, which incorporate multiple functional groups (e.g., reversed-phase paired with cation or anion exchange groups), embodies this principle in a single, convenient format, simplifying methods and reducing the number of required purification steps [66].

Key Sorbent Classes and Their Applications

The following table summarizes the primary sorbent classes used in modern solid-phase extraction, detailing their interaction mechanisms and ideal use cases to guide selection within a multi-sorbent strategy.

Table 1: Key Sorbent Classes for Multi-Sorbent Strategies

Sorbent Class	Primary Interaction Mechanism	Typical Applications	Considerations for Recovery
Reversed-Phase (C18, C8)	Hydrophobic (van der Waals)	Extraction of non-polar to moderate polar analytes from aqueous matrices [2].	Strong retention can require strong elution solvents; potential for irreversible binding of highly hydrophobic analytes.
Hydrophilic-Lipophilic Balance (HLB)	Mixed-mode (Hydrophobic and Hydrogen Bonding)	Broad-spectrum extraction of acidic, basic, and neutral compounds without conditioning [55].	High capacity reduces overloading but requires careful elution optimization to recover analytes with differing polarities.
Ion Exchange (SCX, SAX)	Electrostatic (Cationic or Anionic)	Selective isolation of ionizable compounds at appropriate pH [66].	Recovery is highly pH-dependent; requires proper adjustment of sample and elution solvent pH.
Molecularly Imprinted Polymers (MIPs)	Shape-Selectivity & Chemical Affinity	High-selectivity extraction of specific analyte classes or single compounds [2] [66].	Excellent clean-up but can be too selective for broad-spectrum analysis; template bleeding can contaminate samples.
Restricted Access Materials (RAM)	Size Exclusion & Hydrophobic Interaction	Direct injection of biological fluids; excludes macromolecules like proteins [2].	Protoses the sorbent and analytical instrument from matrix macromolecules, simplifying the protocol and improving recovery of small molecules.
Metal-Organic Frameworks (MOFs)	Complexation, Size Exclusion, Hydrophobic	Extraction of diverse contaminants leveraging high surface area and tunable porosity [68].	High capacity and selectivity, but stability in aqueous/organic solvents can vary; requires matching MOF chemistry to analyte.

Quantitative Comparison of Sorbent Performance

Evaluating sorbent performance through key metrics is essential for rational method development. The data below, compiled from recent studies, illustrates how different sorbents perform in terms of recovery and clean-up efficiency for various analytes.

Table 2: Analytical Performance Metrics for Selected Sorbent Materials

Sorbent Material	Target Analytes	Matrix	Average Recovery (%)	Key Performance Advantage	Citation
Cellulose/PVPP/MDI Composite	Antibiotics (TET, AMP, SMX, etc.)	River Water	84.8 - 97.6%	Low-cost, reusable (5 cycles), broad-spectrum recovery comparable to commercial HLB [55].
Commercial HLB	Antibiotics (TET, AMP, SMX, etc.)	River Water	87.0 - 97.3%	High, consistent recovery for a wide range of pharmaceuticals; considered a benchmark [55].
Fabric Phase Sorptive Extraction (FPSE)	Various multi-class analytes	Biological & Environmental Fluids	Near-exhaustive (per theory)	Combines exhaustive (SPE) and equilibrium (SPME) extraction; fast kinetics and high capacity [65].
Magnetic Solid-Phase Extraction (MSPE) with MOFs	Various organic contaminants	Water	~100% (reported for some)	High efficiency due to dispersive mode and high surface area; rapid separation [68].
Molecularly Imprinted Polymers (MIPs)	Specific target analytes	Complex Matrices	High for target	Exceptional clean-up and selectivity for target compounds, reducing matrix effects significantly [2].

Detailed Experimental Protocols

Protocol 1: Multi-Sorbent SPE for Broad-Spectrum Contaminants in Water

This protocol utilizes a sequential cartridge format for the extraction of pharmaceuticals and other contaminants from water samples, balancing clean-up and recovery through orthogonal sorbent chemistry [2] [66].

Research Reagent Solutions:

• Sorbent A: Hydrophilic-Lipophilic Balance (HLB) cartridge (e.g., 60 mg, 3 mL)
• Sorbent B: Mixed-Mode Cation Exchange (MCX) cartridge (e.g., 60 mg, 3 mL)
• Elution Solvent 1: Methanol (HPLC grade)
• Elution Solvent 2: Methanol with 5% Ammonium Hydroxide (v/v)
• Wash Solvent 1: Deionized Water (with 1% Formic Acid for MCX step)
• Wash Solvent 2: Deionized Water
• Conditioning Solvent: Methanol followed by Deionized Water

Procedure:

• Sample Pre-treatment: Adjust the pH of the water sample (e.g., 100 mL) to ~7. Filter through a 0.45 µm glass fiber filter to remove particulates.
• Sorbent Conditioning: Condition the HLB cartridge with 3 mL of methanol, followed by 3 mL of deionized water. Do not allow the sorbent bed to dry.
• Sample Loading: Pass the pre-treated sample through the HLB cartridge at a steady flow rate of 5-10 mL/min. This step captures a broad range of non-polar and polar contaminants.
• Washing (HLB): Wash the HLB cartridge with 3 mL of deionized water to remove salts and highly polar interferents. Discard the wash.
• Analyte Transfer & Secondary Clean-up: Elute the analytes from the HLB cartridge with 4 mL of methanol directly onto the pre-conditioned MCX cartridge. This transfers the analytes to the second sorbent for orthogonal clean-up.
• Washing (MCX): Wash the MCX cartridge with 3 mL of deionized water acidified with 1% formic acid to remove neutral and basic interferents that were co-eluted from the HLB.
• Elution (MCX): Elute the target acidic and neutral analytes from the MCX cartridge with 4 mL of methanol. For basic analytes, use 4 mL of methanolic ammonia (Elution Solvent 2).
• Analysis: Collect the eluate. Evaporate to dryness under a gentle stream of nitrogen and reconstitute in an appropriate volume of initial mobile phase for LC-MS/MS analysis.

Protocol 2: Evaluation of a Low-Cost Cellulose-Based Sorbent for Antibiotics

This protocol, adapted from Olorunnisola et al. (2025), details the use of a sustainable, cellulose-based sorbent for antibiotic monitoring, demonstrating performance comparable to commercial materials at a reduced cost [55].

Research Reagent Solutions:

• Sorbent: C/PVPP/MDI composite, packed into empty SPE cartridges (100 mg)
• Conditioning Solvent: Methanol, Deionized Water
• Wash Solvent: Deionized Water
• Elution Solvent: Methanol:Acetonitrile (1:1, v/v)
• Standard Solutions: Primary stock solutions (1 mg/mL) of Tetracycline, Ampicillin, Sulfamethoxazole, Penicillin V, and Chloramphenicol in ethanol:water (1:1).

Procedure:

• Sorbent Preparation: Pack 100 mg of the synthesized C/PVPP/MDI composite into an empty 3 mL SPE cartridge between two polyethylene frits.
• Sorbent Conditioning: Condition the cartridge sequentially with 5 mL of methanol and 5 mL of deionized water.
• Sample Loading: Load 100 mL of water sample (spiked with antibiotics or environmental sample) onto the cartridge at a flow rate of 5 mL/min.
• Washing: Rinse the cartridge with 5 mL of deionized water to remove matrix interferences. Discard the effluent.
• Elution: Elute the captured antibiotics with 5 mL of methanol:acetonitrile (1:1, v/v) mixture into a clean collection tube.
• Concentration and Reconstitution: Evaporate the eluate to complete dryness under a gentle nitrogen stream. Reconstitute the residue in 1.0 mL of mobile phase (e.g., 1:1 water:methanol) by vortexing for 1 minute.
• Analysis: Inject the reconstituted solution into the LC-MS/MS system for quantification. The sorbent can be regenerated and reused for up to 5 cycles without significant loss in recovery [55].

Workflow and Strategic Visualization

The following diagrams, generated using DOT language, illustrate the core decision-making workflow for avoiding over-purification and the experimental sequence for a multi-sorbent protocol.

Strategic Workflow for Balanced Purification

Diagram 1: A strategic workflow for developing a balanced sample preparation method, highlighting decision points to avoid over-purification.

Multi-Sorbent Experimental Sequence

Diagram 2: Sequential workflow for a multi-sorbent SPE protocol demonstrating orthogonal clean-up stages.

Achieving an optimal balance between clean-up efficiency and analyte recovery is a deliberate and strategic process, not one of chance. By moving beyond single-sorbent methods and embracing a multi-sorbent strategy founded on the principles of orthogonal selectivity, researchers can systematically overcome the challenge of over-purification. The protocols and data presented herein provide a practical roadmap for developing robust, sensitive, and reliable analytical methods suitable for broad-spectrum contaminant analysis in complex matrices. This approach ensures that the integrity of the sample is preserved throughout the preparation process, leading to analytical data that truly reflects the composition of the original sample.

Analyte loss during sample preparation presents a significant challenge in analytical chemistry, particularly when developing methods for broad-spectrum contaminant analysis that must simultaneously capture diverse chemical structures. Planar molecules and ionic compounds are especially susceptible to losses due to their unique physicochemical properties. Planar structures often experience π-π interactions with surfaces and stationary phases, while ionic species demonstrate unpredictable retention behavior influenced by pH and ionic strength. These interactions lead to incomplete recovery, reduced sensitivity, and compromised data quality in multi-residue methods. This application note systematically addresses these challenges within the context of a comprehensive research thesis on multi-sorbent extraction strategies, providing validated protocols to minimize analyte loss and maximize recovery across diverse compound classes.

Theoretical Foundations of Analyte Loss

Molecular Interactions Driving Analyte Loss

The predominant mechanisms of analyte loss for planar and ionic compounds stem from specific molecular interactions with analytical surfaces. Planar molecules, including many polycyclic aromatic hydrocarbons and planar heterocyclic compounds, exhibit strong π-π stacking interactions with graphitic carbon surfaces and aromatic moieties in stationary phases. These interactions often result in irreversible adsorption or delayed elution. Additionally, charge-transfer complexes can form between electron-deficient and electron-rich planar systems, further complicating their recovery.

Ionic compounds present distinct challenges based on their charge characteristics. Permanent ions experience ionic interactions with charged or ion-exchange sites on surfaces, while ionizable compounds undergo pH-dependent retention shifts that can lead to unexpected elution profiles. The log D (distribution coefficient) value, which varies with pH, fundamentally governs the partitioning behavior of ionizable compounds throughout the extraction process [69]. For zwitterionic compounds containing both acidic and basic functional groups, such as certain angiotensin receptor antagonists, simultaneous ionic interactions can occur at multiple sites, creating complex retention mechanisms that challenge conventional single-mode extraction approaches.

Multi-Sorbent Extraction as a Strategic Framework

Multi-sorbent extraction strategies provide a sophisticated solution to the challenge of broad-spectrum analysis by integrating multiple retention mechanisms within a single workflow. This approach leverages the complementary selectivity of different sorbent chemistries to capture analytes across a wide polarity and ionization range. The fundamental principle involves sequential or parallel utilization of reversed-phase, ion-exchange, and mixed-mode sorbents to address the specific retention needs of different compound classes [69] [3].

This strategy is particularly valuable for complex matrices where planar and ionic compounds coexist with neutral species. By employing a modular cartridge setup with separately packed sorbents, researchers can optimize elution conditions for each retention mechanism independently, maximizing recovery while maintaining the selectivity needed for complex sample analysis [69]. This approach represents a significant advancement over generic polymeric sorbents, which often exhibit limited selectivity and higher matrix effects despite broad retention capabilities.

Experimental Protocols

Modular Solid-Phase Extraction for Multi-Class Compounds

This protocol describes a modular solid-phase extraction (SPE) setup combining three sorbents for effective extraction of neutrals, acids, and bases from complex matrices, adapted from validated methodologies for wastewater analysis [69].

Materials and Equipment

• Sorbent Phases: Oasis HLB (hydrophilic-lipophilic balanced), MCX (mixed-mode cation exchange), WAX (mixed-mode weak anion exchange)
• Cartridge Configuration: Separate cartridges connected in tandem
• Solvents: Methanol (LC-MS grade), acetonitrile (LC-MS grade), formic acid (LC-MS grade), ammonia solution (7 M in methanol)
• Apparatus: Vacuum manifold, glass tubes for eluate collection, adjustable pipettes
• Sample Preparation: Centrifuge, vortex mixer, pH meter

Step-by-Step Procedure

• Sample Pre-treatment:

  • Adjust sample pH to 7.0 using dilute formic acid or ammonia solution
  • Centrifuge turbid samples at 4500 × g for 10 minutes
  • Filter supernatant through 0.45-μm glass fiber filter

• SPE Cartridge Assembly and Conditioning:

  • Connect HLB, MCX, and WAX cartridges in series (HLB first, then MCX, then WAX)
  • Condition with 6 mL methanol followed by 6 mL deionized water at flow rate of 2-3 mL/min
  • Maintain sorbent moisture throughout process

• Sample Loading and Extraction:

  • Load prepared sample (up to 500 mL) under vacuum at steady flow rate of 5 mL/min
  • After loading, dry cartridges under vacuum for 30 minutes
  • Disconnect cartridges for individual elution

• Fractionated Elution:

  • Neutral Fraction (from HLB cartridge): Elute with 8 mL methanol into clean collection tube
  • Basic Fraction (from MCX cartridge): Elute with 8 mL methanolic ammonia (5% v/v)
  • Acidic Fraction (from WAX cartridge): Elute with 8 mL methanol containing 2% formic acid

• Post-Processing:

  • Evaporate eluates to near-dryness under gentle nitrogen stream at 40°C
  • Reconstitute in 500 μL initial mobile phase composition
  • Vortex for 30 seconds and transfer to LC vials for analysis

Table 1: Modular SPE Recovery Performance for Different Compound Classes

Compound Class	Representative Analytes	Average Recovery (%)	CV (%)
Planar Neutrals	Organophosphorus compounds, Neonicotinoids	85-110	3-8
Acidic Compounds	Phenoxy acids, Tetrazolic acids	80-115	4-9
Basic Compounds	Azoles, Tertiary amines	82-112	3-7
Zwitterions	Losartan, Irbesartan	78-105	5-11

Thin-Film Solid-Phase Microextraction for Planar Molecules

Thin-film solid-phase microextraction (TF-SPME) provides enhanced extraction capacity for planar molecules through increased surface area and optimized sorbent chemistry, minimizing π-π interactions that cause analyte loss [3].

