Review Article

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:

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

but their adoption in clinical biochemistry has been slow. These developments have facilitated automated o¡and on-line sample processing. The derivatization during manufacture is incomplete and unbonded hydroxyl (silanol) groups are left on the surface of the silica. emphasizing some of the more recent developments in SPE and its biomedical applications.g. Cyclohexyl phases sorb phenols from aqueous solutions. C 2 phases are useful for very hydrophobic analytes. Here we present an overview of the ¢eld. ethyl acetate or methanol. a long-chain aliphatic hydrocarbon) and a polar portion (e. Trifunctional sorbents are more resistant to acid pH. which are available for secondary ionic interactions with the sample. for acidic analytes. This is a technological growth area. these are used in combination. It involves non-polar interaction of the solute with the stationary phase through lowenergy Van der Waals forces. Modi¢cations used in other formats are discussed later. Examples of ion-pair reagents used commonly for basic analytes are alkylsulphon ic acids (e. Interaction of the polar moiety with the charged group on the solute forms a non-polar ion-pair which is retained by the sorbent. and special syringe holders for SPME. The major classes of sorbents in use. (b) Trifunctional bonded-silica sorbent. there were problems with sample contamination from the polymeric resins used as sorbents. Other sorbents used as HPLC stationary phases were applied to SPE and others have been developed speci¢cally for sample preparation.17.7. Ann Clin Biochem 2002.7. often acetonitrile. These were reliable and have been used extensively in clinical biochemistry. The newer products are used widely in the pharmaceutical industry and for environmental analyses. Increasingly.g.8. The range of sampling formats for SPE has also expanded from simple packed syringes to cartridges. The most widely used bonded phases are C18 sorbents [octadecasilyl (ODS). (a) Monofunctional bonded-silica sorbent. The retention of highly polar compounds on R-P sorbents may be increased by adding an ion-pair reagent to the Figure 1. samples and sorbent when activating the cartridges.7.g.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. The solute is eluted with an organic solvent. methyl or phenyl groups. normal-phase (N-P). Phenyl phases have a high capacity for cyclic hydrocarbons. The following section describes properties of sorbents that are used in column or cartridge format.19 These are hydrophobic siloxanes with hydrocarbon chains or other hydrophobic molecules bound to silanol groups of silica through ^ Si ^ O ^ Si ^ C bonds. This may be exploited to increase the retention of polar analytes such as basic Reversed-phase sorbents5. At pH values above 2¢ 0 these groups dissociate to Si ^ O 7.17 R-P chromatography partitions organic solutes from a polar phase (generally aqueous) to a non-polar phase. (CH 2) 17CH 3] and C8 [octyl. There is now a wide range of high-quality materials available. 39: 464. Waters Associates introduced C18 -derivatized silica sorbents as disposable mini-columns (Sep-Pak 1 cartridges). ion-exchange. are summariz ed in Tables 1 and 2. alkylammonium bromides or alkylammonium hydrogen sulphates (e. Sorbents used for solid-phase extraction The main modes of separation used are reversed-phase (R-P).18 In contrast to HPLC separations. which may be in the form of a hydrocarbon chain or polymeric sorbent. including `designer phases’ developed for targeted extractions of drugs of abuse.5. Addition of methylene chloride to ethyl acetate (1:1. o¡ering considerable versatility. but also trap interferences readily. cyanopropyl ethyl. The a¤nity of the solute for the sorbent depends upon its hydrophobicity. a¤nity and restricted-access. tetramethylammonium bromide and tetramethylammonium sulphate). 1). disks.18 These have a non-polar portion (e. this technique is not used widely for SPE. The most common R-P sorbents for SPE are chemically bonded silica phases. 1octanesulphonic acid) and.g. and trifunctional if it is linked to three oxygen atoms (see Fig. with examples of biomedical applications. SPE pipette tips and 96well plates. Others have bonded cyclohexyl. The sorbents are said to be monofunctional if the bridging silicon is linked to one atom of oxygen. v/v) may elute more hydrophobic compounds. (CH 2) 7CH3] sorbents.15. an acid or base group). C18 phases provide greatest retention.9^14 When SPE was ¢rst introduced in the 1970s.477 .16 In 1978.

2. peptides. bile acids. isopropanol. acetonitrile.carboxylic acid Wide-pore weak: Accell plus CM (Waters). amine. primary and secondary amine. C2. low ionic Acidic buffers. acrylate. pyrimidines. buffers. hexane. purines. poly(divinylbenzene. such as Water. N) Non-polar solutions: hexane. cyclic nucleotides. primary and secondary amine* Silica: silica gel*. O. lipids Methanol. these hydroxyl groups may be inactivated by subsequently blocking them with trimethylsilane during manufacture. carbohydrates. phosphates. end-capped cyanopropyl Wide-pore: C4. ethyl acetate. C6. carboxylic. strength alkaline high ionic sulphonic. phenyl. vicinal (two on adjacent silicon atoms) or geminal (two on the same silicon atom).co-N-vinylpyrrolidone) (Oasis HLB. water-soluble vitamins. polyethyleneimine (JT Baker) Acids. fatty acids. phenols Non-polar Polar solutions: functional groups water. CBX carboxylic acid (JT Baker) Water. may have a wide range of bonded groups and are .3polar extraction dihydroxypropoxypropyl. even the most rigorous endcapping procedures do not de-activate all the silanol groups on the surface of the sorbent. Bonded-silica sorbents are stable to organic solvents and acid solutions (trifunctional sorbents to pH 2¢ 0). buffers. lipid separations. 39: 464. chloroform.divinyl-benzene (PS-DVB) sulphonic acid Weak: propylcarboxylic acid polymers: PS-DVB carboxylic acid. Solid-phase extraction sorbents: traditional separation mechanisms Separation Reversed-phase or non-polar extraction Sorbent Bonded silica: C18. and many others Phenols and pesticides in water. methylene chloride Applications (examples) Drugs of abuse. nucleotides. Waters) Normal-phase or Bonded silica: cyanopropyl. propylsulphonic acid. drug metabolites Pharmaceuticals. kieselguhr (diatomaceous earth) Alumina Florisil Anion-exchange Bonded silica Strong: quaternary amine Weak: aminopropyl.477 strengths may di¡er when the free silanol groups are single (isolated). polymeric pyrimidines polystyrene. C4. C3 Typical analytes Matrix Typical solvents Methanol. chloroform. fatsoluble vitamins. strength acidic high ionic buffers. cyclohexyl. diethylaminopropyl Wide-pore weak: Accell plus QMA (Waters). oils. a process known as end-capping (see Fig. acetone. aminopropyl*. acidic methanol. vitamins. Bonding Ann Clin Biochem 2002. pesticides. hetero-atoms (S. drugs by a mixed adsorption mechanism. biological strength buffers phosphoric acids uids Organic acids. C8. biological strength buffers uids *Also weak ion-exchange.466 Walker and Mills Table 1. antibiotics Catecholamines. amines. Alternatively. methylene chloride. steroids. drugs in blood and urine Hydrophilic groups such as hydroxyl. However. pharmaceuticals Cation-exchange Bonded silica Bases such as Strong: benzene sulphonic acid. ethyl acetate Vitamin D metabolites. 2). complexed copper and cadmium. low ionic Alkaline buffers. are rigid packing materials with good £ow characteristics. biological uids aromatic Graphitized carbon (aromatic carbon) Co-polymers: styrenedivinylbenzene. such as alkyl. therapeutic drugs.

