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Solid-phase extraction in clinical biochemistry
Valerie Walker1 and Graham A Mills2
Addresses 1 Department of Chemical Pathology (NHS) General Hospital Southampton SO16 6YD, UK 2 School of Pharmacy and Biomedical Sciences University of Portsmouth Portsmouth PO1 2DY, UK This article was prepared at the invitation of the Analytical Investigations Standing Committee of the Association of Clinical Biochemists. Correspondence Dr Valerie Walker E-mail: email@example.com
In order to measure low concentrations of analytes in plasma and urine, it is often necessary to extract and concentrate them. With solid-phase extraction (SPE), this is achieved by partitioning the analytes between a solid and a liquid or headspace vapour. A wide range of high-quality materials is now available to do this, offering a variety of separation modes for different applications. These include partitioning using reversed-phase, normal-phase, ion-exchange, restricted-access and immunoaf nity sorbents or molecularly imprinted polymers and, increasingly, combinations of these processes. Solid-phase microextraction was introduced to analyse volatile and semivolatile compounds. The range of sampling formats has expanded from simple packed syringes to cartridges, disks, SPE pipette tips and 96-well plates. These developments have facilitated automated off- and on-line sample processing. The basic principles of SPE and the recent innovations are reviewed here. This is a technological growth area. Some of the developments are nding application in clinical toxicology. However, they could also be of wider value in clinical chemistry for example, for analyses of volatile and non-volatile metabolites, peptides, radioactive elements and trace metal speciation.
Ann Clin Biochem 2002; 39: 464- 477
With increased knowledge of biochemical processes there is a growing need to analyse very low concentrations of compounds that are present with large amounts of interfering substances in blood, urine and other biological samples. Careful sample preparation is often a critical and time-consuming preliminary step and can account for more than 75% of analysis time.1 The aims are to isolate the analytes quantitatively from contaminants, to concentrate them into a measurable range and, ¢nally, to recover them in an appropriate form for analysis. A range of procedures is available, depending on the analyte and the method of quanti¢cation. Examples are centrifugation, chromatography, distillation, ¢ltration, lyophilization and precipitation. Liquid ^ liquid extraction has been the favoured preparative method for gas-chromatographic (GC) and other chromatographic analyses but it is timeconsuming, di¤cult to automate, requires high-purity solvents, is subject to contamination, and recoveries and precision may be poor. Safe disposal of toxic solvents is problematic and expensive.
In solid-phase extraction (SPE), the analytes to be extracted are partitioned between a solid and a liquid. They must have greater a¤nity for the solid phase than for the sample matrix. Interfering compounds are rinsed o¡ and then the analytes are desorbed with a solvent appropriate for instrumental analysis. An alternative approach is to trap interfering compounds on the solid phase, leaving the analytes in the liquid phase;2 however, this does not permit concentration, and is used infrequently. The principles of separation involve intermolecular forces (dipole ^ dipole forces, hydrogen bonding, ionic interactions and Van der Waals forces) between the analyte, active sites on the adsorbent and in the liquid phase or sample matrix, sometimes aided by size exclusion. Although similar sorbents are used for high-performance liquid chromatography (HPLC) and SPE, the particle sizes for SPE are larger (40 mm compared with 5^10 mm for HPLC) and there are fewer theoretical plates per column (5100 for SPE; up to 25 000 for HPLC).3 Whereas HPLC uses continuous elution to separate analytes, SPE uses a sample on/o¡ procedure (on-the-phase retention and o¡-the-phase elution).2^8 Solid-phase microextraction (SPME) is a relatively new application
© 2002 The Association of Clinical Biochemists
tetramethylammonium bromide and tetramethylammonium sulphate). including `designer phases’ developed for targeted extractions of drugs of abuse. 1). but also trap interferences readily. ethyl acetate or methanol. The most widely used bonded phases are C18 sorbents [octadecasilyl (ODS). Here we present an overview of the ¢eld. At pH values above 2¢ 0 these groups dissociate to Si ^ O 7. an acid or base group). with examples of biomedical applications. alkylammonium bromides or alkylammonium hydrogen sulphates (e. This may be exploited to increase the retention of polar analytes such as basic Reversed-phase sorbents5.19 These are hydrophobic siloxanes with hydrocarbon chains or other hydrophobic molecules bound to silanol groups of silica through ^ Si ^ O ^ Si ^ C bonds. methyl or phenyl groups. (a) Monofunctional bonded-silica sorbent. These developments have facilitated automated o¡and on-line sample processing. often acetonitrile.g.17. and trifunctional if it is linked to three oxygen atoms (see Fig. cyanopropyl ethyl. These were reliable and have been used extensively in clinical biochemistry. for acidic analytes. Ann Clin Biochem 2002.16 In 1978. Interaction of the polar moiety with the charged group on the solute forms a non-polar ion-pair which is retained by the sorbent. Others have bonded cyclohexyl. The retention of highly polar compounds on R-P sorbents may be increased by adding an ion-pair reagent to the Figure 1.g. Modi¢cations used in other formats are discussed later. Cyclohexyl phases sorb phenols from aqueous solutions. SPE pipette tips and 96well plates. The following section describes properties of sorbents that are used in column or cartridge format. The sorbents are said to be monofunctional if the bridging silicon is linked to one atom of oxygen. Sorbents used for solid-phase extraction The main modes of separation used are reversed-phase (R-P).g. ion-exchange. but their adoption in clinical biochemistry has been slow. It involves non-polar interaction of the solute with the stationary phase through lowenergy Van der Waals forces. The derivatization during manufacture is incomplete and unbonded hydroxyl (silanol) groups are left on the surface of the silica. The major classes of sorbents in use. There is now a wide range of high-quality materials available.18 These have a non-polar portion (e. this technique is not used widely for SPE. 39: 464. Phenyl phases have a high capacity for cyclic hydrocarbons. o¡ering considerable versatility. v/v) may elute more hydrophobic compounds. Trifunctional sorbents are more resistant to acid pH.5. there were problems with sample contamination from the polymeric resins used as sorbents. a¤nity and restricted-access.17 R-P chromatography partitions organic solutes from a polar phase (generally aqueous) to a non-polar phase. 1octanesulphonic acid) and. emphasizing some of the more recent developments in SPE and its biomedical applications. (CH 2) 17CH 3] and C8 [octyl. a long-chain aliphatic hydrocarbon) and a polar portion (e. Increasingly. normal-phase (N-P).9^14 When SPE was ¢rst introduced in the 1970s. which are available for secondary ionic interactions with the sample.8. Addition of methylene chloride to ethyl acetate (1:1. and special syringe holders for SPME. C 2 phases are useful for very hydrophobic analytes. disks. The a¤nity of the solute for the sorbent depends upon its hydrophobicity. The range of sampling formats for SPE has also expanded from simple packed syringes to cartridges. The most common R-P sorbents for SPE are chemically bonded silica phases. This is a technological growth area. Other sorbents used as HPLC stationary phases were applied to SPE and others have been developed speci¢cally for sample preparation. (CH 2) 7CH3] sorbents.7.18 In contrast to HPLC separations. samples and sorbent when activating the cartridges.477 . which may be in the form of a hydrocarbon chain or polymeric sorbent.Solid-phase extraction in clinical biochemistry 465 of SPE in which volatile and semi-volatile compounds can be partitioned between a solid or liquid stationary phase and a gaseous or liquid phase for subsequent analysis by GC or HPLC. these are used in combination. The newer products are used widely in the pharmaceutical industry and for environmental analyses.7.7. Waters Associates introduced C18 -derivatized silica sorbents as disposable mini-columns (Sep-Pak 1 cartridges). The solute is eluted with an organic solvent. Examples of ion-pair reagents used commonly for basic analytes are alkylsulphon ic acids (e. (b) Trifunctional bonded-silica sorbent. are summariz ed in Tables 1 and 2. C18 phases provide greatest retention.15.g.
