786 SAMPLE PREPARATION 1. Condition: 1-mL MeOH Cyano Bonded SPE Cartridge Silca Gel SPE Cartridge 2. Condition: 1-mL H 2 O 3. Load: 1-mL Plasma 4. Rinse: 1-mL H 2 O 5. Rinse: 1-mL ACN 6. Elute: 2-mL 99.5% MeOH + 0.5% NH 4 OAc 7. Collect & Evaporate to Dryness 8. Reconstitute: MeOH or Figure 16.5 Isolation of albuterol from human plasma by means of solid-phase extraction (SPE). columns that incorporate sample-preparation, and—unlike SPE cartridges—are characterized by high plate numbers and re-usability. RAM are most often selected for the analysis of low-molecular-weight drugs, their impurities, and metabolites [50–52]. Many variations of these packings have been described: (1) internal-surface reversed-phases, (2) shielded hydrophobic phases, (3) semi-permeable surfaces, (4) dual-zone phases, and (5) mixed functional phases. See [50] for a description and tabulation of commercial products. Dual-mode porous packings are used in the most popular RAM columns. These packings are used typically for the analysis of drugs in blood, because proteins present in these samples will accumulate on a RPC column—leading to its failure 16.6 SOLID-PHASE EXTRACTION (SPE) 787 after a few injections. The packing consists of small-pore particles with a C 8 or C 18 layer covering the inside of the pores, and a nonretentive hydrophilic layer covering the exterior of the particle. Proteins are unable to access the pores because of their large size, and are unretained by the particle exterior; consequently they pass through the column with k ≈ 0. Small-molecule analytes can enter the pores and are retained sufficiently to elute after the proteins (often using gradient elution). Because proteins are not retained on these columns, a column can be used for a considerable number of samples. However, some care must be exercised in the choice of mobile-phase pH and organic solvent; otherwise protein precipitation can occur—with fouling of the column. Alternatively, a RAM column can be connected via a switching valve to a conventional RPC column (two-dimensional separation; column-switching, Section 16.9). The valve is initially positioned for elution of the RAM column to waste, and the sample is injected; after the proteins leave the column, the valve is switched to connect the two columns. Analytes are then eluted from the RAM column and enter the RPC column for further processing, usually by means of gradient elution. The RPC column also has a longer lifetime, as plasma proteins never contact this column (e.g., [53]). 16.6.7.3 Molecular-Imprinted Polymers (MIPs) MIPs are among the most selective phases used in SPE, being designed for enhanced retention of a specific analyte. A MIP is a stable polymer with recognition sites that are adapted to the three-dimensional shape and functionalities of an analyte of interest (much like antibody binding). The most common approach involves noncovalent imprinting; this MIP synthesis is shown schematically in Figure 16.6. An analyte is used as a template, and is chemically coupled with a monomer (most often methacrylic acid or methacrylate; Fig. 16.6a,b). After polymerization (Fig. 16.6c), the bound analyte is cleaved to yield a selective binding site (receptor; Fig. 16.6d). The selective interactions between the analyte and the MIP include hydrogen bonding, ionic, and/or hydrophobic interactions. The action of a MIP is based on a ‘‘lock-and-key’’ fit, where a selective receptor or cavity on the surface of a polymer perfectly fits the analyte that was used to prepare the MIP. The concept is similar to immunoaffinity (IA) SPE phases (Section 16.6.7.4), but obtaining a suitable antibody for these IA sorbents can be very time-consuming. An introductory article [54] outlines the basics of MIP technology, while review articles [55–58] and a book [59] provide detailed information on the use and potential of MIPs in SPE. Incomplete removal of analyte template from the MIP during its preparation is one of the main problems. This residual analyte frequently bleeds, resulting in baseline drift and interference with the assay of the desired analyte—especially for low analyte concentrations. There may be some swelling or shrinkage of the MIP with a change in solvent, which can modify the size of the receptor and reduce the retention of the target analyte. A major disadvantage of the MIP approach is that each sorbent must be custom made, either by the user or an outside supplier. Because of the high cost of synthesizing a MIP, their use is restricted to high-volume assays or when there is no other way to perform sample cleanup. Recently several off-the-shelf MIPs have been commercialized (by MTP Technologies, Lund, Sweden) for: • clenbuterol in biological fluids 788 SAMPLE PREPARATION self-assembly wash rebinding + polymerization cross-linker analyte (template) monomers (a) (c) (b) (d ) Figure 16.6 Synthesis of molecular-imprinted polymer (MIP). (a) Analyte plus monomers; (b) formation of analyte-monomer complex; (c) analyte-polymer complex; (d) selective- binding site. • beta agonists, multi-residue extractions in urine and tissue samples • NNAL (4-methylnitrosamino-1-(3-pyridyl)-1-butanol), tobacco-specific