phencyclidine. biological uids Fractionation of complex mixtures Af nity (7. procainamide. internal adsorption uids cortisone. biological Elut PBA (Varian) diol groups uids Dipyridylamide-functionalized polymers Mercury. biological uids ¢9-Carboxytetrahydrocannabinol (cannabinoid metabolite). 8. carbohydrates. hair digests Drugs of abuse: opiates. compounds extracts 7-hydroxycoumarin Dependent on Water. alkaline polar buffers. endogenous Biological uids Oestrogens. prednisolone. acidic and basic metabolites Drugs. Lichospher ADS (Merck). Solid-phase extraction sorbents: newer separation mechanisms Separation (references* ) Sorbent Typical analytes Matrix Applications (examples) Mixed mode (5. bupivacaine. 39. nucleotides Environmental water Silica-bonded phenylboronic acid: Bond. 21. buffers. 35. 37. 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. Antibodies bonded to silica. 39: 464. Ultrabiosep (Hypersil).Water.Solid-phase extraction in clinical biochemistry 467 Table 2.32) Restricted-access matrix adsorption (7. acids or bases if ionic Catecholamines. dexamethasone. non-steroidal anti-in ammatory drugs Non-polar anions or Water cations Cations and anions Water. 36) Miscellaneous Covalent bonding (3) Trace metal bonding (8) Molecularly imprinted polymers ChromSper 5 Biomatrix (ChromPak). LSD. Ann Clin Biochem 2002. a atoxins Single analytes or Biological uids: Theophylline. LSD. tricyclic antidepressants.44. amphetamines. 16.Analytes with vicinal Water. agarose. acidic polar buffers. BioTrap 500 (Chromtech) Catecholamines. benzoylecgonine. 7. biological Plasma cortisol. palladium Water *See also sorbent manufacturers’ literature. salbutamol. anabolic steroids Anions (acidic) non.Water. HiSep (Supelco). R-P ˆ reversed-phase. corticocompounds steroids. 29. biological uids. groups of related water or chloroform propranolol. other soft gels Cations (basic) non. organic acids. morphine.477 . controlled 24) pore glass. benzodiazepines. 41. anabolic steroids. basic drugs. sites: non-polar if Rudrocortisone P.

being smaller for low loading. Capacity and reproducibility are poor if the sorbent is allowed to dry out. Typically. larger molecules (42000 Da) may be excluded from 6-nm pores and adsorb better with 12 ¢5-nm pores. mono. there is substantial variation between bonded phases from di¡erent manufacturers and even between batches.468 Walker and Mills Figure 2. around 250^600 mL of solvent per 100 mg of sorbent is needed. Alternative sorbents used for R-P extractions include highly cross-linked co-polymers (styrene ^ divinylbenz ene).16 graphitiz ed carbon and porous graphitic carbons. more retentive. (c) sorbent-packed cartridge. on the particle size distribution and pore diameter (typically 40 mm and 6 nm.or trifunctional. Loading a¡ects the capacity of the sorbent. However. Ann Clin Biochem 2002. (e) membrane extraction disk suitable for attachment to a syringe.2 Despite improved technology.15. respectively). since the analyte may be eluted from the solidphase more readily with a smaller volume of eluting solvent. acetonitrile or methanol. (d) pipette tip with membrane extraction disk. End-capped bonded-silica sorbent.5. (f) 96-well extraction plate. relatively inexpensive. This may be an advantage. reproduced with permission from 3M Company USA.20 Compared with silica sorbents. the greater the area. pre-treatment is necessary with a solvent such as acetone.8. Bonded-silica sorbents have been used widely in clinical chemistry. (b) syringe barrel assembly with membrane extraction disk. Carbon loading ranges from 12^18% for C18 phases. Wide-pore (427 ¢5 nm) sorbents with bonded short-chain (C3 or C 4) hydrocarbons allow better access for large molecules. these sorbents are generally more hydrophobic. 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. Applications include extractions of drugs. 39: 464.7. reproduced with permission from Alltech Associates UK.477 . to 9^14% for C8 phases and 2¢7^5¢7% for C2 phases. Examples of sampling formats for solid-phase extraction: (a) syringe barrel assembly packed with sorbent. peptides and steroids. Pore size determines the surface area of the sorbent: the smaller the pores. stable over the pH range 0^14 and do not Figure 3. The properties of these sorbents depend on whether they are endcapped.

ionic strength and £ow-rate. weak sorbents have primary or secondary amine or aminopropyl groups. Strong cationexchangers in the Ba2+ or Ag+ forms (from Alltech Associates) are used to remove sulphate and halides. cyclic nucleotides.17 In ion-exchange. Their extraction may be possible with less-retentive silica-bonded phases now available for N-P SPE (aminopropyl-. They are eluted by changing the pH and/or increasing the strength of competing ions.17 In N-P SPE. These sorbents must be stored in a desiccator and are often dried before use. basic compounds are retained better on silica and acidic compounds on alumina. they can be eluted by reducing the pH by 2 units below their pKa. Ionization of weak exchangers is pH-dependent. Very polar compounds such as carbohydrates or amino compounds bind very tightly to silica and alumina and cannot be eluted. they can be eluted by raising the pH by 2 units above their pKa.7. Weak exchangers are used when analytes are so tightly attracted to strong exchangers that they cannot be eluted. carbohydrates and phenols. Chromosorbs 1 and Tenax 1 (diphenyl-pphenylene oxide). citrate may compete for anion-exchange sites. reduces retention of analytes and leads to variable recoveries. Extraction is poor when there are high concentrations of competing ions in the sample matrix. H+ and Na+ are most easily displaced from strong cationexchanger sorbents. purines and pyrimidines). weak sorbents have carboxylic acid groups. polar compounds dissolved in a non-polar solvent such as diethyl ether or n-hexane are extracted by adsorption to a polar sorbent. organic acids and vitamins. Alumina and silica adsorb water. However.8 This is also the case for Abselut Nexus (Varian).Solid-phase extraction in clinical biochemistry 469 have secondary ionic interactions. They are used to extract acidic analytes and there are reported applications for bile acids. the pH of the samples is adjusted so that the solute molecules are ionized. cyanopropyl. This sorbent does not require pre-conditioning and there is no problem from drying out before solvent application.g. can be used in automated on-line combined extraction and analysis systems. another polymeric sorbent. F 7 and C 2H 3O 27 from strong anion-exchanger sorbents.21^23 Ann Clin Biochem 2002. most require pre-conditioning and give poor recoveries if allowed to dry out. Raising the pH by 2 units increases ionization to around 99% and promotes retention. hydrogen bonding and p ^p electron interactions.e. Sorbents (e. Oasis 1 HLB (Waters) combines hydrophilic N-vinylpyrrolid one and lipophilic divinylbenz ene. Ion-exchangers may be silica.or propyldiol-bonded silica). Reducing the pH by 2 units increases protonation to around 99% and promotes retention. Li+. Unfortunately. Silica-bonded phases dissolve at extremes of pH. These can be used to exchange transition metals and divalent cations for Na+. For extraction.17 Selectivity of ionexchange is in£uenced by pH. Basic organic compounds are 50% protonated (i. and OH 7. Florisil 1 (activated magnesium silicate). The analytes are then desorbed with solvents with a high eluting power (eluotropic strength. Before the introduction of bonded phases. Normal-phase sorbents5. They are used to extract basic analytes (e. catecholamines.or resin-based. impurities in these sorbents caused contamination.g. Ions on the sorbent are neutralized by oppositely charged counter-ions. Biomedical applications of N-P SPE have included analysis of vitamin D metabolites. For example. ethylene dimethacrylate (both from Amberlite) and other polymeric materials such as Porapaks 1 . from polar organic solvents. the sorbents used were alumina. Graphitized carbon extracts very polar and water-soluble analytes from aqueous samples. styrene ^ divinylbenzene) are also available that incorporate a chelating group. Newer co-polymers have high retention capacity and. 