bile acids. peptides. antibiotics Catecholamines. C4. acrylate. purines. water-soluble vitamins. fatty acids. methylene chloride. drugs by a mixed adsorption mechanism. N) Non-polar solutions: hexane. phosphates.3polar extraction dihydroxypropoxypropyl. biological strength buffers uids *Also weak ion-exchange. acetonitrile. strength alkaline high ionic sulphonic. Bonded-silica sorbents are stable to organic solvents and acid solutions (trifunctional sorbents to pH 2¢ 0). buffers. and many others Phenols and pesticides in water. C6. acidic methanol. low ionic Acidic buffers. buffers.466 Walker and Mills Table 1. ethyl acetate. O. such as Water. low ionic Alkaline buffers. nucleotides. drug metabolites Pharmaceuticals. Bonding Ann Clin Biochem 2002. therapeutic drugs.divinyl-benzene (PS-DVB) sulphonic acid Weak: propylcarboxylic acid polymers: PS-DVB carboxylic acid. diethylaminopropyl Wide-pore weak: Accell plus QMA (Waters). propylsulphonic acid. hetero-atoms (S. these hydroxyl groups may be inactivated by subsequently blocking them with trimethylsilane during manufacture. Alternatively. hexane. C8. polymeric pyrimidines polystyrene. lipids Methanol. CBX carboxylic acid (JT Baker) Water. aminopropyl*. chloroform. 39: 464. carbohydrates. are rigid packing materials with good £ow characteristics. Solid-phase extraction sorbents: traditional separation mechanisms Separation Reversed-phase or non-polar extraction Sorbent Bonded silica: C18.co-N-vinylpyrrolidone) (Oasis HLB. ethyl acetate Vitamin D metabolites. biological uids aromatic Graphitized carbon (aromatic carbon) Co-polymers: styrenedivinylbenzene. drugs in blood and urine Hydrophilic groups such as hydroxyl. cyclic nucleotides. vicinal (two on adjacent silicon atoms) or geminal (two on the same silicon atom). acetone. a process known as end-capping (see Fig. may have a wide range of bonded groups and are . pesticides. Waters) Normal-phase or Bonded silica: cyanopropyl. methylene chloride Applications (examples) Drugs of abuse. C2. kieselguhr (diatomaceous earth) Alumina Florisil Anion-exchange Bonded silica Strong: quaternary amine Weak: aminopropyl. poly(divinylbenzene. complexed copper and cadmium. polyethyleneimine (JT Baker) Acids. isopropanol. steroids. pyrimidines.carboxylic acid Wide-pore weak: Accell plus CM (Waters). biological strength buffers phosphoric acids uids Organic acids. lipid separations. phenyl. 2). even the most rigorous endcapping procedures do not de-activate all the silanol groups on the surface of the sorbent. such as alkyl.477 strengths may di¡er when the free silanol groups are single (isolated). oils. fatsoluble vitamins. phenols Non-polar Polar solutions: functional groups water. carboxylic. amine. strength acidic high ionic buffers. primary and secondary amine* Silica: silica gel*. primary and secondary amine. pharmaceuticals Cation-exchange Bonded silica Bases such as Strong: benzene sulphonic acid. cyclohexyl. amines. C3 Typical analytes Matrix Typical solvents Methanol. chloroform. vitamins. However. 2. end-capped cyanopropyl Wide-pore: C4.
biological uids. benzoylecgonine. groups of related water or chloroform propranolol.477 . phencyclidine. carbohydrates. 47) Reversed-phaseBond-Elut Certify (Varian): cation-exchange Silica-bonded C8 hydrocarbon Silica-bonded strong cation-exchanger Isolute Con rm HSX (IST): Silica-bonded C8 hydrocarbon Silica-bonded strong cation-exchanger Bakerbond Narc-2 (JT Baker): Silica-bonded C18 hydrocarbon Silica-bonded aromatic sulphonic acid Oasis MCX (Waters): Oasis HLB co-polymer with benzene sulphonic acid Clean Screen (BAS Technology): Silica-bonded hydrophobic and ionexchange groups Reversed-phaseBond-Elut Certify II (Varian): anion-exchange Silica-bonded C8 hydrocarbon Silica-bonded anion-exchanger Isolute Con rm HAX (IST): Silica-bonded C8 hydrocarbon Silica-bonded strong anion-exchanger Oasis MAX (Waters): Oasis HLB co-polymer with anionexchange groups Reversed-phaseIsolute Multimode (IST): cation+anionSilica-bonded C18 hydrocarbon: exchange +strong cation-exchanger +strong anion-exchanger Cation+anionBond-Elut Accucat: exchange Silica-bonded strong cation-exchanger Silica-bonded strong anion-exchanger Immunoaf nity (7. basic drugs. hair digests Drugs of abuse: opiates.Analytes with vicinal Water. acidic and basic metabolites Drugs. internal adsorption uids cortisone. buffers. R-P ˆ reversed-phase. 37. nucleotides Environmental water Silica-bonded phenylboronic acid: Bond. corticocompounds steroids. 41. Solid-phase extraction sorbents: newer separation mechanisms Separation (references* ) Sorbent Typical analytes Matrix Applications (examples) Mixed mode (5. 16. controlled 24) pore glass. LSD. sites: non-polar if Rudrocortisone P. salbutamol. palladium Water *See also sorbent manufacturers’ literature. tricyclic antidepressants. non-steroidal anti-in ammatory drugs Non-polar anions or Water cations Cations and anions Water. procainamide. acids or bases if ionic Catecholamines. compounds extracts 7-hydroxycoumarin Dependent on Water. 29. biological uids Fractionation of complex mixtures Af nity (7. acidic polar buffers. anabolic steroids Anions (acidic) non. organic acids. HiSep (Supelco). LSD. Antibodies bonded to silica. dexamethasone. Ann Clin Biochem 2002. 21. endogenous Biological uids Oestrogens. a atoxins Single analytes or Biological uids: Theophylline.Water. morphine. prednisolone. 8. other soft gels Cations (basic) non. 7. anabolic steroids. benzodiazepines. 39. biological Plasma cortisol.44. Lichospher ADS (Merck). biological uids ¢9-Carboxytetrahydrocannabinol (cannabinoid metabolite). amphetamines. 36) Miscellaneous Covalent bonding (3) Trace metal bonding (8) Molecularly imprinted polymers ChromSper 5 Biomatrix (ChromPak). bupivacaine. biological Elut PBA (Varian) diol groups uids Dipyridylamide-functionalized polymers Mercury. agarose. alkaline polar buffers.Solid-phase extraction in clinical biochemistry 467 Table 2. BioTrap 500 (Chromtech) Catecholamines. 35. 39: 464. Ultrabiosep (Hypersil).32) Restricted-access matrix adsorption (7.Water.
