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EXTRACTION | Solid-Phase Extraction of Drugs and Metabolites
EXTRACTION Solid-Phase Extraction of Drugs and Metabolites N. H. Snow, Seton Hall University, South Orange, NJ, USA & 2007 Elsevier Ltd. All rights reserved.
Introduction Determining drugs and metabolites, especially from biological samples such as blood, urine, or tissues and from complex pharmaceutical formulations is one of the most challenging problems in analytical chemistry. Drugs are often polar, reactive and at low concentrations in the presence of often hundreds of other compounds. High-resolution gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS) are often required if quantitation is needed. Solid-phase extraction (SPE) is a classical sample preparation method and it continues to be the technique of choice for separating drugs and metabolites from complex matrices and preparing them for GC or LC. SPE has its beginnings in the 1950s; organic compounds in water were analyzed by trapping them on a carbon-based sorbent followed by elution with an organic solvent. Commercial devices were first introduced in the 1970s, and SPE has become one of the most popular sample preparation methods for a huge variety of analytes. SPE has myriad applications as a sample preparation technique for chromatographic analysis. The application guides provided by several vendors of SPE equipment are easily obtained online. Nearly all of the major vendors of SPE devices have extensive literature readily available on the World Wide Web (WWW), including application notes, method development guides, and troubleshooting information. A sampling of vendor websites is provided in the Further Reading list; readers should be aware that the SPE vendors often have a wide range of products, so they may have to do some navigating or searching the sites to find the SPE-related materials and that these WWW sites are primarily marketing tools. SPE, generally used for separating compounds of interest from liquids, can be more broadly termed
sorbent extraction, in which the goal is to selectively adsorb analyte(s) onto a surface and then to elute them, while leaving unwanted compounds behind either on the surface or in the original solution. Classically, the sorbent consists of irregular porous particles, usually chemically modified silica gel similar to high-performance liquid chromatography (HPLC)-bonded phases, with average diameter B40 mm. Most commonly, between 100 mg and 1 g of these particles are packed into a cartridge that resembles the barrel of a large syringe. The packing is held in the cartridge using simple frits. A diagram of a classical SPE cartridge is shown in Figure 1. Additional technologies are also available, including filter disks impregnated with selective sorbent material, which allow extraction of trace components from very large volume liquid samples such as drinking water and sorbent-based microextraction, including solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE), in which the sorbent is placed in the liquid sample and removed for desorption of the analytes into an instrument. Along with classical SPE cartridges, these are described in the general text by Fritz. In drug and metabolite analysis, the great majority of methods employ the classical cartridges, so most of the discussion in this article will focus on these. Following a description of the basic processes in SPE, applications of SPE to drug analysis from biological samples, pharmaceuticals, and in the environment will be discussed.
Medical-grade polypropylene
Reservoir
Upper frit (20 µm) Sorbent
Lower frit Luer tip Figure 1 Schematic of an SPE Cartridge. (Reproduced with permission from Grob RL and Barry EF, Modern Practice of Gas Chromatography, 4th Edition, Figure 11.7, p. 560. Copyright 2004, John Wiley and Sons, Inc.
2 III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites
Process and Modes of SPE The basic processes involved in SPE are illustrated in Figure 2 and are described below. Each step is illustrated by an example from a typical method for the SPE of amphetamines from urine using a traditional syringe-like SPE cartridge. More details on this method can be found in the paper by Telapchak and colleagues. Samples and eluents are either pushed through the cartridge using a plunger or drawn through using a vacuum connected to the cartridge outlet. Manifolds that can accommodate several dozen cartridges and sample-collection tubes simultaneously allow for high-sample throughput. Additional (1) Conditioning Water
(2) Sample addition Nonpolar
Methanol
Semipolar Polar
Hexane
C18
(3) Washing
C18
(4) Elution
Methanol
Hexane
C18
C18
Figure 2 SPE process for a C18 cartridge. Step 1: The cartridge is conditioned with an increasingly polar sequence of solvents. Step 2: The aqueous sample is added to the cartridge. Step 3: The cartridge is washed with a polar solvent. Step 4: Analytes are eluted with a nonpolar solvent. (Reproduced with permission from Grob RL and Barry EF, Modern Practice of Gas Chromatography, 4th Edition, Figure 11.8, p. 561. Copyright 2004, John Wiley and Sons, Inc.