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. fatty acids. 20 Early polymeric sorbents used for SPE were a crosslinked polystyrene resin (XAD-1) and styrene ^ divinylbenzene. respectively.5. Acidic compounds are 50% ionized as anions at a pH near the pKa. 39: 464. in clean-up procedures for environmental analyses.7.Weak interactions are involved: dipole ^ dipole. because of their compatibility with C18 HPLC columns.477 . kieselguhr (diatomaceous earth) and silica. eo40¢6) such as methanol (eo ˆ 0 ¢73). Ion-exchange and chelating sorbents2. compounds are extracted by a highenergy ionic interaction with the sorbent. Polar solutes may. These materials are used to manufacture mixed-phase sorbents (see below). In general. as cations) at a pH near their pKa.7. Strong cation-exchanger sorbents have sulphonic acid groups. lipids. Strong anion-exchangers have quaternary amines [^ N+(CH 3) 3] as exchange groups.16. therefore. providing a hydrophilic ^ lipophilic balance. It is used for extracting a range of environmental pollutants from contaminated water samples. They are then adsorbed at oppositely charged sites on the sorbent. They are being used increasingly in drug analyses. Exchange sites of strong ion-exchangers are ioniz ed at any pH. counter-ions. This deactivates hydrogen-bonding sites. usually iminodiacetate in the Na+ form.

Baker).d.35 Another was for plasma cortisol. 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. and morphine and its metabolites in blood.7.7. Samples are applied at pH 5^8.39^44 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.26 There have been few reported biomedical applications of molecularly imprinted polymer SPE. layered on top of each other. Poor recoveries have been a problem with layered sorbents.470 Walker and Mills Immunoa¤nity sorbents have biological antibodies covalently linked to silica. Hydrophilic interferences are removed by washing with bu¡er or water.3. oestrogens.g.7.8. The most popular have an ionexchange group bonded to silica. C8 or C18 ligands.29 propranolol. washed at pH 7 and eluted under mild conditions (e. Subsequent acidi¢cation protonates the drug amino group.37.34 A 2064 mm i. The sorbents may be present as mixed particles within the same cartridge.7. These allow simultaneous extraction and clean-up of blood. agarose or other soft gels. 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]. there is now a new generation of mixedmode polymers available for extraction of a wide range of analytes. plasma and urine prior to con¢rmatory analysis. Silica C18 restricted-access matrix sorbents e¡ectively exclude plasma proteins.g. They include procedures for serum phenytoin. £udrocortisone and prednisolone with an on-line SPE liquid chromatography ^ mass spectrometry (LC ^ MS) procedure.). This problem may be overcome by using a closely related compound to make the template. which can then be separated chromatographically. Matrix interferences are removed with a methanol wash. LSD and morphine in urine. The polymers can then be used for SPE of the analyte and structurally related compounds and for chiral separations.36 Mixed-mode sorbents and multi-modal solid-phase extraction Mixed-mode sorbents have both non-polar and ionexchange functional groups. Finally. respectively.27 They are stable over a wide pH range.7.30 7-hydroxycoumarin31 and the anaesthetic bupivacaine 32 in plasma. including proteins. Bio Trap C18 column (Chromtech) could tolerate up to 15 mL of plasma without changing retention pressure.5. Oasis MAX and MCX (Waters). 3. controlled-size glass particles. Bakerbond Narc-2 (J. The combination is chosen so that an analyte is retained by both mechanisms. which migrates separately from the analyte in the subsequent HPLC analysis. the drugs are eluted with a basic solution to break the ionic bonds.5.16. dexamethasone. reported for restricted-access matrix SPE. In these.24 The cross-reactivity of antibodies can be exploited to extract structurally related compounds (e. or present in di¡erent cartridges that are stacked.37 Around 80% of drugs contain nitrogen that can be protonated. These sorbents are being used increasingly for drug analysis. They are then extracted by R-P hydrophobic interaction with the sorbent.3. with clogging only after 45 mL of plasma. Most immunoextractions are for analysis by HPLC. by selection of pore size or by chemical repulsion by an appropriate hydrophilic coating on the sorbent surface.22. cortisone.25 Sorbents are conditioned with phosphate-bu¡ered saline (PBS) at neutral pH. a drug and its metabolites). a bonded R-P covers the pore surface with C 4. Columns have been re-used up to 50 times for biological samples.5 However. 28 theophylline.38 Mixed-mode sorbents such as Bond-Elut Certify (Varian Analytichem).7 Analysis of bupivacaine is one of the few clinical analyses Ann Clin Biochem 2002. Removal of the template then leaves an imprint sterically complementary to the ligand.33.7 Internal surface reversed-phase supports are the most popular restricted-access matrix sorbents. usually by GC ^ MS.T. It is di¤cult to remove the original template molecule completely and residual material may cause analytical interferences. 21. to the Oasis HLB divinylbenz ene ^ N-vinylpyyrolidone co-polymer.7.7 The pH is adjusted so that the analytes are present in a neutral or negatively charged form. which then binds strongly to the sorbent by ionic interaction.24 Molecularly imprinted polymers are produced by using a target analyte as a template in a casting procedure. ionic or a¤nity interactions. nortestosterone. with PBS ^ methanol or PBS ^ ethanol at pH 2^4). Small molecular mass analytes entering the pores may be concentrated there by conventional hydrophobic. This is useful for extracting lipophilic compounds with an ionizable functional group. 39: 464.21.24.477 . Isolation is therefore possible by cationexchange in addition to R-P adsorption.or 4-vinylpyridine are cross-linked around the template with a cross-linking agent. Oasis MCX and MAX (Waters) are mixed-mode sorbents produced by addition of a strong cation-exchange group or anionexchange group. particularly drugs (see below). SPEC 1 11 (Ansys Diagnostics Inc. The few applications of immunoa¤nity SPE to biological £uids (animal and human) have included anabolic steroids and corticosteroids in faeces and urine. plasma and urine.26 Monomers such as methacrylic acid or 2. Multi-modal SPE uses two or more SPE phases in series to improve clean-up.23.21.