Bonded-silica sorbents have been used widely in clinical chemistry. (f) 96-well extraction plate. (d) pipette tip with membrane extraction disk. Applications include extractions of drugs. these sorbents are generally more hydrophobic. Alternative sorbents used for R-P extractions include highly cross-linked co-polymers (styrene ^ divinylbenz ene).468 Walker and Mills Figure 2. Wide-pore (427 ¢5 nm) sorbents with bonded short-chain (C3 or C 4) hydrocarbons allow better access for large molecules. (e) membrane extraction disk suitable for attachment to a syringe. loading of bonded groups (as a percentage of carbon loading) and surface area (generally 200^600 m 2/g). Because of poor surface contact with aqueous solutions. larger molecules (42000 Da) may be excluded from 6-nm pores and adsorb better with 12 ¢5-nm pores.16 graphitiz ed carbon and porous graphitic carbons. there is substantial variation between bonded phases from di¡erent manufacturers and even between batches. peptides and steroids. Examples of sampling formats for solid-phase extraction: (a) syringe barrel assembly packed with sorbent. Capacity and reproducibility are poor if the sorbent is allowed to dry out. around 250^600 mL of solvent per 100 mg of sorbent is needed. This may be an advantage.or trifunctional.477 . acetonitrile or methanol. However.20 Compared with silica sorbents. Typically.7. being smaller for low loading.5. to 9^14% for C8 phases and 2¢7^5¢7% for C2 phases. (b) syringe barrel assembly with membrane extraction disk. on the particle size distribution and pore diameter (typically 40 mm and 6 nm.2 Despite improved technology. since the analyte may be eluted from the solidphase more readily with a smaller volume of eluting solvent. 39: 464.8. End-capped bonded-silica sorbent. Carbon loading ranges from 12^18% for C18 phases. reproduced with permission from 3M Company USA. reproduced with permission from Alltech Associates UK. relatively inexpensive. respectively). the greater the area. Loading a¡ects the capacity of the sorbent. Pore size determines the surface area of the sorbent: the smaller the pores. The properties of these sorbents depend on whether they are endcapped. mono.15. pre-treatment is necessary with a solvent such as acetone. Ann Clin Biochem 2002. (c) sorbent-packed cartridge. more retentive. stable over the pH range 0^14 and do not Figure 3.
Their extraction may be possible with less-retentive silica-bonded phases now available for N-P SPE (aminopropyl-. polar compounds dissolved in a non-polar solvent such as diethyl ether or n-hexane are extracted by adsorption to a polar sorbent. styrene ^ divinylbenzene) are also available that incorporate a chelating group.17 Selectivity of ionexchange is in£uenced by pH. another polymeric sorbent.17 In ion-exchange. 39: 464.5. organic acids and vitamins.Weak interactions are involved: dipole ^ dipole. the pH of the samples is adjusted so that the solute molecules are ionized. Alumina and silica adsorb water.7. impurities in these sorbents caused contamination. The analytes are then desorbed with solvents with a high eluting power (eluotropic strength. ethylene dimethacrylate (both from Amberlite) and other polymeric materials such as Porapaks 1 .7. Strong cation-exchanger sorbents have sulphonic acid groups. They are then adsorbed at oppositely charged sites on the sorbent. most require pre-conditioning and give poor recoveries if allowed to dry out. basic compounds are retained better on silica and acidic compounds on alumina. Normal-phase sorbents5. However. They are being used increasingly in drug analyses. Silica-bonded phases dissolve at extremes of pH.21^23 Ann Clin Biochem 2002. eo40¢6) such as methanol (eo ˆ 0 ¢73). they can be eluted by reducing the pH by 2 units below their pKa. the sorbents used were alumina. Acidic compounds are 50% ionized as anions at a pH near the pKa. they can be eluted by raising the pH by 2 units above their pKa. For example. can be used in automated on-line combined extraction and analysis systems. They are used to extract basic analytes (e. purines and pyrimidines). because of their compatibility with C18 HPLC columns. in clean-up procedures for environmental analyses. reduces retention of analytes and leads to variable recoveries. Ionization of weak exchangers is pH-dependent. citrate may compete for anion-exchange sites.g. Oasis 1 HLB (Waters) combines hydrophilic N-vinylpyrrolid one and lipophilic divinylbenz ene. These sorbents must be stored in a desiccator and are often dried before use. Ion-exchange and chelating sorbents2.16. be extracted from water and Sorbents based on molecular recognition: immunoaf nity sorbents and molecularly imprinted polymers These sorbents may be highly selective for trace levels of analytes in the presence of high concentrations of interfering substances. providing a hydrophilic ^ lipophilic balance. Before the introduction of bonded phases. fatty acids. Strong cationexchangers in the Ba2+ or Ag+ forms (from Alltech Associates) are used to remove sulphate and halides. and OH 7. catecholamines. cyanopropyl. In general. Weak exchangers are used when analytes are so tightly attracted to strong exchangers that they cannot be eluted. Sorbents (e. Ion-exchangers may be silica. Exchange sites of strong ion-exchangers are ioniz ed at any pH. This deactivates hydrogen-bonding sites. respectively. They are used to extract acidic analytes and there are reported applications for bile acids. usually iminodiacetate in the Na+ form. Graphitized carbon extracts very polar and water-soluble analytes from aqueous samples.or propyldiol-bonded silica). Polar solutes may. ionic strength and £ow-rate. F 7 and C 2H 3O 27 from strong anion-exchanger sorbents. hydrogen bonding and p ^p electron interactions. therefore. Biomedical applications of N-P SPE have included analysis of vitamin D metabolites. Very polar compounds such as carbohydrates or amino compounds bind very tightly to silica and alumina and cannot be eluted.or resin-based.477 . weak sorbents have carboxylic acid groups. These materials are used to manufacture mixed-phase sorbents (see below). cyclic nucleotides. weak sorbents have primary or secondary amine or aminopropyl groups.7.e. kieselguhr (diatomaceous earth) and silica. These can be used to exchange transition metals and divalent cations for Na+. Ions on the sorbent are neutralized by oppositely charged counter-ions. It is used for extracting a range of environmental pollutants from contaminated water samples. Strong anion-exchangers have quaternary amines [^ N+(CH 3) 3] as exchange groups. Unfortunately. from polar organic solvents. counter-ions. Raising the pH by 2 units increases ionization to around 99% and promotes retention. For extraction. Newer co-polymers have high retention capacity and.8 This is also the case for Abselut Nexus (Varian). This sorbent does not require pre-conditioning and there is no problem from drying out before solvent application. as cations) at a pH near their pKa. Chromosorbs 1 and Tenax 1 (diphenyl-pphenylene oxide). Florisil 1 (activated magnesium silicate). H+ and Na+ are most easily displaced from strong cationexchanger sorbents. 20 Early polymeric sorbents used for SPE were a crosslinked polystyrene resin (XAD-1) and styrene ^ divinylbenzene. Li+. Extraction is poor when there are high concentrations of competing ions in the sample matrix. carbohydrates and phenols. They are eluted by changing the pH and/or increasing the strength of competing ions. compounds are extracted by a highenergy ionic interaction with the sorbent. lipids. Reducing the pH by 2 units increases protonation to around 99% and promotes retention.17 In N-P SPE.g.Solid-phase extraction in clinical biochemistry 469 have secondary ionic interactions. Basic organic compounds are 50% protonated (i.