details on these devices can be easily obtained from the vendors of SPE equipment and supplies. 1. First the stationary phase is equilibrated with the elution solvent. This helps to attain maximum analyte extraction recovery by ensuring that the organic moieties on the bonded stationary phase are properly solvated, allowing the analyte molecules to access them and to undergo the intermolecular interactions necessary for separation. Prior to application on the cartridge, the liquid sample may be adjusted to enhance the partition coefficient. For example, in the SPE of amphetamines, in which an ion-exchange copolymer is commonly used in as the sorbent, the liquid sample is generally acidified to pH B6.0 to ionize the amphetamines, while many of the potential organic and acidic interferences will remain neutral. The extraction column is prepared with aliquots of aqueous polar solvents including methanol, water, and pH-6 buffer. Blood or urine may also be centrifuged or filtered to remove cellular material or high-molecular-weight proteins that can foul the cartridge frits. 2. Next, the liquid sample is applied and the liquid is slowly drawn through the sorbent bed either by vacuum suction or by pushing with a plunger. Typically, the analytes are sparingly soluble in the liquid sample matrix, so they are at low concentration. The sorbent surface matrix is chosen to have a high affinity for the analytes. The partition coefficient between the liquid sample and the surface is therefore quite high, often 103 or higher, so the analyte may be nearly completely removed from the sample. In any case, as in classical liquid–liquid extraction (LLE), the amount of analyte that is extracted is dependent on the partition coefficient. In the amphetamine example, a 2 mL urine sample is applied to a 200 mg ion-exchange copolymer extraction column at a flow rate of 1–2 mL min 1. 3. Next, the sorbent, now with analyte and interfering compounds adsorbed, is washed with a solvent that is weak for the analytes, but strong for the interferences. Often, if the original sample matrix was aqueous, this wash solvent might be methanol, a methanol–water mixture, or a sequence of solvents of changing polarity. Washing is analogous to backextraction in LLE; it results in a more pure analyte extract, but reduced analyte recovery. This recovery loss can be estimated if the partition coefficients are known. In the amphetamine example, the washes include water, acetic acid, and methanol to remove acidic and neutral organic compounds and residual watersoluble compounds.
III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites 3
4. Vessels to contain the extracts are placed beneath the cartridges and the analytes are eluted by adding a strong elution solvent to the cartridge and passing it through the sorbent bed. The analytes are usually highly soluble in this solvent. Generally, the solvent is passed through slowly to allow full contact with the surface, and therefore efficient quantitative analyte removal. In the amphetamine example, elution is performed with aliquots of basified dichloromethane/isopropyl alcohol (IPA) (pH 11). By using the basic eluent, the bound amphetamines form the free base are released from the ion-exchange matrix. The free base amphetamines are highly soluble in the dichloromethane–IPA mixture. 5. The extract is then prepared for chromatographic analysis by GC or HPLC, typically by evaporation to dryness, derivatization, reconstitution in an appropriate solvent and injection onto a GC, LC, GC-MS, or LC-MS. Prior to injection, amphetamines are usually derivatized to prevent adsorption in the inlet or column, improving chromatographic behavior.
silica surface, similar to bonded-phase HPLC stationary phases. As in HPLC, the most common sorbent used in SPE involves reacting the silica with octadecyl silane to produce particles nearly completely covered with bound C18 chains. This produces a surface generally considered nonpolar and hydrophobic, generating binding with analytes through hydrophobic interactions between the hydrocarbon portion of the analyte and the hydrocarbon chains on the surface. A summary of common SPE phases and key applications is provided in Table 1. This table is not intended to be comprehensive; complete descriptions and information on all available phases is provided in the literature from the SPE manufacturers referenced in this article. Table 1 Summary of common SPE sorbents Sorbent
Mechanism
Typical applications
C18
Reversed phase
C8
Reversed phase
C2
Reversed phase
Polymeric
Reversed phase
Aminopropyl
Normal or reversed phase
Cyanopropyl
Normal or reversed phase Normal or reversed phase Normal phase
Most common SPE and HPLC phase; drugs of abuse, metabolites, peptides, trace organics, especially acids Similar to C18; generally less hydrophobic; also used commonly for drugs and metabolites Less retention than C8 or C18; applications are similar Used for reversed-phase applications at very high or very low pH, where the silica-based phases might degrade Can be used as a weak ionexchange phase; ionizable compounds; phenols, petroleum fractionation, saccharides, drugs Low hydrophobicity; alternative to silica; drugs, metabolites and pesticides Antibiotics, proteins, peptides, less acidic than silica
Comparison of SPE and LLE In comparison with more classical techniques such as LLE, SPE has several advantages: 1. Significantly reduced solvent volume and usage. 2. Ability to sample in the field. 3. Straightforward sample manipulation and ease of method development using single cartridges. 4. Elimination of emulsion formation, a major drawback of LLE for biological samples. 5. Reduced exposure risk to hazardous solvents and samples. 6. Generally inexpensive cartridges; 5–10 times less expensive than LLE. 7. Contamination is minimized; cartridges are made from medical-grade polypropylene. 8. Excellent flexibility; almost limitless available solvents. 9. SPE method development is straightforward. Often the analyst can find a method of extraction in the application notes provided by SPE vendors or by having knowledge of previously developed HPLC methods for analysis of the compounds.
