Multidimensional Chromatography

Biomedical and Pharmaceutical Applications

273

selectively recognize and bind a specific compound in a similar manner as immunoaffinity sorbents, but can be tailor-made (117). A disadvantage of most current MIPs is that the analyte needs to be dissolved in a non-protic organic solvent and, as a consequence, the use of MIPs in on-line SPE of aqueous biofluids has been very limited so far. In order to circumvent this problem, Boos et al. (118) have proposed an on-line SPE – SPE – LC scheme which comprises a RAM and a MIP precolumn. After sample loading, the RAM sorbent is desorbed with a pure organic solvent which is led to the MIP pre-column for molecular recognition of the target analyte. A practical evaluation of this approach, as well as other multidimensional set-ups involving MIPs, can be expected in the near future.

11.4 LIQUID CHROMATOGRAPHY – GAS CHROMATOGRAPHY

LC is not only a powerful analytical method as such, but it also allows effective sample preparation for GC. The fractions of interest (heart-cuts) are collected and introduced into the GC. The GC column can then be used to separate the fractions of different polarity on the basis of volatility differences. The separation efficiency and selectivity of LC is needed to isolate the compounds of interest from a complex matrix.

Traditionally, LC and GC are used as separate steps in the sample analysis sequence, with collection in between, and then followed by transfer. A major limitation of off-line LC – GC is that only a small aliquot of the LC fraction is injected into the GC (e.g. 1 – 2 l from 1 ml). Therefore, increasing attention is now given to the on-line combination of LC and GC. This involves the transfer of large volumes of eluent into capillary GC. In order to achieve this, the so-called on-column interface (retention gap) or a programmed temperature vaporizor (PTV) in front of the GC column are used. Nearly all on-line LC – GC applications involve normal-phase (NP) LC, because the introduction of relatively large volumes of apolar, relatively volatile mobile phases into the GC unit is easier than for aqueous solvents. On-line LC – GC does not only increase the sensitivity but also saves time and improves precision.

11.4.1NPLC – GC

The first bioanalytical application of LC – GC was presented by Grob et al. (119). These authors proposed this coupled system for the determination of diethylstilbestrol in urine as a replacement for GC – MS. After hydrolysis, clean-up by solidphase extraction and derivatization by pentafluorobenzyl bromide, the extract was separated with normal-phase LC by using cyclohexane 1% tetrahydrofuran (THF) at a flow-rate of 260 l min as the mobile phase. The result of LC – UV analysis of a urine sample and GC with electron-capture detection (ECD) of the LC fraction are shown in Figures 11.8(a) and (b), respectively. The practical detection limits varied between about 0.1 and 0.3 ppb, depending on the urine being analysed. By use of

274	Multidimensional Chromatography

Figure 11.8 (a) LC – UV trace of a typical sample after derivatization of DES to the dipentafluorobenzyl ether. The transferred DES fraction almost corresponds to a peak in the LC trace but, of course, this peak does not represent DES. (b) GC – ECD of the DES fraction from LC separation, involving the pentafluorobenzyl ether derivative of the two isomers of DES. The sample wash spiked with DES (5 ppb) before sample preparation. During passage of the solvent vapour through the column, the column eluent was driven away from the detector cell through the line usually used for feeding the make-up gas into the detector. Reprinted from Journal of Chronatography, 357, K. Grob Jr et al., ‘Coupled high-perfor- mance liquid chromatography – capillary gas chromatography as a replacement for gas chromatography – mass spectrometry in the determination of diethylstilbestrol in bovine urine,’ pp. 416 – 422, copyright 1986, with permission from Elsevier Science.

derivatization and electron-capture detection, the sensitivity of the LC – GC method exceeded that obtained by GC – MS by at least a factor of ten.

Gianesello et al. (120) described the determination of the bronchodilator broxaterol in plasma by on-line LC – GC. After deproteination and extraction, the LC separation was carried out by using a mixture of n-pentane and diethyl ether (55 : 45 (vol/vol) as mobile phase. A small cut of the LC chromatogram (shown in Figure 11.9(a)) was introduced at 85 °C into the GC via so-called concurrent solvent evaporation. Figure 11.9(b) demonstrates that a detection limit of about 0.03 ng/ml was obtained. A fully automated LC – GC instrument was described by Munari and Grob (121) and its applicability was demonstrated by the determination of heroin metabo-

Biomedical and Pharmaceutical Applications

275

(b)

(a)

Broxaterol

Internal standard

0 2 4 6 0 2 4 6 8 10

x	Time (min)

Time (min)

Figure 11.9 (a) LC trace of human plasma with 0.1 g/ml broxaterol, where ‘x’ indicates the cut introduced into the GC run; (b) chromatogram of human plasma with 0.1 ng/ml broxaterol and 5 ng/ml internal standard. Reprinted from Journal of High Resolution Chromatography and Chronatographical Communication, 11, V. Gianesello et al.,

