Advancing
Hyphen
Chro Although expensive, HPLC/NMR/MS may be the best way to unequivocally characterize complex mixtures.
Ian D.Wilson AstraZeneca Pharmaceuticals (U.K.)
John C. Lindon and Jeremy K. Nichol son Imperial College of Science, Technology and Medicine (U.K.)
othing is tougher to analyze than a complex mixture. Yet, hyphenated techniques—in which chromatographic separations are allied with powerful spectroscopic techniques such as MS and NMR spectroscopy—are gradually eliminating the need for isolating unknowns in a pure form before identification. Applications of HPLC/MS are now routine. Systems combining HPLC with NMR, although more recent than HPLC/MS instruments, are also commercially avail-
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able and becoming widely used (1–4). Moreover, practical solutions to the once intractable problems of inefficient solvent signal suppression and (relatively) poor NMR sensitivity are now available. Thus, HPLC/NMR has proved to be very useful in practice. Given the pace of technical innovation in this area, it seems inevitable that, like HPLC/MS, HPLC/ NMR will become widely used for structure determination. Nevertheless, neither HPLC/MS nor HPLC/NMR alone can always provide complete structure determinations.
omat Systems Often, both techniques are required. For example, in cases where MS is unable to provide an unequivocal structure for positional isomers of substituents on an aromatic ring, the solution is trivial by NMR. The converse can also occur: NMR sometimes only provides a partial solution when part of a molecule under investigation has structural moieties that lack NMR resonances. Clearly, NMR and MS provide complementary data, and given that each had been coupled to HPLC separately, it was
inevitable that integrated, doubly hyphenated HPLC/NMR/ MS systems would be constructed. The first published example appeared in 1995 (5). Not long afterward, an HPLC/ NMR instrument with an ion trap mass spectrometer was described for the identification of the urinary-excreted metabolites of acetaminophen (6). Numerous applications have followed rapidly, which now include a range of analytes from such areas as pharmaceuticals, natural products, and drug metabolites (7–20).
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HPLC with spectroscopic detectors?” In this article, we consider the advantages of HPLC/ NMR/MS and briefly discuss the advent of more complex systems that combine UV-diode arrays and IR and circular dicroism.
11.7 T (500 MHz) NMR magnet r=3m
Practical considerations Relatively few chromatographic compromises are needed to implement successful HPLC/NMR and HPLC/NMR/MS Waste experiments. Reversedphase chromatography Peak Solvents with conventional colsampling unit umns and flow rates of 1 mL/min are routinely used. A benefit of Fused silica capillary HPLC/NMR/MS is Mass spectrometer that optimizing the sep95% 5% aration procedure is less Injection Pump UV critical than if the HPLC valve detector Splitter instrument is coupled with less informationrich detectors, such as Column UV. Thus, NMR and MS can still provide a HPLC computer NMR computer MS computer lot of structural information, even when the peaks eluting from the column are composed NMR of more than one component. There are, however, several factors that must be addressed to FIGURE 1. Typical HPLC/NMR/MS system. successfully perform HPLC/NMR/MS experiments. For examThe limited number of publications to date on HPLC/ ple, finding mutually compatible solvent systems is a probNMR/MS probably reflects the scarcity of HPLC/NMR lem. For NMR with reversed-phase eluents, acetonitrile systems available to couple with mass spectrometers rather water/D2O mixtures, which are pH-controlled with inorthan the technical difficulty of modifying the instruments ganic buffers (e.g., phosphate) and do not contribute furinto a single unit. However, it is clear, even from the limited ther signals to the proton NMR spectrum, are ideal. Such published data, that HPLC/NMR/MS represents a viable, involatile buffer systems are less compatible with MS, and readily implemented approach to the analysis of complex alternatives are generally required. However, with changes mixtures. Coupling such powerful techniques in tandem has in MS interface design, this situation is rapidly improving. also led us to ask, “What are the limits to combinations of For the NMR part of the experiment, an acidic modifier Flow probe
