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253

analyte enrichment prior to LC analysis. In LC, a popular way towards enhanced selectivity and sensitivity is the use of small solid-phase extraction (SPE) cartridges filled with a more or less selective sorbent for the preconcentration of samples (3). The application of the on-line combination of SPE and LC for bioanalytical purposes will be discussed to some extent in the next section. Another viable approach towards more chromatographic selectivity is coupled-column LC (LC – LC) in which two or more analytical columns are combined in an on-line fashion to accomplish the isolation of the compound(s) of interest. LC – LC can be used either for the profiling of a complete sample or for the analysis of target compounds (4).

11.2.1COMPREHENSIVE LC – LC

The objective of the profiling mode of LC – LC is to fractionate all components of the analysed mixture. This may be accomplished by so-called comprehensive twodimensional LC in which the entire chromatogram eluting from the primary column is submitted to the secondary column. The secondary instrument must operate fast enough to preserve the information contained in the primary signal. That is, it should be able to generate at least one chromatogram during the time required for a peak to elute from the primary column. Until now, a limited number of studies involving comprehensive LC – LC for the analysis of compounds of biological interest have been reported. Most of these studies were carried out within the group of Jorgenson and mainly deal with the design, construction and implementation of comprehensive LC – LC systems for the separation of either a complex protein mixture or an enzymatic digest of a protein (i.e. a mixture of peptides) (5 – 10). For these purposes, orthogonal on-line combinations of ion-exchange chromatography, size-exclusion chromatography (SEC) or reversed-phase (RP) LC are used. In a recent study, peptide fragments generated in the tryptic digests of ovalbumin and serum albumin are separated by SEC in the first dimension (run time, 160 min) and fast RPLC in the second (run time, 240 s) (7). Following RPLC, the peptides flow to an electrospray mass spectrometer for on-line identification. The complete LC system yields a peak capacity of almost 500, thereby maximizing the chance of completely resolving each peptide of the digest and, thus, permitting highly reliable peptide mapping. In addition, a comprehensive ion-exchange LC – RPLC – mass spectrometry (MS) system for the analysis of proteins was demonstrated in which a 120-min ion-exchange LC run is sampled by 48 RPLC runs of 150 s, leading to a peak capacity of over 2500

(8). The system was succesfully applied to the screening of an Escherichia coli lysate without any prior knowledge of the characteristics (e.g. molecular weight, isoelectric point, hydrophobicity, etc.) of its individual components.

Other bioanalytical applications of systems in which the eluate of a first LC column is sampled in continuous and repetitive intervals and subjected to a second LC dimension are, for example, described by Wheatly et al. (11) and Matsuoka et al. (12). Wheatly coupled gradient affinity LC with RPLC for the determination of the isoenzymaticand subunit composition of glutathione S-transferses in cytosol

254	Multidimensional Chromatography

extracts of human lung and liver tissues. Matsuoaka combined anion-exchange LC with RPLC for the analysis of the enzymatic digests of bovine calmodulin and D59 protein, and for the separation of a crude peptide mixture which was extracted from bovine brain tissue.

With comprehensive two-dimensional LC, indeed truly orthogonal separations with very high efficiency and peak capacity can be obtained, although the interfacing can be quite complicated. Additionally, in bioanalysis large peak capacities are often not needed, especially when the objective is the determination of only a few target compounds (see next section). The usefulness of comprehensive LC – LC lies particularly in the entire profiling of protein and peptide mixtures of unknown composition (protein/peptide mapping). In this respect, comprehensive LC – LC might be an alternative for two-dimensional gel electrophoresis, with LC – LC having the advantage that it can be on-line coupled to MS so that molecular-weight information becomes available fast and without the need for analyte transfer.

11.2.2 HEART-CUT LC – LC

In contrast to comprehensive LC – LC, the goal of the targeted mode of multidimensional LC is to isolate a single analyte or small group of components of a complex mixture. Target analysis by LC – LC is normally carried out by using the principle of ‘heart-cutting’. In this approach, an eluting zone from the first LC column (heart-cut) is switched to a second column for subsequent analysis of the transferred compounds. Thus, the first column is used to extract and enrich analytes, e.g. from complex biological fluids, while the second column is used to separate the molecule(s) of interest. This combination leads to an enhanced selectivity and sensitivity, because the target compounds are preconcentrated and/or can be detected in the absence (or reduced presence) of interfering compounds. As a result, lower detection limits are obtained and the reliability of quantitative analysis is strongly improved. When a very high selectivity is required, the dimensionality (number of subsequent columns) may be increased to three or more by using different retention modes. Of course, on-line LC – LC requires the use of mobile phases which, to some extent, are compatible with the columns involved. Therefore, LC – LC is restricted to certain chromatographic combinations, which often include RPLC, ion-exchange, polar bonded-phase, chiral or affinity columns. It is evident that next to the selection of the proper columns and eluents, correct timing of the column switching and optimization of the transfer volume are essential aspects, and, therefore, systematic method development is an important issue in LC – LC (13 – 15).