Materials and Equipment

• TF-SPME Devices: C18-modified thin films or carbon-based sorbents
• Extraction Chamber: 20 mL headspace vials with magnetic stirrers
• Desorption System: LC desorption chamber or appropriate interface
• Solvents: Methanol, acetonitrile, water (all LC-MS grade)

Step-by-Step Procedure

• Sorbent Conditioning:

  • Immerse TF-SPME device in 5 mL methanol for 30 minutes
  • Transfer to 5 mL water for 10 minutes
  • Do not allow sorbent to dry before extraction

• Sample Preparation:

  • Place 10 mL sample solution in 20 mL headspace vial
  • Add internal standards appropriate for planar targets
  • Adjust ionic strength with 1 g NaCl

• Extraction Process:

  • Immerse conditioned TF-SPME device in sample
  • Stir at 500 rpm for 120 minutes at room temperature
  • Remove device and briefly rinse with deionized water

• Desorption:

  • Place device in desorption chamber with 2 mL methanol:acetonitrile (1:1)
  • Desorb with gentle agitation for 60 minutes
  • Collect eluate and evaporate to 250 μL under nitrogen
  • Transfer to LC vial for analysis

Table 2: TF-SPME Recovery Enhancement for Planar Molecules

Analyte Type	Conventional Fiber SPME Recovery (%)	TF-SPME Recovery (%)	Improvement Factor
Polycyclic Aromatic Hydrocarbons	45-65	78-92	1.7-2.1
Planar Heterocycles	38-60	75-88	1.9-2.3
Chlorinated Planars	42-68	80-95	1.8-2.2

Visualization of Workflows

Multi-Sorbent Extraction Strategy

Sorbent Selection Decision Framework

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Sorbents and Their Applications in Managing Analyte Loss

Sorbent/Reagent	Chemical Composition	Primary Function	Optimal Application
Oasis HLB	Hydrophilic-lipophilic balanced polymer	Broad-spectrum retention of neutrals, acids, and bases	Initial capture in modular SPE; log D range -1 to 7 [69] [3]
Mixed-Mode Cation Exchange (MCX)	Sulfonic acid groups on polymer substrate	Retention of basic compounds via cation exchange	Separation of bases from neutrals and acids; pH < pKa-2 [69]
Mixed-Mode Anion Exchange (WAX)	Quaternary amine groups on polymer substrate	Retention of acidic compounds via anion exchange	Separation of acids from neutrals and bases; pH > pKa+2 [69]
Carbon-Based Sorbents	Graphitized carbon, porous graphite	Selective retention of planar molecules	Targeted capture of planar compounds; minimize π-π interactions [3]
Molecularly Imprinted Polymers (MIPs)	Template-shaped polymer cavities	High-affinity recognition of specific analytes	Selective extraction of target compounds in complex matrices [3]

Data Analysis and Method Validation

Quantitative Performance Metrics

Comprehensive method validation for multi-sorbent strategies requires assessment of recovery, matrix effects, and sensitivity across all target compound classes. The following performance criteria should be established:

• Recvery Limits: Acceptable recovery range of 70-120% with CV < 15% for most analytes
• Linearity: Minimum R² > 0.990 across calibrated concentration range
• Sensitivity: Procedural limits of quantification (LOQs) sufficient for intended application (typically 1-20 ng/L for environmental analysis) [69]

Table 4: Method Validation Metrics for Multi-Sorbent Approach

Performance Indicator	Acceptance Criteria	Typical Performance	Key Influencing Factors
Overall Recovery Range	70-120%	80-115% for 90% of analytes	pH control, solvent strength, elution volume
Precision (Intra-day CV)	< 15%	3-11%	Homogeneity, flow rate control, timing
Matrix Effects (Signal Suppression/Enhancement)	± 20%	± 5-18%	Sample clean-up, selectivity, ion-pairing
Process Efficiency	> 65%	75-105%	Combination of recovery and matrix effects

Advanced Data Processing Techniques

Modern mass spectrometry data requires sophisticated processing to extract meaningful information from complex samples. Implement the following workflow:

• Peak Detection and Alignment: Use retention time correction algorithms to address chromatographic shifts
• Componentization: Group related spectral features (adducts, isotopes) into molecular entities
• Multivariate Analysis: Apply PCA to identify patterns and outliers in the high-dimensional data
• Machine Learning Integration: Utilize supervised learning (Random Forest, SVM) for classification of contamination sources based on chemical fingerprints [8]

For non-target analysis, establish confidence levels for compound identification (Level 1-5) and implement quality control measures including batch-specific QC samples and solvent blanks to ensure data integrity throughout the analytical process.

Troubleshooting and Optimization Guidelines

Common Issues and Solutions

• Low Recovery of Planar Compounds: Replace graphitic carbon sorbents with C18-modified materials; add solvent modifiers like toluene to disrupt π-π interactions
• Incomplete Elution of Ionic Compounds: Increase ionic strength of elution solvent; use competing ions in eluent; optimize pH for ionizable compounds
• Carryover Between Fractions: Implement rigorous cleaning steps between elutions; use separate collection vessels; validate absence of cross-contamination
• Matrix Effects in LC-MS/MS: Enhance sample clean-up; implement isotope-labeled internal standards; use matrix-matched calibration

Method Adaptation Strategies

For new analyte classes, systematically optimize these parameters:

• pH Adjustment: Test sample pH values at pKa ± 2 units for ionizable compounds
• Solvent Strength: Evaluate elution solvents with increasing strength (methanol, acetonitrile, with modifiers)
• Sorbent Sequence: Reconfigure cartridge order in modular systems based on dominant retention mechanisms
• Volume Optimization: Balance sufficient elution volume against excessive dilution requiring reconcentration

The protocols presented herein provide a robust foundation for minimizing analyte loss across diverse compound classes, enabling more comprehensive and reliable broad-spectrum contaminant analysis in complex matrices.

The pursuit of broad-spectrum contaminant analysis presents a significant challenge in modern analytical chemistry, requiring methods capable of isolating and quantifying diverse compounds with varying physicochemical properties from complex matrices. This application note, framed within a broader thesis on multi-sorbent extraction strategies, details optimized workflow parameters for robust analytical methods. The principles of Green Analytical Chemistry (GAC) are integrated throughout, emphasizing the use of sustainable materials and reduced solvent consumption [70] [22]. We provide a comprehensive guide to method development, from systematic parameter optimization using statistical designs to the implementation of detailed protocols for techniques including Solid-Phase Extraction (SPE), Microextraction by Packed Sorbent (MEPS), and QuEChERS. The protocols and data summarized herein are designed to enable researchers to achieve high recovery, superior selectivity, and enhanced sensitivity in the analysis of contaminants in environmental, food, and biological samples.

Workflow Parameter Optimization: A Data-Centric View

Optimizing workflow parameters is a critical step in developing a robust multi-sorbent extraction method. The following table synthesizes optimized parameters from recent research for various extraction techniques, providing a quantitative foundation for method development.

Table 1: Optimized Workflow Parameters for Sorbent-Based Extraction Techniques

Extraction Technique	Target Analytes	Optimal Sorbent(s)	Optimal Solvent & Elution Conditions	Optimal Flow Rates, Volumes & Cycles	Key Performance Metrics
Dual-Sorbent Dynamic Headspace (DHS) [71]	Wine volatiles	Tenax TA & Carbopack B/X	Desorption at high GC inlet temperature	Purge Flow: 16.0 mL/minPurge Volume: 344.3 mLIncubation Temp: 54 °CIncubation Time: 20.18 min	LOD: 0.2–2.0 µg/LLOQ: 0.5–5.0 µg/L
Microextraction by Packed Sorbent (MEPS) [72]	Benznidazole in plasma	C18	Elution Solvent: Acetonitrile (3 x 50 µL)	Sample Volume: 500 µLDraw-Eject Cycles: 10Sample Volume Drawn: 100 µL	Recovery: ~25%Linearity: 0.5–6.0 µg/mL
Solid-Phase Extraction (SPE) [73]	Basic drugs (e.g., atenolol) in plasma	Strata-X-C (Strong Cation Exchange)	Elution Solvent: 5% Ammonium Hydroxide in MethanolWash Solvent: 100% Methanol	Automated protocol; volumes proportional to well-plate format.	Recovery: ~98%Minimal matrix effects
QuEChERS-dSPE [4]	90 Emerging Contaminants in soil/sediment	C18 and/or PSA for clean-up	Extraction Solvent: Acetonitrile with bufferingElution: Solvent exchange for LC-MS	Sample Weight: Not SpecifiedClean-up: d-SPE	Mean Recovery: 70-120%RSD: < 20%MLOQs: 0.25–10 µg/kg
d-SPE Clean-up [74]	35 Mycotoxins in cereals	C18 end-capped	Initial Extraction: Acetonitrile/WaterAnalysis: UPLC-MS/MS with 0.05% formic acid in water/methanol	Not Specified	High recoveries and repeatability per EU regulations

Detailed Experimental Protocols

Protocol: Optimization of Dual-Sorbent Dynamic Headspace (DHS) for Wine Volatiles

This protocol is adapted from the multivariate optimization of a DHS method for the analysis of volatile organic compounds in botrytized wines [71].

1. Principle: Dynamic Headspace Extraction utilizes a continuous flow of inert gas to transfer volatile analytes from a sample onto one or more sorbent traps. The use of two sorbents with different properties (e.g., Tenax TA and graphitized carbon blacks) expands the range of capturable volatiles based on polarity and volatility [71].

2. Reagents and Materials:

• Sorbent Tubes: Tenax TA and Carbopack B/Carbopack X.
• Helium or Nitrogen (carrier gas).
• Internal Standard Solution: e.g., Benzophenone in methanol.
• NaCl (anhydrous).
• Wine samples.

3. Equipment:

• GC-MS system.
• Dynamic Headspace Sampler (e.g., capable of dual-sorbent trapping).
• Central Composite Design (CCD) software for optimization.

4. Step-by-Step Procedure: 1. Sample Preparation: Transfer a measured volume of wine (e.g., 5 mL) into a DHS vial. Add NaCl to saturation to reduce solubility of volatiles. 2. Instrument Setup: Load the dual sorbent trap (Tenax TA and Carbopack B/X) in the DHS unit. 3. Multivariate Optimization: Use a Central Composite Design (CCD) to optimize four key parameters simultaneously: - Incubation Temperature (e.g., 30–70 °C) - Incubation Time (e.g., 10–30 min) - Purge Volume (e.g., 100–500 mL) - Purge Flow Rate (e.g., 10–20 mL/min) 4. DHS Extraction: - Incubate the sample at the optimized temperature (e.g., 54 °C) for the optimized time (e.g., 20.18 min). - Purge the sample with inert gas at the optimized flow (e.g., 16.0 mL/min) and total volume (e.g., 344.3 mL). Volatiles are trapped on the sorbents. 5. Desorption and Analysis: Thermally desorb the trapped volatiles directly into the GC-MS for separation and quantification.

5. Data Assessment:

• Analyze results from the CCD using Pareto charts and response surface methodology to identify statistically significant parameters and their optimal values [71].

Protocol: MEPS for the Determination of Benznidazole in Plasma

This protocol details the extraction of a pharmaceutical compound from a biological matrix using a miniaturized, green approach [72].

1. Principle: Microextraction by Packed Sorbent is a miniaturized form of SPE where a small amount of sorbent (1-2 mg) is packed inside a syringe barrel or needle. The sample is repeatedly aspirated and dispensed over the sorbent to maximize analyte retention, followed by a wash and elution step [75] [72].

2. Reagents and Materials:

• MEPS BIN (Barrel Insert and Needle) packed with C18 sorbent (e.g., from SGE).
• Benznidazole analytical standard.
• Human plasma samples.
• Acetonitrile (HPLC grade).
• Water (HPLC grade).

3. Equipment:

• HPLC-UV or LC-MS system.
• 250 µL syringe compatible with MEPS BIN.

4. Step-by-Step Procedure: 1. Conditioning: Activate the C18 sorbent by aspirating and dispensing 100 µL of acetonitrile, followed by 100 µL of water. Do not let the sorbent dry out. 2. Sample Loading: - Acidify the plasma sample appropriately. - Draw 500 µL of sample into the syringe. - Slowly aspirate and eject the sample through the sorbent for a defined number of cycles (e.g., 10 cycles) at a controlled flow rate to retain the analyte. 3. Washing: Remove weakly bound matrix interferences by passing 100 µL of a weak wash solution (e.g., 5% methanol in water) through the sorbent. 4. Elution: Recover the analyte by aspirating and dispensing a strong elution solvent (e.g., 3 x 50 µL of acetonitrile) into a clean vial. 5. Analysis: Inject the eluate directly into the HPLC-UV system for analysis.

5. Method Optimization:

• A 2⁴ full factorial design should be employed to optimize critical parameters: sample volume, number of draw-eject cycles, sample volume drawn per cycle, and elution volume/scheme [72].

Protocol: Systematic SPE Method Development for LC-MS Analysis

This protocol outlines a streamlined, two-step approach for developing SPE methods that are directly compatible with LC-MS, even when using 100% organic, basified elution solvents [73].

1. Principle: This method uses a structured screening process to rapidly identify the optimal sorbent and solvent conditions for extracting basic drugs from plasma, leveraging modern pH-stable LC columns to allow direct injection of strong eluents [73].

2. Reagents and Materials:

• Strata Method Development Plates (multisorbent plates containing Strata-X, X-C, X-CW, X-AW).
• Single-sorbent Strata-X-C 96-well plates.
• Plasma samples.
• Ammonium hydroxide, formic acid, acetic acid, methanol, acetonitrile.
• pH-stable HPLC column (e.g., Gemini NX C18).