and the list is growing. Both polydimethylsiloxane (PDMS) and polyacrylate phases extract via absorption. the externally coated ¢bre is replaced with a length (50^100 cm) of conventional internally coated fused-silica GC capillary column. amphetamine. The PDMS-Carboxen ¢bre extracts highly volatile solvents and gases. Di¡erent methods are used to attach the coating to the fused-silica core. Some phases have di¡erent thicknesses (e. phencyclidine and tetrahydrocannibol (marijuana). 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. strontium and technetium (Empore 2 Rad disks. 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. With in-tube SPME. It is compatible with GC and HPLC desorption processes. After sampling.51 Several ¢bre coatings are available (from Supelco) 10^14 for the extraction of volatile and semivolatile compounds. metal-loaded sorbents that complex organic compounds have been used for water puri¢cation. The remaining types [Carbowaxdivinylbenzene (DVB).g.T. Usually the thinnest acceptable ¢lm is employed to reduce extraction times. and Blevins and Hall 45 reported the use of SPEC disks in a manual extraction method for a marijuana metabolite. Miscellaneous sorbents Compounds with vicinal (adjacent) diol groups (e. Carbowax-templated resin. Most polymer ¢lms are coated directly (non-bonded types) or are partially cross-linked. 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. PDMS-DVB] are mixed coatings and extract via adsorption. methamphetamine. the ¢bre is withdrawn into the syringe barrel for transfer to a GC injector port 9 or a modi¢ed HPLC Rheodyne valve. The type of ¢bre used a¡ects the selectivity of extraction. In both modes. poisons and their metabolites in blood. 100 mm) and this a¡ects both equilibrium time and sensitivity of the method. Degel39 and de Zeeuw et al. 8 Solid phases built around crown ethers have been used to extract radioisotopes such as radium. Drugs were extracted with WWM-DAU (World-Wide monitoring) solid-phase 100-mg extraction cartridges with acidic and basic elution.). extending the range of applications. cocaine. Polettini et al. 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. In general. catecholamines. PDMS-Carboxen. opiates. There are as yet no reported applications for biological samples. phencyclidine. from 3M). methadone. 39: 464. The bi-polar PDMSCarboxen ¢bre has a mixed carbon coating with small micro-pores and a surface area of approximately 1000 m 2/g.41 reported a fully automated procedure for systematic analysis of drugs. available from Gerstel GmbH & Co. and mercury (Hg 2+) and palladium (Pd 2+) have been extracted from environmental water with dipyridylamine -functionalized polymers. including antidepressants. The technique involves either the equilibrium or non-equilibrium partitioning of analytes between the stationary phase and sample. 30 mm.Solid-phase extraction in clinical biochemistry 471 Substance Abuse and Mental Health Services Administration: amphetamines. antipsychotics and various over-the-counter preparations. with analytes dissolving and di¡using into the bulk of the coating. IsoluteCon¢rm 1 HSX (IST) cartridges were used in an automated GC ^ MS procedure for drugs of abuse in hair [cocaine. ecstasy.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. codeine.g.44 The eluted drugs were derivatized and analysed by GC ^ MS in full scan mode. carbohydrates and nucleotides) may be extracted by covalent bonding with silica-bonded phenylboronic acid (Bond-Elut PBA from Varian). derivatized and analysed by GC ^ MS.12. agitation of the sample by stirring or sonication improves transfer of analytes to the ¢bre.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. 6-monoacetylmorphine and morphine].477 .3 In industry. eve. as with conventional GC stationary phases. benzoylecgonine (cocaine metabolite). Two other variations of the SPME technique have become available recently. They can be damaged if exposed to high levels of organic analyte or strong acid or alkali. plasma and urine using the Hewlett-Packard HP 7686 PrepStation. morphine and codeine.3. 7 mm. Ann Clin Biochem 2002.40 achieved good recoveries with SPEC disks in general screens for drugs in urine.13 In stir-bar sorptive extraction (Twister 2. SPE phases coated with derivatizing agent were used for in situ derivatization of analytes as they passed through SPE cartridges. with analytes attaching to the surface of the ¢bre as a monolayer.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.5 Preliminary hydrolysis is needed to release opiates from glucuronide conjugates. Bakerbond Narc-2 (J.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. followed by a semi-automated identi¢cation procedure.

The bed volume is 125 mL per 100 mg of sorbent.52 analysis of urinary organic acids. kinetic and mass transfer processes.12. Usually they are supported on a fritted glass ¢lter in a standard ¢ltration apparatus.2. Empore 2 extraction disks (3M). Channels in the sorbent bed may cause disturbances of solvent £ow through the sorbent and reduce the retention of analytes. or in 96-well plates.14.7. but other solid phases have since been introduced. with pressure or with centrifugation. Generally they require only small volumes of eluting solvent and this can help in concentrating the eluted analyte before analysis. 4^96 mm in diameter (most often 47 mm) and typically 0¢5 mm thick. Samples and solvents may pass through the column under gravity or may be aspirated by vacuum applied to the syringe outlet.3.65. with 10% (w/w) of PTFE and approximately 90% sorbent. are irregular and have a pore size of 6 nm.54. Alternatively.13. up to 50 or more times). which can a¡ect GC analyses. SPEC disks (Ansys Inc. They are usually polypropylene or polyethylene open syringe barrels ranging in size from 1 to 25 mL. in both direct immersion and headspace modes.62 isoprene 63 and formaldehyde in breath 64 and volatile fatty acids in faeces.45. tricyclic antidepressants. phenothiazines. 500 mg or 1 g) is sandwiched between 2620 mm fenestrated polyethylene plates (frits).6. The packing materials generally have a mean particle diameter of 40^50 mm.65^68 Particle-loaded membranes [e. or suction from a vacuum manifold.58 So far.472 Walker and Mills Bonded ¢bres are more resistant. Ann Clin Biochem 2002. including styrene ^ divinylbenz ene. Reported applications include quanti¢cation of trimethylamine in urine for diagnosis of inherited trimethylaminuria (¢sh odour syndrome). Because of their .38. Problems with SPE columns and cartridges are that sample processing rates are slow because of their small cross-sectional area. ion-exchange. sorbent packing density is variable and sorbent impurities may cause contamination.g. few have used SPME to analyse endogenous compounds in biological matrices. They can be used under vacuum. Applications have been reviewed recently. Benzodiazepines.66 Many of these problems are lessened by the use of SPE disks or membranes.8. has been discussed in depth. 39: 464. Fibres can be re-used several times (e. Sorbent (generally 100 mg. The ¢rst disks manufactured contained C18-bonded silica particles. The mass of sorbent used determines the sample capacity. 