Diol
Silica Alumina
Florisil
SPE Sorbents and Chemistries
Acrylic acid
There is a large variety of sorbents and chemistries available for SPE. Selectivity is generally obtained by covalently bonding a chosen organic moiety to a
Acrylamide
Used to extract compounds from organic solvents Normal phase Similar to silica; can be adjusted for acidic, basic, or neutral analytes Normal phase Pesticides (Association of Official Analytical Chemists (AOAC) and Environmental Protection Agency (EPA) methods), polychlorinated biphenyls (PCBs) Cation exchange Anionic proteins, pigments, phenols, peptides Anion exchange Aldehydes, ketones, carbonyls (EPA applications)
4 III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites
Extraction of Drugs and Metabolites
of drugs from biological matrices has certainly become a mainstream technique.
Biological Systems
Analysis of drugs and metabolites from biological systems is both a common and highly challenging application of SPE. Biological systems often contain both soluble and insoluble matrix components that can easily foul the cartridges or extract along with the analyte(s) of interest, generating interference with the extraction or with subsequent chromatographic analysis, or reduce the activity of the analyte(s) of interest, also reducing extraction recovery. However, owing to the advantages cited above, SPE is the technique of choice in the common clinical, forensic, and toxicological analysis of drugs, especially drugs of abuse, from urine and blood. Methods and procedures for SPE of nearly all drugs of abuse are available; these are detailed in the text references included in this article. Further, nearly all of the SPEequipment vendors have extensive databases of drugs-of-abuse application notes available on the WWW. For example, Varian lists over 160 applications in a search of its database using ‘drugs of abuse’ as a keyword. Since the basic procedures are readily searchable and obtainable online from the vendor websites listed herein, the remainder of this section focuses on recent advances and new techniques from 2005 and early 2006. Research on the development of new methods and applications for SPE of drugs and metabolites is surprisingly increasing, given the large number of available methods in the literature. Recently, analytes in biological matrices well beyond urine and blood, which are still most commonly described in the literature methods, such as brain tissue, oral fluid, hair, milk, and beverages, have been extracted. A literature search, using SciFinder, with ‘solid-phase extraction’ and ‘drugs of abuse’ included about 80 papers in a 15-month period beginning January 2005. The variety of drugs described is also much wider than the traditional drugs of abuse, including muscle relaxants, thyroid medications, and mescaline. There has also been strong interest in g-hydroxybyturate (GHB) and ketamine (K), which have become notorious in use by young people as the ‘date rape’ and a popular ‘club’ drug, respectively. Finally, the instrumental choices used in combination with SPE are evolving; LC-MS and LC-MS/MS are becoming more popular, often supplanting the traditional GC-MS, especially when the most complex sample matrices, such as blood are involved. Also interesting is that most of the papers were written by authors outside the traditional analytical chemistry and separation science communities; SPE
Pharmaceuticals
Pharmaceutical analysis using SPE is mostly focused in the discovery and development processes. As the sample matrices of finished products and raw materials, such as tablets, gels, ointments devices, and capsules are well known, with relatively high analyte concentration or mass, they are generally straightforward to dissolve in common solvents. Therefore, SPE is generally not required for quality assurance assays. In drug discovery, however, the analytes and matrices are not always as well known, and the analytes may be at low concentration, requiring very high selectivity and making SPE an important tool. Recently, to enhance selectivity further than traditional SPE sorbents allow, molecular-imprinted polymers have been synthesized and used as highly selective stationary phases for SPE analysis of very specific compound classes and for chiral separations. MIPs, for instance, have been used with SPE in 96-well plate configuration for the screening of a compound undergoing pharmaceutical development. The high selectivity of the molecular imprinted polymer (MIP) stationary phase allowed highly