‘Determination of broxaterol in plasma by coupled HPLC – GC,’ pp 99 – 102, 1988, with permission from Wiley-VCH.

lites. The LC eluent consisted of ethyl ether/methanol/diethylamine (91.5 : 8 : 0.5, vol/vol) at a flow-rate of 400 l/min and a fraction of 500 l was transferred to the GC unit through a loop-type interface. Ghys et al. (122) have used the same set-up for the coupling of micro-SEC and GC. The interesting aspect of a micro LC column of 320 m i.d. with a flow-rate of about 1 l/min is that a relatively large fraction can be introduced into the GC unit. For the analysis of steroid esters in a pharmaceutical formulation, a volume of only 4 l THF was transferred to the GC system. Figure 11.10 shows the potential of this system. The heart-cut chromatogram of the sample is very similar to the chromatogram of a standard solution of the steroid esters.

276	Multidimensional Chromatography

1

(a)

	3-4
	6
	5
	2
30	40	50
	Time (min)
(b)	(c)
	0.82	2.25

			25.67						17.33	25.75
12.88	14.66	17.20		26.77	2.04	2.55 2.66	11.91	16.37		26.86	26.09

Figure 11.10 (a) Micro-SEC – UV trace of Sustanon, where peak 2-5 were transferred to the GC unit. Peak identification is as follows: 1, benzylalcohol; 2, testosterone propionate; 3, testosterone isocaproate; 4, testosterone phenylpropionate; 5, testosterone decanoate; 6, oil matrix; (b) GC analysis of the transfer (4 l) from the micro SEC system; (c) Direct GC analysis of a standard solution of the steroid esters. Reprinted from Proceedings of the 10th Symposium on Capillary Chromatography, M. Ghys et al., ‘On-line micro size-exclusion chromatography – capillary gas chromatography,’ 1989, with permission from Wiley-VCH.

Presently, the on-line coupling of NPLC and GC via heart-cutting is an established procedure which has been used successfully for several bioanalytical applications. Obviously, direct analysis of aqueous samples is not possible by NPLC, and therefore, a solvent switch by a sample pretreatment step (e.g. liquid – liquid extraction or SPE) is always required when biological samples are analysed by NPLC – GC.

Biomedical and Pharmaceutical Applications

277

11.4.2RPLC – GC

For pharmaceutical and biomedical analysis, RPLC is much more important than NPLC. However, the interfacing techniques used in NPLC – GC do not generally work well when used for RPLC – GC. The main difficulties encountered when transferring water or water-containing eluents to a GC unit are due to the large vapour volume of water, its high surface tension, poor wetting characteristics, high boiling point and aggressive hydrolytic reactivity. Two approaches have been described for interfacing RPLC and GC on-line, i.e. (i) direct introduction of the aqueous LC fraction by miniaturization of the LC step or by use of special retention gaps, and (ii) phase-switching techniques, i.e. the analytes are first transferred to an organic solvent and subsequently introduced into the GC system.

For drug analysis, Goosens et al. (123) used a Carbowax-deactivated retention gap to transfer eluents from the LC unit to the GC part of the system. Up to 200 l of eluent (acetonitrile-water) were introduced into a Carbowax-coated retention gap by using an on-column interface and solvent vapour exit (124). It was found that the water content of the eluent should not exceed that of the azeotropic mixture, or otherwise water, which is left in the gap after evaporation of the azeotropic mixture, will mar the analysis. In order to deal with the presence of buffers or ion-pairing agents, an anion-exchange micromembrane device was inserted between the LC and GC parts of the system to remove the ion-pairing agent methanesulphonic acid from an acetonitrile – water LC eluent (125). The applicability of the on-line LC – micromembrane – GC system was illustrated for the potential drug eltroprazine (125) and for an impurity profile of the drug mebeverine (126). Before the LC fraction was introduced on-line into the GC – MS system, acetonitrile was added to achieve an azeotropic acetonitrile/water ratio, and, therefore only a part of the LC peak could be transferred. Nevertheless, electron impact and chemical ionization spectra of an impurity could be obtained at a level of 0.1 % with respect to the drug.