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. leading to poor recovery and reproducibility. Glass or Te£on syringes with stainless-steel frits are also available to avoid contamination by plasticizer. ENVIDisks (Alltech Associates).) and Resprep2 (Restek)] contain 10^30 mm chemically bonded silica particles incorporated into a glass ¢bre matrix. nicotine and cotinine.g. Cartridges have a polypropylene body with female and male luer tips to apply pressure from a syringe. chelating and mixed-mode sorbents.14 Quanti¢cation is usually achieved by external calibration techniques. The theory of the thermodynamic. depending on the sample matrix.9. but they are also available in preloaded syringe barrel or cartridge holders. Rigid particle-embedded glass bre disks [e. pethidine (meperidine). They are £at disks. Sorbent material is packaged between 20-mm frits. They are available in a range of sizes and often give faster £ow-rates with less clogging compared with loaded PTFE membranes.55 Drug analyses include amphetamines.g.66 Syringe barrels have been most popular.5. The technique is being used increasingly to analyse drugs and their metabolites.53 In some SPME applications the analytes have been derivatized during the extraction procedure. volatile anaesthetic gases and solvents and pesticides in blood and urine.61 volatile compounds in blood.65. they may be pushed through by applying pressure to the top of the inlet. antidepressants and analgesics were identi¢ed in body £uids using SPME and GC ^ MS. They consist of 8^12 mm sorbent particles embedded in a web of inert polytetra£uoroethylene (PTFE or Te£on) micro¢brils.477 which is usually around 5% of the sorbent mass. or pulled through by centrifugation.13.17.53 SPME is used widely for analysis of environmental pollutants. they become blocked by particles and adsorbed matrix.14. 3). antihistamines and phencyclidine. 5. in pipette tips.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.53 Volatile sulphur compounds were extracted from urine headspace vapour with PDMS-Carboxen SPME ¢bres and identi¢ed by GC ^ MS.59 steroids 60 and volatile compounds in a variety of metabolic disturbances. foodstu¡s and pharmaceutical preparations.9. They have a male luer ¢tting so that they are interchangeable with di¡erent SPE vacuum manifolds. SPME is a complex multi-phase equilibrium process.61 These compounds are notoriously di¤cult to analyse.52. Novo Clean disks (Alltech Associates)] look like ¢lters. Solid-phase extraction sampling formats There is now a variety of formats available (see Fig. 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.

smaller solvent volumes are needed for conditioning and elution. Plates are available for a full range of chemistries. Anachem Ltd) with retention by an ion-exchange mechanism. Instead of using a closed well format.or 24-place vacuum manifolds or with 96-well plates. The SPE packing is Separan S-hydroxyethylmethacrylate -B10 sulphobutyl (HEMA-SB polymer.70. Precision was found to be better for positive pressure £ow systems than for vacuum-controlled £ow. reducing the risk of crosscontamination. The sample -processing speed depends upon the application and analyser.70 enable rapid preparation with high sample throughput. or in parallel.g. usually an HPLC with computer-controlled column switching. recovery and accuracy with fewer operator errors. A throughput of up to 40 samples/day is possible.74^76 The SPE cartridge is regenerated and may be used several hundred times. 576 or 1152 wells) for highthroughput screening applications. to 25^50 samples/ h and up to 400 samples/ h for the fastest serial. Use of the smallest mass of sorbent needed for the application reduces solvent require ment and elution time. The disks are thin and packed with particles with a large surface area for reasonable e¤ciency.7 are conventional pipette tips ¢tted with an SPE disk (Ansys Diagnostics).477 .70 In some cases. Since £ow-rate is proportional to the square of diameter. with 12. and improved mechanical stability of the sorbent bed reduces channelling. since the analyser can resolve a wide range of analytes e¤ciently. Samples may be processed with a vacuum manifold or with a centrifuge with special micro-plate carriers.65 Ninety-six-well extraction plates 3. and safer operation because of reduced exposure to hazardous solvents and biological samples.70. LC/MS ^ MS). Over typical £ow rates of 10^100 mL/min for a 47-mm disk. The large cross-sectional area reduces the risk of plugging by contaminants. Because only a fraction of the sorbent used for conventional SPE is incorporated into glass micro¢bre disks. 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.e. Automation of solid-phase extraction The bene¢ts of automating SPE are improved precision. Rates vary from around 3 samples/ h with simpler instruments. The degree of sample puri¢cation needed depends on the analysis to be undertaken. 39: 464.70 The SPE procedure may be automated on work-stations o¡-line and the sample extracts then injected onto an analyser. Some require only a simple cleanup procedure (e. to remove salts and non-volatile matrix components). The ASTED system has also been used to Ann Clin Biochem 2002. enabling assessment of di¡erent separation protocols simultaneously.73 An innovative application for automated SPE is HPLC analysis of urinary free catecholamines using an automated sequential trace enrichment of dialysates (ASTED) system. Alternatively. respectively. with numerous samples being processed simultaneously ^ for example. Liquid can be drawn upwards or forced down through the disk. SPE pipette tips 2.73 Some systems automate SPE disk procedures. several 96-well plate blocks are used together (i.70 Possible disadvantages are risks of carry-over and problems from sample instability if there is delay between sample preparation and analysis. or PTFE or glass ¢bre disks providing 9^15 mg of sorbent.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. Sample clean-up and enrichment are achieved by SPE in association with a Gilson cartesian robotic sampler. an automated SPE system may be interfaced directly to an analyser. particle-loaded membranes have been reported to provide between 4^966 and 9^1467 theoretical plates.73 Samples may be processed serially.3.69. They consist of moulded polystyrene or polypropylene plates with 96 individual columns arranged in rows of 8612. The tips can be used for single samples. Unwanted wells may be covered. This allows higher £ow-rates than for conventional SPE columns.5.Solid-phase extraction in clinical biochemistry 473 rigidity. with one sample being analysed as the next undergoes SPE.66 it may be increased by using larger disks. Ninety-six-well SPE method development kits are available with a di¡erent sorbent in each row. the SPE plates have a £ow-through system and they are set on top of a 96-well collection system. They can be used in manual and automated liquid-handling operations. Disks (4 mm) preloaded into SPE pipette tips are available. they do not need external support but may be used in conventional SPE columns. A stainless -steel enrichment cartridge replaces the loop on the Rheodyne injection valve of the HPLC system.5. as packed beds (generally 10^200 mg). including the newer mixed-mode sorbents.and fastest parallel-processing equipment. They can be used in many automated and robotic systems and are used extensively for sample preparation in the pharmaceutical industry. which allows for a more concentrated sample for subsequent analysis. These may have short packed sorbent beds or disks.70 O¡line processing o¡ers £exibility and avoids interface problems with the analytical devices. Memsep 2 disks (Waters) are stacked cellulose disks compressed into a continuous bed with pores functionalized with ion-exchange groups. possible cost savings from reduced analysis time and the possibility of overnight runs.