Removal of the template then leaves an imprint sterically complementary to the ligand. Oasis MCX and MAX (Waters) are mixed-mode sorbents produced by addition of a strong cation-exchange group or anionexchange group. nortestosterone.21. Bio Trap C18 column (Chromtech) could tolerate up to 15 mL of plasma without changing retention pressure. In these. LSD and morphine in urine. Silica C18 restricted-access matrix sorbents e¡ectively exclude plasma proteins.24.g. Oasis MAX and MCX (Waters). Subsequent acidi¢cation protonates the drug amino group.16.7. oestrogens. dexamethasone.27 They are stable over a wide pH range. including proteins. £udrocortisone and prednisolone with an on-line SPE liquid chromatography ^ mass spectrometry (LC ^ MS) procedure. These sorbents are being used increasingly for drug analysis.3. plasma and urine.39^44 Applications were recently reviewed and methods presented for analysis of ¢ve drug classes recognized by the US Restricted-access matrix sorbents These are porous silica particles that exclude large matrix components.T. which then binds strongly to the sorbent by ionic interaction. washed at pH 7 and eluted under mild conditions (e.37 Around 80% of drugs contain nitrogen that can be protonated.7.7.24 Molecularly imprinted polymers are produced by using a target analyte as a template in a casting procedure. They include procedures for serum phenytoin.34 A 2064 mm i. with PBS ^ methanol or PBS ^ ethanol at pH 2^4). there is now a new generation of mixedmode polymers available for extraction of a wide range of analytes.38 Mixed-mode sorbents such as Bond-Elut Certify (Varian Analytichem). The few applications of immunoa¤nity SPE to biological £uids (animal and human) have included anabolic steroids and corticosteroids in faeces and urine. with clogging only after 45 mL of plasma. Most immunoextractions are for analysis by HPLC. and morphine and its metabolites in blood.23. Isolation is therefore possible by cationexchange in addition to R-P adsorption. reported for restricted-access matrix SPE. plasma and urine prior to con¢rmatory analysis. The polymers can then be used for SPE of the analyte and structurally related compounds and for chiral separations. 39: 464.).7.33. the drugs are eluted with a basic solution to break the ionic bonds.477 .26 There have been few reported biomedical applications of molecularly imprinted polymer SPE. This is useful for extracting lipophilic compounds with an ionizable functional group.7.37. The sorbents may be present as mixed particles within the same cartridge.35 Another was for plasma cortisol. ionic or a¤nity interactions. particularly drugs (see below). cortisone. or present in di¡erent cartridges that are stacked. which migrates separately from the analyte in the subsequent HPLC analysis.30 7-hydroxycoumarin31 and the anaesthetic bupivacaine 32 in plasma. Poor recoveries have been a problem with layered sorbents. Small molecular mass analytes entering the pores may be concentrated there by conventional hydrophobic. 3. layered on top of each other. Clean Screen DAU (World-Wide Monitoring) and Isolut 1 Conform HAX and HSX (IST) are being used increasingly to isolate drugs of abuse from blood.5. Baker). SPEC 1 11 (Ansys Diagnostics Inc. a bonded R-P covers the pore surface with C 4. Hydrophilic interferences are removed by washing with bu¡er or water. which can then be separated chromatographically. C8 or C18 ligands.8. It is di¤cult to remove the original template molecule completely and residual material may cause analytical interferences. Bakerbond Narc-2 (J.d.7 Internal surface reversed-phase supports are the most popular restricted-access matrix sorbents.7 The pH is adjusted so that the analytes are present in a neutral or negatively charged form. respectively. controlled-size glass particles.470 Walker and Mills Immunoa¤nity sorbents have biological antibodies covalently linked to silica. These allow simultaneous extraction and clean-up of blood. 28 theophylline.3. to the Oasis HLB divinylbenz ene ^ N-vinylpyyrolidone co-polymer. Matrix interferences are removed with a methanol wash. Finally.29 propranolol.5.g. by selection of pore size or by chemical repulsion by an appropriate hydrophilic coating on the sorbent surface.7 Analysis of bupivacaine is one of the few clinical analyses Ann Clin Biochem 2002. The most popular have an ionexchange group bonded to silica. This problem may be overcome by using a closely related compound to make the template.25 Sorbents are conditioned with phosphate-bu¡ered saline (PBS) at neutral pH.5 However.24 The cross-reactivity of antibodies can be exploited to extract structurally related compounds (e. Multi-modal SPE uses two or more SPE phases in series to improve clean-up. The combination is chosen so that an analyte is retained by both mechanisms.7. a drug and its metabolites).21. usually by GC ^ MS. 21.5. They are then extracted by R-P hydrophobic interaction with the sorbent. Samples are applied at pH 5^8. Columns have been re-used up to 50 times for biological samples.22. R-P bonded silicas that are not end-capped provide mixed-mode sorption [hydrogen-bonding to silanol (O 7) groups and R-P adsorption to alkyl chains]. agarose or other soft gels.26 Monomers such as methacrylic acid or 2.36 Mixed-mode sorbents and multi-modal solid-phase extraction Mixed-mode sorbents have both non-polar and ionexchange functional groups.or 4-vinylpyridine are cross-linked around the template with a cross-linking agent.