3. Equipment:

• Automated liquid handler.
• LC-MS/MS system.

4. Step-by-Step Procedure: Step 1: Sorbent and Condition Screening 1. Condition the multisorbent plate with methanol and then water. 2. Load plasma samples diluted with 0.1 M acetic acid under three different condition sets: - NN (Neutral Load/Neutral Wash): Load and wash with water, elute with methanol. - AB (Acidic Load/Basic Elute): Load and wash-1 with pH 2.5 ammonium formate buffer, wash-2 with 70-100% methanol, elute with 5% NH₄OH in methanol. - BA (Basic Load/Acidic Elute): Load with pH 5.5 ammonium acetate, elute with 2% formic acid in methanol. 3. Analyze eluates to determine which sorbent/condition pair yields the best recovery.

Logical Workflow for Multi-Sorbent Method Development

The following diagram illustrates a systematic, decision-based workflow for developing an optimized multi-sorbent extraction method, integrating principles from the cited protocols.

Diagram 1: A logical workflow for developing a multi-sorbent extraction method, highlighting iterative optimization and key decision points.

The Scientist's Toolkit: Research Reagent Solutions

Successful implementation of the protocols above requires carefully selected materials. The following table catalogs key reagents and their functions in sorbent-based extraction.

Table 2: Essential Research Reagents for Sorbent-Based Extraction Method Development

Reagent / Material	Function / Application	Examples from Literature
C18 Sorbent	Reversed-phase retention of non-polar to moderately polar analytes via hydrophobic interactions. Widely used in SPE, MEPS, and d-SPE.	MEPS for Benznidazole [72], d-SPE clean-up for mycotoxins [74].
Mixed-Mode Sorbents (e.g., Strata-X-C)	Combine reversed-phase and ion-exchange mechanisms. Allow strong organic washes to remove phospholipids before eluting basic analytes with basified organic solvent.	SPE of basic drugs from plasma [73].
Tenax TA & Graphitized Carbons	Porous polymer sorbents ideal for trapping a broad spectrum of volatile organic compounds (VOCs) in headspace and air sampling applications.	Dual-sorbent DHS for wine volatiles [71].
Primary Secondary Amine (PSA)	A d-SPE sorbent used to remove various polar interferences including fatty acids, sugars, and some pigments from food matrices.	QuEChERS for emerging contaminants [4].
Sustainable/Natural Sorbents	Eco-friendly alternatives such as cellulose-based materials, cork, or wood, used as efficient, low-cost sorbents in line with Green Analytical Chemistry.	Extraction of environmental, food, and biological samples [70].
Molecularly Imprinted Polymers (MIPs)	Synthetic polymers with tailor-made recognition sites for a specific analyte or class, offering high selectivity.	Selective sample preparation in food analysis [22].
Ammonium Hydroxide in Methanol	A strong, basic elution solvent used to disrupt ionic and hydrophobic interactions, particularly effective for eluting basic analytes from mixed-mode cation exchangers.	Elution in Strata-X-C SPE [73].
Deep Eutectic Solvents (DES)	Green, biodegradable solvents considered as alternatives to traditional organic solvents in extraction processes.	Green synthesis and extraction [70].

This application note provides a consolidated resource for optimizing the critical workflow parameters of solvent selection, flow rates, and elution conditions within a multi-sorbent extraction strategy. The synthesized data and detailed, experimentally-validated protocols demonstrate that a systematic approach—leveraging statistical experimental design and modern, sustainable materials—is key to developing robust, sensitive, and green analytical methods. By adhering to the workflows and protocols outlined herein, researchers and drug development professionals can effectively navigate the complexities of broad-spectrum contaminant analysis across diverse sample matrices.

Matrix effects represent a significant challenge in the chemical analysis of complex biological and environmental samples, often leading to inaccurate quantification, reduced sensitivity, and compromised data quality. These effects occur when co-extracted matrix components interfere with the detection or quantification of target analytes, either suppressing or enhancing their analytical signal [76] [77]. In the context of a broader thesis on multi-sorbent extraction strategies for broad-spectrum contaminant analysis, understanding and mitigating these effects is paramount for method development and validation.

The complexity of samples such as fatty tissues, plants, and blood arises from their diverse composition of lipids, proteins, carbohydrates, pigments, and other endogenous compounds that can co-extract with target analytes [76] [22]. For instance, in gas chromatography-mass spectrometry (GC-MS) analysis, matrix components can mask active sites in the GC system, leading to the matrix-induced enhancement effect, where analyte response is increased due to reduced adsorption or degradation [76]. Similarly, in liquid chromatography-tandem mass spectrometry (LC-MS/MS), matrix components can cause ion suppression or enhancement during ionization [69].

This application note provides a comprehensive overview of matrix effect management strategies, with specific protocols and data for analyzing complex samples. The focus is on practical approaches that can be implemented in routine laboratory workflows to improve the accuracy and reliability of analytical results.

Matrix Effects in Different Sample Types

Blood-Derived Matrices

Blood and its derivatives (plasma, serum, whole blood) present unique challenges due to their complex composition of cells, proteins, lipids, and metabolites. A 2025 study systematically evaluated fatty acid methyl ester (FAME) concentrations across different blood matrices, revealing significant variations in compound detectability and concentration [78].

Table 1: Comparison of FAME Concentrations in Blood Matrices

FAME Compound	Highest Concentration Matrix	Lowest Concentration Matrix	Relative Concentration Pattern
Undecanoate	Serum	Plasma	Serum > Whole Blood > Plasma
Pentadecanoate	Serum	Plasma	Serum > Whole Blood > Plasma
Linolenate	Serum	Plasma	Serum > Whole Blood > Plasma
Palmitate	Serum	Plasma	Serum > Whole Blood > Plasma
Stearate	Whole Blood	Plasma	Whole Blood > Serum > Plasma
Heptadecanoate	Whole Blood	Plasma	Whole Blood > Serum > Plasma
Myristate	Serum	Whole Blood	Serum > Plasma > Whole Blood
Dodecanoate	Plasma	Whole Blood	Plasma > Serum > Whole Blood
Docosahexaenoate	All Comparable	All Comparable	Plasma ≈ Serum ≈ Whole Blood

The study found that 9 out of 37 FAME compounds were detected in all three matrices (whole blood, serum, and plasma), with strong correlations observed between serum and plasma concentrations. However, whole blood FAME concentrations appeared significantly different, leading to the conclusion that plasma serves as the most ideal matrix for detection and quantification of circulating fatty acids [78]. This highlights the critical importance of matrix selection in study design.

Plant-Based Matrices

Plant materials introduce diverse matrix effects due to their varying contents of water, acids, starch, protein, and oils. A 2023 investigation into matrix effects during GC-MS/MS analysis of pesticide residues in four plant-based matrices revealed substantial differences in matrix effects across commodity groups [76].

Table 2: Matrix Effects in Plant Commodity Groups During Pesticide Analysis

Matrix Type	Commodity Group Characteristics	Strong Enhancement (%)	Strong Suppression (%)	Dominant Matrix Effect
Apples	High water content	73.9	<5	Enhancement
Grapes	High acid and water content	77.7	<5	Enhancement
Spelt Kernels	High starch/protein, low water and fat	<10	82.1	Suppression
Sunflower Seeds	High oil content, very low water	<15	65.2	Suppression

The study demonstrated that matrices with high water content (apples, grapes) predominantly exhibited signal enhancement, while those with high starch/protein or high oil content (spelt kernels, sunflower seeds) showed primarily signal suppression [76]. These findings underscore the need for matrix-specific method optimization and validation.

Fatty Tissues and Oils

Fatty tissues and oils present particular challenges due to their high lipid content, which can co-extract with target analytes and cause significant interference. The evaluation of lipid extraction methods for fatty acid quantification revealed that chloroform-based extraction methods generally outperformed those without chloroform for most sample types [79]. Direct fatty acid transmethylation protocols that omitted the lipid extraction step performed poorly, highlighting the importance of appropriate sample preparation in managing matrix effects.

The analysis of cold-pressed oils from 50 different plant materials demonstrated considerable variation in saturated fatty acids (7.87–36.04%), monounsaturated fatty acids (10.17–80.25%), and polyunsaturated fatty acids (non-detectable to 78.25%) [80]. This diversity in composition necessitates tailored approaches to matrix effect compensation.

Analytical Strategies for Matrix Effect Management

Multi-Sorbent Extraction Approaches

Multi-sorbent extraction strategies offer a powerful solution for managing matrix effects by selectively retaining either target analytes or interfering matrix components. A modular solid-phase extraction (SPE) setup combining three sorbents successfully extracted neutrals, acidic, and basic micropollutants from wastewater, with subsequent elution in three independent fractions [69].

This approach utilized:

• RP OASIS HLB: For reversed-phase retention of neutral compounds
• MCX (Mixed-mode Cation Exchange): For acids and basic compounds
• WAX (Weak Anion Exchange): For acidic compounds

The method achieved recoveries between 80-120% for 57-60 compounds in raw and treated wastewater, with procedural limits of quantification from 1-20 ng L⁻¹ [69]. The fractionation approach yielded cleaner extracts and reduced matrix effects compared to generic polymer sorbents.

Another innovative approach used a magnetic core-shell metal-organic framework (MOF) adsorbent for selective adsorption of interfering substances from wastewater samples prior to analyte extraction [77]. The magnetic responsiveness allowed for simple and rapid phase separation without centrifugation, followed by vortex-assisted liquid-liquid microextraction (VA-LLME) of the purified analytes.

Matrix-Matched Calibration

Matrix-matched calibration is a widely adopted strategy for compensating for matrix effects, particularly in GC-MS analysis [76]. This approach involves preparing calibration standards in blank matrix extracts that are free of the target analytes but contain the interfering matrix components.

A study on pesticide residues in plant materials demonstrated that although strong matrix effects were observed across all investigated matrices, the use of matrix-matched calibration for each sample type enabled satisfactory method performance, with recoveries for the majority of analytes within acceptable limits [76]. The approach was externally validated through proficiency testing with z-scores within the acceptable range of ≤ |2|.

Chemical Derivatization

Chemical derivatization can improve analyte stability, chromatographic behavior, and detection sensitivity while simultaneously reducing matrix effects. For phenolic compounds, acylation with acetic anhydride in the presence of sodium carbonate converts hydrophilic phenols into less polar derivatives with better extraction efficiency and chromatographic performance [77].

This approach addressed the challenges of broad peaks and poor chromatographic behavior caused by hydrogen bonding of phenolic hydroxyl groups with GC stationary phases. The derivatization method, combined with matrix cleanup, enabled accurate quantification of phenolic pollutants at trace levels in complex wastewater samples [77].

Detailed Experimental Protocols

Protocol 1: Multi-Sorbent SPE for Broad-Spectrum Contaminant Analysis

This protocol describes a modular SPE approach for the extraction of neutral, acidic, and basic compounds from complex aqueous matrices, adapted from recent research [69].

Materials:

• RP OASIS HLB cartridges (60 mg)
• Mixed-mode MCX cartridges (150 mg)
• Mixed-mode WAX cartridges (150 mg)
• Methanol (LC-MS grade)
• Acetonitrile (LC-MS grade)
• Formic acid (LC-MS grade)
• Ammonia solution (7 M in methanol)
• Ammonium fluoride
• Ultrapure water (18.2 MΩ cm⁻¹)

Procedure:

• Sample Pre-treatment: Centrifuge wastewater samples at 7000 × g for 10 minutes to remove particulate matter. Adjust pH to 7.0 ± 0.5.
• SPE Cartridge Assembly: Connect the HLB, MCX, and WAX cartridges in series, with HLB at the top and WAX at the bottom.
• Conditioning: Condition the cartridge assembly with 5 mL methanol followed by 5 mL ultrapure water at a flow rate of 2-3 mL/min.
• Sample Loading: Load 100 mL of sample through the cartridge assembly at a flow rate of 3-5 mL/min.
• Washing: Wash with 5 mL of 5% methanol in water.
• Fractionated Elution:
  • Neutral Fraction: Elute with 5 mL methanol and collect.
  • Acidic Fraction: Elute MCX and WAX cartridges with 5 mL methanol containing 2% formic acid.
  • Basic Fraction: Elute MCX and WAX cartridges with 5 mL methanol containing 2% ammonia.
• Concentration: Evaporate each fraction to near dryness under a gentle nitrogen stream at 40°C.
• Reconstitution: Reconstitute in 1 mL of initial mobile phase for LC-MS/MS analysis.

Quality Control:

• Process procedural blanks with each batch to monitor contamination.
• Use internal standards to correct for volume variations and recovery losses.
• Validate recovery for each compound class using spiked samples.

Protocol 2: GC-MS Analysis of FAMEs in Blood Matrices

This protocol details the analysis of fatty acid methyl esters in blood-derived matrices, optimized from recent methodology [78].

Materials:

• Methanolic KOH (0.5 M)
• n-Hexane (GC grade)
• Fatty acid methyl ester standards (RESTEK Food Industry FAME Mix)
• Anhydrous sodium sulfate
• GC-MS system with RT-2560 capillary column (100 m × 0.25 mm ID × 0.20 μm)

Sample Preparation:

• Alkali Trans-esterification: Add 300 μL of blood, plasma, or serum to 3 mL of methanolic KOH in a glass tube.
• Incubation: Heat at 50°C for 30 minutes with occasional vortexing.
• Extraction: Add 4 mL n-hexane, vortex for 2 minutes, and centrifuge at 7000 × g for 7 minutes.
• Collection: Transfer the hexane (upper) layer to a clean vial.
• Drying: Add anhydrous sodium sulfate to remove residual water.
• Concentration: Evaporate to approximately 500 μL under nitrogen stream.