sensitive determination of the analyte at levels far below traditional SPE. A SciFinder search using ‘solid-phase extraction’ and ‘drug discovery’ as keywords provides about 50 references in the 15 months beginning January 2005. As was the case with biological samples, most of the recent papers involved LC and LC-MS as the instrumental method. Also, most papers involving the use of SPE in drug discovery and development focus more on the instrumental technique (often LC-MS or LC-NMR-MS) than on the SPE, which may in the end be the most difficult portion of the analysis, and therefore it is often difficult to find all the details of the extraction process. A second focus has been on high-throughput methods for screening of large numbers of drug candidates at very early stages in the discovery process. Environmental
Analysis of drugs in environmental samples, especially natural and wastewater, has recently become a major application for SPE. The premise is clear: the drugs and metabolites manufactured and consumed by humans and animals are eventually passed into the environment, plus it is possible that drug residues may be present in the wastewater streams from manufacturing facilities. Endocrine disruptors and steroids are of special concern as these are quite common and
III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites 5
have many toxicological effects from long-term human or animal exposure to small doses. The analytical problems are very similar to those involved with drugs of abuse detection from biological fluids, except that the analyte concentrations are often lower. Interestingly, most of the work in this area has occurred in Europe, with relatively little in the United States. Typical studies of this type include the analysis of several pharmaceutical compounds using a standard environmental method for acidic or basic contaminants, or straightforward modifications of these methods. As in the previous discussions, SPE is traditionally combined with GC-MS but is now more often combined with LC-MS. A SciFinder literature search using ‘solid-phase extraction,’ ‘drugs’ and ‘environmental’ as keywords produced about 80 references. A sampling of the drugs examined in these papers includes nonsteroidal antiinflammatory drugs, analgesics, steroids, antibiotics, estrogens, antidepressants, lipid regulators, and b-blockers. Typically, the studies are designed to track the progress of the drug or its degradation products through the wastewater stream and into the environment. Many of the authors note that there is, to date, little literature describing the fate of pharmaceuticals in the environment, although the research interest in this is increasing rapidly. Environmental SPE methods for drugs are either based on traditional environmental methods for other compounds or on clinical or pharmaceutical methods. Further, environmental methods for pharmaceuticals are often performed in combination with traditional methods for other compounds such as organic acids and pesticides. Most work to date is related to analysis of common drug classes such as painkillers, antioxidants, and antibacterial agents.
Conclusions SPE is a staple technique for the preparation of samples, especially for GC and LC and MS. There is a large variety of equipment configurations, including sorbent cartridges, impregnated disks, phase-coated fused-silica fibers and phase-coated stir-bars, and others. This combined with an even larger variety of sorbent chemistries shows that there is an SPE technique appropriate to nearly any sample preparation analyte and matrix combination. SPE continues to be used throughout the analytical chemistry community. See also: II/Extraction: Solid-Phase Extraction. III/Drugs of Abuse: Solid-Phase Extraction.
Further Reading Baker JT. http://www.jtbaker.com (accessed on June 2006). Cole MD (2003) The Analysis of Controlled Substances. Chichester: Wiley. Fritz JS (2003) Solid Phase Extraction. New York: Wiley. Pawliszyn J (1998) Solid Phase Micro-extraction Theory and Practice. New York: Wiley. Phenomenex. http://www.phenomenex.com (accessed on June 2006). Supelco. http://www.sigmaaldrich.com/Brands/Supelco_ Home.html (accessed on June 2006). Telapchak MJ, August TF and Chaney G (2004) Forensic and Clinical Applications of Solid Phase Extraction. Totowa, NJ: Humana Press. United Chemical. http://www.unitedchem.com (accessed on June 2006). Varian. http://www.varianinc.com (accessed on June 2006). Waters. http://www.waters.com (accessed on June 2006).