In biomedical analysis, LC – LC has been used most extensively and successfully in the heart-cut mode for the analysis of drugs and related compounds in matrices such as plasma, serum or urine. Table 11.1 gives an overview of analytes in biological matrices which have been determined by heart-cut LC – LC systems. A typical example of such an approach is the work of Eklund et al. (16) who determined the free concentration of sameridine, an anaesthetic and analgesic drug, in blood plasma

Table 11.1 Biomedical applications of on-line heart-cut LC–LC

		Sample	First	Subsequent
Analyte(s)	Sample matrix	preparationa	LC modeb	LC mode(s)b	Detectionc	Reference
Drugs and Related Compounds
Ampicillin	human plasma	deproteination	C18	C18	post-Flu	28
	human urine	buffer dilution	C18	C18	post-Flu	28
-agonists	human/bovine urine	none	C18	C18	UV	22
	bovine urine	none	C18	C18	MS–MS	23
Basic drugs	human serum	liq – liq extraction	ion-pair C18	C18	UV	37
Bupivacaine	human plasma	ultrafiltration	C8	CIEX	UV	24
Chloramphenicol	pig tissue	liq –liq extraction	PRP	C18	UV	32
Dexamethasone	bovine tissue	liq – liq extraction	phenyl	silica; CN	UV	29
Dopa and its	human/rat plasma	deproteination	CIEX	C18	EC	66
metabolite
Efletirizine	human plasma	SPE	C18	C18	UV	69
	human urine	dilution	C18	C18	UV	69
Glycyrrhizin and its	human plasma	SPE	C18	C18	UV	39
metabolite
Ibuprofen	human serum	liq – liq extraction	ion-pair C18	C18; C18	UV	34
Manidipine and its	human serum	liq – liq extraction	ion-pair C18	C18	UV	38
metabolite
Mefenamic acid	human serum	liq – liq extraction	C18	C18; C18	UV	34
Melengestrol	bovine tissue	liq – liq extraction	phenyl	silica; silica	UV	31
acetate

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		Table 11.1	(continued)
		Sample	First	Subsequent
Analyte(s)	Sample matrix	preparation a	LC modeb	LC mode(s) b	Detection c	Reference
Methandrostenolone	equine	liq – liq extraction	phenyl	C18; C8	UV and	30
and its metabolites	plasma/urine				MS–MS
Metyrapone	human	liq – liq extraction	silica	Chiralcel OJ	UV	42
	plasma/urine
Phenyl-	human	liq – liq extraction	ion-pair C18	C18	UV	36
propanololamine	plasma/urine
Probenicid	rat plasma	deproteination	C18	ion-pair C18	UV	63
Propanolol	human plasma	liq – liq extraction	ion-pair C18	C18	UV	35
Methotrexate	human urine	SPE	AIEX	C18	UV	26
Remoxipride	human plasma	liq – liq extraction	CN	C18	EC	17
metabolite
Ro 23-7637	dog plasma	SPE	CIEX	C18	UV	27
Ro 24-0238	human plasma	SPE	CIEX	C8	UV	25
Ropivacaine	human plasma	ultrafiltration	C8	CIEX	UV	24
Sameridine	human plasma	ultrafiltration	C18	CIEX; CIEX	UV	16
TCV-116 and	human	liq – liq extraction	C18	C18	Flu	64
its metabolites	serum/urine
Tipredane	rat urine	none	CN	C18	UV	18
metabolites
	human urine	none	C18	C1	UV	19
Vanillylmandelic	human urine	acidification,	ion-pair C18	AIEX	EC	65
acid		centrifugation
Various drugs	human	none	micel C8	C18	UV or Flu	33
	serum/plasma		or CN

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Zidovudine- -D-	rat plasma	deproteination	C18	ion-pair C18	UV	63
glucuronide
Enantiomers of Drugs and Related Compounds
Amino acids	protein hydrolysates,	various	CIEX	Crownpak	post-Flu	43
	bacterial cultures,			CR( )
	food, urine
Artilide fumarate	human/anim.	SPE	C8	C18; Pirkle	pre-Flu	44
	plasma
Bupivacaine	human plasma	liq – liq extraction	AGP	C18; C8	UV	54
Chlortalidone	human whole	liq – liq extraction	CN	-CD phenyl	UV	51
	blood
Dihydropyridine	dog plasma	liq –liq extraction	AIEX	Ovomucoid	EC	49
calcium blocker
Ibutilide fumarate	human/animal	SPE	C8	C18; Pirkle	pre-Flu	44
	plasma
Ifosamide	human plasma	liq – liq extraction	D,L-naphthyl-	Chiralcel OD	UV	55
			alanine
Ketoprofen	human plasma	deproteination	C18	C18; Ovomucoid	UV	47
Leucovorine	dog plasma	deproteination	BSA-silica	C18	UV	53
	human plasma	deproteination	phenyl	BSA-silica	UV	56
Manidipine	human serum	liq – liq extraction	C18	C18	UV	45
		Chiralcel OJ
Mefloquine	human plasma/whole	liq – liq extraction	CN	silica; (S)-	UV	52
	blood			naphthylurea
Metoprolol	human plasma	SPE	AGP	C18; C18	Flu	54
Metyrapol	human	liq – liq extraction	silica	Chiralcel OJ	UV	42
	plasma/urine