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such as trifluoroacetic acid (TFA), which has no interfering protons, might be used. Unfortunately, because of ionization suppression, TFA causes problems with the MS of acidic analytes—sometimes molecules are undetected, even when present in high-microgram quantities in the HPLC effluent. The best compromise is probably formic acid (ammonium formate buffers are generally compatible with both NMR and MS), although formate produces a signal in the 1 H NMR spectrum. Such considerations restrict the choice of solvent system that may be used for HPLC/NMR/MS, but they do not create insuperable difficulties. Although highly efficient solvent-suppression methods have been developed for NMR and deuterated solvents are no longer mandatory for HPLC/NMR, it is still convenient to prepare mobile phases from acetonitrile/D2O, methanold4 /D2O, or acetonitrile-d3. The use of deuterated acetonitrile/methanol solutions makes solvent suppression easier and allows for the detection of peaks, such as acetylated drugs and their metabolites, near the acetyl methyl resonance. This option is more expensive than using ordinary acetonitrile, but the increased expenditure is small compared with the cost of the experiment. Even where protio-organic solvents are used, D2O is recommended, because it significantly reduces solvent-suppression problems and is relatively inexpensive. However, in practice, the use of D2O can cause some problems with MS interpretation, as exchangeable protons will be replaced by deuterons. This difficulty can be eliminated by mixing the eluent with H2O or methanol before MS to re-exchange the protons (7, 8). This phenomenon can be advantageous because, by recording MS with and without deuterium exchange, the number of exchangeable protons can be measured, which can be helpful in determining, for example, the number of hydroxyls in an unknown. A typical HPLC/NMR/MS system (Figure 1) consists of a UV detector, a conventional liquid chromatograph, and a sampling unit for collecting and storing peaks for subsequent analysis. The NMR and MS spectrometers are connected in parallel via a splitter at the outlet of the UV detector. The NMR and MS spectrometers are not connected in-line—there are several technical reasons for such an arrangement. In particular, placing the mass spectrometer after the NMR spectrometer results in the pressurization of the NMR flow probe. With current designs, if the inline coupling of the mass spectrometer is not considered, the pressure can cause leaks because the NMR flow probes were not meant to operate under such conditions. This problem will be solved with the next generation of probes.
One advantage of the parallel operation of the two spectrometers is the different time scales in which NMR and MS operate. Thus, when it is necessary to operate the NMR spectrometer in stopped-flow mode to perform spectral acquisition over an extended period, the mass spectrometer can be used on a different problem. For example, the NMR acquisition may require several minutes or hours to ensure that a good spectrum of a low-concentration analyte in the chromatographic eluent is obtained, or it might be desirable to obtain time-consuming, two-dimensional NMR spectra. This is less of an issue if the samples are concentrated sufficiently to allow the acquisition of direct on-flow NMR spectra in real time. An additional technical problem is the adverse effect that high-NMR magnetic fields can have on the performance of a mass spectrometer. The latest generation of actively shielded NMR magnets, with their much-reduced stray fields, will help resolve this problem. Given the high sensitivity of the MS detector, we generally split the flow from the UV detector—95% directly into the flow probe of the NMR and 5% to the mass spectrometer. As this suggests, the limiting sensitivity for the overall combination is generally, but not always, the amount of material needed to obtain the required NMR spectrum. The amount of sample needed depends on residence time
Locate the instrumentation in such a way that a double hyphenated
system can be assembled quickly. in the probe (i.e., on-flow or stopped-flow techniques), design of the flow probe, field strength, nucleus (e.g., 1H, 19F), and the type of NMR experiment being performed (i.e., one-or-two dimensional). However, for a “typical” lowmass molecule, which uses a 500-MHz NMR spectrometer with flow rates of 0.5–1.0 mL/min, ~5–10 µg would be expected to give a useful spectrum. With flow rates of 0.1–0.5 mL/min, this amount could be as small as ~1 µg. There is a further reduction in the amount of material needed for a one-dimensional spectrum if stopped-flow techniques are used. A two-dimensional NMR experiment, such as a 1H-1H total correlation spectroscopy, might be completed in a few hours with 1 µg of material. The nature of the problem to be solved is one of the main factors that must be considered before installing a complex and capital-intensive HPLC/NMR/MS instrument. As indicated, many problems in structure identification and elucidation can be solved simply using either HPLC/MS or HPLC/NMR. It would make little sense
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to tie up expensive spectrometers for problems that do not require them. The approach we advocate is to locate the instrumentation in such a way that a doubly hyphenated system can be assembled rapidly in response to an identified need. When not linked, the individual spectrometers can then be used more efficiently to solve unrelated problems.
