Chiral Metabonomics: 1H NMR-Based Enantiospecific Differentiation

Article pubs.acs.org/ac

Chiral Metabonomics: 1H NMR-Based Enantiospecific Differentiation of Metabolites in Human Urine via Direct Cosolvation with βCyclodextrin Míriam Pérez-Trujillo,† John C. Lindon,‡ Teodor Parella,† Hector C. Keun,‡,§ Jeremy K. Nicholson,*,‡,§ and Toby J. Athersuch*,‡,§ †

Servei de Ressonància Magnètica Nuclear, Facultat de Ciències i Biociències, Universitat Autònoma de Barcelona, Bellaterra 08193, Spain ‡ Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7 2AZ, U.K. § MRC-HPA Centre for Environment and Health, Department of Epidemiology and Biostatistics, School of Public Health, Faculty of Medicine, Imperial College London, Norfolk Place, London, W2 1PG, U.K. S Supporting Information *

ABSTRACT: Differences in molecular chirality remain an important issue in drug metabolism and pharmacokinetics for the pharmaceutical industry and regulatory authorities, and chirality is an important feature of many endogenous metabolites. We present a method for the rapid, direct differentiation and identification of chiral drug enantiomers in human urine without pretreatment of any kind. Using the well-known anti-inflammatory chemical ibuprofen as one example, we demonstrate that the enantiomers of ibuprofen and the diastereoisomers of one of its main metabolites, the glucuronidated carboxylate derivative, can be resolved by 1H NMR spectroscopy as a consequence of direct addition of the chiral cosolvating agent (CSA) β-cyclodextrin (βCD). This approach is simple, rapid, and robust, involves minimal sample manipulation, and does not require derivatization or purification of the sample. In addition, the method should allow the enantiodifferentiation of endogenous chiral metabolites, and this has potential value for differentiating metabolites from mammalian and microbial sources in biofluids. From these initial findings, we propose that more extensive and detailed enantiospecific metabolic profiling could be possible using CSA-NMR spectroscopy than has been previously reported.

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administered to humans as mixtures of isomeric substances whose biological activity may well reside predominantly in one form, and stereoselective metabolism of racemic mixtures may also contribute to the toxicity or adverse effects encountered with drugs. Chirality considerations in drug metabolism and pharmacokinetic analyses remain an important issue for the pharmaceutical industry and the regulatory authorities,8,9 and therefore approaches that provide a chiral perspective on the composition of biofluids are of considerable value. Currently, most of the methods and analytical techniques used to discriminate metabolite enantiomers in biofluids involve chromatographic techniques with chiral stationary phases and/or the derivatization of the metabolites under study.10 Such analyses tend to be labor-intensive, involve significant manipulation of the original sample, and may suffer from deleterious matrix effects and kinetic resolution leading to erroneous results. NMR spectroscopy has also been widely used in the enantiodifferentiation of chiral small molecules, and several approaches exist.11 The use of chiral cosolvating agents

igh-field nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for the quantitative compositional analysis of biofluids and can be used for the monitoring of metabolic events relating to drug toxicity and disease.1−6 It typically provides a rich source of structural and dynamic chemical information about a biofluid sample in a few minutes. Such analyses have numerous practical advantages including a minimal requirement for sample preparation and their nondestructive nature. NMR spectroscopy is particularly useful for defining metabolic changes, because many metabolites can be detected simultaneously and the collection of these fluids is relatively noninvasive (e.g., urine). NMR spectroscopy is also a valuable tool for the de novo identification of both endogenous and xenobiotic metabolites. Chirality is a property inherent to all biological systems, with a large number of biochemical processes exhibiting stereospecificity, involving endogenous compounds and also xenobiotics, including drugs and microbial metabolites. For example, receptors and enzymes which are the targets of drug action frequently show enantioselectivity and/or prochiral selectivity7 (i.e., they are able to differentiate enantiomers or distinguish between prochiral groups/atoms within the same molecule, respectively). About one in four of all therapeutic agents are © 2012 American Chemical Society