with analytes dissolving and di¡using into the bulk of the coating. Some phases have di¡erent thicknesses (e. benzoylecgonine (cocaine metabolite). With in-tube SPME. methamphetamine.). Usually the thinnest acceptable ¢lm is employed to reduce extraction times. The type of ¢bre used a¡ects the selectivity of extraction. antipsychotics and various over-the-counter preparations. 30 mm. There are as yet no reported applications for biological samples.5 Preliminary hydrolysis is needed to release opiates from glucuronide conjugates. the ¢bre is withdrawn into the syringe barrel for transfer to a GC injector port 9 or a modi¢ed HPLC Rheodyne valve. phencyclidine. agitation of the sample by stirring or sonication improves transfer of analytes to the ¢bre. It is compatible with GC and HPLC desorption processes. Ann Clin Biochem 2002. They can be damaged if exposed to high levels of organic analyte or strong acid or alkali.49 The method uses a modi¢ed syringe assembly that houses a short fused-silica micro-¢bre coated with a 7^100 mm ¢lm or layer of stationary phase material. cocaine. Most polymer ¢lms are coated directly (non-bonded types) or are partially cross-linked.T. and Blevins and Hall 45 reported the use of SPEC disks in a manual extraction method for a marijuana metabolite. available from Gerstel GmbH & Co. poisons and their metabolites in blood. Polettini et al. from 3M). Miscellaneous sorbents Compounds with vicinal (adjacent) diol groups (e. Both polydimethylsiloxane (PDMS) and polyacrylate phases extract via absorption. including antidepressants. Degel39 and de Zeeuw et al. 7 mm. extending the range of applications.Solid-phase extraction in clinical biochemistry 471 Substance Abuse and Mental Health Services Administration: amphetamines. SPME can be operated in two modes: either with direct immersion into the sample matrix or with exposure to the headspace vapour above the sample in a heated sealed vial.3 In industry. 39: 464. plasma and urine using the Hewlett-Packard HP 7686 PrepStation. methadone. 6-monoacetylmorphine and morphine]. as with conventional GC stationary phases.477 . the externally coated ¢bre is replaced with a length (50^100 cm) of conventional internally coated fused-silica GC capillary column. amphetamine. with analytes attaching to the surface of the ¢bre as a monolayer. In both modes. a small magnetic stirring bar coated with a 0¢3^1 ¢ 0 mm layer of suitable stationary phase material extracts the analytes directly from solution.40 achieved good recoveries with SPEC disks in general screens for drugs in urine. strontium and technetium (Empore 2 Rad disks. codeine.46 Other clinical applications of mixed-mode sorbents include analysis of anticonvulsant drugs 47 and diclofenac 48 in plasma. polar ¢bres are used for polar analytes and non-polar types for non-polar analytes. derivatized and analysed by GC ^ MS.44 The eluted drugs were derivatized and analysed by GC ^ MS in full scan mode. The PDMS-Carboxen ¢bre extracts highly volatile solvents and gases. PDMS-Carboxen. followed by a semi-automated identi¢cation procedure. morphine and codeine.7 Solid-phase microextraction SPME was introduced in 1990 by Arthur and Pawliszyn as a rapid extraction technique for the analysis of volatile and semi-volatile compounds from a variety of matrices. Drugs were extracted with WWM-DAU (World-Wide monitoring) solid-phase 100-mg extraction cartridges with acidic and basic elution. opiates. ecstasy. carbohydrates and nucleotides) may be extracted by covalent bonding with silica-bonded phenylboronic acid (Bond-Elut PBA from Varian). and the list is growing. SPE phases coated with derivatizing agent were used for in situ derivatization of analytes as they passed through SPE cartridges.51 Several ¢bre coatings are available (from Supelco) 10^14 for the extraction of volatile and semivolatile compounds.g. In general. metal-loaded sorbents that complex organic compounds have been used for water puri¢cation. 100 mm) and this a¡ects both equilibrium time and sensitivity of the method. eve. The technique involves either the equilibrium or non-equilibrium partitioning of analytes between the stationary phase and sample. Baker) columns were used in an automated procedure to analyse urine for drugs of abuse and their metabolites and a wide range of other drugs.12. The bi-polar PDMSCarboxen ¢bre has a mixed carbon coating with small micro-pores and a surface area of approximately 1000 m 2/g. and mercury (Hg 2+) and palladium (Pd 2+) have been extracted from environmental water with dipyridylamine -functionalized polymers. phencyclidine and tetrahydrocannibol (marijuana). catecholamines. Di¡erent methods are used to attach the coating to the fused-silica core. Bakerbond Narc-2 (J. After sampling.41 reported a fully automated procedure for systematic analysis of drugs. Carbowax-templated resin. Two other variations of the SPME technique have become available recently. IsoluteCon¢rm 1 HSX (IST) cartridges were used in an automated GC ^ MS procedure for drugs of abuse in hair [cocaine.50 The ¢bre is then exposed and the analytes are desorbed into the hot GC injector or eluted by mobile phase into an HPLC column. Analytes are adsorbed as the sample is passed through the column and then desorbed by a separate desorption phase (usually the mobile phase) for HPLC analysis. The remaining types [Carbowaxdivinylbenzene (DVB).13 In stir-bar sorptive extraction (Twister 2. PDMS-DVB] are mixed coatings and extract via adsorption.3.g. 8 Solid phases built around crown ethers have been used to extract radioisotopes such as radium.
or in 96-well plates.9.6. The packing materials generally have a mean particle diameter of 40^50 mm. but other solid phases have since been introduced. up to 50 or more times). chelating and mixed-mode sorbents. Rigid particle-embedded glass bre disks [e. few have used SPME to analyse endogenous compounds in biological matrices. Ann Clin Biochem 2002.13. depending on the sample matrix.66 Many of these problems are lessened by the use of SPE disks or membranes. Generally they require only small volumes of eluting solvent and this can help in concentrating the eluted analyte before analysis. they may be pushed through by applying pressure to the top of the inlet.55 Drug analyses include amphetamines.13.54. tricyclic antidepressants. They are £at disks.3. antihistamines and phencyclidine.12.7.2. Problems with SPE columns and cartridges are that sample processing rates are slow because of their small cross-sectional area. They have a male luer ¢tting so that they are interchangeable with di¡erent SPE vacuum manifolds. kinetic and mass transfer processes. Empore 2 extraction disks (3M). ENVIDisks (Alltech Associates). Applications have been reviewed recently. The mass of sorbent used determines the sample capacity.8. which can a¡ect GC analyses. They can be used under vacuum. volatile anaesthetic gases and solvents and pesticides in blood and urine.61 volatile compounds in blood. 4^96 mm in diameter (most often 47 mm) and typically 0¢5 mm thick. Deuterated or C13 analogues for a number of compounds are available for use as internal standards for quantitative selective-ion monitoring analyses when using MS detection. Sorbent (generally 100 mg.53 SPME is used widely for analysis of environmental pollutants.) and Resprep2 (Restek)] contain 10^30 mm chemically bonded silica particles incorporated into a glass ¢bre matrix. Fibres can be re-used several times (e. leading to poor recovery and reproducibility. They are usually polypropylene or polyethylene open syringe barrels ranging in size from 1 to 25 mL. or suction from a vacuum manifold.61 These compounds are notoriously di¤cult to analyse.17. in both direct immersion and headspace modes.477 which is usually around 5% of the sorbent mass. Cartridges have a polypropylene body with female and male luer tips to apply pressure from a syringe. including styrene ^ divinylbenz ene. SPME is a complex multi-phase equilibrium process.65.14.53 Volatile sulphur compounds were extracted from urine headspace vapour with PDMS-Carboxen SPME ¢bres and identi¢ed by GC ^ MS.38. Channels in the sorbent bed may cause disturbances of solvent £ow through the sorbent and reduce the retention of analytes. 39: 464. with 10% (w/w) of PTFE and approximately 90% sorbent.59 steroids 60 and volatile compounds in a variety of metabolic disturbances.45.g. Glass or Te£on syringes with stainless-steel frits are also available to avoid contamination by plasticizer.66 Syringe barrels have been most popular. in pipette tips. Benzodiazepines. antidepressants and analgesics were identi¢ed in body £uids using SPME and GC ^ MS.9. Sorbent material is packaged between 20-mm frits. Because of their . ion-exchange.126.96.36.1992 Walker and Mills Bonded ¢bres are more resistant. with pressure or with centrifugation.56 SPME has been used for speciation of arsenic (as monomethylarsonic acid and dimethylarsinic acid) in urine 57 and to monitor di¡erences in urinary organic mercury among subjects with and without mercury amalgam tooth ¢llings. The technique is being used increasingly to analyse drugs and their metabolites. nicotine and cotinine. Alternatively. The bed volume is 125 mL per 100 mg of sorbent. 5.65^68 Particle-loaded membranes [e. The ¢rst disks manufactured contained C18-bonded silica particles.g. they become blocked by particles and adsorbed matrix. Reported applications include quanti¢cation of trimethylamine in urine for diagnosis of inherited trimethylaminuria (¢sh odour syndrome).65.52 analysis of urinary organic acids. or pulled through by centrifugation. They consist of 8^12 mm sorbent particles embedded in a web of inert polytetra£uoroethylene (PTFE or Te£on) micro¢brils. pethidine (meperidine). They are available in a range of sizes and often give faster £ow-rates with less clogging compared with loaded PTFE membranes.58 So far.14 Quanti¢cation is usually achieved by external calibration techniques. SPEC disks (Ansys Inc. 3). Samples and solvents may pass through the column under gravity or may be aspirated by vacuum applied to the syringe outlet. foodstu¡s and pharmaceutical preparations. The theory of the thermodynamic. has been discussed in depth. phenothiazines.53 In some SPME applications the analytes have been derivatized during the extraction procedure. Usually they are supported on a fritted glass ¢lter in a standard ¢ltration apparatus.62 isoprene 63 and formaldehyde in breath 64 and volatile fatty acids in faeces. sorbent packing density is variable and sorbent impurities may cause contamination. Novo Clean disks (Alltech Associates)] look like ¢lters.g. 500 mg or 1 g) is sandwiched between 2620 mm fenestrated polyethylene plates (frits). but they are also available in preloaded syringe barrel or cartridge holders. SPE disks are available commercially in at least four di¡erent types and can a¡ord a number of advantages over packed barrel and cartridge formats. Solid-phase extraction sampling formats There is now a variety of formats available (see Fig. are irregular and have a pore size of 6 nm.