of a 500-MHz NMR spectrometer, while the remainder went into the particle beam interface of a mass spectrometer operating in the chemical ionization mode. The NMR and mass spectra obtained on-flow were perfectly adequate and demonstrated the potential of such systems (5). This was soon followed by the first application of gradient reversed-phase HPLC/NMR/MS using the well-known metabolites of acetaminophen, which were present in the Applications solid-phase extracts of human urine following a normal Most applications of HPLC/NMR/MS have been for phar- therapeutic dose of the drug (6). Acetaminophen provides maceuticals and drug metabolite determination, but applia useful “proof of concept” test because a major metabocations are also appearing in the natural products field—an lite is acetaminophen sulfate. The HPLC/NMR spectrum area that will benefit greatly from this combination. of an acetaminophen sulfate metabolite clearly shows a peak The idea of combining an NMR spectrometer and a in the chromatogram that is characteristic of the metabolite, mass spectrometer into a single HPLC system was discussed but this experiment cannot unequivocally identify the comby several groups soon after the introduction of the first repound. Moreover, sulfate does not have a diagnostic 1H liable HPLC/NMR probes. The first published demonstraNMR signal. MS, on the other hand, provides the importion of an HPLC/NMR/MS system tackled the analysis of tant information that the molecular mass has increased by a mixture of fluconazole and two related triazole structures 80 amu, consistent with sulfation. Besides confirming the using on-flow MS and NMR data for all three components. presence of the expected acetaminophen metabolites (sulAfter the isocratic separation of analytes on a C18 column fate and glucuronide), HPLC/NMR/MS identified an “unusing acetonitrile/D2O (25:75), the flow of eluent (1 known” endogenous component that is not normally obmL/min) from the column was directed into a splitter and served in urine—N-acetylphenylglycine. split 60:40. The major portion was sent to the flow probe More recently, Burton and co-workers published similar studies identifying acetaminophen metabolites in human urine (9). A CF3 CF3 single quadrupole inCF3 strument was used to obtain the mass spectral HOOC Cl Cl O HO data. They also convertN N HO HO3S H H Cl ed the analytes to their OH COOH protio forms by mixing N OH H O O Sulfation the eluent directed toOH CF3 HO Glucuronidation ward the mass spectroCF3 meter with methanol:1% Glucuronidation Oxidation acetic acid. Or Cl Although ion trap N Cl H OH and MS/MS instruCF3 ments have advantages NH2 in some circumstances, Rearrangement ? in many cases, a single HOOC Cl Oxidation ? O quadrupole mass specHO N CF3 HO OH trometer is sufficient OH to obtain the required mass spectral informaCF3 HO Cl tion. We have used one, Sulfation CF 3 Glucuronidation along with a 500-MHz NH2 NMR spectrometer, to Cl HO3S OH obtain excellent results HO O Cl NH2 HO O for the HPLC/NMR/ HOOC NH2 MS of ibuprofen metabolites in an extract of FIGURE 2. Metabolic profile of 2-trifluoromethyl-4-chloroaniline in the rat determined by HPLC/UV/NMR/MS. human urine (10).