GC-MS Parameters:

• Injector Temperature: 250°C (split mode, split ratio 4:1)
• Carrier Gas: Helium, constant flow 1.02 mL/min
• Oven Program:
  • Initial temperature: 40°C held for 2 min
  • Ramp: 4°C/min to 240°C
  • Hold: 15 min at 240°C
  • Total run time: 67 min
• Ion Source Temperature: 200°C
• Interface Temperature: 250°C
• Detection Mode: Selective ion monitoring

Quantification:

• Prepare calibration curve using serial dilutions of FAME standard mix
• Use internal standardization for quantification
• Apply matrix-matched calibration for each blood matrix type

Visualization of Workflows

Multi-Sorbent Extraction Strategy

Multi-Sorbent Extraction Workflow

Matrix Effect Management Strategies

Matrix Effect Management Strategies

The Scientist's Toolkit: Essential Research Reagents

Table 3: Essential Reagents for Managing Matrix Effects

Reagent/Sorbent	Function	Application Examples
OASIS HLB	Hydrophilic-lipophilic balanced polymer for broad-spectrum retention	Extraction of neutral, acidic, and basic compounds from water samples [69]
Mixed-mode MCX	Cation exchange + reversed-phase for acids and bases	Fractionation of basic compounds from complex matrices [69]
Mixed-mode WAX	Anion exchange + reversed-phase for acidic compounds	Fractionation of acidic compounds from complex matrices [69]
Primary Secondary Amine (PSA)	Removal of fatty acids, sugars, and organic acids	Clean-up in QuEChERS method for pesticide analysis [76]
Graphitized Carbon Black (GCB)	Removal of pigments and sterols	Clean-up of plant extracts containing chlorophyll and carotenoids [76]
Magnetic Core-Shell MOFs	Selective adsorption of matrix interferences	Matrix cleanup prior to phenolic compound extraction [77]
Methanolic KOH	Alkali trans-esterification catalyst	Conversion of fatty acids to FAMEs for GC analysis [78]
Acetic Anhydride	Acylation reagent for derivatization	Derivatization of phenolic compounds for improved GC behavior [77]

Effective management of matrix effects is essential for accurate analytical measurement in complex samples such as fatty tissues, plants, and blood. The strategies outlined in this application note, including multi-sorbent extraction, matrix-matched calibration, and chemical derivatization, provide robust approaches for mitigating these effects. The detailed protocols and comparative data presented here offer practical guidance for researchers developing analytical methods for broad-spectrum contaminant analysis. Implementation of these approaches will enhance data quality and reliability in chemical analysis across diverse sample types.

Validating Methods and Comparing Sorbent Performance

The demand for comprehensive broad-spectrum contaminant analysis is rapidly increasing in fields such as environmental monitoring, food safety, and pharmaceutical development. Establishing robust analytical figures of merit—recovery, limit of detection (LOD), limit of quantification (LOQ), and linearity—is fundamental to validating any analytical method, ensuring its reliability, accuracy, and precision [81]. These parameters are particularly crucial when developing multi-sorbent extraction strategies designed to isolate a wide range of analytes with diverse physicochemical properties from complex matrices.

Such strategies are essential for moving beyond single-contaminant analysis toward efficient multi-residue methods. This protocol outlines the establishment of these critical analytical figures of merit within the context of a broader research thesis on multi-sorbent extraction for broad-spectrum contaminant analysis, providing detailed application notes for researchers and scientists.

Core Definitions and Regulatory Importance

Key Figures of Merit

• Recovery: Expresses the efficiency of an extraction and clean-up process, calculated as the percentage of a known amount of analyte added to a sample that is measured after extraction. High recovery (ideally 70-120% with precision RSD <20%) indicates minimal analyte loss and low matrix effects [81] [82].
• Limit of Detection (LOD): The lowest concentration of an analyte that can be reliably detected by the method, though not necessarily quantified with acceptable precision. It is typically defined as a signal-to-noise ratio of 3:1 [81].
• Limit of Quantification (LOQ): The lowest concentration of an analyte that can be quantified with acceptable levels of precision and accuracy. It is typically defined as a signal-to-noise ratio of 10:1 or as the lowest point on a validated calibration curve [81].
• Linearity: The ability of the method to obtain test results that are directly proportional to the concentration of the analyte within a given range. It is demonstrated via a calibration curve, and the goodness-of-fit is expressed by the correlation coefficient (R²), which should be >0.99 [82] [67].

The Multi-Sorbent Strategy Context

Traditional single-sorbent extraction can struggle with the broad-spectrum analysis of contaminants with varying polarities and chemical structures. A multi-sorbent strategy leverages the complementary selectivity of different sorbents to achieve wider analyte coverage and superior sample clean-up [82]. For instance, a method for 34 endocrine-disrupting compounds utilized a dual-cartridge SPE approach combining a reversed-phase sorbent for hydrophobic compounds and a weak anion-exchange sorbent for polar, ionizable analytes [82]. Establishing reliable figures of merit for each analyte within such a complex method is paramount for its successful application.

Experimental Protocols for Determination

Protocol for Determining Recovery

• Preparation of Fortified Samples: Prepare a series of blank samples (matrix free of the target analytes). Fortify these samples with known concentrations of the analyte(s) of interest across the expected concentration range (low, mid, high).
• Extraction and Analysis: Process the fortified samples through the entire multi-sorbent extraction and analytical method (e.g., UHPLC-MS/MS).
• Calculation: For each concentration level, calculate the recovery (%) using the formula:
  • Recovery (%) = (Measured Concentration / Fortified Concentration) × 100
• Validation: Determine the mean recovery and the relative standard deviation (RSD%) of replicate measurements (n ≥ 5). The results should meet predefined criteria (e.g., 70-120% recovery with RSD < 20%) [83] [81].

Protocols for Determining LOD and LOQ

Several approaches can be used; the laboratory fortified blank is often considered the most reliable [81].

• Approach 1: Signal-to-Noise Ratio (SN)
  • Procedure: Analyze low-concentration samples and measure the signal response of the analyte (S) and the background noise (N) from a blank sample.
  • Calculation:
    • LOD = Concentration giving (S/N) ≥ 3
    • LOQ = Concentration giving (S/N) ≥ 10
• Approach 2: Calibration Curve Slope (CCS)
  • Procedure: Generate a calibration curve in the low concentration range. The standard deviation of the response (σ) can be estimated from the standard error of the regression.
  • Calculation:
    • LOD = 3.3 × (σ / S)
    • LOQ = 10 × (σ / S)
    • where S is the slope of the calibration curve.
• Approach 3: Laboratory Fortified Blank (LFB)
  • Procedure: Prepare and analyze replicate blank samples (n ≥ 7) fortified with a very low concentration of the analyte.
  • Calculation:
    • LOD = t-value × Standard Deviation of the fortified replicates
    • where the t-value is based on n-1 degrees of freedom (e.g., 3.14 for 7 replicates).
    • LOQ = 3.3 × LOD (or a multiple defined by the laboratory's quality assurance plan) [81].

Protocol for Establishing Linearity

• Calibration Standards: Prepare a series of calibration standards at a minimum of five concentration levels, spanning the expected range in real samples.
• Analysis and Plotting: Analyze the standards and plot the instrument response against the known concentration of the analyte.
• Statistical Analysis: Perform linear regression analysis on the data. The correlation coefficient (R²) should be >0.99 to demonstrate acceptable linearity [82] [67]. The residuals should be randomly scattered, indicating a good fit.

Data Presentation and Analysis

The following tables summarize experimental data from recent studies employing sorbent-based extraction for the determination of various contaminants, illustrating typical performance characteristics for these figures of merit.

Table 1: Analytical Figures of Merit for Mycotoxin Determination in Maize Using a Modified QuEChERS Method with C18 Sorbent and UHPLC-MS/MS [83]

Analyte Class	Number of Analytes	Recovery Range (%)	Precision (RSD%)	LOD (ng/g)	LOQ (ng/g)	Linearity (R²)
Regulated Mycotoxins	23	70 - 120	< 15%	< 21.10	< 37.49	> 0.99
Emerging Mycotoxins	(included in 23)	55.25 - 129.48	< 15.03%	Not specified	Not specified	> 0.99

Table 2: Performance Data for Pharmaceutical Analysis in Water Using Different Sorbent-Based Methods

Analytes	Matrix	Extraction Method	Recovery (%)	LOD	LOQ	Linearity (R²)	Source
5 Antibiotics (e.g., Tetracycline, Ampicillin)	Water	Cellulose-Composite SPE	84.8 - 97.6	0.03 - 2.07 ng/L	Not specified	> 0.99	[55]
34 EDCs (Pharmaceuticals, Plasticizers, Hormones)	River Water	Dual-Cartridge SPE	> 70% for all	0.1 - 50 ng/L	0.3 - 200 ng/L	> 0.99 for all	[82]
Carbamazepine, Caffeine, Ibuprofen	Water/Wastewater	SPE (evaporation step omitted)	77 - 160	100 - 300 ng/L	300 - 1000 ng/L	≥ 0.999	[67]

Workflow and Strategy Visualization

Multi-Sorbent Method Development Workflow

Interrelationship of Analytical Figures of Merit

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Multi-Sorbent Extraction and Method Validation

Item / Reagent	Function / Application	Example from Literature
Hydrophilic-Lipophilic Balance (HLB) Sorbent	A versatile polymeric sorbent for extracting a broad range of acidic, basic, and neutral compounds. Often used as a core sorbent in multi-sorbent strategies.	Oasis HLB and Oasis PRiME HLB are widely used for pharmaceuticals and contaminants in water and biological samples [84] [82].
C18 Bonded Silica	A reversed-phase sorbent for extracting non-polar to moderately polar compounds. Used for fractionation or as part of a mixed sorbent clean-up.	Used in QuEChERS for pesticide and mycotoxin analysis [22] [83].
Mixed-Mode Cation/Anion Exchange Sorbents	Provide both reversed-phase and ion-exchange interactions. Essential for selectively isolating ionizable analytes from complex matrices.	Oasis WAX (Weak Anion Exchange) used in dual-cartridge SPE for polar EDCs [82].
Molecularly Imprinted Polymers (MIPs)	"Smart sorbents" with high selectivity for a specific target molecule or class, improving recovery and reducing matrix effects.	Used for selective extraction of veterinary drugs, mycotoxins, and other contaminants from complex food and biological matrices [22] [84].
Dispersive SPE (d-SPE) Sorbents	Used for quick clean-up of sample extracts by removing matrix interferences like lipids, organic acids, and pigments.	Central to the QuEChERS method; sorbents like C18, PSA, and graphitized carbon black are common [22] [83].
Cellulose-Based Composite Sorbents	Low-cost, sustainable alternative to commercial polymeric sorbents. Can be chemically modified for enhanced performance.	C/PVPP/MDI composite showed excellent recovery for antibiotics in water, comparable to Oasis HLB [55].

Solid-phase extraction (SPE) remains a cornerstone technique in modern analytical laboratories for the purification, separation, and concentration of analytes from complex matrices. The fundamental principle governing SPE involves the differential affinity of analytes between a liquid sample and a solid sorbent phase [2]. While traditional single-phase sorbents have served as the workhorse for decades, a paradigm shift toward multi-sorbent strategies is emerging to address the challenge of analyzing compounds with diverse physicochemical properties in a single method [55]. This application note provides a comparative analysis of these approaches, framed within broader research on multi-sorbent strategies for broad-spectrum contaminant analysis, to guide researchers and drug development professionals in selecting and implementing optimal sample preparation protocols.

The evolution of SPE sorbents has progressed from basic silica-based phases (C18, C8, C2) to advanced materials including hydrophilic-lipophilic balance (HLB) polymers, molecularly imprinted polymers (MIPs), and metal-organic frameworks (MOFs) [7] [85]. This expansion of available sorbent chemistries enables the development of more sophisticated extraction systems capable of simultaneous isolation of multiple analyte classes, which is particularly valuable in pharmaceutical analysis and environmental monitoring where complex mixtures are commonplace.

Theoretical Background and Sorbent Principles

SPE techniques can be broadly classified into exhaustive methods (e.g., traditional SPE) and equilibrium methods (e.g., solid-phase microextraction, SPEM) [86]. Exhaustive methods aim for complete transfer of analytes to the sorbent phase, while equilibrium methods rely on partitioning between the sample and sorbent until equilibrium is established. The selection between single-phase and multi-sorbent approaches depends fundamentally on the scope of analysis, sample matrix, and target analytes.

Traditional single-phase sorbents operate primarily through a single dominant interaction mechanism. Reverse-phase sorbents (C18, C8, phenyl) retain analytes via hydrophobic interactions, while ion-exchange sorbents (SCX, SAX) utilize electrostatic forces, and normal-phase sorbents employ polar interactions [2]. The limitation of this approach emerges when analyzing complex samples containing analytes with varied polarity, acidity, and functional groups, as a single sorbent chemistry may not provide adequate retention for all compounds of interest [7].

Multi-sorbent strategies integrate multiple extraction mechanisms within a single system, either through physical mixing of sorbents, sequential cartridge arrangements, or use of hybrid materials with multifunctional properties. These approaches can provide complementary retention mechanisms, enabling broader analyte coverage and potentially reducing the number of separate sample preparation procedures required [55].

Table 1: Fundamental SPE Sorbent Classes and Their Properties

Sorbent Class	Primary Mechanisms	Typical Applications	Strengths	Limitations
Reverse Phase (C18, C8, C2)	Hydrophobic interactions	Moderate to non-polar compounds; biological fluids	Well-characterized, widely available	Poor retention of highly polar compounds
Ion Exchange (SCX, SAX)	Ionic interactions, electrostatic forces	Ionic or ionizable compounds; biological matrices	High selectivity for charged analytes	pH-dependent, requires control of ionic strength
Mixed-Mode	Multiple mechanisms (e.g., reverse phase + ion exchange)	Compounds with diverse properties; forensic toxicology	Broader spectrum retention	More complex method development
Hydrophilic-Lipophilic Balance (HLB)	Balanced polar and non-polar interactions	Broad spectrum; pharmaceutical contaminants	Retains both hydrophilic and lipophilic compounds	May require optimization for specific analytes
Molecularly Imprinted Polymers (MIPs)	Shape-selective cavities, chemical complementarity	Specific target compounds; complex matrices	High specificity, customizability	Limited to pre-defined analytes, synthesis complexity
Metal-Organic Frameworks (MOFs)	Coordination, π-π stacking, pore size selection	Trace contaminants; environmental samples	Ultra-high surface area, tunable functionality	Stability concerns in certain solvents

Comparative Experimental Analysis

Performance Evaluation of Single-Phase Sorbents

A comprehensive study evaluating 17 different single-phase sorbents for the extraction of 13 pharmaceutical compounds from human urine provides valuable insights into the performance variability among traditional SPE materials [7]. The research employed UHPLC-UV analysis with a Poroshell 120 EC-C18 column and acetonitrile −0.05% TFA in water mobile phase under gradient elution. Among the sorbents tested, phenyl (C6H5) phase demonstrated superior performance for this particular application, providing the most effective clean-up and analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%. The method validation showed linearity across 0.01–30.0 μg/mL range, with limits of detection at 0.003–0.217 μg/mL and precision of 0.8–7.1% [7].