Instead of using a closed well format. Because only a fraction of the sorbent used for conventional SPE is incorporated into glass micro¢bre disks.and fastest parallel-processing equipment. smaller solvent volumes are needed for conditioning and elution. and safer operation because of reduced exposure to hazardous solvents and biological samples. Sample clean-up and enrichment are achieved by SPE in association with a Gilson cartesian robotic sampler.g.3. SPE pipette tips 2. Over typical £ow rates of 10^100 mL/min for a 47-mm disk. They can be used in many automated and robotic systems and are used extensively for sample preparation in the pharmaceutical industry. The sample -processing speed depends upon the application and analyser. to remove salts and non-volatile matrix components).70 In some cases.70 The SPE procedure may be automated on work-stations o¡-line and the sample extracts then injected onto an analyser. as packed beds (generally 10^200 mg).69. with numerous samples being processed simultaneously ^ for example. Use of the smallest mass of sorbent needed for the application reduces solvent require ment and elution time. Liquid can be drawn upwards or forced down through the disk.5. The large cross-sectional area reduces the risk of plugging by contaminants. reducing the risk of crosscontamination. Since £ow-rate is proportional to the square of diameter. since the analyser can resolve a wide range of analytes e¤ciently. Speedisks2 (JT Baker) consist of a thin sandwich of 10-mm sorbent particles held without any PTFE support between two glass ¢bre ¢lters held in place by a screen. They consist of moulded polystyrene or polypropylene plates with 96 individual columns arranged in rows of 8612.65 Ninety-six-well extraction plates 3. possible cost savings from reduced analysis time and the possibility of overnight runs.70. the SPE plates have a £ow-through system and they are set on top of a 96-well collection system. Memsep 2 disks (Waters) are stacked cellulose disks compressed into a continuous bed with pores functionalized with ion-exchange groups. recovery and accuracy with fewer operator errors.73 Samples may be processed serially. Plates are available for a full range of chemistries. Ninety-six-well SPE method development kits are available with a di¡erent sorbent in each row. Precision was found to be better for positive pressure £ow systems than for vacuum-controlled £ow.70 Possible disadvantages are risks of carry-over and problems from sample instability if there is delay between sample preparation and analysis. The SPE packing is Separan S-hydroxyethylmethacrylate -B10 sulphobutyl (HEMA-SB polymer. A throughput of up to 40 samples/day is possible. or PTFE or glass ¢bre disks providing 9^15 mg of sorbent. enabling assessment of di¡erent separation protocols simultaneously. which allows for a more concentrated sample for subsequent analysis.70. The tips can be used for single samples. an automated SPE system may be interfaced directly to an analyser.73 An innovative application for automated SPE is HPLC analysis of urinary free catecholamines using an automated sequential trace enrichment of dialysates (ASTED) system. This allows higher £ow-rates than for conventional SPE columns. to 25^50 samples/ h and up to 400 samples/ h for the fastest serial. A stainless -steel enrichment cartridge replaces the loop on the Rheodyne injection valve of the HPLC system. they do not need external support but may be used in conventional SPE columns. Unwanted wells may be covered. They can be used in manual and automated liquid-handling operations. The disks are thin and packed with particles with a large surface area for reasonable e¤ciency.70 enable rapid preparation with high sample throughput.e. Some require only a simple cleanup procedure (e. These may have short packed sorbent beds or disks. including the newer mixed-mode sorbents. 576 or 1152 wells) for highthroughput screening applications. Rates vary from around 3 samples/ h with simpler instruments. usually an HPLC with computer-controlled column switching. and improved mechanical stability of the sorbent bed reduces channelling.or 24-place vacuum manifolds or with 96-well plates.73 Some systems automate SPE disk procedures. or in parallel. 39: 464. Automation of solid-phase extraction The bene¢ts of automating SPE are improved precision. Disks (4 mm) preloaded into SPE pipette tips are available.7 are conventional pipette tips ¢tted with an SPE disk (Ansys Diagnostics).70 O¡line processing o¡ers £exibility and avoids interface problems with the analytical devices. The degree of sample puri¢cation needed depends on the analysis to be undertaken. The ASTED system has also been used to Ann Clin Biochem 2002. with one sample being analysed as the next undergoes SPE.477 . Anachem Ltd) with retention by an ion-exchange mechanism. Samples may be processed with a vacuum manifold or with a centrifuge with special micro-plate carriers. Alternatively.Solid-phase extraction in clinical biochemistry 473 rigidity. particle-loaded membranes have been reported to provide between 4^966 and 9^1467 theoretical plates.74^76 The SPE cartridge is regenerated and may be used several hundred times.71 Zhang and Henion 72 developed a robotic 96-well plate system using 3M Empore 2 C18 extraction disks to extract oestrogen sulphates from human urine. with 12.5. several 96-well plate blocks are used together (i.66 it may be increased by using larger disks. LC/MS ^ MS). respectively.