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2000
(a)
(d)
13
C-acetyl CH3 = 6.2 Hz) C-H
2J
(
MeOH-d4 ACN
MeOH
(CH3)2CH
mass
1500 1000
13
C-acetyl CH3
2′ 3′ 4′
500 2.15
3000
2.10
3
0 0.0
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HDO ArOCH2 1′ 5′ CHOH
6 5
(b)
CHCH2NH
mV
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6.0
5.0
3.0
2.0
289 100
1000
O
(e)
OD DOOC DO DO
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4.0
CH(CH3)2
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40.0
O
O
OD D
N
(c)
C O
%
C N H D
CH3
[M+D]
+
468
-179 (d3-anhydroglucuronic acid)
% 288 0 100
0 0.0
10.0
20.0
30.0
290 467
469 m/z
200
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40.0
Time (min)
FIGURE 3. Detection of radiolabeled b-blocker metabolites in rat urine by HPLC/UV/NMR/MS with an in-line radioactivity detector. (a) UV, (b) radioactivity, and (c) mass chromatograms and (d) NMR and (e) mass spectra of the hydroxyglucuronide metabolite.
Both NMR and MS readily detected the major glucuronide metabolites, but MS, which is more sensitive to certain minor metabolites and degradation products, directed the NMR spectroscopists to peaks of interest that would otherwise have been missed. On the other hand, good NMR spectra were obtained for metabolites that, due to poor ionization, were undetected by MS, which provided insight into the ratio of diastereoisomers in the glucuronide conjugates of ibuprofen (administered as a racemic mixture). This information would not have been produced had MS been the only characterization mode. The results we obtained on the metabolic fate of a series of fluoroanilines in rats are perhaps more interesting, given that the identities of the metabolites were not known. We completed work on several of these compounds, including 2-trifluoromethyl-4-bromoaniline (11, 12) and the related 4-chloro (13) substituted analog (Figure 2). HPLC with 1 H NMR and MS facilitated the rapid identification of the
major metabolites of the two halogenated anilines, which were found to be sulfate conjugates of a ring-hydroxylated compound. Numerous minor metabolites (sulfates and glucuronides) were also identified. Had these anilines been candidate drugs, being able to rapidly elucidate their metabolic fates could be of great value for future synthesis cycles aimed at modifying either the biological activity or pharmacokinetic properties, because the data indicates the metabolically labile sites on these molecules. Other investigators have used HPLC/NMR/MS to study drug metabolism. Dear and co-workers recently described studies, using a 600-MHz NMR spectrometer and an ion trap mass spectrometer, of the novel non-nucleoside reverse transcriptase inhibitor GW 420867 (8). Eluents corresponding to peaks of interest were collected in a peak-sampling unit for NMR. Once a suitable NMR spectrum had been obtained, the sample was introduced into the ion source of the mass spectrometer (with or without back exchange
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with a protio solvent). Several hydroxylated and glucuronidated metabolites were identified. To collect this data, the group used an in-line configuration, with MS subsequent to NMR, and reported no problems due to leakage from the NMR probe. Studies of candidate drugs frequently use radiolabeled compounds to facilitate the detection of metabolites in excreta. Typical chromatographic and spectroscopic spectra obtained with an HPLC/UV/NMR/MS system incorporating a radioactivity monitor are shown in Figure 3. This example is for a 14C-labeled b-blocker dosed to a rat (14). NMR and MS data for the minor hydroxyglucuronide metabolite clearly enables the structure to be determined. In addition, the drug was dosed as the racemate, but the NMR spectrum clearly shows that the metabolism (or excretion) was enantioselective based on the slightly different intensities of the anomeric proton resonances of the two diastereoisomeric glucuronides. In this experiment, we also investigated the metabolic deacetylation and subsequent reacetylation of the b-blocker drug and its metabolites. This “futile acetylation” is surprisingly common in N-acetyl-containing compounds, but it is often not observed in conventional metabolism studies because the products and starting materials are the same.
Although superheated water is not suitable for every compound, the early promise shown
OH
O
OH
CH3
HO HO
CH3
Intregral
OH
ppm
O
2,000 8
OH
1,786 7
6
6,412 5
4
3
2
1
FIGURE 4. The 1H-NMR spectrum of hypericin obtained during HPLC/UV/NMR/MS studies on an extract of Hypericum preforatum L. On the basis of limited NMR data and the chromatographic retention time, identification is still considered tentative. However, combined with MS data on the same chromatographic peak (for both proton- and deuterium-exchanged hyperecin (m/z 503 and 508, respectively), it seems more reasonable to claim unequivocal identification.