nitrosamine in biological matrices • riboflavin (vitamin B2) in aqueous samples • triazines, multi-residue extraction in water, soil, and food products • chloramphenicol, antibiotic in biological matrices • beta blockers, multi-residue extractions in water and biological samples 16.6.7.4 Immunoaffinity Extraction of Small Molecules Immunoaffinity packings are based on antibodies that are attached to a particle. As in the case of MIPs, the analyte is retained by a receptor that is highly complementary, so as to provide a ‘‘lock-and-key’’ fit. As a result immunoaffinity packings are quite specific for an individual analyte, and are used for the selective extraction and concentration of individual compounds or classes of compounds from the sample—often in a one-step process. Antibodies for large biomolecules are readily available and have been used for many years in immunology and medical research (affinity chromatography). Because antibodies for small molecules are more difficult to obtain, the development of small-molecule immunoaffinity extraction is more recent and less developed. Some excellent review articles describe immunoaffinity extractions in more detail [60–64]. As long as an antibody can be prepared, the numbers of immunoaffinity packings can be almost unlimited. However, a great deal of time and effort is required in their production, so their use is restricted for the same reasons as for MIP packings (Section 16.6.7.3). Nevertheless, several commercial immunoaffinity 16.6 SOLID-PHASE EXTRACTION (SPE) 789 packings have become available. Class-specific packings are available for a variety of pharmaceutical, food, and environmental applications [65]. As an example of the use of an immunoaffinity packing, a procedure for the separation of four aflatoxins as a group has been reported [66]. 16.6.7.5 QuEChERS and Dispersive SPE QuEChERS ( Quick, Easy, Cheap, Effective, Rugged, and Safe) is an extraction technique for the sample preparation of pesticides in high-moisture samples such as vegetables [67]. The technique uses simple glassware and a minimal amount of organic solvent, followed by the successive addition of salt plus buffer and a SPE packing (Fig. 16.7). The initial addition of a hydrophilic solvent such as acetonitrile or acetone to a homogenized portion of a vegetable sample allows the extraction of pesticides into the solvent (step 2 of Fig. 16.7). Subsequent addition of salt (step 3) leads to separation of the organic solvent from water associated with the sample, and promotes extraction of pesticides into the organic solvent. The internal standard is added next (step 4), and after shaking and centrifugation, an aliquot of the organic phase is subjected to further cleanup using dispersive SPE: the addition of small amounts of bulk SPE packing (e.g., C 18 , graphitized carbon, amino) to the extract for the purpose of removing interferences from the extract (step 5). After sample cleanup the supernatant is sampled and analyzed (step 7). Step 6 is an optional step for pesticides that are unstable at intermediate pH values. QuEChERS has been found particularly useful for screening the food supply for multiple pesticides. Official QuEChERS methods from American Association of Official Analytical Chemists (AOAC). Method 2007.01 and the DIN-adopted European Standard Method EN 15662 are now available. QuEChERS has been investigated for over 500 pesticides in a variety of fruit and vegetable matrices [63, 65, 68]; analyte recoveries (for concentrations of 100 ng/g) generally range from 70 to 110%, with a variability of less than 10%. The technique has been extended to new matrices such as meat and fish products, as well as analytes such as antibiotics and other drugs [69–70]. 16.6.7.6 Class-Specific SPE Cartridges Over the years, specialty phases have been introduced that are compound- or class-specific. While MIP and immunoaffinity packings (Sections 16.6.7.3, 16.6.7.4) can provide extreme specificity or selectivity, specialty packings with special func- tional groups can selectively interact with certain compound classes. Under basic conditions immobilized phenylboronic acid (PBA) selectively binds analytes that possess vicinol diols (e.g., sugars and catechols). Other compounds that are also selectively retained include alpha-hydroxy acids, aromatic o-hydroxy acids and amides, and aminoalcohol-containing compounds. Covalent bonds between pack- ing and analyte are formed, allowing interfering compounds to be washed from the packing with a variety of different solvents. Once washed, the covalent bonds can be broken by washing the phase with an acidic buffer/solvent that hydrolyzes the covalent bonds. A popular application of the PBA phases is the isolation of catecholamines in biological fluids [41]. 790 SAMPLE PREPARATION 1. Weigh 10-g sample into 50-mL tube 2. Add 10-mL ACN 3. Add 4-g MgSO 4 + 1-g NaCl 4. Add IS solution 5. Take aliquot, add MgSO 4 + sorbent (dispersive SPE) 6. Adjust pH or stabilize (optional) 7. Inject shake 1-min shake 1-min shake 30-s; centrifuge shake 30-s; centrifuge QuEChERS Figure 16.7 Application of QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction of pesticides from high-moisture vegetable samples. 