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Figure 11.3 Typical configuration for the on-line coupling of an achiral and chiral chromatographic system by means of a switching valve. The non-enantio-resolved solute is isolated on the achiral phase and then stereochemically separated on the chiral phase. Reprinted from G. Subramanian, A Practical Approach to Chiral Separation by Liquid Chromatography, 1994, pp. 357 – 396, with permission from Wiley-VCH.

determination of metyrapone (a steroid biosynthesis inhibitor) and the enantiomers of its chiral metabolite metyrapol in human plasma and urine (42). A short silica column was used to separate metyrapone and metyrapol, followed by a Chiralcel OJ column for the enatioselective separation of ( ) and ( )-metyrapol (Figure 11.4). The assay was validated for metabolic studies which indicated that the enzymatic reduction of myterapone is enantiospecific. Some authors have circumvented the poor performance (e.g. broad asymmetric peaks) of particular chiral columns by reversing the order of columns, i.e. carrying out a chiral – achiral coupling and exploiting peak compression effects. After chiral resolution in broad zones, the enantiomers are reconcentrated into sharply defined bands using a separate hydrophobic column for each enantiomer, thereby substantially improving the overall sensitivity and selectivity. This concept has been applied for the enantioseparation of substances such as leucovorine (53), manidipine (45), bupivacaine, metoprolol, oxazepam and terbutaline (54) in plasma.

LC – LC of endogenous compounds in biological fluids has also been reported (see Table 11.1) and does not differ essentially from the bioanalysis of drugs by coupled-column LC. For example, Tagesson et al. (59) determined the DNA adduct 8-hydroxydeoxyguanosine in human urine by on-line injecting a sample fraction eluting from a first polymeric RPLC column into a second ODS column which was connected to an electrochemical detector. This system was used for assaying in vivo oxidative DNA damage in cancer patients. High levels of the urinary adduct were found in patients subjected to body irradiation and chemotherapy.

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Figure 11.4 Chromatograms of plasma samples on a silica – chiralcel OJ coupled column system: (a) plasma spiked with oxprenolol (internal standard); (b) plasma spiked with 0.40 g/ml metyrapone and 0.39 g/ml metyrapol (racemate); (c) plasma sample obtained after oral administration of 750 mg metarypone. Peaks are as follows: 1, metyrapone; 2, metyrapol enantiomers; 3, oxprenolol. Reprinted from Journal of Chromatography, 665, J. A. Chiarotto and I. W. Wainer, ‘Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral – chiral liquid chromatography,’ pp. 147 – 154, copyright 1995, with permission from Elsevier Science.

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Considering the numerous applications, heart-cut LC – LC has convincingly proven its value. Nevertheless, in LC – LC specific method development is generally needed for each analyte. Moreover, heart-cut procedures require accurate timing and, therefore, the performance of the first analytical column in particular should be highly stable to thus yield reproducible retention times. This often means that in LC – LC some kind of sample preparation remains necessary (see Table 11.1) in order to protect the first column from proteins and particulate matter, and to guarantee its lifetime.

11.3 SOLID-PHASE EXTRACTION – LIQUID CHROMATOGRAPHY

Today, solid-phase extraction (SPE) is probably the most popular sample preparation method used for sample enrichment and/or clean-up (3). During the last decade, many improvements in the SPE field such as new formats (e.g. sophisticated cartridges and discs, pipette tips and 96-well plates), new sorbents and the development of automated systems, have led to an extensive use of SPE. Moreover, the on-line combination of SPE and LC using column switching techniques is now routine. SPE, can in principle, be described as a simple liquid chromatographic process in which the sorbent is the stationary phase. Therefore, the direct coupling of SPE and LC can be considered to be a multidimensional chromatographic technique. However, in contrast to LC – LC, in SPE – LC no continuous elution is carried out during the first dimension. Ideally, the analytes are fully retained on the SPE phase during the sorption and washing steps, and, subsequently, quickly and completely desorbed in a small volume during the elution step. In an on-line SPE – LC set-up the desorption solvent is entirely injected on to the LC column.