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has been born out. By incorporating a 13C-labeled acetyl group, both NMR and MS can be used to probe these “metabolically silent” reactions. In this instance, ~10% of the 13C-acetyl group was removed and replaced with endogenous 12C-material. HPLC/NMR/MS metabolism studies have not been confined to animals. Recently, we examined the fate of 5trifluoromethyl pyridone in hydroponically grown maize (15). Analysis of the aqueous extracts by gradient, reversedphase HPLC rapidly revealed that the major biotransformation products are the N-glucoside and O-malonylglucoside conjugates of the parent pyridone. This technology can also be used in combinatorial chemistry. Holt and co-workers investigated applying HPLC/ NMR/MS to a model peptide library of 10 compounds (9). A 500-MHz NMR spectrometer was linked to a mass spectrometer with an electrospray interface; the eluent was split 20:1 from the column, in favor of the NMR side. A reversed-phase gradient achieved the separation using an acetonitrile TFA/D2O-based eluent. Mass and NMR spectra that unequivocally identified all the test compounds were obtained, despite coelution of two compounds. Approximately 100 µg of each analyte was used on-column, enabling both NMR and MS data to be obtained on-flow. Within the pharmaceutical industry, there is a growing body of experience with HPLC/NMR/MS systems, and we anticipate many more publications from this field. Natural products are also a source of drug candidates, but in many biologically active extracts, compounds that are already known are responsible for the activity and are therefore of no interest. These extracts are usually profiled by HPLC/MS, although there is an increasing number of examples with HPLC/NMR. The ability to rapidly screen extracts and eliminate known compounds (“dereplication”) enables resources to be concentrated on identifying novel structures. Thus, this is another area where the application of HPLC/NMR/MS may prove fruitful. As a “proof of concept”, HPLC/NMR/MS was used to identify phytoecdysteroids—polyhydroxylated steroids used as plant defense substances against phytophagous insects—in a plant extract. In this work, an extract of the plant Silene otites was subjected to reversed-phase HPLC using a gradient of D2O and deuteroacetonitrile (16). On-flow 1 H NMR/MS confirmed the identity of the three major ecdysteroids (20-hydroxyecdysone, 2-deoxy-20-hydroxy-
ecdysone, and 2-deoxyCH CN CH CO ecdysone) in the extract. HOD MeOH t-Bu Careful examination ArMe of the MS data also sugAr gested the presence of a Impurity hitherto unsuspected CHCl ecdysteroid as a minor 7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm component in this mixC ture. From the molecumV 0.8 lar mass of the unknown 1600 0.6 A compound, it was possi219 0.4 100 ble to narrow its likely [M-D] 0.2 identity down to three 1200 % 1 known compounds. H 0.0 2000 1000 4000 3000 0 NMR spectroscopy fi200 300 Wavenumbers (cm ) m/z nally revealed its identity 800 as integristerone A. This work was a particularly nice application of the 400 double hyphenation of NMR and MS because a previous HPLC/ 0 NMR-only study missed 0.0 10.0 20.0 the minor component. Time (min) The mass spectral data obtained in this second FIGURE 5. HPLC/NMR/IR/MS analysis of a polymer additives mixture. study would have been The UV chromatogram obtained for a mixture of polymer additives separated by size-exclusion chromatography showing the on-line NMR and MS spectra and the off-line FT-IR spectra of 2,6-di-tert-butyl-4-methoxyphenol. insufficient on its own to identify the new ecdysteroid, but it was useful in directing further investigations toward the minor perheated water for HPLC/NMR and HPLC/NMR/MS component in the extract. (18, 19). The HPLC/NMR/MS experiment used salicyAnother natural products application of HPLC/NMR/ lamide as a model analyte to demonstrate that this unusual MS has identified compounds in extracts of Hypericum eluent did not cause any problems for the spectrometers. preforatum L (17). Among the compounds characterized Although superheated water is not suitable for every comwere the arabinoside and galacturonide of quercetin, which pound, the early promise shown by these results has been had not previously been described for this species. In addi- born out in subsequent studies. tion, the rutinoside, glucoside, rhamnoside, and galactoside Although the coupling of HPLC, NMR, and MS repreof quercetin, hypericin (Figure 4), protohypericin, pseudosents, perhaps, the