16.7 MEMBRANE TECHNIQUES IN SAMPLE PREPARATION With the exception of filtration- and SPE-membranes, membrane separation tech- niques have not been widely used for HPLC sample preparation. Microporous, semi-permeable membranes permit selective filtration because of the size of their micropores. Compared to SPE or LLE, membrane separations are slower and less likely to increase the analyte concentration by orders of magnitude. A successful application of membrane separation requires either removal of analyte from the acceptor side by trapping (trace enrichment) or by a change in the chemical state of the analyte (e.g., change from a charged to an uncharged species). An advantage of membrane separation techniques for RP-HPLC analysis is that both the donor and acceptor liquids are usually water or buffer. Analytes can also be moved across a membrane by chemical or electrochemical gradients. Membrane separations can be carried out in a static system or in a flowing system, with the latter more amenable to automation. Ultrafiltration, reverse osmosis, dialysis, microdialysis, and electrodial- ysis are examples of techniques that use membranes for concentration, purification, and separation of analytes. For more information on membrane separations, see [71]. 16.8 SAMPLE PREPARATION METHODS FOR SOLID SAMPLES 791 16.8 SAMPLE PREPARATION METHODS FOR SOLID SAMPLES A sample must be in a liquid state prior to HPLC analysis. Some insoluble solids contain soluble analytes, such as additives in a solid polymer, fats in food, and polyaromatic hydrocarbons in soil. Contacting the sample with solvent allows the extraction of analytes into the solvent. The solvent is separated from the solid residue by decanting, filtration, or centrifugation, and the filtrate is further treated, if necessary, prior to HPLC analysis. Tables 16.8 and 16.9 summarize some techniques used for the extraction (‘‘leaching’’) of soluble analytes from an insoluble solid matrix. Table 16.8 Traditional Methods for Sample Preparation of Solid Samples Sample Preparation Method Principles of Technique Comments Solid–liquid extraction Sample and solvent are placed in a stoppered container and agitated; the resulting solution is separated from the solid by filtration (sometimes called ‘‘shake-flask’’ method). Solvent is sometimes boiled or refluxed to improve solubility; sample should be in a finely divided state to aid the leaching process. Soxhlet extraction Sample is placed in an extraction thimble; refluxing solvent flows through the thimble and dissolves analytes that are continuously collected in a boiling flask (Sections 16.8.1, 16.8.2.1). Extraction occurs by contact with the pure solvent; sample must be stable at the boiling point of the solvent; a slow process, but extraction is carried out unattended until complete; excellent recoveries (used as standard to which other solid extraction methods are compared). Homogenization a Sample and solvent are homogenized in a blender or mechanical homogenizer to a finely divided state; solvent is removed for further workup. Used for plant and animal tissue, food, environmental samples; organic or aqueous solvent can be used; dry ice or diatomaceous earth can be added to make sample flow more freely. Sonication a Use of ultrasound to create vigorous agitation at the surface of a finely divided solid material; either a sonotrode probe or ultrasonic bath can be used. Heat can be added to increase rate of extraction; safe; rapid; best for coarse, granular materials. Dissolution a Sample is treated with dissolving solvent and taken directly into solution with or without chemical change. Some samples may not dissolve directly (e.g., digestion or other pretreatment needed); filtration may be required after dissolution. a Not discussed in text. 792 SAMPLE PREPARATION Table 16.9 Modern Extraction Methods for Solid Samples Method of Sample Pretreatment Principles of Technique Comments Supercritical fluid extraction (SFE) Sample is placed in a flow-through container, and supercritical fluid (e.g., CO 2 ) is passed through the sample; after depressurization, the extracted analyte is collected (Section 16.8.2.2). To affect the ‘‘polarity’’ of SFE fluid, density can be varied and solvent modifiers added. Pressurized fluid extraction (PFE)/accelerated solvent extraction (ASE) Sample and extraction solvent are placed in a sealed container and heated above the solvent’s boiling point, causing pressure in the vessel to rise; extracted sample is removed and transferred to a vial for further treatment (Section 16.8.2.3). Greatly increases speed of liquid–solid extraction process; may be automated; extracted sample is diluted and requires further concentration. Automated Soxhlet extraction (ASE) Combines hot solvent leaching and Soxhlet extraction; sample thimble is first immersed in boiling solvent, then thimble is raised for conventional Soxhlet extraction/rinsing (Section 16.8.2.1). Uses less solvent than traditional Soxhlet; decreased extraction time due to the two-step process. Microwave-assisted extraction (MAE) Sample is placed in an open or closed container and heated by microwave energy (Section 16.8.2.4). Closed container MAE allows use of pressure to improve extraction. Matrix solid-phase dispersion (MSPD) Bonded-phase support is used as an abrasive to produce disruption of sample-matrix architecture and as a ‘‘bound’’ solvent to aid complete sample disruption during the sample blending process. Solid or viscous sample (≈0.5g) is homogenized in a mortar, or other suitable container, with about 2 g of SPE sorbent (e.g., C 18 ). Blend is transferred to an empty column and analytes are eluted with solvent. 