Introduction Determining drugs and metabolites, especially from biological samples such as blood, urine, or tissues and from complex pharmaceutical formulations is one of the most challenging problems in analytical chemistry. Drugs are often polar, reactive and at low concentrations in the presence of often hundreds of other compounds. High-resolution gas chromatography (GC) and liquid chromatography (LC) combined with mass spectrometry (MS) are often required if quantitation is needed. Solid-phase extraction (SPE) is a classical sample preparation method and it continues to be the technique of choice for separating drugs and metabolites from complex matrices and preparing them for GC or LC. SPE has its beginnings in the 1950s; organic compounds in water were analyzed by trapping them on a carbon-based sorbent followed by elution with an organic solvent. Commercial devices were first introduced in the 1970s, and SPE has become one of the most popular sample preparation methods for a huge variety of analytes. SPE has myriad applications as a sample preparation technique for chromatographic analysis. The application guides provided by several vendors of SPE equipment are easily obtained online. Nearly all of the major vendors of SPE devices have extensive literature readily available on the World Wide Web (WWW), including application notes, method development guides, and troubleshooting information. A sampling of vendor websites is provided in the Further Reading list; readers should be aware that the SPE vendors often have a wide range of products, so they may have to do some navigating or searching the sites to find the SPE-related materials and that these WWW sites are primarily marketing tools. SPE, generally used for separating compounds of interest from liquids, can be more broadly termed
sorbent extraction, in which the goal is to selectively adsorb analyte(s) onto a surface and then to elute them, while leaving unwanted compounds behind either on the surface or in the original solution. Classically, the sorbent consists of irregular porous particles, usually chemically modified silica gel similar to high-performance liquid chromatography (HPLC)-bonded phases, with average diameter B40 mm. Most commonly, between 100 mg and 1 g of these particles are packed into a cartridge that resembles the barrel of a large syringe. The packing is held in the cartridge using simple frits. A diagram of a classical SPE cartridge is shown in Figure 1. Additional technologies are also available, including filter disks impregnated with selective sorbent material, which allow extraction of trace components from very large volume liquid samples such as drinking water and sorbent-based microextraction, including solid-phase microextraction (SPME) and stir-bar sorptive extraction (SBSE), in which the sorbent is placed in the liquid sample and removed for desorption of the analytes into an instrument. Along with classical SPE cartridges, these are described in the general text by Fritz. In drug and metabolite analysis, the great majority of methods employ the classical cartridges, so most of the discussion in this article will focus on these. Following a description of the basic processes in SPE, applications of SPE to drug analysis from biological samples, pharmaceuticals, and in the environment will be discussed.
Medical-grade polypropylene
Reservoir
Upper frit (20 µm) Sorbent
Lower frit Luer tip Figure 1 Schematic of an SPE Cartridge. (Reproduced with permission from Grob RL and Barry EF, Modern Practice of Gas Chromatography, 4th Edition, Figure 11.7, p. 560. Copyright 2004, John Wiley and Sons, Inc.
2 III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites
Process and Modes of SPE The basic processes involved in SPE are illustrated in Figure 2 and are described below. Each step is illustrated by an example from a typical method for the SPE of amphetamines from urine using a traditional syringe-like SPE cartridge. More details on this method can be found in the paper by Telapchak and colleagues. Samples and eluents are either pushed through the cartridge using a plunger or drawn through using a vacuum connected to the cartridge outlet. Manifolds that can accommodate several dozen cartridges and sample-collection tubes simultaneously allow for high-sample throughput. Additional (1) Conditioning Water
(2) Sample addition Nonpolar
Methanol
Semipolar Polar
Hexane
C18
(3) Washing
C18
(4) Elution
Methanol
Hexane
C18
C18
Figure 2 SPE process for a C18 cartridge. Step 1: The cartridge is conditioned with an increasingly polar sequence of solvents. Step 2: The aqueous sample is added to the cartridge. Step 3: The cartridge is washed with a polar solvent. Step 4: Analytes are eluted with a nonpolar solvent. (Reproduced with permission from Grob RL and Barry EF, Modern Practice of Gas Chromatography, 4th Edition, Figure 11.8, p. 561. Copyright 2004, John Wiley and Sons, Inc.