Numerous studies involving the on-line coupling of SPE columns to LC analytical columns have been reported, including a significant number of biomedical applications (70). Various drugs and endogenous compounds have been analysed in biological matrices by employing this technique, including antibiotics (71), retinoids (72, 73), methotrexate (74), codenie (75), aspirin (76), psilocin (77), almokalant (78), anabolic steroids (79), morphine (80), ceftazidime (81), terbinafine (82), clozapine (83), mizolastine (84), cebaracetam (85) and piroxicam (86). In many of these applications ODS sorbents are used for SPE. Typically, 0.1 – 1 ml of plasma or ca. 1 – 10 ml of urine are loaded on a preconditioned SPE cartridge (2 – 15 mm long, 1 – 4.6 mm i.d., packed with 15 – 40 m particles) which is mounted on a sixor tenport valve and replaces the conventional injection loop. After washing (clean-up), the pre-column is desorbed with mobile phase which is led to the analytical column (often packed with an RPLC phase) for separation of the analytes. Quantification and/or identification is frequently carried out using UV or MS detection. During analysis, the SPE cartridge may be cleaned, reconditioned and loaded with the next sample. Figure 11.5 shows a typical example of on-line SPE – LC for the determination of drugs in human plasma (87). Many of the on-line SPE – LC applications for bioanalysis described in the literature are quite similar and we will not further discuss these studies. In the remaining part of this section, some selected at-line and

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Figure 11.5 Chromatograms of plasma samples obtained with fully automated on-line SPE – LC: (a) drug-free human plasma; (b) human plasma spiked with omeprazole (100 ng/ml) and phenacetin (internal standard; 1000 ng/ml). Reprinted from Journal of Pharmaceutical and Biomedical Analysis, 21, G. Garcia-Encina et al., ‘Validation of an automated liquid chromatographic method for omeprazole in human plasma using on-line solidphase extraction,’ pp. 371 – 382, copyright 1999, with permission from Elsevier Science.

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on-line applications will be treated in which special formats and/or selective SPE sorbents are used in combination with LC.

11.3.1AUTOMATED SPE – LC

In order to achieve more cost-effective analyses, biomedical laboratories aim for a higher sample throughput and, thus, also for faster sample pretreatment procedures. This aspiration has led to an increasing interest in the automation of sample preparation by using robotic devices. The whole off-line SPE sequence can be automated with instruments such as the ASPEC (Gilson), Microlab (Hamilton) and Rapid Trace (Zymark). Some of these devices can be coupled to LC in an at-line fashion, as they provide the possibility for automated injection of the final extract. A good illustration of the high degree of automation that can be accomplished today is the determination of the glucocorticosteroid budesonide in plasma samples, as described by Kronkvist et al. (88). Their automated method comprised a Tecan robot for initial sample manipulation (e.g. pipetting and mixing), an ASPEC XL for SPE of the samples, an on-line trace enrichment system for further purification and concentration of the sample extracts, and a gradient LC – MS – MS system for separation and selective detection of the analyte and the internal standard. The total system can analyse up to 800 samples a week with satisfactory accuracy, yielding an LOQ of 15 pM for 1 ml plasma samples.

In 1996, the 96-wells SPE technology was introduced for high-throughput analysis; this allows the simultaneous processing of 96 samples in a standard microtiter plate format (89). Such technology, in combination with a pipetting robot, was used to design a high-throughput SPE system followed by RPLC with UV detection for the determination of cimetidine (a histamine H2-receptor antagonist) in plasma (90). Validation results were found to be adequate and a good correlation between the results obtained with the automated method and with a manual method was demonstrated. The average sample preparation time decreased from 4 min to 0.6 min per sample and a sample throughput of 160 samples a day was achieved. Recently, Jemal et al. (91) made a comparison of plasma sample pretreatment by manual liquid – liquid extraction, automated 96-well liquid – liquid extraction and automated 96-well SPE for the analysis by LC – MS – MS. The 96-well methods were three times faster than the manual method. The time required for the 96-well SPE could be further reduced by 50 % when the extracts were injected directly on to the LC (i.e. not incorporating drying and reconstitution steps in the SPE procedure).

In principle, on-line SPE – LC can be automated quite easily as well, for instance, by using such programmable on-line SPE instrumentation as the Prospekt (Spark Holland) or the OSP-2 (Merck) which have the capability to switch to a fresh disposable pre-column for every sample. Several relevant applications in the biomedical field have been described in which these devices have been used. For example, a fully automated system comprising an autosampler, a Prospekt and an LC with a UV

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detector was applied for the analysis of omeprazole, a drug for the treatment of gastric ulcers, in human plasma (87). The extraction was carried out on-line by using disposable SPE cartridges filled with an ODS sorbent, which, after washing, yielded sufficient sample clean-up and enrichment to permit measurement of drug levels at the low ng/ml level (see Figure 11.5). The automated system was validated and used for a large number of plasma samples from bioequivalence studies. In another study, adapalene (a retinoid) and retinol were simultaneously determined in human plasma and mouse tissue by combining an autosampler and an OSP-2 unit with a gradient LC with UV and fluorescence detection (92). The high degree of automation of the on-line SPE – LC system, its high sensitivity (LOQ of 0.25 ng/ml for adapalene) and its good reproducibility, made this method convenient for the determination of pharmacokinetic drug profiles.