Ogorka et al. (127) have coupled RPLC and GC via on-line liquid – liquid extraction of the aqueous mobile phase and used the system for the impurity profiling of drugs. The instrumental set-up is shown in Figure 11.11, where a main critical part is the phase separator. These authors optimized the extraction for mobile phases consisting of methanol – water and acetonitrile – water by using n-pentane, n-hexane and dichloromethane as extraction solvents. The extraction yield depended on the water content of the mobile phase and the polarity of the organic phase. Transfer volumes of 500 l of aqueous mobile phase have been used. The usefulness was extended via on-column derivatization by the introduction of a reagent via the loop-type interface or by derivatization during the extraction (128). By the use of MS as the GC detector, the identification of various unknown impurities in pharmaceutical products was achieved. Contrary to direct LC – MS, the composition of the LC eluent is less limited because non-volatile buffers can also be chosen. The same system was also applied for the analysis of biological samples, i.e. the determination of -blockers in human serum and urine (129) and the determination of morphine and its analogues in urine (130). In the latter case, the analytes were silylated with bis (trimethylsilyl)

278				Multidimensional Chromatography
	Mobile phase
Pump 1	RP column			UV
				R2
	UV	CB	Organic		R3 W
	TP1			PS	FR
			Aqueous W
		PTFE coil			PR
			TP2
	R1	3	2	1	Helium
Extraction solvent
				GC unit
pump 2	MS

Early vapour exit

Figure 11.11 Schematic diagram of the instrumental set-up used for on-line coupled LC – GC – MS: 1, retention gap; 2, retaining pre-column; 3, analytical capillary; R1, LC column (restriction); FR, flow regulator; R2, needle-valve restrictor; R3, capillary (75 m i.d.); PS, phase separator (sandwich type); CB, cooling bath; PR, pressure regulator; W, waste; TP1 and TP2, T-pieces; UV, UV detector. (Note that the UV detector* can be positioned either before or after the liquid – liquid extraction unit.) Reprinted from Journal of Chromatography, 626, J. Ogorka et al., ‘On-line coupled reversed-phase high-performance liquid chromatography – gas chromatography – mass spectrometry. A powerful tool for the identification of unknown impurities in pharmaceutical products’, pp. 87 – 96, copyright 1992, with permission from Elsevier Science.

acetamide in the retention gap before the GC separation. After the reaction, the solvent vapour exit was closed and the GC run was started. The yield of the on-line derivatization was comparable with off-line derivatization. It should be noted that the liquid – liquid extraction step in the applied system can offer extra selectivity. The extraction yield was increased by the use of higher temperatures. The high total selectivity is illustrated by Figure 11.12. The total analysis time was less than 60 min, which is much shorter than with more traditional analytical methods. The limits of quantification were 61 – 92 ng/ml.

11.5 SOLID-PHASE EXTRACTION – GAS CHROMATOGRAPHY

In LC – LC and SPE – LC, the presence of water is commonly no problem at all. Actually, the reverse is true because eluents in RPLC are typically water – methanol or water – acetonitrile mixtures, and a high water content is mandatory during trace enrichment in order to ensure strong retention. However, when such an SPE precolumn or analytical column is coupled to a GC system, the introduction of water should be avoided completely or, at best, be permitted under strictly controlled conditions (see above). It will be clear that on-line trace enrichment (and clean-up) by SPE

Biomedical and Pharmaceutical Applications

279

135

Response (mV)	80
	26

0

135

80

Response (mV)

26

0

2

3

1

4 5

10	20	30	40
	Time (min)

10	20	30	40
	Time (min)

Figure 11.12 GC analysis of (a) urine sample spiked with opiates 3 g/ml) and (b) blank urine sample. Peak identification is as follows: 1, dihydrocodeine; 2, codeine; 3, ethylmorphine; 4, morphine; 5, heroin. Reprinted from Journal of Chromatography, A 771, T. Hyötyläinen et al., ‘Determination of morphine and its analogues in urine by on-line coupled reversed-phase liquied chromatography – gas chromatography with on-line derivatization,’ pp. 360 – 365, copyright 1997, with permission from Elsevier Science.

280	Multidimensional Chromatography

for GC analysis, i.e. SPE – GC, is a highly interesting approach for the rapid tracelevel detection and quantitation of the wide range of GC-amenable compounds that have to be monitored. This convincingly justifies the rather special interest in this particular area of on-line LC – GC, but so far the applications have been mainly in the environmental field and not many bioanalytical applications have been described.

On-line dialysis – SPE – GC was developed for the determination of drugs in plasma, with benzodiazepines as model compounds (131). Clean-up was based on dialysis of 100 l samples for 7 min by using water as the acceptor, and trapping the diffused analytes on a PLRP-S column. After drying, the analytes were desorbed with 375 l of ethyl acetate, which were injected on-line into the GC via a loop-type interface. This system provides a very efficient clean-up and offers the possibility of adding chemical agents which can help to reduce drug – protein binding. In order to demonstrate the potential of the approach, benzodiazepines were determined in plasma at their therapeutic levels. Flame-ionization (FID), nitrogen – phosphorus (NPD) and MS detection were used.