details on these devices can be easily obtained from the vendors of SPE equipment and supplies. 1. First the stationary phase is equilibrated with the elution solvent. This helps to attain maximum analyte extraction recovery by ensuring that the organic moieties on the bonded stationary phase are properly solvated, allowing the analyte molecules to access them and to undergo the intermolecular interactions necessary for separation. Prior to application on the cartridge, the liquid sample may be adjusted to enhance the partition coefficient. For example, in the SPE of amphetamines, in which an ion-exchange copolymer is commonly used in as the sorbent, the liquid sample is generally acidified to pH B6.0 to ionize the amphetamines, while many of the potential organic and acidic interferences will remain neutral. The extraction column is prepared with aliquots of aqueous polar solvents including methanol, water, and pH-6 buffer. Blood or urine may also be centrifuged or filtered to remove cellular material or high-molecular-weight proteins that can foul the cartridge frits. 2. Next, the liquid sample is applied and the liquid is slowly drawn through the sorbent bed either by vacuum suction or by pushing with a plunger. Typically, the analytes are sparingly soluble in the liquid sample matrix, so they are at low concentration. The sorbent surface matrix is chosen to have a high affinity for the analytes. The partition coefficient between the liquid sample and the surface is therefore quite high, often 103 or higher, so the analyte may be nearly completely removed from the sample. In any case, as in classical liquid–liquid extraction (LLE), the amount of analyte that is extracted is dependent on the partition coefficient. In the amphetamine example, a 2 mL urine sample is applied to a 200 mg ion-exchange copolymer extraction column at a flow rate of 1–2 mL min 1. 3. Next, the sorbent, now with analyte and interfering compounds adsorbed, is washed with a solvent that is weak for the analytes, but strong for the interferences. Often, if the original sample matrix was aqueous, this wash solvent might be methanol, a methanol–water mixture, or a sequence of solvents of changing polarity. Washing is analogous to backextraction in LLE; it results in a more pure analyte extract, but reduced analyte recovery. This recovery loss can be estimated if the partition coefficients are known. In the amphetamine example, the washes include water, acetic acid, and methanol to remove acidic and neutral organic compounds and residual watersoluble compounds.
III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites 3
4. Vessels to contain the extracts are placed beneath the cartridges and the analytes are eluted by adding a strong elution solvent to the cartridge and passing it through the sorbent bed. The analytes are usually highly soluble in this solvent. Generally, the solvent is passed through slowly to allow full contact with the surface, and therefore efficient quantitative analyte removal. In the amphetamine example, elution is performed with aliquots of basified dichloromethane/isopropyl alcohol (IPA) (pH 11). By using the basic eluent, the bound amphetamines form the free base are released from the ion-exchange matrix. The free base amphetamines are highly soluble in the dichloromethane–IPA mixture. 5. The extract is then prepared for chromatographic analysis by GC or HPLC, typically by evaporation to dryness, derivatization, reconstitution in an appropriate solvent and injection onto a GC, LC, GC-MS, or LC-MS. Prior to injection, amphetamines are usually derivatized to prevent adsorption in the inlet or column, improving chromatographic behavior.
silica surface, similar to bonded-phase HPLC stationary phases. As in HPLC, the most common sorbent used in SPE involves reacting the silica with octadecyl silane to produce particles nearly completely covered with bound C18 chains. This produces a surface generally considered nonpolar and hydrophobic, generating binding with analytes through hydrophobic interactions between the hydrocarbon portion of the analyte and the hydrocarbon chains on the surface. A summary of common SPE phases and key applications is provided in Table 1. This table is not intended to be comprehensive; complete descriptions and information on all available phases is provided in the literature from the SPE manufacturers referenced in this article. Table 1 Summary of common SPE sorbents Sorbent
Mechanism
Typical applications
C18
Reversed phase
C8
Reversed phase
C2
Reversed phase
Polymeric
Reversed phase
Aminopropyl
Normal or reversed phase
Cyanopropyl
Normal or reversed phase Normal or reversed phase Normal phase
Most common SPE and HPLC phase; drugs of abuse, metabolites, peptides, trace organics, especially acids Similar to C18; generally less hydrophobic; also used commonly for drugs and metabolites Less retention than C8 or C18; applications are similar Used for reversed-phase applications at very high or very low pH, where the silica-based phases might degrade Can be used as a weak ionexchange phase; ionizable compounds; phenols, petroleum fractionation, saccharides, drugs Low hydrophobicity; alternative to silica; drugs, metabolites and pesticides Antibiotics, proteins, peptides, less acidic than silica
Comparison of SPE and LLE In comparison with more classical techniques such as LLE, SPE has several advantages: 1. Significantly reduced solvent volume and usage. 2. Ability to sample in the field. 3. Straightforward sample manipulation and ease of method development using single cartridges. 4. Elimination of emulsion formation, a major drawback of LLE for biological samples. 5. Reduced exposure risk to hazardous solvents and samples. 6. Generally inexpensive cartridges; 5–10 times less expensive than LLE. 7. Contamination is minimized; cartridges are made from medical-grade polypropylene. 8. Excellent flexibility; almost limitless available solvents. 9. SPE method development is straightforward. Often the analyst can find a method of extraction in the application notes provided by SPE vendors or by having knowledge of previously developed HPLC methods for analysis of the compounds.