most important combination of powerhypericin, pseudohypericin and protopseudohypericin were ful spectroscopic instruments with HPLC, there are other positively identified. These results were obtained using repossibilities. We recently used HPLC/NMR with an online interface to collect eluent for subsequent FT-IR analyversed-phase gradient HPLC, a 500-MHz NMR, and a ses (20). This combination has also been extended to entriple quadrupole mass spectrometer. Recently a similar compass the triple hyphenation of HPLC/NMR/IR/MS, study on the glycosides present in apple peel has also been which was used to analyze a mixture of polymer additives. reported (21). Representative spectra for on-flow NMR and MS and offline FT-IR of 2,6-di-tert-butyl-4-methoxyphenol, which A suite of possibilities were obtained from the third peak in the chromatogram, Superheated water has been shown to be a suitable eluent are shown in Figure 5. for performing certain reversed-phase HPLC separations. Selecting compatible solvent systems for NMR and MS This is also of interest for HPLC/NMR experiments, beis sometimes complex, and the addition of IR (even offcause there could be considerable economic and practical line) places even more constraints on solvent composition. advantages to using D2O as the sole eluent. Our prelimiNevertheless, as the previous example illustrates, such sysnary experiments using superheated D2O are very promistems are technically feasible. Recently, studies have been ing, and we recently performed several studies using su3
3
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Absorbance
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–
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performed using an HPLC system incorporating a UV diode array, FT-IR (using an attenuated total reflection flow cell to obtain on-flow IR spectra), NMR, and timeof-flight MS. These studies demonstrated the feasibility of obtaining a relatively complete set of spectroscopic data for components of drugs (22) and plant extract mixtures. Other spectroscopic detectors are available for HPLC (e.g., circular dichroism) that could be used in combination with NMR and MS detectors as the basis for powerful multiply hyphenated systems. It is possible to envisage a system that, in the near future, could enable an analyst to program separation conditions and select from a suite of applicable post-separation spectroscopic techniques. Given the increasing use of “open access” systems for providing MS and NMR data to organic chemists, this type of system could be the basis (following the removal of the column) for an open-access flow-injection analysis system. An area that is currently receiving considerable attention in HPLC/NMR is miniaturization, which has been achieved for microbore HPLC and for capillary-scale separations (CE and capillary electrochromatography) (23–27). These systems enable measurement of picomole (nanogram) amounts of material by NMR spectroscopy (4) and may evolve into commercially produced equipment. There seems good reason to suppose that they will eventually be used in HPLC/ NMR/MS systems. Thus, although it may be overkill for many problems, for those samples that require it, combining HPLC with both NMR and MS represents a readily implemented analytical strategy. Moreover, given the relative ease with which this double hyphenation can be implemented, it is inevitable that HPLC/NMR/MS systems will become available for routine applications.
References (1) (2) (3) (4) (5) (6) (7) (8)
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Lindon, J. C.; Nicholson, J. K.; Wilson, I. D. Drug Metab. Rev. 1997, 29, 705–746. Lindon, J. C.; Nicholson, J. K.; Wilson I. D. Prog. Nucl. Magn. Reson. Spectrosc. 1996, 29, 1–49. Albert, K. J. Chromatogr., A. 1996, 856, 199–212. Gfrorer, P; Schewitz, J.; Puseker, K.; Bayer, E. Anal. Chem. 1999, 71, 315 A–321 A. Pullen, F. S.; Swanson, A. G.; Newman, M. J.; Richards, D. S. Rapid Comm. Mass Spectrom. 1995, 9, 1003–1006. Shockcor, J. P.; Unger, S. E.; Wilson, I. D.; Foxall, P. J. D.; Nicholson, J. K.; Lindon, J. C. Anal. Chem. 1996, 68, 4431–4435. Burton, K. I.; Everett, J. R.; Newman, M. J.; Pullen, F. S.; Richards, D. S.; Swanson, A. G. J. Pharm. Biomed. Anal. 1997, 5, 1903–1912. Dear, G. J.; Ayrton, J.; Plumb, R.; Sweatman, B. C.; Ismail, I. M.; Fraser, I. J.; Mutch, P. J. Rapid Comm. Mass Spectrom. 1998, 12, 2023–2030. Holt, R. M.; Newman, M. J.; Pullen, F. S.; Richards, D. S.; Swanson, A. G. J. Pharm. Biomed. Anal. 1997, 15, 1903–1912.