The nature of the matrix: its pH. whether it is aqueous or organic. it is helpful to carry out a literature search when planning a new procedure. are extracted by R-P sorbents.5. analytes from organic solvents. The approach to method development is still largely empirical and based on trial and error. non-polar.7. extending the versatility of SPE. It is a measure of the solvent’s elution strength of solutes from a particular adsorbent. theophylline and serum total glucocorticoids. its solubility in aqueous and organic solvents. More automated systems are required . For 40^60 mm particles it is about 120 mL per 100 mg of sorbent.17. For example.66 The capacity will be reduced if other compounds in the sample are also retained. In practice. ethyl acetate or chloroform for GC and GC ^ MS.474 Walker and Mills analyse phenobarbitone. carbonyl. or replaced by. a number of issues still need to be addressed. This is a rapidly developing ¢eld. A wider range of sorbents can be expected. miscible.5 In some applications the eluting solvent has been mixed with. but this is at the expense of larger eluting volumes and hence greater dilution of the analyte. There are recommended experimental protocols for an ordered approach. if a very non-polar eluting solvent such as n-hexane is to be used. methanol.77 Alternative computer-based strategies have been proposed based on theoretical considerations. Capacity is increased by using more Ann Clin Biochem 2002. Alternatively. in response to the expansion in applications. For biological applications. Sorbent manufacturers have large databases of methods in use. This is the maximum volume of sample that can be applied before the sorbents become saturated and cannot retain additional analyte.g. currently driven by combinatorial chemistry methods employed by the pharmaceutical industry with the associated need to extract and analyse large sample numbers rapidly.79 Their use will increase. and slightly polar analytes. There is still substantial variation between silica-bonded sorbents from di¡erent manufacturers and even between batches. with a high eo of 0 ¢73. or the sorbent may be dried under vacuum. Those with very polar functional groups which are not ionized (amino. is selected to elute moderately and strongly polar analytes from polar sorbents.78 However. The aim is for the smallest cartridge volume possible. but uncharged.66 The suitability of a device for an application is indicated by the breakthrough volume. The search should extend to other industrial applications to gain advantage of the newer sorbent technologies. More standardization of sorbents is required with better reproducibility and recoveries. . It can be measured experimentally or predicted from a variety of experimental methods. Analytes that can be ionized may be extracted by ion-exchange. It indicates the capacity for the analyte. The future Solid-phase extraction is a rapidly developing ¢eld. miniaturized. solvent to a non-polar eluting solvent to wet the sorbent bed e¡ectively. . The nature of likely contaminants.74^76 Smith and Lloyd 73 reported on 20 automated SPE systems available commercially in 1998. A sorbent is required that retains the analyte but not interfering substances. Choice is directed by the polarity of the analytes. The solvent must be compatible with the ¢nal analytical method.17. The degree of concentration and puri¢cation needed. rapid procedures that can be automated. Selection of a solid-phase extraction method It is necessary to consider: . Mixtures are often best.17 Because of water sorbed to silica phases. it is common to add a polar. N-P sorbents are used to extract polar. whether it can be ionized. phenylbutazone. Methylene chloride (eo ˆ 0¢32) elutes non-polar analytes e¡ectively from non-polar bonded phases. . The analytical detection limit and its sensitivity. . 39: 464. a derivatizing reagent enabling simultaneous elution and derivatization of the analytes.477 sorbent. The amount of sorbent used must be su¤cient to retain all the analytes in the sample. The elution volume should be 2^5 times the volume required to ¢ll the interstitial spaces and pores of the sorbent. sulphydryl) will be retained on polar sorbents. Retention of matrix components reduces the e¡ective capacity of the sorbent. The concentration of analyte in the sample. The chemistry of the analyte: its functional groups. Methanol or acetonitrile are often selected for HPLC.77 The eluotropic strength (eo) is often a useful parameter for selecting eluting solvents. they are usually dissolved in an aqueous matrix. ionic strength.2. ethyl acetate) may be added to displace water before eluting. The eluting solvent should disrupt analyte ^ sorbent interactions. a bridging solvent (e. phenytoin. Disks enable the development of simpli¢ed. Existing methods may be adaptable to the application. hydroxyl. but not yet for use with HPLC. . 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.

48 Ann Clin Biochem 2002.37 23 Andersson LI. J Chromatogr A 2000. Bonn GK.82 ion-exchangers.g. Some of the developments should be of value to clinical chemistry and it is important to keep abreast of them. Careful selection of SPE sorbents should facilitate analysis of newly recognized peptide `messengers’ (e. our knowledge of their metabolism is still very incomplete. Polymeric reversed-phase SPE sorbents: characterization of a hydrophilic.lipophilic balanced SPE sorbent. Immunoaf nity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices. New York: Marcel Dekker. J Chromatogr A 2000. 1999: 54. high-throughput SPE methods. Solid Phase Extraction: Principles and Practice. Biological/pharmaceutical applications. easy to operate and interface with di¡erent standard laboratory analysers. Molecular imprinting: developments and applications in the analytical chemistry eld. 39: 464. Recent developments in polymer-based sorbents for solid-phase extraction. References 1 Alpendurada M de F. Wilson ID.95 21 Stevenson D.g. New York: Marcel Dekker. versatile. McDowall RD. SPE has been used to extract mercury and palladium 8 and cadmium and copper80 from environmental water. the sampling ¢bre would need to be made more robust. particularly in speciation studies. New York: Wiley. Chichester: Wiley VCH. attract commercial development.94 15 Bouvier ESP. ed. Automated on-line systems are needed to interface SPME with HPLC. Hennion MC. 745: 3. Applications of Solid Phase Microextraction.50 19 Martin P.g. ed. Intube SPME or even the new `Twister’ device might be useful alternatives. cytokines) and important metabolites that are present in low concentrations in biological £uids. LC-GC Int 1998. Some of the new developments taking place in the pharmaceutical industry could be of bene¢t to diagnostic biochemistry. Ion-pair solid-phase extraction. McDonald PD. if ¢nancially justi¢ed. Walker V. Covalent a¤nity chromatography with thiol ^ disulphide interchange SPE was used to extract metallothionein and metals from physiological £uids. 885: 343. It is time to revisit SPE and its use in clinical analyses. J Chromatogr A 2000. 