miniaturized. There is still substantial variation between silica-bonded sorbents from di¡erent manufacturers and even between batches. Capacity is increased by using more Ann Clin Biochem 2002. The solvent must be compatible with the ¢nal analytical method. The nature of the matrix: its pH. There are recommended experimental protocols for an ordered approach.g. a derivatizing reagent enabling simultaneous elution and derivatization of the analytes. For biological applications. and slightly polar analytes. or replaced by. The search should extend to other industrial applications to gain advantage of the newer sorbent technologies. In practice.17 Because of water sorbed to silica phases. For example. phenytoin. is selected to elute moderately and strongly polar analytes from polar sorbents. hydroxyl. Disks enable the development of simpli¢ed. Mixtures are often best. The amount of sorbent used must be su¤cient to retain all the analytes in the sample. Sorbent manufacturers have large databases of methods in use.78 However. It is a measure of the solvent’s elution strength of solutes from a particular adsorbent. but not yet for use with HPLC.7. This is the maximum volume of sample that can be applied before the sorbents become saturated and cannot retain additional analyte. ethyl acetate or chloroform for GC and GC ^ MS.477 sorbent.474 Walker and Mills analyse phenobarbitone. The analytical detection limit and its sensitivity. they are usually dissolved in an aqueous matrix. . It indicates the capacity for the analyte. It can be measured experimentally or predicted from a variety of experimental methods. miscible. methanol. analytes from organic solvents. The nature of likely contaminants. Retention of matrix components reduces the e¡ective capacity of the sorbent. carbonyl. The eluting solvent should disrupt analyte ^ sorbent interactions. whether it is aqueous or organic. Existing methods may be adaptable to the application.74^76 Smith and Lloyd 73 reported on 20 automated SPE systems available commercially in 1998. The concentration of analyte in the sample.66 The suitability of a device for an application is indicated by the breakthrough volume. The future Solid-phase extraction is a rapidly developing ¢eld. More automated systems are required .17. it is helpful to carry out a literature search when planning a new procedure. More standardization of sorbents is required with better reproducibility and recoveries. in response to the expansion in applications.17. Those with very polar functional groups which are not ionized (amino. its solubility in aqueous and organic solvents. a bridging solvent (e. ethyl acetate) may be added to displace water before eluting. it is common to add a polar. 39: 464. but this is at the expense of larger eluting volumes and hence greater dilution of the analyte. . Alternatively. Choice is directed by the polarity of the analytes. The chemistry of the analyte: its functional groups.5 In some applications the eluting solvent has been mixed with. Methanol or acetonitrile are often selected for HPLC. theophylline and serum total glucocorticoids. For 40^60 mm particles it is about 120 mL per 100 mg of sorbent. The approach to method development is still largely empirical and based on trial and error. Methylene chloride (eo ˆ 0¢32) elutes non-polar analytes e¡ectively from non-polar bonded phases. .66 The capacity will be reduced if other compounds in the sample are also retained. The elution volume should be 2^5 times the volume required to ¢ll the interstitial spaces and pores of the sorbent. phenylbutazone. solvent to a non-polar eluting solvent to wet the sorbent bed e¡ectively. or the sorbent may be dried under vacuum. currently driven by combinatorial chemistry methods employed by the pharmaceutical industry with the associated need to extract and analyse large sample numbers rapidly. A sorbent is required that retains the analyte but not interfering substances. rapid procedures that can be automated. extending the versatility of SPE. ionic strength. sulphydryl) will be retained on polar sorbents. if a very non-polar eluting solvent such as n-hexane is to be used. N-P sorbents are used to extract polar. . Autosamplers that permit both temperature-controlled headspace and agitated direct immersion SPME sampling have recently become available for use with a range of GC instruments.77 Alternative computer-based strategies have been proposed based on theoretical considerations. Selection of a solid-phase extraction method It is necessary to consider: . a number of issues still need to be addressed. Analytes that can be ionized may be extracted by ion-exchange.2. A wider range of sorbents can be expected.77 The eluotropic strength (eo) is often a useful parameter for selecting eluting solvents. with a high eo of 0 ¢73. The degree of concentration and puri¢cation needed. non-polar. are extracted by R-P sorbents.79 Their use will increase. . This is a rapidly developing ¢eld.5. The aim is for the smallest cartridge volume possible. but uncharged. whether it can be ionized.
Covalent a¤nity chromatography with thiol ^ disulphide interchange SPE was used to extract metallothionein and metals from physiological £uids. Graphitized carbons for solid-phase extraction. Capparella M. ed. J Chromatogr B 2000. Introduction of new stationary phases (e. Recent developments in polymer-based sorbents for solid-phase extraction. particularly in speciation studies. LC-GC Int 1998. attract commercial development. Wilson ID. Phillipsburg: J. 39: 464. Anal Chem 1997.72 9 Pawliszyn J. ed. It may be possible to replace other extraction procedures (e. J Chromatogr A 2000. 745: 39. Some of the developments should be of value to clinical chemistry and it is important to keep abreast of them.82 ion-exchangers. 3M Empore Rad disks are very selective for radium. J Chromatogr A 2000. 902: 267. Shef eld: Academic Press. Ion-pair solid-phase extraction. strontium and technetium 3 and might be applied to monitoring exposure to these elements. Extraction Methods in Organic Analyses. Iraneta PC. et al. Pichon V. J Chromatogr B 2000. SPME might ¢nd a clinical role for analysis of volatile and semi-volatile compounds.58 There may be a place for SPE and SPME in clinical trace-metal analyses.477 . Headspace solid-phase microextraction procedures for gas chromatographic analysis of biological uids and materials. New York: Wiley. 11: 8.. Kiser R.g. Solid Phase Microextraction: Theory and Practice. which is standard for metabolic laboratories. 1999 7 Hennion M-C. Some of the new developments taking place in the pharmaceutical industry could be of bene¢t to diagnostic biochemistry.lipophilic balanced SPE sorbent. Solid-phase microextraction: a promising technique for sample preparation in environmental analysis. Newer SPE phases may enhance sample enrichment of trace metabolites or peptides and thereby improve analytical sensitivity. Molecular imprinting: developments and applications in the analytical chemistry eld.16 4 Simpson NJK.73 3 Majors RE. Use of mixed-mode SPE is already well established for drugs. Finally. Pb and Sn as tetraethylborate complexes. LC ^ MS and capillary electrophoresis. Miller S. Baker Inc. McDowall RD. 745: 3.54 8 Huck CW. quantifying and investigating the metabolism of volatile substances and as an additional tool to investigate inherited and acquired metabolic disturbances. 1999: 194. J Chromatogr A 2000. ed. Neue UD. McDonald PD. Mills MS. 69: 2972.95 21 Stevenson D. particularly for detecting. Bonn GK. 745: 15. Solid Phase Extraction for Sample Preparation. chirally active phases) would extend the analytical range. Applications of Solid Phase Microextraction. Solid-phase extraction: method development. 1997 10 Pawliszyn J. J Chromatogr A 2000. Immunoaf nity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices. Biological/pharmaceutical applications. Highly selective immuno sorbent or molecularly imprinted polymer phases might be appropriate for some analytes and. LC-GC Int 1998. J Chromatogr A 2000. References 1 Alpendurada M de F. For clinical laboratories. Strategies and Applications. New York: Marcel Dekker. 889: 3. Chichester: Wiley VCH. 1988 18 Carson MC.T. J Chromatogr A 2000.5 20 Hennion MC.81 and headspace SPME was used in a GC/MS ^ MS method to measure Hg2+ and alkylated Hg. J Chromatogr B 2000.37 23 Andersson LI. ed. sorbents. Fifty years of solid-phase extraction in water analysis: historical development and overview. ed. polypyrrole polymers. Cambridge: Royal Society of Chemistry. 1999: 54. 885: 51. 885: 343. Extraction Methods in Organic Analyses. Devices introduced for environmental monitoring might be adopted for the analysis of clinical samples. Solid phase extraction in organic analyses. solvent extraction of urinary organic acids) with simpler.g. A review of modern solid-phase extraction.g. 885: 73. our knowledge of their metabolism is still very incomplete.63 13 Mills GA. 11: 35. 902: 17. Solid Phase Extraction: Principles and Practice.94 15 Bouvier ESP. Because analysis of volatile compounds has been problematic. Solid-phase Extraction: Principles. Pawliszyn J. 856: 3. 885: 3. J Chromatogr A 2000.87 14 Ulrich S. cytokines) and important metabolites that are present in low concentrations in biological £uids. Headspace SPME is versatile. versatile. Hennion MC. Intube SPME or even the new `Twister’ device might be useful alternatives. simple to carry out and does not require special equipment other than a GC ^ MS system. Solid-phase microextraction in biomedical analysis. Microextraction of drugs. Polymeric reversed-phase SPE sorbents: characterization of a hydrophilic.48 Ann Clin Biochem 2002. Effect of carbon loading on the extraction properties of C-18 bonded silica used for solid-phase extraction of acidic and basic analytes. Walker V. 1998 6 Fritz IS. J Chromatogr A 2000. 1999 12 Lord H.40 Ï 16 Lis ka I. Solid Phase Microextraction: A Practical Guide.16 17 Zief M. Morgan ED. and coupling with liquid chromatography.50 19 Martin P. J Chromatogr A 1999. Automated on-line systems are needed to interface SPME with HPLC. Analytical Solid-phase Extraction. Chichester: Wiley. New York: Marcel Dekker. SPE has been used to extract mercury and palladium 8 and cadmium and copper80 from environmental water. if ¢nancially justi¢ed. easy to operate and interface with di¡erent standard laboratory analysers. For example. Wilson ID. Shef eld: Academic Press. high-throughput SPE methods. 2000 5 Thurman EM.Solid-phase extraction in clinical biochemistry 475 that are robust. 902: 167. Phillips DJ. It is time to revisit SPE and its use in clinical analyses. In: Handley AG.220 22 Delaunay N.14 2 Handley AG. the sampling ¢bre would need to be made more robust. Immuno-af nity solid-phase extraction. In: Handley AG.13 24 Stevenson D. 1999 11 Scheppers Wercinski SC. Careful selection of SPE sorbents should facilitate analysis of newly recognized peptide `messengers’ (e.