Received: December 22, 2011 Accepted: February 8, 2012 Published: February 8, 2012 2868

dx.doi.org/10.1021/ac203291d | Anal. Chem. 2012, 84, 2868−2874

Analytical Chemistry

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Figure 1. Expanded regions of the 1H NMR spectra of (a) racemic ibuprofen 1 in D2O, (b) 1 in D2O with 10 equiv of βCD and (c) sample enriched in S-1 (S-1/R-1, 90:10) with the same conditions as part b.

nucleus as it is found almost ubiquitously in metabolites and has favorable properties including high natural abundance (99.985%), a spin quantum number of 1/2, and a high gyromagnetic ratio (267.513 × 106 s−1 T−1). To date, there are no studies that have illustrated the use of direct enantioseparation of metabolites in a complex biological matrix such as urine by 1H NMR spectroscopy. In this work, we illustrate the direct enantiodifferentiation in urine of the nonsteroidal anti-inflammatory drug (NSAID), ibuprofen, and one of its major metabolites,19 the glucuronidated carboxylate derivative (2-[4-(2-carboxy-2methylpropyl)phenyl]propionic acid), by 1H NMR spectroscopy after the direct addition of the chiral solvating agent βCD. The metabolism of ibuprofen in humans has been studied extensively, including investigations using 1H NMR as the primary analytical platform.20 The pharmacology and pharmacokinetics of this compound has been extensively reviewed,21 and the disposition of ibuprofen has been shown to be stereoselective,22,23 with this selectivity varying with physiological factors.24 Ibuprofen is characterized by a single chiral center adjacent to the carboxylic acid moiety and is marketed as a racemic mixture though it is known that the pharmacological activity resides almost exclusively in the (S)-(+)-enantiomer.25 One characteristic of the metabolic fate is the incomplete chiral inversion of the largely pharmacologically inactive R- isomer to that of the active S-isomer.23−28

(CSA) for enantiodifferentiation can be attractive as there is no need for derivatization and/or purification prior to analysis as is the case for chiral derivatizing agents (CDA) and does not usually suffer from broadening of the signals in the NMR spectrum associated with chiral lanthanide shift reagents (CLSR).12 CSA are chiral single enantiomers able to differentiate enantiomers by the formation of diastereoisomeric complexes (via noncovalent interactions) with the enantiomers of the chiral analyte. Such complexes are in fast exchange with the individual components on the NMR time scale, and thus a population-weighted average set of chemical shifts results, causing differences in the observed NMR chemical shifts (δ). These differences are usually denoted as ΔΔδ and reported as the absolute difference in the observed change in the δ of one enantiomer (e.g., ΔδR) with respect to the observed change in the δ of the other (e.g., ΔδS), where these changes are relative to the chemical shift of the enantiomers in the absence of a chiral auxiliary. While the majority of CSA are soluble in organic compounds,13 the range of water-soluble CSA is limited, including native and modified cyclodextrins (CD),14 some calixresorcarenes,15 and calixarenes.16 Cyclodextrins have previously been used for chiral 19F and 13C NMR analysis of drug metabolism directly within intact biological samples, in the study of two model compounds, p-trifluoromethylmandelic acid and Mosher’s acid (α-methoxy-α-trifluoromethylphenylacetic acid), and in the analysis of 13C labeled ketoprofen.17,18 These methods are useful in the context of drug metabolism studies but less so in metabolic profiling of endogenous metabolites, mainly due to the absence of suitably sensitive or specific heteronuclei (e.g., 19F), or the limited opportunity for the routine use of labeled substrates. NMR-based metabolic profiling is largely conducted using 1H as the observation

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EXPERIMENTAL SECTION Materials. Racemic ibuprofen, (S)-(+)-ibuprofen, β-cyclodextrin, 3-(trimethylsilyl)-[2,2,3,3-2H4]-propionic acid sodium salt (TSP), sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased from Sigma-Aldrich Company, Ltd. (Gillingham, Dorset, U.K.). NMR grade 2869