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		Table 11.1	(continued)
		Sample	First	Subsequent
Analyte(s)	Sample matrix	preparation a	LC mode b	LC mode(s) b	Detection c	Reference
Oxazepam	human plasma	SPE	BSA-silica	C18; C18	UV	54
p-HPPH d	rat liver	liq – liq extraction	C18	C18 with	UV	46
	microsomes			L-prolinamide
Pimobendan and its	human plasma	liq – liq extraction	silica	Chiralcel OD	UV	48
metabolite
Terbutaline	human plasma	SPE	phenyl	-CD C18	EC	51
	human plasma	SPE	AGP	C18; C18	EC	54
	human plasma	SPE	phenyl	-CD	EC or MS	57
Verapamil and	human plasma	liq – liq extraction	C18	Ovomucoid	UV	50
its metabolites
Endogenous Compounds
Creatinine	human	buffer dilution	SEC	SEC; CIEX	UV	58
	serum urine			CIEX;
Glutathione	cytosol of human	homogenization,	affinity	C8 or C18	UV	11
S-transferases	lung tissue	centrifugation
Hydroxy-	human urine	SPE	PRP	C18	EC	59
deoxyguanosine
Hydroxytryptamine	rabbit blood	centrifugation	CIEX	C18	EC	68
and its metabolite		on-line SPE
Immunoglobulin G	bovine serum	none	SEC	affinity	UV	60
and its multimers
Neopterin	human serum	centrifugation	C18	ion-pair C18	Flu	62
Proinsulin fusion	E. coli cells	sulfitolysis	SEC	AIEX	UV	61
protein
Riboflavin	human	deproteination	PRP	C18	Flu	67
	blood/plasma

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Thiamineand	human	deproteination	PRP	ion-pair PRP	post-Flu	67
pyridoxal-phoshates	blood/plasma
Uric acid	human	buffer dilution	SEC	AIEX; SEC	UV	58
	serum/urine

aliq – liq, liquid – liquid; SPE, solid-phase extraction.

baffinity, affinity chromatography; AGP, immobilized 1-acid glycoprotein; AIEX, anion-exchange; -CD, -cyclodextrines; BSA, bovine serum albumin; C1, methylsilica; C8, octylsilica; C18, octadecylsilica; CIEX, cation-exchange; Crown pak CR( ), octadecyl silica coated with chiral crown ether; CN, cyanopropylsilica; ion pair, ion-pair chromatography; micel, micellar chromatography; phenyl, phenylsilica; Pirkle, 3,5-dinitrobenzoyl-D-phenylglycine-bonded silica; PRP, polymeric reversed-phase; SEC, size-exclusion chromatography.

cEC, electrochemical detection; Flu, fluorescence detection; MS, mass spectrometric detection; pre-Flu, fluorescence detection after pre-column derivatization; postFlu, fluorescence detection after post-column derivatization; UV, UV absorbance detection.

dp-HPPH, p-hydroxyphenyl phenylhydantoin.

Applications Pharmaceutical and Biomedical

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260	Multidimensional Chromatography

by coupling RPLC and ion-exchange LC. After ultrafiltration of the blood sample, 400 l of the ultrafiltrate was injected into an LC – LC system (Figure 11.1(a)) without further pretreatment. A heart-cut (ca. 1 ml) of the eluate of the first column (octadecylsilica (ODS)) which contained the sameridine, was transferred to and enriched on, a cation-exchange extraction column. In the next step, the concentrated sample was desorbed into the second analytical column (cation-exchange) for the final separation and subsequent UV detection at 205 nm. In this way the interfer- ence-free and sensitive determination of sameridine could be achieved (Figures 11.1(b) and (c)) with a limit of quantitation (LOQ) of 1 nM and a within-day precision of 2 – 8 %. In a similar approach, Baker et al. (18) determined the major

Figure 11.1 (a) Schematic representation of a coupled-column LC system for sameridine analysis consisting of a reversed-phase analytical column (C1), a cation-exchange extraction column (C2) and a cation-exchange analytical column (C3); (b) chromatogram of plasma sample after intrathecal administration of sameridine (plasma concentration, 11.2 nM); (c) chromatogram of a blank plasma sample. Reprinted from Journal of Chromatography, B 708, E. Eklund et al. ‘Determination of free concentration of sameridine in blood plasma by uttrafiltration and coupled-column liquid chromatography,’ pp. 195 – 200, copyright 1998, with permission from Elsevier Science.