The selectivity of the trace-enrichment procedure can be improved by using an immunoaffinity precolumn: 19- -nortestosterone was used as the test compound (132). Desorption from the antibody-loaded pre-column had to be carried out with about 2 ml of methanol – water (95 : 5, vol/vol), which obviously could not be transferred to the retention gap. The eluate was therefore diluted with HPLC-grade water and the mixture led through a conventional ODS pre-column. As a result of so-called reconcentration by dilution – which means that the gain in breakthrough volume due to increased retention caused by the decrease of the modifier percentage distinctly outweighs the volume increase – the analyte was quantitatively trapped on this second pre-column. Desorption and transfer to the GC system were coried out in a similar way to that described above. The method was applied to the determination of steroid hormones in 5 – 25 ml human urine. The detection limit for 19- -nortestos- terone was about 0.1 ppb with an RSD of 6 % (see Figure 11.13).

Examples of SPE – GC of biological samples are few, while the usefulness of SPE – GC for the analysis of surface and drinking water has been demonstrated many times (133). This might be due to the fact that biological samples are often considerably more complex than environmental water samples. In addition, various biomedically and pharmaceutically interesting analytes will not be amenable to GC. Nevertheless, because many of the initial SPE – GC interfacing problems have now been solved (133), it seems appropriate and worthwhile to explore its utility in the bioanalytical field more thoroughly.

11.6 SOLID-PHASE MICROEXTRACTION COUPLED WITH GAS OR LIQUID CHROMATOGRAPHY

Although solid-phase microextraction (SPME) has only been introduced comparatively recently (134), it has already generated much interest and popularity. SPME is based on the equilibrium between an aqueous sample and a stationary phase coated on a fibre that is mounted in a syringe-like protective holder. For extraction, the fibre

Biomedical and Pharmaceutical Applications

(a)

(b)

FIDResponse	NE NG
(c)

281

ß-19-NT

28	29	30	31	32	33	34	35
(d)		Time (min)

0	5	10	15	20	25	30	35
			Time (min)

Figure 11.13 (a – c) Immunoaffinity extraction-SPE – GC-FID traces of (a) HPLC-grade water (b) urine (c) urine spiked with -19-nortestostrone (0.5 g/l) or norethindrone and norgestrel (both 4 g/l); (d) SPE – GC-FID trace of urine. Reprinted from Analytical Chemistry, 63, A. Farjam et al., ‘Direct introduction of large-volume urine samples into an on-line immunoaffinity sample pretreatment-capillary gas chromatography system,’ pp. 2481 – 2487, 1991, with permission from the American Chemical Society.

is exposed to the sample by suppressing the plunger. Sorption of the analytes on the fibre takes place in either the sample by direct-immersion or the headspace (HS) of the sample. After equilibrium or a well-defined time, the fibre is withdrawn in the septum piercing needle and introduced into the analytical instrument where the

282	Multidimensional Chromatography

Figure 11.14 Analysis of amphetamines by GC-NPD following HS-SPME extraction from human hair: (a) Normal hair; (b) normal hair after addition of amphetamine (1.5 ng) and methamphetamine (16.1 ng); (c) hair of an amphetamine abuser. Peak identification is as follows: 1, -phenethylamine (internal standard); 2, amphetamine; 3, methamphetamine; 4, N- propyl- -phenethyamine (internal standard). Reprinted from Journal of Chronatography, B 707, I. Koide et al., ‘Determination of amphetamine and methamphetamine in human hair by headspace solid-phase microextraction and gas chromatography with nitrogen-phosphorus detection,’ pp. 99 – 104, copyright 1998, with permission from Elsevier Science.

analytes are either thermally desorbed into the GC unit or re-dissolved in a proper solvent for LC. Coupling to the LC system requires an appropriate interface and was first reported in 1995. The technique was commercialized in 1993 by Supelco. The initial work was exclusively done with SPME – GC (135 – 137), due to the direct and convenient sample introduction into the GC system, while the main application area being environmental analysis. Recently, SPME is being increasingly used in bioanalysis. Successful coupling with LC systems enables the analysis of pharmaceuticals, proteins and surfactants that cannot be analysed by GC. Up until now, only a few papers have described the use of direct-immersion SPME for plasma analysis (138 – 141). For plasma and blood samples, the relevant drug partitions between the fibre, sample and proteins. Models for the relationship between the total amount of the drug present in the plasma and the amount of drug extracted have been developed (138, 139). In this way, a good approximation of the drug – protein binding can also be obtained. A few other typical bioanalytical examples will be discussed below.

In a recent report, HS-SPME was used for the extraction of amphetamines from human hair (142). Human hair analysis is gaining interest in the analysis of drugs of abuse, since it offers attractive features: easy and ‘unlimited’ sampling, and as the