745: 39. Extraction Methods in Organic Analyses.477 . Solid-phase Extraction: Principles. 885: 3. J Chromatogr A 2000. ed. Devices introduced for environmental monitoring might be adopted for the analysis of clinical samples. Miller S. Because analysis of volatile compounds has been problematic.220 22 Delaunay N. Strategies and Applications. Wilson ID. 69: 2972. Chichester: Wiley. Extraction Methods in Organic Analyses. ed. Graphitized carbons for solid-phase extraction. 856: 3. Headspace SPME is versatile. Solid-phase microextraction in biomedical analysis. Solid-phase extraction: method development. particularly for detecting. 1999 11 Scheppers Wercinski SC. 3M Empore Rad disks are very selective for radium.13 24 Stevenson D. Fifty years of solid-phase extraction in water analysis: historical development and overview. ed. Phillips DJ. J Chromatogr B 2000. polypyrrole polymers. strontium and technetium 3 and might be applied to monitoring exposure to these elements. J Chromatogr B 2000. Solid phase extraction in organic analyses. A review of modern solid-phase extraction.63 13 Mills GA.14 2 Handley AG.. Analytical Solid-phase Extraction.T.54 8 Huck CW. Finally. et al. J Chromatogr B 2000. J Chromatogr A 2000. Anal Chem 1997. Capparella M. For clinical laboratories. J Chromatogr A 2000. 745: 15. Phillipsburg: J. In: Handley AG. Pb and Sn as tetraethylborate complexes.5 20 Hennion MC. 1988 18 Carson MC. Solid Phase Microextraction: A Practical Guide. Mills MS. For example.72 9 Pawliszyn J. SPME might ¢nd a clinical role for analysis of volatile and semi-volatile compounds. In: Handley AG. Kiser R. which is standard for metabolic laboratories. 902: 267. Microextraction of drugs. quantifying and investigating the metabolism of volatile substances and as an additional tool to investigate inherited and acquired metabolic disturbances. simple to carry out and does not require special equipment other than a GC ^ MS system.40 Ï 16 Lis ka I.16 4 Simpson NJK. Solid Phase Extraction for Sample Preparation. sorbents. Pawliszyn J. J Chromatogr A 2000. 1998 6 Fritz IS. It may be possible to replace other extraction procedures (e. Effect of carbon loading on the extraction properties of C-18 bonded silica used for solid-phase extraction of acidic and basic analytes. 889: 3. solvent extraction of urinary organic acids) with simpler. J Chromatogr A 1999.81 and headspace SPME was used in a GC/MS ^ MS method to measure Hg2+ and alkylated Hg. chirally active phases) would extend the analytical range. 11: 35. Shef eld: Academic Press. J Chromatogr A 2000.58 There may be a place for SPE and SPME in clinical trace-metal analyses. LC-GC Int 1998. Solid-phase microextraction: a promising technique for sample preparation in environmental analysis.73 3 Majors RE. Newer SPE phases may enhance sample enrichment of trace metabolites or peptides and thereby improve analytical sensitivity. Baker Inc. 885: 73.Solid-phase extraction in clinical biochemistry 475 that are robust.87 14 Ulrich S. LC ^ MS and capillary electrophoresis. Headspace solid-phase microextraction procedures for gas chromatographic analysis of biological uids and materials. Shef eld: Academic Press. Morgan ED. Highly selective immuno sorbent or molecularly imprinted polymer phases might be appropriate for some analytes and.16 17 Zief M. Use of mixed-mode SPE is already well established for drugs. 1999 12 Lord H. Neue UD. 902: 17. 885: 51. 2000 5 Thurman EM. 11: 8. Cambridge: Royal Society of Chemistry. Immuno-af nity solid-phase extraction. 902: 167. and coupling with liquid chromatography. Pichon V. 1997 10 Pawliszyn J. Introduction of new stationary phases (e. 1999: 194. 1999 7 Hennion M-C. Iraneta PC. Solid Phase Microextraction: Theory and Practice.

J Chromatogr B 1998. Koster EH. Simultaneous determination of Hg(II) and alkylated Hg.476 Walker and Mills 43 Lee MR.GC/MS. GC/EI/MS and GC ion trap/CI/MS.8 59 Liebich HM.8 50 Chen J.53 28 Bereczki A. J Chromatogr A 2000. Solid-phase extraction in amphetamine and methamphetamine analysis of urine. Hajimiragha H. van der Greef J. a novel extraction technique for aqueous samples: theory and principles. Ann Clin Biochem 2001. David F. J Chromatogr B 2000. Walker V. 34: 45. urine. 11: 17. McCulloch S. Sandra P. Hofte AJ.8 29 Mullet WM. 71: 1009. Solid-phase extraction procedures in systematic toxicological analysis.94 34 Yu Z. Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography. LC-GC Int 1998.3 51 Baltussen E.15 39 Degel F. J Chromatogr A 1996. Lanza F. J Chromatogr B 1998. Ann Clin Biochem 2000.35 58 Dunemann L. 22: 389.40 40 de Zeeuw RA. Improved solid-phase extraction of methadone and its two major metabolites from whole blood.7 31 Walshe M. Vignali C.7 33 Yu Z. 739: 153. 713: 265. Yu SC.gas chromatography. Dickson SJ. Stoddart RW. 24: 685. 930: 31.5 53 Mills GA. Anal Chem 1999. 885: 445. Mughal H. 730: 113. Anal Commun 1997.304 35 Yu Z.25 32 Andersson LI. Chromatographia 1998. 713: 427. Horvath V. Hall DC.ion trap mass spectrometry. 765: 45. Solid-phase micro-extraction of drugs from biological matrices. Evaluation of molecular-imprinted polymers for use in the solid phase extraction of propranolol from biological uids.90 48 Arcelloni C.47 52 Mills GA.full scan mass spectrometry. Kelly MT. Analyst 2000. Solid-phase microextraction for the analysis of biological samples. Urinary organic acid screening by ¨ solid-phase microextraction of the methyl esters. J Anal Toxicol 1997. 11: 1926. High-performance liquid chromatographic determination of diclofenac in human plasma after solid-phase extraction. Lanzi R.55 56 Mosaddegh MH. 745: 49. Thurman EM.71 47 Speed DJ. J Chromatogr B 2001. 70: 3636. de Zeeuw RA. Pawliszyn J. and metabolites in whole blood.477 .101 41 Polettini A. Determination of theophylline in serum by molecularly imprinted solid-phase extraction with pulsed elution. J Chromatogr B 1999. McClure J. Fermo I. J Chromatogr A 2000. 38: 541. Wijsbeek J. J Chromatogr B 1999.6 26 Andersson LI. and Sn species in human body uids using SPME.mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection. Liquid chromatography. Begerow J.73 27 Hwang C-C. 713: 51. ´ ´ Determination of phenytoin in plasma by molecularly imprinted solid-phase extraction. Horvai G. II. Fully-automated systematic toxicological analysis of drugs. Staub C. Chromatographic resolution of the enantiomers of phenylpropanolamine by using molecularly imprinted polymer as the stationary phase. Morgan DE. 363: 466. 