11.3.2 RESTRICTED-ACCESS SORBENTS IN SPE – LC

In on-line SPE – LC, deproteination of plasma and serum is often required before extraction, especially when the same SPE cartridge has to be used for repeated analyses. However, today there is strong interest in on-line sample pretreatment techniques which permit the handling of untreated biological samples. For this purpose, special SPE sorbents (so-called restricted-access materials (RAMs)) have been developed which allow sorption (enrichment) of low-molecular-weight analytes at the inner pore surface of the particles, but at the same time exclude high- molecular-weight matrix compounds (e.g. proteins) from the pores (Figure 11.6(a)). In recent years, RAMs have become quite popular for the direct injection of biological fluids into on-line SPE – LC systems (93 – 98). For instance, Yu and Westerlund (93) selected a RAM pre-column filled with an alkyl-diol silica to rapidly separate bupivocaine (a local anaesthetic) from proteins and polar endogenous compounds in human plasma. A 500 l plasma sample was directly introduced on to the SPE column and the fraction containing the analyte was on-line transferred to an RPLC column for final separation. A single alkyl-diol silica pre-column could withstand over 50 ml of plasma injections. The same type of RAM pre-columns appeared to be very useful for on-line clean-up and enrichment during the determination of several drugs and metabolites in biological fluids (e.g. serum, urine, intestinal aspirates and supernatants of cell cultures) which could be directly injected (94).

LC – MS with on-line SPE using a RAM pre-column with an internal ODS phase was described by van der Hoeven et al. (95) for the analysis of cortisol and prednisolone in plasma, and arachidonic acid in urine. The samples were injected directly and the only off-line pretreatment required was centrifugation. By using the on-line SPE – LC – MS system, cortisol and related compounds could be totally recovered and quantified in 100 l plasma within 5 min with a typical detection of 2 ng/ml (Figure 11.6(b)). The RAM-type of sorbents, in which the outer surface of the particles is covered with 1-acid glycoprotein, also appear to be useful for direct SPE of

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Figure 11.6 (a) Schematic illustration of a restricted-access sorbent particle. (b) On-line SPE – LC – MS using an alkyl-diol RAM pre-column for the determination of cortisol, cortisone, prednisolone and fludrocortisone (100 ng/ml each) in untreated plasma. Part (a) reprinted from Journal of Chromatography, A 797, J. Hermansson et al., ‘Direct injection of large volumes of plasma/serum of a new biocompatible extraction column for the determination of atenolol, propanolol and ibuprofen. Mechanisms for the improvement of chromatographic performance,’ pp 251 – 263, copyright 1998, with permission from Elsevier Science. Part (b) reprinted from Journal of Chromatography, A 762, R. A. M. van der Hoeven et al., ‘Liquid chromatography – mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection,’ pp. 193 – 200, copyright 1997, with permission from Elsevier Science.

relatively large volumes (200 – 500 l) of plasma and serum. By using these precolumns in an on-line SPE – LC set-up with fluorescence detection, Hermansson et al. (96) could quantitatively determine atenolol and propanolol in serum down to the low ng/ml level. The stability and performance of the system was good, even

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after the injection of 200 serum samples of 500 l. An on-line sample clean-up using a Pinkerton-GFF2 RAM pre-column was combined with LC on a non-porous ODS phase for the analysis of six cardiovascular drugs in serum (97). Matrix interferents could be removed efficiently and were not detected in the serum chromatograms.

11.3.3 IMMUNOAFFINITY SORBENTS IN SPE – LC

In the large majority of all SPE – LC applications, SPE pre-columns are packed with a hydrophobic packing material such as an ODS phase or a polystyrene – divinylbenzene copolymer. With these packing materials, in principle, sensitivity can be increased dramatically (high analyte retention), but selectivity may only be slightly improved (non-selective hydrophobic interaction). If enhanced selectivity is a major requirement, packing materials with immobilized antibodies (immunoaffinity sorbents) can be used which provide extraction based on molecular recognition. After some exploration work of others (99, 100), Farjam and co-workers were the first to extensively study the on-line application of immunoaffinity pre-columns in combination with LC for the selective determination of low-molecular-weight compounds (steroids and aflatoxins) in biological samples (101 – 105). A system consisting of a pre-column packed with Sepharose-immobilized polyclonal antibodies against -19-nortestorenone ( -19-NT), a second ODS-filled pre-column, an analytical ODS column and a UV detector (Figure 11.7(a)), was used for the determination of -19-NT and -19-NT in calf urine which was injected directly (101). The analytes were subsequently trapped on the immunoaffinity pre-column, selectively desorbed by using the cross-reacting steroid norgestrel, reconcentrated on the ODS precolumn and transferred to the analytical column. Recoveries of -19-NT were over 95% and the detection limit was 50 ng/l for a 25 ml urine injection (Figure 11.7(b)). A similar procedure was also used for the determination of -19- NT and -19-NT in calf bile, liver, kidney and meat samples (102), and for the analysis of clenbuterol in calf urine (106). In a subsequent study, an anti-aflatoxin immunoaffinity pre-column was used on-line for the selective preconcentration of aflatoxin M1 from urine and milk samples (103, 104). By using LC with fluorescence detection following the immuno-SPE step, detection limits of 20 ng/ml for aflatoxin M1 were found if 2.4-ml milk samples are used. The lifetime of the immunoaffinity pre-column was strongly reduced when milk samples were analysed, due to proteolic enzymes in milk which degrade the antibodies. This problem was circumvented by adding an on-line dialysis unit to the system to prevent interfering macromolecular compounds from entering the pre-column (104). A single immunoaffinity pre-column could now be used for over 70 milk analyses. Another example of the use of on-line immunoaffinity-SPE – LC is the selective quantification of 9-tetrahydrocannabinol, the major indicator of cannabis intoxication, in human saliva (107).