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each of the urine samples described above (prepared with and without βCD, molar ratio of βCD:1 corresponding to 10:1). The ibuprofen concentration was chosen so as to be in the same order of magnitude as endogenous metabolites. Resonances corresponding to ibuprofen were easily identified by comparison with the nonspiked spectrum (Figure 2a,b) and

deuterium oxide (D2O) was purchased from Goss Scientific Instruments Ltd. (Nantwich, Cheshire, U.K.). Sample Collection and Preparation. Human urine was obtained from three healthy volunteers, two males and one female, before and 7 h after the oral administration of 400 mg of ibuprofen. Samples were centrifuged (16 000g, 10 min), aliquoted (2 mL), and stored frozen at −80 °C until analysis. NMR sample preparation details are described in the Supporting Information. NMR Spectroscopy. 1H NMR spectra were acquired at 14.1 T (600.14 MHz 1H resonance frequency) on a Bruker DRX600 spectrometer (Bruker Biospin, Rheinstetten, Germany) equipped with a 5 mm CP TCI probe (1H, 13C, 15N) with a Z-gradient. The probe temperature was maintained at 300.0 K. Unless otherwise stated, NMR spectra were acquired using the 1D 1H NMR pulse sequence commonly termed 1D NOESY-presat.1,29 The 1H−1H 2D J-resolved (JRES) spectrum was acquired with presaturation of the water peak during the relaxation delay. Further details are provided in the Supporting Information. The identification of ibuprofen metabolites and endogenous urinary components in the urine samples was carried out primarily by reference to the existing literature.20,30,31

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RESULTS AND DISCUSSION Enantiodifferentiation of R- and S-Ibuprofen in Aqueous Media. We selected βCD as the CSA for enantiodifferentiation of racemic ibuprofen 1 in pure aqueous media (D2O), since it has been previously used for the enantiorecognition of 1.32 A titration experiment was conducted (0−12 equiv of βCD added to the sample, as described in the Supporting Information) with the greatest enantiodifferentiation (as measured by ΔΔδ) obtained after addition of 10 equiv. No further enantiodifferentiation was observed on further addition of this CSA. The NMR peaks remained sharp and indicated that all interacting systems were in the fast exchange limit. Figure 1 shows the three peaks of ibuprofen 1 that were most affected by the βCD. The intense doublet at δ 1.32, corresponding to the protons of the methyl group, Hg-1, was enantiodifferentiated with a maximum ΔΔδ of 4.4 Hz. The multiplet of the aromatic protons at δ 7.22, He1, was also resolved, with a maximum ΔΔδ of 2.8 Hz. A large shift in the δ of the aromatic protons at δ 7.15, Hd-1, was observed (102.0 Hz) after the addition of 10 equiv of βCD, but no resolution of the peak took place. The shifts induced in the δs of these protons by the addition of the βCD indicates that there is a complexation between the ibuprofen and the βCD, under these experimental conditions, and that the interaction involves the polar end (propionic acid tail) and the aromatic ring of the molecule. Identification of R- and S-Ibuprofen by Standard Addition. To identify the resonances attributable to each of the enantiomers, a sample containing a solution of racemic ibuprofen 1 was spiked with S-1. For both resolved peaks (Hg1 and He-1, Figure 1),the S-ibuprofen resonance was less shielded (higher δ) than the R-ibuprofen (Figure 1). Although the enantiodiscrimination of 1 by βCD has been reported previously,32 a greater enantiodifferentiation was obtained in D2O using the conditions described above. Enantiodifferentiation of R- and S-Ibuprofen in Intact Human Urine. The interaction of βCD and racemic ibuprofen 1 in a human urine matrix was studied (see the Supporting Information for experimental details). Racemic 1 was added to

Figure 2. Expanded region of 1H NMR spectra of (a) human urine of a healthy volunteer, (b) after the addition of racemic ibuprofen 1, (c) the former sample in the presence of βCD, (d) after spiking the sample in part c with S-1 and (e) the JRES spectrum of the sample in part c.