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metabolite of the glucocorticoid tipredane in rat urine which was injected directly into the first LC column. In this case, the initial separation was performed on a cyanopropylsilica column and a portion of the eluate was switched to an ODS column where the final separation was carried out. This method allowed quantification of the metabolite down to 25 ng/ml, showed acceptable linearity, precision and accuracy in the 25 – 5000 ng/ml range, and was successfully used for a long-term toxicology study. Lanbeck-Vallén et al. (28) used post-column derivatization with fluorescamine for the analysis of the penicillin ampicillin in plasma by LC with fluorescence detection. However, since fluorescamine reacts with most primary amines, the selectivity of the LC system had to be increased. This was achieved by the use of a coupled column system (Figure 11.2(a)). The total combination yielded a highly selective system which permitted the sensitive determination of ampicillin (Figure 11.2(b)) with a detection limit of 14 nM for 0.5 ml plasma samples.

Figure 11.2 (a) LC – LC system with post-column reaction detection for the determination of ampicillin in plasma; (b) Chromatogram of plasma sample (collected 10 min after oral administration of 670 mol of ampicillin) containing 1.26 M ampicillin (amp). Reprinted from Journal of Chromatography, 567, K. Lanbeck-Vallén et al., ‘Determination of ampicillin in biological fluids by coupled-column liquid chromatography and post-column derivatization,’ pp. 121 – 128, copyright 1991, with permission from Elsevier Science.

262	Multidimensional Chromatography

In its true sense, the term ‘multidimensional’ refers to LC systems in which there is a distinct difference in retention mechanisms in the columns used. Still, column switching without change in retention mode can be a relatively easy to implement approach for separating complex mixtures. This is a practical substitute for lineargradient elution and is particularly useful for analyte enrichment and sample cleanup, as has been extensively demonstrated by Hoogendoorn and co-workers (14, 20, 21) for environmental analysis. The concept is also useful in bioanalysis, as was shown by Polettini et al. (22) who applied coupled-column RPLC using two identical ODS columns for the automated analysis of -agonists in urine samples. With direct injection of large volumes (1.5 ml) of human urine and careful adjustment of transfer volumes and mobile phase conditions, LC – LC with UV detection allowed the determination of clenbuterol at the low-ng/ml level. In a subsequent study (23), the RPLC – RPLC system was coupled to a tandem MS system in order to gain further selectivity and sensitivity. With this set-up, -agonists could be analysed in bovine urine with a LOQ of 0.1 ng/ml. For the determination of clenbuterol at the 1 ng/ml level, the inter-day reproducibility was 8.4 %. With similar hydrophobic columns, still different retention modes can be established by using one in the ionpairing mode and the other in the RP mode. This approach has been used by Yamashita and co-workers (34 – 38) for the analysis of basic and acidic drugs in biological fluids. For example, phenylpropanololamine was determined in human plasma and urine by first pre-separating the basic drug from endogenous compounds on an ODS column using a low-pH eluent containing butanesulfonate as the ion-pair reagent. Subsequently, after column switching of the heart-cut containing the analyte, further separation is carried out on a second, identical column in the absence of butanesulfonate. The selectivity of the method was such that short-wavelength UV detection could be used, thus yielding a detection limit of 0.4 ng/ml in plasma. The method was applied to the determination of phenylpropanololamine in the plasma of human volunteers after oral administration of 25 mg of the drug.

The on-line coupling of achiral and chiral columns is now a proven concept for the bioanalytical separation of enantiomers (40, 41). Table 11.1 summarizes a number of these LC – LC applications. Frequently, first an achiral column is used to separate enantiomeric pairs from matrix components, and then the enantiomers are transferred to a chiral column for selective separation (Figure 11.3). In this way, the chiral separation is often enhanced by excluding interfering substances from the second column. Van de Merbel et al. (43) used two-dimensional LC for the determination of D- and L-enantiomers of amino acids in biological samples. The amino acids are first separated by ion-exchange chromatography and subsequent enantioseparation is achieved by injection of 3 l heart-cuts on to a second column with a chiral crown ether stationary phase. The selectivity and sensitivity of the system was further increased by using fluorescence detection after on-line post-column labelling of the amino acids with o-phthalaldehyde. In this way, small quantities of D-enan- tiomers could be determined in complex biological samples such as protein hydrolysates, urine, bacterial cultures and yoghurt, with hardly any additional pretreatment. Another typical example of achiral – chiral coupled LC is the