Another comparative study investigated the extraction efficiency of inflammatory bowel disease drugs with varying polarities (logP 1.66-2.92) using different sorbent materials [86]. The research characterized sorbent performance through breakthrough volume, retention volume, hold-up volume, retention factor, and theoretical plate number. The results demonstrated significant variability in sorbent performance based on analyte properties, highlighting that no single sorbent universally excelled for all compounds.

Table 2: Quantitative Performance Comparison of Selected Sorbent Materials

Sorbent Material	Analyte Classes	Recovery Range (%)	Reproducibility (RSD%)	Limit of Detection Range	Key Advantages
Phenyl (C6H5)	13 diverse drugs [7]	>85.5%	<10%	0.003–0.217 μg/mL	Effective clean-up, high reproducibility
Cellulose Composite (C/PVPP/MDI)	5 antibiotics [55]	84.8–97.6%	Not specified	0.03–2.07 ng/L	Low-cost, reusable (5 cycles), broad-spectrum
HLB	5 antibiotics [55]	87.0–97.3%	Not specified	Comparable to cellulose composite	High recovery for broad compound classes
Urea-modified MOF	Clonazepam [85]	94.9–99.0%	1.4%	0.030 μg/L	High selectivity, excellent sensitivity
Sol-gel CW 20M	IBD drugs [86]	Variable by analyte	Not specified	Method-dependent	Enhanced polar compound retention

Multi-Sorbent and Advanced Material Approaches

Innovative sorbent materials demonstrate the potential for enhanced performance through integrated functionality. A low-cost cellulose-based composite (C/PVPP/MDI) was developed by cross-linking cellulose with poly(vinyl-polypyrrolidone) and 4,4-methylenebisphenyldiisocyanate, creating a dual-functionalized sorbent with both hydrophilic (PVPP) and hydrophobic (MDI) characteristics [55]. This material achieved antibiotic recoveries of 84.8–97.6% in water matrices, comparable to commercial HLB sorbents (87.0–97.3%), while offering cost reduction of approximately 50% and reusability for up to five cycles without significant performance loss [55].

Similarly, a urea-modified metal-organic framework (MIL-101(Fe)-Urea) was specifically designed for selective clonazepam extraction from water samples [85]. The functionalization with urea groups created tailored interaction sites, resulting in high recovery rates (94.9–99.0%), excellent linearity (R² = 0.997) across 20–1500 μg/L range, and remarkable precision (RSD = 1.4%). This approach demonstrates how strategic sorbent design can yield highly selective extraction materials for specific analyte classes [85].

Detailed Experimental Protocols

Protocol 1: Multi-Sorbent SPE for Broad-Spectrum Pharmaceutical Analysis

This protocol describes an approach for extracting analytes with diverse physicochemical properties using a multi-sorbent cartridge, adapted from methodologies for pharmaceutical compound extraction [7] [55].

Research Reagent Solutions:

• Sorbent Materials: Mixed-mode cartridge (e.g., C8/SAX) or layered sorbents (C18 + SCX)
• Extraction Solvents: Methanol, acetonitrile, acidified water (0.05% TFA)
• Conditioning Solution: 5 mL methanol followed by 5 mL acidified water (0.05% TFA)
• Wash Solution 1: 3 mL 5% methanol in water (remove weakly retained interferents)
• Wash Solution 2: 3 mL 2% formic acid in methanol (remove interfering bases)
• Elution Solution: 5 mL mixture of acetonitrile:methanol:ammonium hydroxide (80:15:5 v/v/v)

Procedure:

• Sample Pretreatment: Adjust sample pH to 7.0 ± 0.5. Centrifuge if particulate matter is present. For biological samples, protein precipitation may be required prior to SPE.
• Cartridge Conditioning: Pass 5 mL methanol through sorbent bed at 1 mL/min flow rate. Follow with 5 mL acidified water (0.05% TFA) without allowing the bed to dry.
• Sample Loading: Load prepared sample at controlled flow rate of 1-2 mL/min to ensure optimal analyte retention.
• Wash Steps: Pass 3 mL Wash Solution 1 through cartridge, followed by 3 mL Wash Solution 2. Dry cartridge under vacuum for 5-10 minutes after wash steps.
• Analyte Elution: Elute with 5 mL Elution Solution into a clean collection tube at reduced flow rate (0.5-1 mL/min).
• Sample Reconstitution: Evaporate eluent under gentle nitrogen stream at 40°C. Reconstitute dry residue in 100-500 μL mobile phase compatible with subsequent analysis.

Protocol 2: Cellulose-Based Composite SPE for Antibiotic Monitoring

This protocol utilizes a cost-effective, sustainable sorbent material for monitoring antibiotic contaminants in water samples, based on research demonstrating comparable performance to commercial HLB sorbents [55].

Research Reagent Solutions:

• Sorbent Material: C/PVPP/MDI composite sorbent (cellulose cross-linked with PVPP and MDI)
• Conditioning Solution: 5 mL methanol followed by 5 mL deionized water
• Elution Solution: 5 mL methanol with 2% acetic acid
• Storage Solution: Methanol:water (50:50 v/v)

Procedure:

• Sorbent Preparation: Synthesize C/PVPP/MDI composite by cross-linking cellulose with PVPP and MDI in dimethylformamide at 70°C for 6 hours [55].
• Cartridge Packing: Pack 200 mg of C/PVPP/MDI composite into empty SPE cartridges between two frits.
• Conditioning: Condition cartridge with 5 mL methanol followed by 5 mL deionized water at flow rate of 1-2 mL/min.
• Sample Loading: Load 100-500 mL water sample (pH adjusted to 7.0) through cartridge at 3-5 mL/min flow rate.
• Cartridge Drying: Apply vacuum for 10 minutes to dry sorbent bed completely.
• Analyte Elution: Elute antibiotics with 5 mL methanol with 2% acetic acid into collection tube.
• Concentration: Evaporate eluent to near-dryness under nitrogen stream at 40°C. Reconstitute in 1.0 mL mobile phase for analysis.
• Sorbent Regeneration: For reuse, wash cartridge with 5 mL methanol:water (50:50 v/v) and store in this solution at 4°C.

Troubleshooting Common SPE Challenges

Implementation of both single-phase and multi-sorbent SPE approaches can encounter technical challenges that affect method performance. The most common issues include poor recovery, lack of reproducibility, and impure extracts [87] [88].

Poor Recovery: When analyte recovery is suboptimal, systematic investigation is required to identify the point of analyte loss [88].

• Analyte present in loading fraction: Indicates insufficient retention. Potential solutions include adjusting sample pH to enhance analyte affinity for sorbent, diluting sample with weaker solvent, decreasing flow rate during loading, or selecting a sorbent with greater affinity.
• Analyte present in wash fraction: Suggests wash solvent is too strong. Decrease wash solvent strength or volume, and ensure complete cartridge drying before wash step.
• Analyte not fully eluting: Elution conditions may be inadequate. Increase elution solvent strength or volume, incorporate soak steps, decrease flow rate during elution, or consider changing to a less retentive sorbent.

Lack of Reproducibility: Inconsistent extractions often stem from procedural variability [88].

• Maintain consistent sample pre-treatment protocols
• Ensure proper cartridge conditioning without drying before sample loading
• Control flow rates throughout the process (typically 1 mL/min recommended)
• Use appropriate soak times (1-5 minutes) during critical steps
• Avoid cartridge overload by matching sample volume to sorbent capacity

Impure Extractions: When interfering compounds co-elute with target analytes [87] [88]:

• Optimize wash solvent strength to maximize impurity removal while retaining analytes
• Implement sample pre-treatment (e.g., pH adjustment, precipitation, filtration)
• Select sorbents with greater selectivity for target analytes
• Consider multi-sorbent approaches or mixed-mode cartridges for enhanced selectivity

The comparative analysis demonstrates that both traditional single-phase and multi-sorbent SPE approaches have distinct advantages depending on application requirements. Single-phase sorbents provide well-characterized, specific interactions suitable for targeted analysis of compounds with similar properties, with phenyl sorbents showing particular effectiveness for multiple drug classes [7]. Multi-sorbent strategies, whether through mixed-mode cartridges, layered sorbents, or advanced composite materials, offer expanded capabilities for broad-spectrum analysis of compounds with diverse physicochemical properties [55].

The emerging generation of functionalized sorbents, including MOFs with tailored functionalities [85] and cellulose-based composites [55], demonstrates the potential for enhanced selectivity and sustainability in sample preparation. These materials address key limitations of traditional sorbents while maintaining or improving analytical performance. The integration of multi-sorbent approaches with modern chromatographic techniques represents a powerful combination for comprehensive contaminant analysis in complex matrices.

For researchers engaged in broad-spectrum contaminant analysis, multi-sorbent strategies provide a flexible framework that can be adapted to specific analytical challenges. The protocols presented herein offer practical starting points for method development, while the troubleshooting guidelines address common implementation barriers. As SPE technologies continue to evolve, the principles of selective retention and efficient elution remain central to effective sample preparation across diverse application domains.

Within the framework of a multi-sorbent extraction strategy for broad-spectrum contaminant analysis, benchmarking the performance and cost-effectiveness of individual commercial sorbents is a critical foundational step. The selection of an appropriate sorbent directly governs the sensitivity, selectivity, and overall success of an analytical method, particularly when targeting diverse contaminant classes in complex matrices [20]. Modern analytical science demands protocols that are not only effective but also efficient, minimizing solvent consumption and sample volume while aligning with the principles of Green Analytical Chemistry (GAC) [89]. This application note provides detailed methodologies and standardized benchmarks to facilitate the direct comparison of commercial sorbents, enabling researchers to make informed decisions for developing robust multi-sorbent workflows in drug development and environmental analysis.

Sorbent Performance Benchmarking Data

A critical first step in designing a multi-sorbent strategy is understanding the performance characteristics of individual materials. The following tables summarize key performance metrics for commercial sorbents in different application contexts, providing a basis for initial selection.

Table 1: Performance Benchmarks for Specialty Application Sorbents

Sorbent / Technology	Key Performance Metrics	Application Context
eLivate DLE Sorbent [90]	>98% Li recovery; >95% impurity rejection; 10,000 cycles with <5% degradation; Lifespan: 5-8 years.	Direct Lithium Extraction (DLE) from brines.
High-Performance Mercury Sorbents [91]	85-95% mercury removal efficiency; Target: >90% removal for regulatory compliance.	Mercury emission control in industrial flue gas.
Carbon-Based Mercury Sorbents [91]	Dominant 64.2% market share by material type.	Cost-effective mercury capture in flue gas treatment.

Table 2: Quantitative Market and Cost Projections for High-Performance Mercury Sorbents (2025-2035) [91]

Metric	Value
Market Value (2025)	USD 349.8 Million
Market Forecast Value (2035)	USD 517.8 Million
Forecast CAGR (2025-2035)	4.0%
Leading Material Type (2025)	Carbon-based Material (64.2% share)
Leading Application (2025)	Mercury Removal from Industrial Flue Gas (56.8% share)

Experimental Protocols for Sorbent Benchmarking

Protocol: Batch Adsorption for Capacity and Efficiency

This protocol is designed to determine the fundamental adsorption capacity and contaminant removal efficiency of a sorbent for a specific analyte.

• 1. Objective: To quantify the adsorption capacity (Qe) and removal efficiency (%E) of a commercial sorbent for a target contaminant under controlled conditions.
• 2. Materials:
  • Stock solution of target contaminant (e.g., an organophosphorus pesticide in solvent).
  • Commercial sorbent under test.
  • Orbital shaker or end-over-end mixer.
  • Centrifuge and microcentrifuge tubes.
  • Analytical instrument for quantification (e.g., GC-MS, LC-MS/MS).
• 3. Procedure:
  • Prepare a series of concentrations of the contaminant stock solution.
  • Precisely weigh a fixed mass of sorbent (e.g., 10 ± 0.1 mg) into each microcentrifuge tube.
  • Add a fixed volume (e.g., 1 mL) of each contaminant solution to the tubes. Include a blank (solvent only with sorbent) and a control (contaminant solution without sorbent).
  • Seal the tubes and agitate for a predetermined time (e.g., 60 minutes) at a constant temperature to reach adsorption equilibrium.
  • Centrifuge the tubes to separate the sorbent.
  • Analyze the supernatant to determine the equilibrium concentration (Ce) of the contaminant.
• 4. Data Analysis:
  • Removal Efficiency (%E): %E = [(C₀ - Cₑ) / C₀] × 100, where C₀ is the initial concentration.
  • Adsorption Capacity (Qe, mg/g): Qₑ = [(C₀ - Cₑ) × V] / m, where V is the solution volume (L) and m is the sorbent mass (g).

Protocol: Microextraction by Packed Sorbent (MEPS) for Analytical Sample Preparation

MEPS is a miniaturized, efficient sample preparation technique ideal for evaluating sorbent performance in a format that aligns with modern analytical workflows [89].