the effect of βCD addition was then assessed (Figure 2c). The identity of the R-1 and S-1 were then confirmed by addition of a S-1. The split of the methyl protons, Hg-1, (ΔΔδ of 3.7 Hz) and of the aromatic protons, He-1, (ΔΔδ of 6.7 Hz) and a significant broadening of the doublet of methyl protons Ha-1 were observed. The aromatic protons, Hd-1 multiplet, were considerably shielded (106.3 Hz). The sample containing βCD was then spiked with S-1 for the identification of each enantiomer. As a consequence, it could be seen that Hg-1 and He-1 of the S-1 enantiomer were shielded with respect to the corresponding resonances of the R-1 enantiomer. These results are in agreement with those obtained with racemic ibuprofen and βCD in pure D2O, with only slight differences in the ΔΔδ values obtained for Hg-1 and He-1, and the broadening observed for Ha-1. Therefore, under these conditions, the enantiodifferentiation behavior and capacity of βCD with ibuprofen (that means the complexation between the βCD and the enantiomers of ibuprofen) is not altered significantly between pure D2O solution and a typical human urine matrix. Figure 2 shows the expanded region (from δ 0.85 to 1.51) of the 1H NMR spectra of the urine sample (a), of urine spiked with racemic 1 (b), of the former sample in the 2870



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Figure 3. Ibuprofen metabolites identified in human urine collected 7 h after intake of 400 mg of racemic ibuprofen and enantiodifferentiation caused by addition of CSA. Expanded region (0.85−1.60 ppm) of 1H NMR spectra of urine samples (a) predose, (b) postdose (without βCD), and (c) postdose (with βCD).

presence of βCD (c), and finally spiking the sample from part c with S-1 (d). J-resolved (JRES) spectroscopy was also performed on the sample from part c (Figure 2e). Spectra obtained from JRES experiments are much less overlapped in the F2 dimension as, after tilting, they do not contain information about the H−H scalar coupling (J), which is manifest in the F1 dimension. In this instance, the JRES spectra confirmed the underlying multiplet structure of the Hg-1 resonance in the 1D 1H spectrum (two overlapped doublets relating to the R- and S-enantiomers of 1). JRES spectra have been shown to have a number of advantages for metabolic profiling studies through the simplification of complex 1H NMR spectra, with the F2 projection peaks minimizing peak overlap.29,33−35 We suggest that use of JRES spectra may be of considerable benefit when CSA are used, as the increased spectral overlap resulting from enantiodifferentiation of multiplets can be partially overcome. Differentiation of Diastereoisomers of Ibuprofen Metabolites in Intact Human Urine. In previous studies, ibuprofen metabolites have been identified and characterized by NMR spectroscopy in human urine, typically preceded by solidphase extraction chromatography30 or utilizing hyphenated HPLC−NMR.36 Here we go further, identifying ibuprofen metabolites and differentiating their diastereoisomers by performing a rapid 1H NMR experiment. The experiments described above provided an initial knowledge of the system and of the optimum conditions for experiments with real samples of human urine after the intake of the racemic drug. Healthy human volunteers (n = 3) provided spot urine samples before and after (0−7 h) the intake of 400 mg of racemic ibuprofen (see the Supporting Information for

preparation of sample details). The absence of the ibuprofen parent 1 was confirmed by spiking of the standard compound. Initial identification of the ibuprofen metabolites was carried out using one of the three postdose samples and by the comparison of the obtained 1D 1H NMR data with the extensive data described in previous work.20,30,36 These data led us to identify ibuprofen glucuronide, 2, and two further glucuronidated metabolites, the carboxylate derivative (2-[4-(2-carboxy-2-methylpropyl)phenyl]propionic acid), 3, and the hydroxy derivative (2-[4-(2-hydroxy-2methylpropyl)phenyl]propionic acid), 4 (see Figure 3). Other new signals observed after dosing include a broad envelope of overlapping aromatic resonances at δ ∼7.25 and three overlapped doublets at δ ∼5.56 (data not shown). These doublets are from the glucuronide β-anomeric protons of compounds 2, 3, and 4. The region from δ 0.8 to δ 1.6 contained several signals of drug origin, including a doublet at δ 0.87, Ha-2, a doublet at δ 1.08, Ha-3, and a singlet at δ 1.21, Ha-4.19,25 A fourth ibuprofen metabolite was identified, the nonglucuronidated hydroxyl derivative (2-[4-(2-hydroxy-2methylpropyl)phenyl]propionic acid), 5 (see Figure 3), that shows a singlet at δ 1.21, corresponding to Ha-5. This peak is overlapped with the Ha-4 signal, also a singlet. These two different resonances were resolved when βCD was added to the sample (explained below), causing different shifts in their δs, which made evident the existence of a fourth metabolite. The signal corresponding to Hg-5 was identified at 1.53 ppm, partially overlapped with the doublet of Hg-2. These peaks were assigned based on assignments made previously in the literature.19 The metabolism of ibuprofen has previously been studied in healthy human volunteers where between 60 and 2871