762: 193. Frenay M. Lai EPC. J Pharm Biomed Anal 1997.700 Ann Clin Biochem 2002. poisons. Analyst 1998. Tolokan A.41 30 Martin P. Tjaden UR. 67: 2530. Chen YL.bre derivatization for analysis of faecal short-chain fatty acids. Westerlund D. 24: 97. et al. Gesele E. Franke JP. J Chromatogr A 2001. Pedercini S. deuterated internal standards. The preparation of a molecular imprinted polymer to 7-hydroxycoumarin and its use as a solid-phase extraction material.200 37 Franke JP. Yeh YC. 11: 737.mass spectrometry. Anal Chem 1990. Hall AJ. 123: 2501. Richardson T. Pawliszyn J. Anal Chem 1998. Rapid determination of corticosteroids in urine by combined solid phase microextraction/liquid chromatography/ mass spectrometry. Analysis of drugs of abuse in hair by automated solid-phase extraction. 21: 278. 29: 529. J Chromatogr B 2001. et al. J Anal Toxicol 2000. Oliver JS. Groppi A. Westerlund D.32 60 Volmer DA. J Microcolumn Sep 1999. Mughal H. Lin CL. Howarth J. SPEC disc solid-phase extraction for rapid broad-spectrum drug screening in urine. Solid-phase microextraction coupled to highperformance liquid chromatography. Westerlund D. Cordeo R. Walker V. Molteni G. Anal Chem 1995. Stir bar sorptive extraction (SBSE). Cramers C.7 57 Mester Z. Immunoaf nity extraction in veterinary residue analysis: a regulatory viewpoint.82 44 Cooper GA. Comparison of new solid-phase extraction methods for chromatographic identi cation of drugs in clinical toxicological analysis. Fresen J Anal Chem 1999. Ef cient sample pre-concentration of bupivacaine from human plasma by solid-phase extraction on molecularly imprinted polymers. Direct injection of large volumes of plasma in a column-switching system for the analysis of local anaesthetics. Recent advances in disc-format solid-phase extraction.mass spectrometry. Headspace solid-phase microextraction with 1-pyrenyldiazomethane in.79 42 Paterson S.mass spectrometry. 763: 195. Speciation of dimethylarsinic acid and monomethylarsonic acid by solid-phase microextraction. Clin Biochem 1996. Pb. and plasma by gas chromatography.MS. 16: 319. 39: 464. J Anal Toxicol 2000. Hui JP. Rapid Commun Mass Spectrom 1997.9 38 Ferrer I. Analysis of urine for drugs of abuse using mixed-mode solid-phase extraction and gas chromatography. 62: 2145. Jones K. Chromatographia 1997.200 49 Arthur C.92 45 Blevins DD. Lee W-C. J Chromatogr B 2000.22 54 Theodoridis G. 47: 299. 725: 149. Characterization of the precolumn BioTrap 500 C-18 for direct injection of plasma samples in a column-switching system. J Anal Toxicol 1998. Application of solid-phase micro-extraction technology to drug screening and identi cation. Molecular imprinting for drug bioanalysis. Irth H. Jones GR. Wilson ID. Barcelo D. Smyth MR. Double-disk solid-phase ´ extraction: simultaneous cleanup and trace enrichment of herbicides and metabolites from environmental samples. 125: 1515. 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39: 464. 71: 3955.68 62 Cardinali FL.39 67 Fernando WPN.8 72 Zhang Waters Chromatography Chemical Division www.sigma-aldrich.82 77 Penton Fouda HG. Gunatilleka AD. Turnell DC. Morrison GM. Manufacturers and suppliers of solid-phase sorbents and apparatus International Sorbent Technology Ltd (ISL) www. Sample preparation for gas chromatography with solidphase extraction and solid-phase microextraction. Application of solid-phase extraction to the preconcentration of metallothionein and metals from physiological Lemire SW. 689: 856: 55. 65: 58895 68 Lingemann Zhang N.52 Accepted for publication 23 April 2002 Appendix 1. Pawliszyn J. Determination of copper speciation in ¨ freshwater samples through SPE. Green B.31 82 Wu J.7 76 Van de Merbel NC.477 . Miniaturized solid-phase extraction with resin disks. Balasova V. J Chromatogr B 2000. 456: 53. Preparation and applications of polypyrrole lms in solid-phase microextraction.spark-holland. Anal Chem 1998. A technique for the preparation of biological samples prior to high-performance liquid chromatography. Pawliszyn J.15 70 Rossi DT.bohdan. Adv Chromatogr 1997.54 63 Hyspler 74 Cooper JDH. LC. Wooten JV. An automated method for the analysis of urinary free catecholamines using ASTED and highpressure liquid chromatography. 26: 361. Poole CF. Anal Chem 1993. Determination of isoprene in human expired breath using solidphase microextraction and gas chromatography. Turnell Bohdan Automation (Mettler Toledo Bohdan) www. Anal Chim Acta 1997. LC-GC 1998. 70: 2311. Sampling and determination of formaldehyde using solid-phase microextraction with on-line derivatisation.zinsser-analytic.waters. 885: Tomtec Instruments www. 37: 205. 909: 37.varianinc. Anal Chem 1994.66 81 Kabzinski AK. 909: 79. J Chromatogr A 2000. Ann Clin Biochem 1989. 885: 305. Crhova S. Quantitative and qualitative determination of estrogen sulfates in human urine by liquid chromatography/tandem mass spectrometry using 96-well technology. 11: 21. 885: 17.GC Eur Restek Corp www. J Chromatogr A 2000. Swyden MC. High-throughput method development approaches for bioanalytical mass spectrometry. J Chromatogr A Henion J. J Chromatogr 1988. Automating solid-phase extraction: current aspects and future JT Baker www. 12: 704. Ashley DL. Anal Chem 1999. Hoekstra-Oussoren Jones Chromatography www. Gasparic J. Cooper JDH. J Chromatogr B 1997. Walker V.19 477 79 Fritz Anachem Ltd www. J Chromatogr B Gilson Inc www. 38: 255. 753: 259. Poole CF.37 69 Wells DA.appliedseparations.mmm. Cizkova M. 766: 121.64 73 Smith GA.36 78 Nilsson 3M Empore2 Applied Separations www.tecan-uk. J Chromatogr A 2001. 739: 183. Masso JJ.46 66 Poole CF.69 75 Green B. Lloyd TL.20 65 Larrivee ML. 66: 139.htm Tecan www. Larrivee ML. Automated solid-phase extraction and sample preparation: nding the right solution for your Ann Clin Biochem 2002.htm Cyberlab Inc www. Sethuraman R.restekcorp. Zadak Z. J Chromatogr A 1997.gilson.90 64 Martos PA. J Chromatogr A 2001. 343: 259.asp Sagian Inc (see Beckman Coulter) ANSYS Inc www. 38: Supelco (Sigma-Aldrich Co Ltd) Zinsser Analytic Spark Holland BV www. J Chromatogr A 1999. Headspace solid-phase microextraction pro ling of volatile compounds in urine: application to metabolic investigations.113 71 Janiszewski JS.gilson.htm Beckman Coulter Inc www. Solid-phase extraction for combinatorial Varian Inc www.beckman.mass spectrometry. The use of solid-phase microextraction in conjunction with a benchtop quadrupole mass spectrometer for the analysis of volatile organic compounds in human blood at the low parts-per-trillion level. McCraw JM. J Chromatogr Sci J Chromatogr Sci 2000.85 80 Bjorkland LB.Solid-phase extraction in clinical biochemistry 61 Mills GA. 96-Well plate products for solid-phase extraction. Automated sequential trace enrichment of dialysates and robotics. Contributions of theory to method development in solid-phase Membrane-based sample preparation coupled on-line to chromatography electrophoresis. Investigation of the kinetic properties of particle-loaded membranes for solid-phase extraction by forced ow planar chromatography. Solvation parameter model for the prediction of breakthrough volumes in solid-phase extraction with particle-loaded membranes. Particle-loaded membranes for sample concentration and/or clean-up in Alltech Associates Inc www.