• 1. Objective: To implement and benchmark the performance of a sorbent packed in an MEPS syringe for the extraction of trace contaminants from a complex liquid sample.
• 2. Materials:
  • MEPS syringe (e.g., 100-500 µL capacity) packed with 0.5 - 10 mg of the sorbent under test [89].
  • Sample (e.g., biological fluid, water).
  • Conditioning solvent, wash solvent, and elution solvent.
  • HPLC or GC system coupled with MS for analysis.
• 3. Procedure: [89]
  • Conditioning: Pass 2-3 volumes of conditioning solvent (e.g., methanol) through the sorbent bed, followed by 2-3 volumes of the sample's solvent (e.g., water) to activate the sorbent.
  • Sample Loading: Draw and dispense the sample solution through the sorbent bed slowly for 5-10 cycles to allow analyte adsorption.
  • Washing: Pass a small volume (e.g., 50-100 µL) of a wash solvent to remove weakly adsorbed matrix interferences.
  • Elution: Analyze the sample by drawing a small volume (e.g., 20-100 µL) of a strong elution solvent and dispensing it into an autosampler vial for analysis. The elution can be performed in one step or multiple steps.
  • Reconditioning: The same sorbent bed can be re-used for multiple extractions (up to 100 or more depending on the matrix) after a cleaning and reconditioning step [89].
• 4. Data Analysis: Calculate key performance indicators including extraction recovery, precision (%RSD), and detection limits to compare different sorbents.

Workflow Visualization: Multi-Sorbent Strategy Screening

The following diagram outlines a logical workflow for the systematic benchmarking of commercial sorbents, informing the development of a comprehensive multi-sorbent strategy.

Sorbent Benchmarking and Integration Workflow

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for Sorbent Benchmarking

Item	Function / Application
C18-Bonded Silica Sorbent	Reversed-phase extraction for non-polar to medium-polarity analytes; a benchmark for lipophilic contaminants.
Mixed-Mode Sorbents (e.g., MCX, WAX)	Combine reversed-phase and ion-exchange mechanisms for broad-spectrum extraction of acidic, basic, and neutral compounds [20].
Polymeric Sorbents (e.g., HLB)	Hydrophilic-Lipophilic Balanced copolymers for a wide log P range; effective without pre-conditioning and resilient to run dry [20].
Molecularly Imprinted Polymers (MIPs)	Synthetic sorbents with high selectivity for pre-defined target analytes, reducing matrix effects [20].
Carbon-Based Sorbents (e.g., Activated Carbon)	High surface area for efficient capture of small molecules and metals (e.g., mercury); cost-effective [91].
Microextraction by Packed Sorbent (MEPS) Syringe	A miniaturized, reusable device for solid-phase extraction that integrates sample preparation with analytical instrumentation, minimizing solvent and sample consumption [89].
Selective Elution Solvents	Solvents of varying strength and pH (e.g., Methanol, Acetonitrile, Ammonium Hydroxide) used to selectively desorb different contaminant classes from mixed-mode sorbents.

The Role of LC-MS/MS and HRMS in Confirming Selectivity and Sensitivity

The demand for robust analytical methods capable of detecting trace-level contaminants and drugs in complex matrices has never been greater. Within this landscape, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (HRMS) have emerged as pivotal technologies. The core of their utility lies in two fundamental analytical figures of merit: selectivity, the ability to distinguish the target analyte from interfering matrix components, and sensitivity, which defines the lowest concentration of an analyte that can be reliably measured [92] [93]. The strategic choice between LC-MS/MS and HRMS platforms significantly influences the reliability, scope, and efficiency of broad-spectrum contaminant analysis, particularly when coupled with advanced sample preparation techniques like multi-sorbent extraction.

This application note delineates the distinct and complementary roles of these platforms, providing a structured comparison and detailed protocols to guide researchers in selecting and validating the appropriate technology for their specific needs within multi-residue analytical frameworks.

Comparative Performance of LC-MS/MS and HRMS

A critical evaluation of LC-MS/MS and HRMS reveals a nuanced trade-off between sensitivity, selectivity, and the analytical scope, which must be carefully balanced based on application requirements.

Selectivity: The Resolving Power of HRMS

Selectivity is paramount in minimizing false positives and false negatives. HRMS provides superior selectivity through its ability to perform exact mass measurement at high resolution. A comprehensive study comparing selectivity demonstrated that HRMS, operated at a resolution of 50,000 FWHM (full width at half maximum), exceeded the selectivity provided by LC-MS/MS [92]. In one instance, an LC-MS/MS analysis of a honey sample produced a false positive for a banned nitroimidazole drug, where an endogenous matrix compound perfectly matched the retention time and two MRM transitions of the drug. However, HRMS clearly resolved the interfering matrix compound from the analyte, unmasking the false finding [92].

Low-resolution MS instruments, such as linear ion traps, can struggle with co-eluting interferences. As shown in a comparison of platforms for zeranol analysis, concomitant ions unresolved in a unit mass window were mistakenly included in quantitative analyses by low-resolution instruments. In contrast, HRMS platforms (Orbitrap and ToF) differentiated between an analyte mass of 319.1551 and an interfering peak at 319.1915, which would have been concealed within the same nominal mass window in a low-resolution scan [94].

Table 1: Comparison of Selectivity in Resolving Interferences

Analytical Scenario	LC-MS/MS Performance	HRMS Performance	Key Finding
Nitroimidazole in Honey	False positive due to matching retention time and MRM ratios [92]	Interfering matrix compound resolved, false positive uncovered [92]	HRMS provides higher selectivity to avoid false positives
Zeranol Analysis in Urine	Concomitant ions unresolved within unit mass window [94]	Exact mass differentiated analyte (319.1551) from interference (319.1915) [94]	High resolution and mass accuracy prevent integration of interfering peaks

Sensitivity and Instrument Performance

Sensitivity, often defined by the lower limit of quantification (LLOQ), is a key driver in method development, especially for quantifying endogenous compounds at ultra-trace levels.

Direct comparisons between platforms show that sensitivity can be compound and instrument-dependent. In the analysis of zeranols, the Orbitrap demonstrated the highest sensitivity (lowest LODs and LOQs), followed by the linear ion traps, with the time-of-flight (ToF) instrument showing the highest variation [94]. Conversely, in the quantification of large molecules like peptides, a head-to-head comparison revealed that a HRMS instrument used in a targeted "quant/quant" mode showed equivalent or better sensitivity for all six model peptides tested compared to a standard triple quadrupole [95]. This demonstrates that the historical sensitivity gap for HRMS in quantitative analysis has closed.

Exemplifying high sensitivity, a validated LC-MS/MS method for oxytocin in plasma achieved an impressive LLOQ of 1 ng/L, which is essential for measuring the peptide's low endogenous levels [96]. Similarly, a LC-HRMS method for cannabinoids in human plasma achieved an LLOQ of 0.2 ng/mL for all analytes, matching the sensitivity of a previously reported LC-MS/MS method but with the added selectivity benefits of exact mass [97].

Table 2: Analytical Sensitivity Achieved in Recent Applications

Analyte	Matrix	Technique	LLOQ	Citation
Oxytocin	Plasma	LC-MS/MS	1 ng/L [96]
Cannabinoids	Plasma	LC-HRMS	0.2 ng/mL [97]
78 Cardiovascular Drugs	Oral Fluid	LC-HRMS/MS	Compound-dependent LOI [98]
Zeranols	Urine	Orbitrap HRMS	Highest sensitivity vs. other platforms [94]

Essential Method Validation Characteristics

For any quantitative LC-MS method, rigorous validation is required to ensure data reliability. The following eight characteristics are essential, whether using LC-MS/MS or HRMS [93]:

• Accuracy: The closeness of the measured value to the true value. Assessed by comparing the measured concentration in a quality control (QC) sample to its known concentration.
• Precision: The degree of agreement between repeated measurements of the same sample. Evaluated by calculating the coefficient of variation (%CV) across replicates.
• Specificity: The ability to unequivocally assess the analyte in the presence of other components like matrix, metabolites, or degradants. For LC-MS/MS, this is confirmed via MRM transitions; for HRMS, via exact mass measurement [93] [97].
• Quantification Limit: The lowest concentration that can be quantified with acceptable accuracy and precision (LLOQ).
• Linearity: The ability of the method to elicit results that are directly proportional to analyte concentration across a defined range.
• Recovery: The efficiency of the sample preparation/extraction process, determined by comparing the analyte response after extraction to that of a non-extracted standard.
• Matrix Effect: The suppression or enhancement of analyte ionization by co-eluting matrix components. It is evaluated by comparing the analyte response in a post-extraction spiked matrix to its response in a pure solution [96] [93].
• Stability: The integrity of the analyte under specific conditions (e.g., during storage, processing, in the autosampler) must be demonstrated.

Experimental Protocols for Broad-Spectrum Analysis

The integration of a multi-sorbent extraction strategy with LC-MS analysis is critical for successful broad-spectrum contaminant analysis. The following protocol outlines a comprehensive workflow.

Sample Preparation: Multi-Sorbent Solid Phase Extraction (SPE)

Principle: Multi-sorbent SPE utilizes a combination of sorbents with complementary chemical properties (e.g., reversed-phase, ion-exchange) to achieve a broad-range extraction of analytes with diverse physicochemical properties from a complex matrix [8].

Materials:

• Oasis HLB sorbent: A hydrophilic-lipophilic balanced copolymer for retaining a wide range of acidic, basic, and neutral compounds.
• ISOLUTE ENV+ sorbent: A modified styrene-divinylbenzene polymer designed for extracting polar compounds from water.
• Strata WAX (Weak Anion Exchange) & WCX (Weak Cation Exchange): Mixed-mode sorbents for selective clean-up and extraction of acids and bases, respectively [8].
• SPE vacuum manifold.
• LC-MS grade solvents: Methanol, acetonitrile, water.
• Ammonium hydroxide and formic acid (for pH adjustment).

Procedure:

• Conditioning: Sequentially condition the multi-sorbent SPE plate/column with 3-5 mL of methanol followed by 3-5 mL of water or an appropriate buffer. Do not let the sorbent dry out.
• Loading: Acidify or basify the sample supernatant (e.g., plasma, urine, tissue extract) as required to optimize analyte retention. Load the sample onto the SPE cartridge at a controlled, slow flow rate (1-3 mL/min).
• Washing: Remove weakly retained matrix interferences with 2-3 mL of a weak solvent. A common wash is 5% methanol in water, optionally containing a low percentage of acid or base to remove specific interferences.
• Elution: Elute the retained analytes into a clean collection tube using 2-4 mL of a strong organic solvent or solvent mixture. Typical eluents include pure methanol, acetonitrile, or a mixture with a modifier (e.g., 5% ammonium hydroxide in methanol for basic compounds, or 5% formic acid in methanol for acidic compounds).
• Reconstitution: Evaporate the eluate to dryness under a gentle stream of nitrogen at 40°C. Reconstitute the dry residue in 100-200 µL of initial mobile phase compatible with the subsequent LC-MS analysis, vortex mix thoroughly, and centrifuge before transfer to an autosampler vial.

LC-HRMS Analysis for Untargeted Screening

Instrumentation: UHPLC system coupled to a Q-Orbitrap or Q-TOF mass spectrometer.

Chromatographic Conditions:

• Column: C18 reverse-phase column (e.g., 100 x 2.1 mm, 1.7 µm).
• Mobile Phase A: Water with 0.1% formic acid.
• Mobile Phase B: Acetonitrile with 0.1% formic acid.
• Gradient: 5% B to 95% B over 10-15 minutes, hold for 2 minutes, re-equilibrate.
• Flow Rate: 0.4 mL/min.
• Injection Volume: 5-10 µL.

Mass Spectrometric Conditions:

• Ionization Mode: Electrospray Ionization (ESI), positive and/or negative polarity switching.
• Resolution: ≥ 50,000 FWHM (at m/z 200) [92].
• Scan Range: m/z 100-1000.
• Source Parameters: Capillary voltage (3.5 kV), sheath gas, auxiliary gas, and heater temperature optimized for the specific instrument and flow rate.
• Data Acquisition: Full-scan MS (for quantification and exact mass) with data-dependent MS/MS (dd-MS2) for confirmatory fragmentation.

LC-MS/MS Analysis for Targeted Quantification

Instrumentation: UHPLC system coupled to a triple quadrupole (QqQ) mass spectrometer.

Chromatographic Conditions:

• Similar to the LC-HRMS method, but can be optimized for faster cycle times.

Mass Spectrometric Conditions:

• Ionization Mode: ESI, positive or negative mode.
• Data Acquisition: Multiple Reaction Monitoring (MRM).
• Optimization: For each target analyte, the precursor ion and two specific product ions are defined. Dwell times and collision energies for each transition are optimized for maximum sensitivity.
• Scheduled MRM can be employed to monitor each MRM trace in a narrow retention time window, allowing for the simultaneous quantification of hundreds of analytes in a single run [92].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials for Multi-Sorbent Extraction and LC-MS Analysis

Item	Function/Application	Example from Literature
Oasis HLB Sorbent	Broad-spectrum retention of acidic, basic, and neutral compounds; core sorbent in multi-strategy approaches [8].	Used in combination with other sorbents for comprehensive extraction [8].
Strata WAX & WCX	Mixed-mode ion-exchange sorbents for selective clean-up of acidic and basic analytes, respectively, reducing matrix effects [8].	Implemented in multi-sorbent strategies to enhance selectivity [8].
Stable Isotope-Labeled Internal Standards (SIL-IS)	Compensates for analyte loss during sample preparation and corrects for matrix suppression/enhancement during ionization [96].	Oxytocin-d5 used for oxytocin quantification [96].
Phosphate Buffered Saline (PBS) with 0.1% BSA	Serves as a surrogate matrix for preparing calibration standards when the biological matrix contains endogenous levels of the analyte [96].	Used to create calibration curves for endogenous oxytocin in plasma [96].
LC-MS Grade Solvents	High-purity solvents minimize background noise and ion suppression, crucial for achieving high sensitivity.	Used in all protocols for mobile phase and sample preparation [96] [97].

Workflow Visualization

The following diagram illustrates the integrated decision-making process for method development, from sample preparation to the strategic choice of detection platform, based on the analytical goals.