16.8.1 Traditional Extraction Methods No one solvent-extraction technique can be used for all samples. Table 16.8 lists several traditional methods for the pretreatment of solid samples. Soxhlet extraction has been used for more than a hundred years, is time tested, and accepted by most scientists. Regulatory agencies such as the EPA, the Food and Drug Administration (FDA), and their equivalents in other countries readily approve these classical methods of extracting solid samples. However, older methods often use large 16.8 SAMPLE PREPARATION METHODS FOR SOLID SAMPLES 793 amounts of organic solvent, which has led to miniaturization in recent years. As the oldest form of efficient extraction, Soxhlet extraction is the accepted standard for comparison with newer extraction technologies for solid samples. Solvent extraction can assume many forms. The shake-flask method, whereby a solvent is added to the sample followed by agitation, works well when the ana- lyte is highly soluble in the extraction solvent and the sample is quite porous. For fast extraction, the sample should be finely divided. Heating or refluxing the sample-plus-solvent can speed extraction. For faster and more complete extraction, ultrasonic agitation (sonication) often allows more effective solid–liquid contact, while providing gentle heating that further aids extraction. Sonication is a recom- mended procedure for the pretreatment of many solid environmental samples, such as US EPA Method 3550 [72] for extracting non-volatile and semi-volatile organic compounds from solids such as soils, sludges, and wastes. For this method different extraction solvents and sonication conditions are recommended, depending on the type of analytes and their concentration in the solid matrix. Soxhlet extraction has been the most widely used method for the extraction of solids. In this procedure the solid sample is placed in a Soxhlet thimble (a disposable porous container made of stiffened filter paper), and the thimble is placed in the Soxhlet apparatus (Fig. 16.8). The extraction solvent is refluxed; it subsequently condenses into the thimble and extracts the soluble analytes. The apparatus is designed to siphon the extract each time the chamber holding the thimble fills with solution. The siphoned solution containing the dissolved analytes returns to the boiling flask, and the process is repeated until the analyte has been transferred from the solid sample to the flask. Because the sample is contained in the boiling extraction solvent, it must be stable at elevated temperature. Only clean (distilled) warm solvent is used to extract the solid in the thimble, which increases the efficiency of the extraction vs. the simple shake-flask method—a key advantage of classical Soxhlet extraction. Method development consists of finding a volatile solvent (e.g., boiling point <100 ◦ C) that has a high solubility for the analyte and a low solubility for the solid sample matrix. Soxhlet extractions are usually slow (12–24 hr, or more), but the process takes place unattended. Modern Soxhlet extractors can speed up extraction 8- to 10-fold as described below (Section 16.8.2.1). The most common extractors use hundreds of milliliters of very pure (and expensive!) solvent, but small-volume extractors and thimbles are available for mg-size samples. The analyte concentration, the necessary mass to obtain a representative sample, and the chromatographic detector sensitivity, together determine the required sample size. 16.8.2 Modern Methods for Extracting Solids Table 16.9 lists several modern methods used for extracting solid samples. 16.8.2.1 Modern Soxhlet Extraction Classical Soxhlet extraction has been improved to reduce extraction time 8- to 10-fold [73]. An automated apparatus lowers and raises the sample thimble, so as to either place the thimble in the boiling solvent or raise the thimble for conventional Soxhlet extraction. The sample is first totally immersed in the boiling solvent, where the higher temperature of the boiling solvent speeds up extraction of the analyte by 794 SAMPLE PREPARATION Condenser Siphon tube Solvent reservoir Extraction thimble Sample Figure 16.8 Apparatus for Soxhlet extraction. increasing both its solubility and diffusion rate. Then, to flush residual extract from the sample, the thimble is raised above the boiling solvent, and conventional Soxhlet extraction proceeds. Finally, the solvent is boiled off to concentrate the analyte. Automated Soxhlet extraction is approved by the EPA for the extraction of organic analytes from soil, sediment, sludges, and waste solids (Method 3541 [74]). Further improvements in the above procedure for faster Soxhlet extraction have been proposed. Focused microwave-assisted Soxhlet extraction can reduce extraction time further for environmental solid samples, by using microwave absorbing solvents [75, 76]. The use of water as an extraction solvent makes the process much more environmentally friendly [77]. 