Diol
Silica Alumina
Florisil
SPE Sorbents and Chemistries
Acrylic acid
There is a large variety of sorbents and chemistries available for SPE. Selectivity is generally obtained by covalently bonding a chosen organic moiety to a
Acrylamide
Used to extract compounds from organic solvents Normal phase Similar to silica; can be adjusted for acidic, basic, or neutral analytes Normal phase Pesticides (Association of Official Analytical Chemists (AOAC) and Environmental Protection Agency (EPA) methods), polychlorinated biphenyls (PCBs) Cation exchange Anionic proteins, pigments, phenols, peptides Anion exchange Aldehydes, ketones, carbonyls (EPA applications)
4 III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites
Extraction of Drugs and Metabolites
of drugs from biological matrices has certainly become a mainstream technique.
Biological Systems
Analysis of drugs and metabolites from biological systems is both a common and highly challenging application of SPE. Biological systems often contain both soluble and insoluble matrix components that can easily foul the cartridges or extract along with the analyte(s) of interest, generating interference with the extraction or with subsequent chromatographic analysis, or reduce the activity of the analyte(s) of interest, also reducing extraction recovery. However, owing to the advantages cited above, SPE is the technique of choice in the common clinical, forensic, and toxicological analysis of drugs, especially drugs of abuse, from urine and blood. Methods and procedures for SPE of nearly all drugs of abuse are available; these are detailed in the text references included in this article. Further, nearly all of the SPEequipment vendors have extensive databases of drugs-of-abuse application notes available on the WWW. For example, Varian lists over 160 applications in a search of its database using ‘drugs of abuse’ as a keyword. Since the basic procedures are readily searchable and obtainable online from the vendor websites listed herein, the remainder of this section focuses on recent advances and new techniques from 2005 and early 2006. Research on the development of new methods and applications for SPE of drugs and metabolites is surprisingly increasing, given the large number of available methods in the literature. Recently, analytes in biological matrices well beyond urine and blood, which are still most commonly described in the literature methods, such as brain tissue, oral fluid, hair, milk, and beverages, have been extracted. A literature search, using SciFinder, with ‘solid-phase extraction’ and ‘drugs of abuse’ included about 80 papers in a 15-month period beginning January 2005. The variety of drugs described is also much wider than the traditional drugs of abuse, including muscle relaxants, thyroid medications, and mescaline. There has also been strong interest in g-hydroxybyturate (GHB) and ketamine (K), which have become notorious in use by young people as the ‘date rape’ and a popular ‘club’ drug, respectively. Finally, the instrumental choices used in combination with SPE are evolving; LC-MS and LC-MS/MS are becoming more popular, often supplanting the traditional GC-MS, especially when the most complex sample matrices, such as blood are involved. Also interesting is that most of the papers were written by authors outside the traditional analytical chemistry and separation science communities; SPE
Pharmaceuticals
Pharmaceutical analysis using SPE is mostly focused in the discovery and development processes. As the sample matrices of finished products and raw materials, such as tablets, gels, ointments devices, and capsules are well known, with relatively high analyte concentration or mass, they are generally straightforward to dissolve in common solvents. Therefore, SPE is generally not required for quality assurance assays. In drug discovery, however, the analytes and matrices are not always as well known, and the analytes may be at low concentration, requiring very high selectivity and making SPE an important tool. Recently, to enhance selectivity further than traditional SPE sorbents allow, molecular-imprinted polymers have been synthesized and used as highly selective stationary phases for SPE analysis of very specific compound classes and for chiral separations. MIPs, for instance, have been used with SPE in 96-well plate configuration for the screening of a compound undergoing pharmaceutical development. The high selectivity of the molecular imprinted polymer (MIP) stationary phase allowed highly