Both LC-MS/MS and HRMS are powerful platforms for confirmatory analysis, each with distinct strengths. LC-MS/MS on triple quadrupole instruments remains the gold standard for high-sensitivity, high-throughput targeted quantification of a predefined set of analytes, offering robust performance and a wide dynamic range. In contrast, HRMS on Orbitrap or Q-TOF instruments provides superior selectivity through exact mass measurement, effectively minimizing false positives and is uniquely suited for untargeted screening, metabolite identification, and broad-scope residue analysis.

The integration of a multi-sorbent extraction strategy is a critical enabler for both techniques, effectively preparing complex samples for broad-spectrum analysis. The choice between LC-MS/MS and HRMS should be guided by the specific analytical objectives: LC-MS/MS for focused, quantitative tasks where speed and sensitivity are paramount, and HRMS for exploratory research and methods requiring the highest level of analytical confidence and comprehensiveness.

Assessing Reusability and Green Metrics for Sustainable Method Development

The integration of green chemistry principles into analytical methodologies is paramount for advancing sustainable scientific practices. This application note delineates a robust framework for assessing the reusability and environmental impact of sorbent-based extraction techniques, situating this evaluation within a broader multi-sorbent extraction strategy for broad-spectrum contaminant analysis. The paradigm has shifted from traditional metrics focused solely on analytical performance to a holistic view that harmonizes greenness with practical utility, a concept often referred to as White Analytical Chemistry (WAC) [99] [65]. This approach is essential for developing methods that are not only effective but also environmentally responsible and economically viable, particularly when dealing with complex matrices such as environmental, food, and biological samples [99] [100] [20].

Sample preparation is frequently the most critical and environmentally impactful stage of the analytical workflow, often consuming over two-thirds of the total analysis time and generating significant waste [99] [100]. The emergence of miniaturized, sorbent-based techniques presents a viable alternative to conventional methods like Liquid-Liquid Extraction (LLE) and Solid-Phase Extraction (SPE), which are characterized by high consumption of hazardous solvents, energy, and time [99] [20]. This document provides a detailed protocol for applying a dual-tool evaluation strategy—using the Complementary Modified Green Analytical Procedure Index (ComplexMoGAPI) for environmental impact and the Blue Applicability Grade Index (BAGI) for practical utility—to benchmark and optimize sustainable method development [99].

Greenness and Practicality Assessment Framework

Key Evaluation Metrics

A comprehensive assessment requires metrics that quantitatively evaluate both environmental footprint and practical application. The following two tools are designed to be used complementarily.

• ComplexMoGAPI: This metric is an advanced version of the Green Analytical Procedure Index (GAPI). It provides a visual diagram that scores the greenness of an analytical method across multiple stages, including sample collection, storage, transportation, preparation, and analysis. A key differentiator of ComplexMoGAPI is its consideration of pre-analytical steps, such as the synthesis of sorbent materials, which contribute significantly to the overall environmental footprint [99]. The output is a colored pictogram where green indicates low environmental impact, yellow moderate, and red a significant impact [99] [101].

• BAGI (Blue Applicability Grade Index): This tool complements greenness metrics by quantitatively assessing the practicality of an analytical method. It evaluates critical parameters such as analysis time, cost, operational complexity, sample throughput, energy consumption, and analytical performance (e.g., accuracy, detection limits) [99]. A higher BAGI score indicates a more user-friendly, efficient, and economically feasible method, ensuring that green methods are also practically viable for routine laboratories [99].

The Assessment Protocol

The following protocol offers a step-by-step guide for applying the dual-tool framework to evaluate sample preparation methods.

Protocol Title: Dual-Tool Assessment of Reusability and Sustainability in Sorbent-Based Extraction Methods

1. Method Selection and Definition:

• Identify the sorbent-based extraction method to be evaluated (e.g., FPSE, CPME, DSPE using biomass-based sorbents).
• Clearly define the entire analytical procedure, from sample collection to final instrumental analysis.
• Document all parameters, including sample volume, sorbent type and mass, solvents (type and volume), energy consumption, extraction time, and instrument settings.

2. Sorbent Reusability Testing:

• Procedure: After the initial extraction and analyte desorption, regenerate the sorbent according to the established protocol (e.g., washing with an appropriate solvent). Re-use the same sorbent device for multiple extraction-desorption cycles using a freshly spiked sample.
• Data Collection: For each cycle, calculate the analyte recovery percentage. Monitor for a significant drop (e.g., >15%) in recovery or signs of physical degradation in the sorbent.
• Output: Determine the maximum number of uses without significant performance loss. This data is a critical input for both greenness (waste reduction) and practicality (cost-effectiveness) evaluations [100] [19].

3. Application of ComplexMoGAPI:

• Procedure: Input the documented method parameters into the ComplexMoGAPI framework. This involves evaluating each of the 15+ criteria covering reagents, waste, energy, instrumentation, and operator safety [99] [101].
• Output: Generate the final ComplexMoGAPI pictogram and an overall greenness score. The diagram provides an immediate visual summary of the method's environmental performance across its entire lifecycle.

4. Application of BAGI:

• Procedure: Score the method against the BAGI criteria, which include figures of merit, practical and economic aspects, and safety parameters [99].
• Output: Calculate a final BAGI score on a standardized scale. A high score confirms that the green method maintains high analytical performance and is readily applicable in a real-world laboratory setting.

5. Data Interpretation and Strategy Optimization:

• Compare the ComplexMoGAPI and BAGI scores against benchmark methods or target values.
• Identify specific areas with poor scores (e.g., high solvent use in ComplexMoGAPI or long analysis time in BAGI) as targets for optimization in the multi-sorbent strategy.
• Iteratively refine the method (e.g., by switching to a greener solvent, reducing steps, or choosing a more robust sorbent) and re-run the assessment until an optimal balance between greenness and practicality is achieved.

The logical relationship between the assessment components and the overarching goal of sustainable method development is visualized below.

Diagram 1: Workflow for Sustainable Method Assessment. This diagram outlines the iterative process of using reusability data, ComplexMoGAPI, and BAGI to develop an optimal method.

Application to Sorbent-Based Extraction Techniques

The following tables and protocols detail the application of this framework to specific, modern sorbent-based extraction techniques.

Fabric Phase Sorptive Extraction (FPSE)

FPSE utilizes a fabric substrate coated with a sol-gel-derived sorbent, combining the equilibrium extraction of SPME with the exhaustive extraction principles of SPE. Its key advantage is the ability to handle complex samples directly with minimal pretreatment [99] [65].

Experimental Protocol for FPSE:

• Membrane Preparation: Cut the sol-gel coated FPSE membrane to the required size (e.g., 2 cm²).
• Activation (Conditioning): Immerse the membrane in a suitable solvent (e.g., methanol) for a brief period (e.g., 5 minutes), then condition in the sample matrix solvent or water for another 5 minutes.
• Extraction: Place the activated FPSE membrane directly into the sample solution (10-50 mL). Stir for a predetermined extraction time (15-60 minutes) to facilitate mass transfer of analytes to the sorbent.
• Rinsing: Remove the membrane and briefly rinse with ultrapure water to remove loosely adsorbed matrix components. Gently blot dry.
• Desorption (Back-Extraction): Immerse the membrane in a small volume (0.5-2 mL) of a desorption solvent (e.g., acidified methanol, acetonitrile) in a vial. Stir or sonicate for 5-15 minutes to release the analytes.
• Analysis: Recover the desorption solvent. It can be directly injected into an LC or GC system, or evaporated and reconstituted in an instrument-compatible solvent.
• Reusability: The membrane is regenerated by washing with the desorption solvent and then a conditioning solvent, ready for the next use. Studies show FPSE membranes can be reused over 20 times without significant loss in performance [99].

Capsule Phase Microextraction (CPME)

CPME encapsulates a sol-gel sorbent within a porous polypropylene capsule that houses a magnetic stirrer. This design integrates sample stirring with filtration, allowing for direct analysis of samples with particulate matter [99] [65].

Experimental Protocol for CPME:

• Capsule Activation: Prime the Microextraction Capsule (MEC) by passing ~1 mL of methanol or acetonitrile through it, followed by an equivalent volume of water.
• Loading and Extraction: Place the MEC into a vial containing the sample. The capsule serves as a stir bar, mixing the solution for a set extraction time (e.g., 20-90 minutes) while the analytes are simultaneously extracted.
• Washing: Remove the MEC, rinse externally with water, and dry with a kimwipe.
• Elution: Transfer the MEC to a new vial. Pass a small volume (100-500 µL) of a strong organic solvent (e.g., methanol with 1% formic acid) through the capsule to elute the adsorbed analytes. This can be done using a syringe or gentle pressure.
• Analysis: The eluent is collected and analyzed directly.
• Reusability: The MEC is regenerated by repeating the activation step. CPME devices are known for their high chemical and mechanical stability, often exceeding 30 extractions per capsule [99].

Biomass-Based Sorbents in Dispersive (Micro) Solid-Phase Extraction

These sorbents, derived from agricultural waste (e.g., coconut shell, rice husk) or other natural materials, are celebrated for their sustainability, low cost, and unique surface chemistries [100] [19].

Experimental Protocol for DSPE using Biomass-Based Sorbents:

• Sorbent Preparation: Synthesize and characterize the biomass-based sorbent (e.g., biochar, activated carbon). Ground and sieve to a specific particle size.
• Dispersion: Weigh a small amount (5-50 mg) of the sorbent and add it directly to the sample solution (e.g., water).
• Extraction: Vortex or stir the mixture vigorously for a set time to ensure good dispersion and contact between the sorbent and analytes.
• Separation: Centrifuge the sample to pellet the sorbent. If using magnetic composites (MSPE), collect the sorbent with an external magnet.
• Washing: Discard the supernatant. The sorbent pellet may be washed with a small volume of water to remove co-adsorbed interferences.
• Desorption: Add a small volume of organic solvent (e.g., 1 mL acetone) to the sorbent, then vortex or sonicate to desorb the analytes.
• Analysis: Separate the solvent from the sorbent (via centrifugation or magnetism) and analyze.
• Reusability: The sorbent can be regenerated by washing and drying, though performance may vary depending on the biomass material and analyte [100].

Table 1: Comparison of Modern Sorbent-Based Extraction Techniques

Technique	Key Characteristics	Typical Reusability (Cycles)	Key Greenness Advantages	Key Practicality Advantages
Fabric Phase Sorptive Extraction (FPSE) [99] [65]	Sol-gel sorbent bonded to a flexible fabric substrate; direct immersion extraction.	>20	Minimal solvent consumption; no sample pretreatment; reusable.	Simplified workflow; handles complex matrices; high chemical stability.
Capsule Phase Microextraction (CPME) [99] [65]	Sol-gel sorbent encapsulated in a stirring membrane capsule.	>30	Integrated stirring/filtration reduces steps; very low solvent volume.	High-throughput potential; excellent batch-to-batch reproducibility; easy to operate.
Biomass-Based Sorbents (DSPE/MSPE) [100] [19]	Sorbents from renewable/waste sources (e.g., biochar); used in dispersion mode.	5-15 (varies)	Utilizes waste materials; biodegradable; very low cost.	Highly tunable surface chemistry; effective for diverse contaminants.

Table 2: Greenness and Practicality Scores for Representative Methods

Analytical Method	ComplexMoGAPI Score (Greenness)	BAGI Score (Practicality)	Primary Strengths	Primary Limitations
Traditional SPE [99]	Low (Significant red/yellow sectors)	Moderate (High consumption & cost)	Exhaustive extraction; well-established.	High solvent use; multi-step; single-use cartridges.
FPSE for Forensic Analytes in Blood [99]	High (Predominantly green)	High	Excellent green profile; robust for complex matrices.	Requires custom fabrication of membranes.
CPME for Pesticides in Water [99]	High (Predominantly green)	High	Superior practicality; integrates multiple steps.	---
DSPE with Biochar for Water Pollutants [100] [19]	High (Predominantly green)	Moderate to High	Outstanding green credentials; very low cost.	Reusability can be limited; potential for analyte carryover.

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Materials for Sustainable Method Development

Item	Function/Description	Application Note
Sol-Gel Sorbents [99] [65]	Organic-inorganic hybrid polymers chemically bonded to a substrate. Provide high pH stability, porosity, and extraction efficiency for a wide range of analytes.	Core coating technology for FPSE and CPME; enables custom selectivity and reusability.
Biomass-Based Sorbents (Biochar) [100] [19]	Carbon-rich materials produced from pyrolysis of agricultural waste (e.g., coconut shells, wood chips). Offer high surface area and tunable surface functional groups.	Sustainable, low-cost alternative to synthetic sorbents in DSPE/MSPE; ideal for pollutant removal and analysis.
Green Desorption Solvents [99] [101]	Solvents with favorable environmental, health, and safety profiles (e.g., ethanol, ethyl acetate, acetone). Used to elute analytes from the sorbent.	Critical for reducing the overall environmental impact of the method; minimizes hazardous waste.
ComplexMoGAPI & BAGI Software/Templates [99] [101]	Metric tools for the standardized evaluation of method greenness and practicality.	Essential for quantitative assessment and justification of a method's sustainability claims.

The systematic assessment of reusability and green metrics is no longer optional but a critical component of modern analytical method development. The application of the dual-tool framework comprising ComplexMoGAPI and BAGI provides an objective and comprehensive strategy to benchmark and optimize sorbent-based extraction techniques. As demonstrated, methods like FPSE, CPME, and those employing biomass-based sorbents consistently score highly on both greenness and practicality scales, validating their role in a sustainable multi-sorbent strategy for broad-spectrum contaminant analysis. By adopting this rigorous assessment protocol, researchers and drug development professionals can make informed decisions that align analytical excellence with environmental stewardship and economic feasibility.