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(10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27)
Clayton, E.; Taylor, S.; Wright, B.; Wilson, I. D. Chromatographia 1998, 47, 264–270. Scarfe, G. B.; Wilson, I. D.; Spraul, M.; Hofmann, M.; Braumann, U.; Lindon, J. C.; Nicholson, J. K. Anal. Commun. 1997, 34, 37–39. Scarfe, G. B.; Wright, B.; Clayton, E.; Taylor, S.; Wilson, I. D.; Lindon, J. C.; Nicholson, J. K. Xenobiotica 1998, 28, 373–388. Scarfe, G. B.; Wright, B.; Clayton, E.; Wilson, I. D.; Lindon, J. C.; Nicholson, J. K. Xenobiotica 1999, 29, 77–91. Scarfe, G.B., et al. Xenobiotica 2000, in press. Bailey, N. J. C, et al. J. Ag. Food Chem. 2000, 48, 42–46. Wilson, I. D., et al Chromatographia 1999, 49, 374–378. Hansen, S. H., et al. Anal. Chem. 1999, 71, 5235–5241. Smith, R. M.; Chienthavorn, O.; Wilson, I. D.; Wright, B. Anal. Commun. 1998, 35, 261–263. Smith, R. M.; Chienthavorn, O.; Wilson, I. D.; Wright, B.; Taylor, S. D. Anal. Chem. 1999, 71, 4493–4497. Ludlow, M.; Louden, D.; Handley, A.; Taylor, S.; Wright, B.; Wilson, I. D. J. Chromatogr., A 1999, 857, 89–96. Lommen, A.; Godejohann, M.; Venema, D. P.; Hollman, P. C. H.; Spraul, M. Anal. Chem. 2000, 72, 1793–1797. Wilson, I.D., et al. Anal. Chem. in press. Behnke, B., et al. Anal. Chem. 1996, 68, 1110–1115. Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. J. Am. Chem. Soc. 1994,116, 7929–7930. Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849–3857. Wu, N.; Webb, A. G.; Peck, T. L.; Sweedler, J. V. Anal. Chem. 1995, 67, 3101–3107. Schewitz, J., et al. Analyst 1999, 123, 2835–2837.
Ian Wilson is head of the Bioanalytical Spectroscopy Unit at AstraZeneca’s Alderley Park research site. Wilson’s research interests are in most branches of separation science, particularly sample preparation, planar chromatography, and hyphenated techniques, as well as the application of these methods to problems in biology, particularly for drug metabolism and toxicology. John C. Lindon is professor in the biological section of the medical school of Imperial College of Science, Technology and Medicine. His research interests encompass the development and application of magnetic resonance methods to biomedical research and the allied use of chemometric methods for interpreting the resulting complex data sets. Jeremy Nicholson is head of the biological chemistry section of the medical school at Imperial College of Science, Technology and Medicine. His research interests center on molecular processes in metabolism, toxicology, and medicine, which include NMR studies and pattern recognition analyses of bodily fluids and living tissue, developing new methods for investigating molecular mechanisms of drug toxicity, pattern recognition and computational chemistry in drug design and toxicology, and investigations of liver and kidney dysfunction and environmental toxicology. Address correspondence about this article to Wilson at the Department of Drug Metabolism and Pharmacokinetics, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, U. K. (ian.wilson@astrazeneca. alderley.com).