Kim ND.5 53 Mills GA. Jones GR. Forensic Sci Int 2000. Molecular imprinting for drug bioanalysis. Anal Chem 1990. Sandra P. Gesele E.47 52 Mills GA. 123: 2501.94 34 Yu Z. Yeh YC. J Chromatogr A 2000. 107: 261. Fermo I.82 55 Snow NH.22 54 Theodoridis G. Oliver JS. J Chromatogr B 2001. and metabolites in whole blood. Urinary organic acid screening by ¨ solid-phase microextraction of the methyl esters. 765: 45.90 48 Arcelloni C. Application of solid-phase micro-extraction technology to drug screening and identi cation. The preparation of a molecular imprinted polymer to 7-hydroxycoumarin and its use as a solid-phase extraction material. van der Greef J.71 47 Speed DJ. Simultaneous determination of Hg(II) and alkylated Hg. Hu SH. Solid-phase extraction in amphetamine and methamphetamine analysis of urine. 39: 464.8 59 Liebich HM. J Anal Toxicol 2000. Wilson ID. Characterization of the precolumn BioTrap 500 C-18 for direct injection of plasma samples in a column-switching system. and gas chromatography. Solid-phase extraction procedures in systematic toxicological analysis. Lee W-C. 930: 31.700 Ann Clin Biochem 2002. Mughal H. 762: 193. Mughal H. McCulloch S. Lanza F.gas chromatography. Wijsbeek J. Analyst 2000.304 35 Yu Z. 71: 1009. Cordeo R. A review on the application of imprinted polymers to solid-phase extraction and binding assay. Analysis of urine for drugs of abuse using mixed-mode solid-phase extraction and gas chromatography.477 . Hajimiragha H. Fully-automated systematic toxicological analysis of drugs. Hofte AJ. Pedercini S.mass spectrometry. Yu SC. 37: 690. Walker V. urine. 29: 529. 11: 1926. Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution. Pontiroli A. SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine.55 56 Mosaddegh MH. Hall DC. GC/EI/MS and GC ion trap/CI/MS. Ann Clin Biochem 2001.6 26 Andersson LI.9 38 Ferrer I. 34: 45.40 40 de Zeeuw RA. Koster EH. 885: 445.55 36 Van der Hoeven RA. Anal Chem 1998. 44: 589. Chen YL. 47: 299. Jones K. Determination of bupivacaine in human plasma with an alkyldiol silica precolumn. Woll J. Frenay M.3 51 Baltussen E. Staub C.35 58 Dunemann L. Richardson T. 16: 319. ´ ´ Determination of phenytoin in plasma by molecularly imprinted solid-phase extraction. Lanzi R. Kelly MT. Morgan DE. J Chromatogr A 1997.7 33 Yu Z. deuterated internal standards. et al. 363: 466. 763: 195. Dickson SJ. Chromatographia 1997. Solid-phase microextraction coupled to highperformance liquid chromatography. Headspace solid-phase microextraction with 1-pyrenyldiazomethane in. J Chromatogr A 2001. 745: 49.33 25 Godfrey MAJ. David F. Houldsworth P. J Anal Toxicol 2000. de Zeeuw RA. 873: 129.ion trap mass spectrometry. and plasma by gas chromatography.79 42 Paterson S. McClure J. Pawliszyn J. J Microcolumn Sep 1999. Clin Biochem 1996.32 60 Volmer DA. Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. 723: 281. Lai EPC.bre derivatization for analysis of faecal short-chain fatty acids. 125: 1515. Westerlund D. 67: 2530. Cairns ER. Anal Chem 1995. 739: 153. and Sn species in human body uids using SPME. Lin CL. 725: 149. de Jong GJ. Analyst 1998. Horvai G. J Chromatogr B 2000. Tjaden UR. LC-GC Int 1998. J Chromatogr A 1996. 21: 278. O’Kennedy R. Fresen J Anal Chem 1999. 11: 17. Stir bar sorptive extraction (SBSE).15 39 Degel F. Recent advances in disc-format solid-phase extraction. 24: 97. Westerlund D.200 37 Franke JP.8 29 Mullet WM. J Chromatogr B 2001. J Chromatogr B 1999. Rapid Commun Mass Spectrom 1997. 713: 51.full scan mass spectrometry. Speciation of dimethylarsinic acid and monomethylarsonic acid by solid-phase microextraction.25 32 Andersson LI. Immunoaf nity extraction in veterinary residue analysis: a regulatory viewpoint. et al. J Chromatogr B 2000. 22: 389. Barcelo D. Cramers C. Pawliszyn J. Tolokan A. et al. 713: 427. Molteni G.20 46 Girod C.MS. Ef cient sample pre-concentration of bupivacaine from human plasma by solid-phase extraction on molecularly imprinted polymers. Analysis of drugs of abuse in hair by automated solid-phase extraction. Groppi A. Westerlund D. Rapid determination of corticosteroids in urine by combined solid phase microextraction/liquid chromatography/ mass spectrometry.7 57 Mester Z. Vignali C.200 49 Arthur C. Improved solid-phase extraction of methadone and its two major metabolites from whole blood. Liquid chromatography.53 28 Bereczki A.mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.7 31 Walshe M. J Chromatogr B 1999.82 44 Cooper GA. Anal Chem 1999. Montagna M. J Anal Toxicol 1997. 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