Conclusion

Multi-sorbent extraction strategies represent a paradigm shift in sample preparation, effectively addressing the critical need for broad-spectrum contaminant analysis in increasingly complex samples. By intelligently combining sorbents with complementary retention mechanisms, these methods achieve superior clean-up and higher analyte recoveries for a diverse chemical space than is possible with single-phase extractions. The integration of novel materials like MIPs and the adoption of miniaturized, automated formats enhance both selectivity and analytical throughput. Future progress will be driven by the development of even more selective 'smart' sorbents, the deeper integration of machine learning for method optimization and data analysis, and a stronger focus on green, cost-effective solutions to make advanced analytical techniques accessible worldwide. For biomedical and clinical research, these advances promise more reliable therapeutic drug monitoring, robust toxicological screening, and a greater capacity to characterize the human exposome.

References

• 1. Sorbent-based extraction procedures [https://www.sciencedirect.com/science/article/pii/B9780443161223000111]
• 2. A review of the modern principles and applications of solid ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9659506/]
• 3. Application of Sorbent-Based Extraction Techniques in ... [https://www.mdpi.com/1420-3049/28/24/7985]
• 4. Optimization and validation of an extraction method for the ... [https://www.sciencedirect.com/science/article/abs/pii/S0021967323005125]
• 5. Embedding Mixed Sorbents in Binder: Solid-Phase ... [https://pubmed.ncbi.nlm.nih.gov/38127806/]
• 6. Comparison of Different d-SPE Sorbent Performances ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8588138/]
• 7. Comparison of different sorbent materials for solid-phase ... [https://www.sciencedirect.com/science/article/abs/pii/S1570023214001858]
• 8. Integrating non-target analysis and machine learning [https://www.nature.com/articles/s41545-025-00504-z]
• 9. A wide range of toxic VOCs measured by dual-sorbent ... [https://www.sciencedirect.com/science/article/abs/pii/S0269749122014154]
• 10. Comparative study of the adsorption performance of a multi ... [https://www.sciencedirect.com/science/article/abs/pii/S0039914010000512]
• 11. Practical Application of a Multi-Bed Adsorbent Tube ... [https://www.mdpi.com/2073-4433/13/9/1457]
• 12. status and future Current on automated multidimensional... trends [https://scispace.com/papers/current-status-and-future-trends-on-automated-3m2b7f2u9y]
• 13. HLB Index in Surfactants – Meaning and Practical ... [https://vichem.vn/en/hlb-index-in-surfactants-meaning-and-practical-applications/]
• 14. HLB - what is it? Hydrophilic-lipophilic balance [https://www.products.pcc.eu/en/blog/hydrophilic-lipophilic-balance-hlb/]
• 15. HLB- A Deeper Look + Application [https://www.yourcosmeticchemist.com/post/hlb-a-deeper-look-application]
• 16. Development of a multi-residue analytical method, based ... [https://pubmed.ncbi.nlm.nih.gov/20832069/]
• 17. How to Purify Your Protein With Ion Exchange ... [https://www.goldbio.com/blogs/articles/develop-the-best-protocol-for-protein-purification-with-ion-exchange-chromatography-iex?srsltid=AfmBOoqGrI3p7upjQw8yj6wuh6b9lkbSe8PJIau5vO54QCGCp9MGL2rd]
• 18. Sample Preparation in Ion Exchange Chromatography [https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-purification/appendix1?srsltid=AfmBOopQt4VZ0qkEInFSXBccAbOxvyb0deGPUDbv0FpGblfH09fkj9mf]
• 19. Greenness of extraction procedures involving biochar ... [https://www.sciencedirect.com/science/article/pii/S2772582025000567]
• 20. Analytical Extraction Methods and Sorbents' Development ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8464751/]
• 21. Dual sorbent coating based magnet-integrated fabric ... [https://www.sciencedirect.com/science/article/pii/S0048969723059806]
• 22. Application of Sorbent-Based Extraction Techniques in Food ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10745519/]
• 23. Advancements in Microextraction by Packed Sorbent [https://www.mdpi.com/2297-8739/12/1/11]
• 24. Recent Insights into Sample Pretreatment Methods for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10053610/]
• 25. Performance comparison of MOF and other sorbent ... [https://www.nature.com/articles/srep31283]
• 26. Universal PFAS Sorbent Performance Comparison | ADE Group [https://ade.group/universal-pfas-sorbent-performance-comparison/]
• 27. Analysis of the effect of artificial aging on the phase ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12310116/]
• 28. Recent advances in dental zirconia: 15 years of material ... [https://www.sciencedirect.com/science/article/abs/pii/S0109564124000447]
• 29. Paving the way towards green contaminant analysis [https://www.sciencedirect.com/science/article/pii/S2772577425000187]
• 30. Aptamer-functionalized Metal-organic frameworks (MOFs) for biosensing - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC7855766/]
• 31. Next-Generation Sorbent-Based Extractions with Metal ... [https://www.chromatographyonline.com/view/next-generation-sorbent-based-extractions-with-metal-organic-frameworks]
• 32. Development of a dispersive micro solid phase extraction ... [https://www.sciencedirect.com/science/article/abs/pii/S0026265X24005678]
• 33. Recent Advances in Affinity MOF-Based Sorbents with ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7571043/]
• 34. Adsorbents Market 2025: Artificial Intelligence Revolution & ... [https://www.linkedin.com/pulse/adsorbents-market-2025-artificial-intelligence-revolution-8efcf/]
• 35. The Open DAC 2025 Dataset for Sorbent Discovery in ... [https://arxiv.org/html/2508.03162v1]
• 36. A review of the modern principles and applications of solid ... [https://link.springer.com/article/10.1007/s44211-022-00190-8]
• 37. Miniaturized solid-phase extraction techniques in sample ... [https://www.sciencedirect.com/science/article/pii/S0026265X25011488]
• 38. 3 Reasons to Switch to a Tip-based Sample Preparation ... [https://dpxtechnologies.com/3-reasons-to-switch-to-a-tip-based-sample-preparation-method/]
• 39. Advantages of Solid Phase Extraction - Hawach - SPE Cartridge [https://www.specartridge.com/advantages-of-solid-phase-extraction/]
• 40. Solid Phase Extraction SPE Disk in the Real World: 5 Uses ... [https://www.linkedin.com/pulse/solid-phase-extraction-spe-disk-real-world-5-uses-tpjvc/]
• 41. Advanced Topics in Solid-Phase Extraction: Chemistries [https://www.chromatographyonline.com/view/advanced-topics-solid-phase-extraction-chemistries-1]
• 42. An improved and validated solid-phase extraction ... [https://www.sciencedirect.com/science/article/pii/S0026265X25028103]
• 43. Versatile sorbent materials in sample preparation [https://www.sciencedirect.com/science/article/pii/S2772582025000634]
• 44. A fully automated, high-throughput electro-extraction and ... [https://www.sciencedirect.com/science/article/pii/S000326702500618X]
• 45. Waste-derived sorbents in analytical extraction processes [https://www.sciencedirect.com/science/article/abs/pii/S0165993625002973]
• 46. HPLC Update Day 2: Modern Method Development [https://www.chromatographyonline.com/view/hplc-update-day-2-modern-method-development]
• 47. Polystyrene foam-based pipette-tip Micro-solid phase ... [https://www.sciencedirect.com/science/article/abs/pii/S0308814625023520]
• 48. A Dual-Column Solid Phase Extraction Strategy for Online ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3518407/]
• 49. Recent trends in sampling and sorbent-based ... [https://www.sciencedirect.com/science/article/pii/S0026265X24020150]
• 50. Comparison between 5 extractions methods in either ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10200056/]
• 51. Assessment of human plasma and urine sample preparation ... [https://pubs.rsc.org/en/content/articlehtml/2020/an/d0an01319f]
• 52. Guidelines for preventing and reporting contamination in ... [https://www.nature.com/articles/s41564-025-02035-2]
• 53. A fast bioanalytical method based on microextraction by ... [https://pubmed.ncbi.nlm.nih.gov/28738608/]
• 54. Ten years of monitoring pharmaceuticals and pesticides in ... [https://www.sciencedirect.com/science/article/abs/pii/S0269749124022139]
• 55. A low-cost cellulose-based composite as an efficient solid ... [https://pubs.rsc.org/en/content/articlehtml/2025/ra/d5ra02296g]
• 56. A comprehensive review of emerging environmental ... [https://link.springer.com/article/10.1007/s44274-025-00259-x]
• 57. Simultaneous analysis of multi-class antibiotics in bottled ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12091094/]
• 58. Quantifying Antibiotic Distribution in Solid and Liquid ... [https://www.mdpi.com/2079-6382/11/12/1735]
• 59. Multiresidue antibiotic-metabolite quantification method ... [https://link.springer.com/article/10.1007/s00216-021-03573-4]
• 60. Comprehensive review of emerging contaminants [https://www.sciencedirect.com/science/article/pii/S0147651324004962]
• 61. Sorbent-based sampling methods for volatile and semi-volatile organic compounds in air. Part 2. Sorbent selection and other aspects of optimizing air monitoring methods - PubMed [https://pubmed.ncbi.nlm.nih.gov/20106482/]
• 62. Sorbent-based sampling methods for volatile and semi-volatile organic compounds in air. Part 2. Sorbent selection and other aspects of optimizing air monitoring methods - ScienceDirect [https://www.sciencedirect.com/science/article/abs/pii/S002196731000052X]
• 63. How to Select a Sorbent | Products [https://www.glsciences.com/product/spe_columns/about_a_spe/01043.html]
• 64. Specialty Sorbents 2025 Market Trends and 2033 Forecasts [https://www.marketreportanalytics.com/reports/specialty-sorbents-58714]
• 65. Novel Sorptive Sample Preparation Techniques for ... [https://www.chromatographyonline.com/view/novel-sorptive-sample-preparation-techniques-for-separation-science]
• 66. Solid-phase extraction: method development, sorbents ... [https://www.sciencedirect.com/science/article/abs/pii/S0021967399008328]
• 67. Development and validation of a green/blue UHPLC-MS ... [https://www.nature.com/articles/s41598-025-15614-4]
• 68. Overview of Liquid Sample Preparation Techniques for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11477957/]
• 69. Solid-phase extraction and fractionation of multiclass ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9124662/]
• 70. Sustainable materials for sorptive extraction techniques [https://www.sciencedirect.com/science/article/pii/S0165993625003541]
• 71. Multivariate optimization of dual-sorbent dynamic ... [https://www.sciencedirect.com/science/article/abs/pii/S0308814621014552]
• 72. Microextraction by packed sorbent and high-performance ... [https://www.sciencedirect.com/science/article/abs/pii/S1570023223000508]
• 73. A Simplified Approach to Optimize SPE Method ... [https://www.chromatographyonline.com/view/simplified-approach-optimize-spe-method-development-downstream-lc-ms-analysis-allowing-100-organic-b]
• 74. Optimization, Validation, and Application of Cleanup ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11594120/]
• 75. Application of microextraction by packed sorbent to ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3281198/]
• 76. Matrix Effect Evaluation in GC/MS-MS Analysis of Multiple ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10650748/]
• 77. Matrix cleanup assisted extraction of phenolic pollutants ... [https://www.nature.com/articles/s41598-025-24462-1]
• 78. Assessment of Fatty Acid Concentrations Among Blood ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12298658/]
• 79. Evaluation of lipid extraction methods for fatty acid ... [https://www.sciencedirect.com/science/article/pii/S0003269723004207]
• 80. GC-MS and HPLC-DAD analysis of fatty acid profile ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7907780/]
• 81. Limit of detection and limit of quantification development ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3630005/]
• 82. Simultaneous extraction and determination of 34 multiclass ... [https://www.sciencedirect.com/science/article/abs/pii/S0026265X25012263]
• 83. Comprehensive assessment of different extraction ... [https://www.sciencedirect.com/science/article/pii/S2772753X22001332]
• 84. Innovative Solid-Phase Extraction Strategies for Improving the... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11124106/]
• 85. Highly selective solid phase extraction of clonazepam from ... [https://www.nature.com/articles/s41598-025-07127-x]
• 86. Comparison between Exhaustive and Equilibrium ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6385024/]
• 87. Three Most Common Problems Regard to Solid Phase ... [https://www.hawach.com/news/three-most-common-problems-regard-to-spe.html]
• 88. How to Solve Common Challenges in Solid-Phase Extraction [https://www.silicycle.com/articles/how-to-solve-common-challenges-in-solid-phase-extraction/]
• 89. Microextraction by packed sorbents (MEPS) [https://www.sciencedirect.com/science/article/abs/pii/S0165993624005260]
• 90. Summit Nanotech Validates Sorbent with... | Summit Nanotech [https://www.summitnanotech.com/post/industry-leading-sorbent-reliability]
• 91. High Performance Mercury Sorbent Market | Global Market Analysis... [https://www.futuremarketinsights.com/reports/high-performance-mercury-sorbent-market]
• 92. Comprehensive comparison of liquid chromatography ... [https://www.sciencedirect.com/science/article/abs/pii/S0003267010006136]
• 93. 8 Essential Characteristics of LC-MS/MS Method Validation [https://www.resolian.com/bioanalytics/8-essential-characteristics-of-lc-ms-ms-method-validation/]
• 94. A comparison of four liquid chromatography-mass ... - PubMed [https://pubmed.ncbi.nlm.nih.gov/37432442/]
• 95. Large molecule quantification: sensitivity and selectivity ... [https://www.bioanalysis-zone.com/spotlight-large-molecule-quantification-sensitivity-and-selectivity-head-to-head-comparison-of-triple-quadrupole-with-q-tof-2/]
• 96. Development and validation of a highly-sensitive ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11919332/]
• 97. Development and validation of a LC-HRMS method for the ... [https://www.sciencedirect.com/science/article/abs/pii/S0928098721000075]
• 98. Development and application of a LC-HRMS/MS method ... [https://pubmed.ncbi.nlm.nih.gov/31103169/]
• 99. Modern sorbent-based sample preparation methods for the ... [https://www.sciencedirect.com/science/article/abs/pii/S0165993624006125]
• 100. Recent advances in biomass-based sorbents for the ... [https://www.sciencedirect.com/science/article/abs/pii/S0165993625002456]
• 101. Greenness evaluation metric for analytical methods and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12318332/]