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produced by the presence of the glucuronic acid moiety in these conjugated metabolites. The assignment of Ha/Ha′-4 and Ha-5 after the addition of the βCD was done based on the above discussion. These preliminary experiments demonstrate the rapid and simple differentiation of enantiomers, diastereoisomers, and prochiral groups of drugs and drug metabolites within an intact human urine sample by a simple 1H NMR experiment and the use of a suitable CSA. A titration was conducted using a fresh aliquot of the same urine sample by incremental addition of CSA to derive the molar ratio that afforded both the maximum and optimal ΔΔδ values for the Ha-3 signals of metabolite 3 (see Table S-1 in the Supporting Information). These data confirm that, as might be expected, the βCD/substrate molar ratio for optimum enantioselective differentiation is not similar for different compounds. We repeated the βCD titration with samples provided by three healthy volunteers, two males and one female, before and after the intake of 400 mg of racemic ibuprofen (see the Supporting Information). All postdose samples contained the ibuprofen metabolites described above. However, the concentration of these metabolites and the molar ratio between them differed between individuals (Figure 4a−c). Interindividual

70% inversion was routinely observed, leaving only a small proportion of the excreted metabolites with an R-configuration at the α-carboxylic carbon.23,24 Such epimerization reactions are characteristic of the Profen class of NSAIDs. Considering the four ibuprofen metabolites identified (see Figure 3), all of them retain the chiral center about the original α-carboxylic carbon and just one of them, the glucuronide carboxylate derivative 3, contains a second chiral center (Cb-3), making possible in this case the existence of two diastereoisomers, assuming the aryl propionic acid moiety has only the S configuration. βCD was added to a fresh aliquot of the same postdose sample, following the procedure described previously (see the Supporting Information for experimental details), in order to observe what happens when enantiospecificity was induced using the described method. The 1H NMR spectral region [δ 0.85−1.60] was selected due to the presence of only minimal signal overlap. Figure 3 shows this region for the predose (a), postdose without βCD (b), and postdose with βCD (c) samples. A split of the peaks corresponding to the methyl protons Ha-2, Ha-3, and Ha-4 of the three glucuronidated metabolites was observed, whereas the singlet Ha-5 presented a shift but not a split. A shift of peaks relating to drug metabolites in other regions of the spectrum, such as the aromatic resonances and protons Hg-2 and Hg-4, was also observed. For 2, 3, and 4, the aromatic signals and those of the protons closest to the original α-carboxylic carbon are difficult to study because of the overlapping with other signals in the sample matrix. The doublet corresponding to metabolite 3, Ha-3, was split into two doublets (ΔΔδ of 2.2 Hz) when enantiospecificity was induced in the sample by adding the βCD (see Figure 3). This indicated the presence of two diastereoisomers of metabolite 3 in the urine, with each doublet corresponding to Ha-3 protons of one diastereoisomer. Furthermore, the intensities of these signals were similar (∼1:1 molar ratio). In the case of metabolites 2 and 4, a prochiral selectivity by the addition of βCD was observed. In the case of metabolite 2, a splitting of the Ha-2 doublet was observed (ΔΔδ of 4.8 Hz). This reflected differential interaction of the prochiral Ha/Ha′ protons in this metabolite with the βCD. A similar observation was made for metabolite 4, with the Ha/Ha′-4 singlet split on addition of the βCD (ΔΔδ of 2.5 Hz). In each case, the Ha/Ha′ molar ratio was 1:1. The singlet corresponding to Ha-5 (initially at δ 1.22 and overlapped with Ha-4) was shifted to δ 1.23 after the addition of the βCD and became resolved from the Ha-4 and Ha′-4 singlets. As observed, the behavior of the nonglucuronidated species (free ibuprofen parent, metabolite 5) and those of the glucuronidated species (metabolites 2, 3, and 4) differed in the presence of the chiral agent βCD. In the case of the glucuronides, there was a split of the peaks of the methyl protons originating from the isopropyl chain (Ha-2, Ha-3, and proposed Ha-4), while no split of Hg was observed. In the case of the free ibuprofen, it is the Hg-1 peak that is split, while Ha-1 presented just a broadening (Figure 2). It is probable that this different behavior is due to different parts of the molecule interacting with the βCD between glucuronidated substrates and nonglucuronidated substrates. For the glucuronidated species, the complex formation with the βCD occurs at the isopropyl chain, whereas for the nonglucuronidated species, the complexation occurs at the propionic acid end of the molecule. These differences in complexation/geometry may result from the steric hindrance