16.8.2.2 Supercritical Fluid Extraction (SFE) When first introduced in the early 1990s, supercritical fluid extraction (SFE) was thought to be preferred for all solid-sample extraction problems. SFE was viewed as a safe, solvent-free technique that allowed easy removal of the carbon dioxide used for analyte solubilization. Moreover SFE was expected to allow faster, more complete extraction of diverse samples such as soil, sand, sludge, fly ash, foods, and polymers. New applications of SFE are still being reported, but no new instruments or major accessories for existing instruments have been commercialized since 2000. While SFE 16.8 SAMPLE PREPARATION METHODS FOR SOLID SAMPLES 795 is an important process for industrial-scale purification, its analytical applications (including sample pretreatment) are today relatively unimportant. Numerous reasons can be cited for the continuing decrease in SFE for sample pretreatment, but these are outside the scope of the present book. For details, on the use of SFE, see [78–82]. 16.8.2.3 Pressurized Fluid-Extraction (PFE)/Accelerated Solvent Extraction (ASE) Originally introduced by Dionex (Sunnyvale, CA) as ASE, the technique was generalized to PFE by the EPA. PFE is now well accepted as a modern alternative to Soxhlet extraction; it achieves analyte recoveries equivalent to those from Soxhlet extraction, but with less solvent and in only 10 to 20 minutes. Sample weights of 1 to 30 g are possible, with extraction cells that range in size from 1 to 100 mL. PFE uses the same solvents as classical Soxhlet and sonication methods, and method development is therefore easy. The finely divided sample is placed in an extraction cell located in an oven, and a pump transfers solvent from one or more reservoirs into the extraction cell. Only one sample at a time can be extracted, but an autosampler that can handle up to 24 samples for unattended operation is available. Extractions can be performed in a static and/or dynamic mode. Once the extraction is completed in the static mode, a nitrogen purge is used to transfer the extract to a collection vial. In the PFE process the sample is contained in a large volume of solvent, so an additional step is needed to concentrate the analyte of interest (e.g., RPC, evaporation). The EPA method 3545A [83], entitled ‘‘Pressurized Fluid Extraction’’ provides for the extraction of water-insoluble or slightly water-soluble organic compounds from soils, clays, sediments, sludges, and waste solids. This method is applicable to the extraction of semi-volatile organic compounds such as pesticides, herbicides, polychlorinated biphenyls (PCBs), and dioxins. PFE, sometimes referred to as pressurized solvent extraction (PSE), uses elevated temperatures (100–180 ◦ C) and pressures (1500–2000 psi). An example of PFE is the extraction of active ingredients from natural-product pharmaceuticals [84] at 100 ◦ C and 1500 psi. Samples, in capsule form, are opened, the ground material is collected and 1 to 3 g are loaded into the extraction cells. Only ≈15 mL of the extraction solvent, ACN, is used for each sample, with a 14-minute cycle time per sample. Analysis is performed by RPC, with external calibration. Results (3–4% CV) are comparable to those obtained by Soxhlet and sonication, but were faster, used less solvent and were fully automated. 16.8.2.4 Microwave-Assisted Solvent Extraction (MAE) Microwave-assisted extraction (sometimes referred to an microwave-accelerated solvent extraction) of organic analytes [85] is an alternative to the traditional liquid-solid extractions described earlier. Microwave heating is advantageous in that the extraction solvent is heated internally rather than by convective heating. The temperature in the extraction container is determined by the solvent used for extraction (not by the set point of an external oven or heater), and as the temperature of the solvent in the closed container rises, the pressure also increases. As a result extraction takes place faster than with convection heating. Water has a high dielectric constant and readily absorbs microwave energy, but organic solvents, such as hydrocarbons, do not absorb microwave energy and there- fore are not heated. Microwave extractions with these non–microwave-absorbing . as acetonitrile or acetone to a homogenized portion of a vegetable sample allows the extraction of pesticides into the solvent (step 2 of Fig. 16.7). Subsequent addition of salt (step 3) leads to. extractors and thimbles are available for mg-size samples. The analyte concentration, the necessary mass to obtain a representative sample, and the chromatographic detector sensitivity, together. the vessel to rise; extracted sample is removed and transferred to a vial for further treatment (Section 16.8.2.3). Greatly increases speed of liquid solid extraction process; may be automated; extracted