Immunoaffinity extraction combined on-line with LC in conjunction with MS (108 – 110) or tandem MS (111, 112) has also been demonstrated for the determination of analytes in biological fluids. Obviously, such systems offer a very high

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Figure 11.7 (a) Set-up used for immunoaffinity SPE – LC. (b) The analysis of -19- nortestosterone ( -19-NT) and -19-nortestosterone ( -19-NT) (300 ng/l each) in 26.5 ml calf urine by using on-line SPE – LC with an immunoaffinity pre-column against -19-NT. Norgestrel (NG) is used for desorption of the immunoaffinity pre-column. Reprinted from Journal of Chromatography, 452, A. Farjam et al., ‘Immunoaffinity pre-column for selective on-line sample pre-treatment in high-performance liquid chromatography determination of 19-nortestosterone,’ pp. 419 – 433, copyright 1988, with permission from Elsevier Science.

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selectivity which may be needed when analytes have to be determined at the trace level and or characterization and confirmatory information is required. Cai and Henion (111) used an immunoaffinity-SPE – SPE – LC – MS – MS system for the analysis of LSD and its analogues and metabolites in human urine. The on-line chromatographic system involved an immunoaffinity pre-column, a packed capillary trapping column and a packed capillary analytical column. With the trapping column as intermediate the immuno pre-column could be operated at high flow rates, while the analytical column was maintained at 3.5 l/min, thereby facilitating the coupling with MS by electrospray. The urine of LSD users was analysed and concentrations of LSD and analogues at the low-ppt level could be measured. A similar system was used for the analysis of five -agonists in bovine urine yielding LOQs in the 10 – 50 ppt range, and applied to the determination of the bovine renal elimination of clenbuterol over a period of 15 days after administration (112). For the selective extraction of propanolol from urine, a protein G pre-column was primed with drug-specific antibodies and coupled on-line to an SPE – LC – MS system which comprised a CN analytical column for separation and an ion-spray source for LC – MS interfacing. By using the single-ion-monitoring mode, propanolol could be specifically detected down to 1 ng/ml in 20 ml urine. Creaser et al. (108) used on-line immunoaffinitySPE – LC – ion-trap MS with a particle beam interface for the determination of corticosteroids in equine urine.

11.3.4 METAL-LOADED SORBENTS AND MOLECULARLY IMPRINTED POLYMERS IN SPE – LC

An interesting – but not often used – option to increase SPE selectivity is the application of metal-loaded sorbents that are able to bind certain organic compounds which form specific complexes with the immobilized metal ions such as Cu(II), Ag(I), Hg(II) and Pd(II). Nielen et al. (113) showed that metal-loaded phases can be used for selective on-line sample handling and trace enrichment in LC. Some bioanalytical applications were reported by Irth and co-workers 114 – 116). A pre-column with a silver-loaded thiol phase was used in an on-line fashion for the extraction of 5-fluo- rouracil (a chemotheurapetic agent) from plasma (114). SPE sorption of the analyte by complexation with Ag(I) occured at high pH, while desorption to the ODS analytical column was based on analyte protonation at low pH. For plasma samples, either a second pre-column packed with PLRP-S was placed before the Ag(I)-thiol precolumn in order to remove macromolecular and apolar components, or the samples were deproteinated before analysis. By using UV detection, a detection limit of 3 nM was obtained for 5-fluorouracil for direct injection of 1 ml samples. The same SPE – LC – UV system with a silver-loaded pre-column was used for the trace-level determination of 3´-azido-3´-deoxythymidine (AZT), a potential drug for the treatment of AIDS, in plasma (115), and also for the analysis of four barbiturates in plasma (116).

Recently, molecularly imprinted polymers (MIPs) have gained attention as new, selective sorbents for chromatography and SPE. The cavities in the polymer