sensitive determination of the analyte at levels far below traditional SPE. A SciFinder search using ‘solid-phase extraction’ and ‘drug discovery’ as keywords provides about 50 references in the 15 months beginning January 2005. As was the case with biological samples, most of the recent papers involved LC and LC-MS as the instrumental method. Also, most papers involving the use of SPE in drug discovery and development focus more on the instrumental technique (often LC-MS or LC-NMR-MS) than on the SPE, which may in the end be the most difficult portion of the analysis, and therefore it is often difficult to find all the details of the extraction process. A second focus has been on high-throughput methods for screening of large numbers of drug candidates at very early stages in the discovery process. Environmental
Analysis of drugs in environmental samples, especially natural and wastewater, has recently become a major application for SPE. The premise is clear: the drugs and metabolites manufactured and consumed by humans and animals are eventually passed into the environment, plus it is possible that drug residues may be present in the wastewater streams from manufacturing facilities. Endocrine disruptors and steroids are of special concern as these are quite common and
III/EXTRACTION / Solid-Phase Extraction of Drugs and Metabolites 5
have many toxicological effects from long-term human or animal exposure to small doses. The analytical problems are very similar to those involved with drugs of abuse detection from biological fluids, except that the analyte concentrations are often lower. Interestingly, most of the work in this area has occurred in Europe, with relatively little in the United States. Typical studies of this type include the analysis of several pharmaceutical compounds using a standard environmental method for acidic or basic contaminants, or straightforward modifications of these methods. As in the previous discussions, SPE is traditionally combined with GC-MS but is now more often combined with LC-MS. A SciFinder literature search using ‘solid-phase extraction,’ ‘drugs’ and ‘environmental’ as keywords produced about 80 references. A sampling of the drugs examined in these papers includes nonsteroidal antiinflammatory drugs, analgesics, steroids, antibiotics, estrogens, antidepressants, lipid regulators, and b-blockers. Typically, the studies are designed to track the progress of the drug or its degradation products through the wastewater stream and into the environment. Many of the authors note that there is, to date, little literature describing the fate of pharmaceuticals in the environment, although the research interest in this is increasing rapidly. Environmental SPE methods for drugs are either based on traditional environmental methods for other compounds or on clinical or pharmaceutical methods. Further, environmental methods for pharmaceuticals are often performed in combination with traditional methods for other compounds such as organic acids and pesticides. Most work to date is related to analysis of common drug classes such as painkillers, antioxidants, and antibacterial agents.
Conclusions SPE is a staple technique for the preparation of samples, especially for GC and LC and MS. There is a large variety of equipment configurations, including sorbent cartridges, impregnated disks, phase-coated fused-silica fibers and phase-coated stir-bars, and others. This combined with an even larger variety of sorbent chemistries shows that there is an SPE technique appropriate to nearly any sample preparation analyte and matrix combination. SPE continues to be used throughout the analytical chemistry community. See also: II/Extraction: Solid-Phase Extraction. III/Drugs of Abuse: Solid-Phase Extraction.
Further Reading Baker JT. http://www.jtbaker.com (accessed on June 2006). Cole MD (2003) The Analysis of Controlled Substances. Chichester: Wiley. Fritz JS (2003) Solid Phase Extraction. New York: Wiley. Pawliszyn J (1998) Solid Phase Micro-extraction Theory and Practice. New York: Wiley. Phenomenex. http://www.phenomenex.com (accessed on June 2006). Supelco. http://www.sigmaaldrich.com/Brands/Supelco_ Home.html (accessed on June 2006). Telapchak MJ, August TF and Chaney G (2004) Forensic and Clinical Applications of Solid Phase Extraction. Totowa, NJ: Humana Press. United Chemical. http://www.unitedchem.com (accessed on June 2006). Varian. http://www.varianinc.com (accessed on June 2006). Waters. http://www.waters.com (accessed on June 2006).