Figure 4. Expanded region (0.70−1.30 ppm) of a 1H NMR spectra of urine sample of three individuals after the intake of 400 mg of racemic ibuprofen 1. Spectra correspond to the samples prepared without βCD (a−c) and with βCD (d−f) in the sample.

variation in the pharmacokinetic and enantiomeric disposition of ibuprofen has been explored previously.19,21−23 By addition of βCD to the postdose samples, it was possible to achieve a similar degree of diastereoseparation (as measured by ΔΔδ) for all individuals (Figure 4d−f) and thus produce easily comparable 1H NMR spectra. The optimal molar ratio βCD/ 3 was consistent (2:1) across all three individuals. Interestingly, although the ΔΔδ values observed for Ha-3 in the three samples remained constant, the intensity of each of the two 2872



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doublets (each corresponding to different diastereoisomer) appeared to be different between individuals (Figure 4d−f). Thus, and by visual inspection, the molar ratio observed between the resolved diastereoisomers of the glucuronide carboxylate derivative 3 seems similar for the two male subjects, where one of the doublets (higher frequency) is clearly more intense than the other. While this is likely to result from a different diastereoisomeric molar ratio of 3 between individuals, we cannot rule out other causes, e.g., overlap with endogenous metabolite signals. Taken together, these results illustrate the robustness of the method in the presence of different endogenous backgrounds and utility in assessing enantiomeric and/or diastereomeric metabolite ratios. CSA-Induced Chemical Shift Changes in Endogenous Urinary Components. The 1H NMR spectra of human urine samples from a healthy volunteer in the presence and absence of βCD were compared (see the Supporting Information). One of the main advantages of using a cyclodextrin, besides its solubility in aqueous media, is its narrow chemical shift range (Figure S-1 in the Supporting Information). This is of benefit when perdeuterated CSA cannot be used to avoid the interference (overlapping) in the 1H NMR spectrum between the signals of the CSA and those of the analyte.37 Comparison of the spectra indicated βCD-induced changes in chemical shift for several endogenous metabolites including histidine, phenylalanine, tryptophan, valine, hippurate, citrate, and dimethylamine (Figure S-1 in the Supporting Information). This confirms that in addition to the complexation with ibuprofen and its metabolites, there are additional, competing interactions of βCD with other components of the biological matrix. Interactions of βCD and several of the metabolites listed above have previously been reported.38−41 Furthermore, the ability of cyclodextrins to form both inclusion and noninclusion complexes as well forming soluble aggregates42 indicates that the specific interactions observed for ibuprofen and its metabolites have the potential to be modulated by the presence of a number of different competing interactions.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.K.N.); toby.athersuch@ imperial.ac.uk (T.J.A.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.P.-T. and T.P. acknowledge support from the Ministerio de Ciencia e Innovación (Project CTQ 2009-08328). M.P.-T. acknowledges the support of a PAS mobility grant (2009) from the Agència de Gestió d’Ajuts Universitaris i de Recerca who provided funding for collaborative visits. H.C.K. acknowledges the EU FP7 EnviroGenomarkers: Genomics Biomarkers of Environmental Health (Grant Agreement 226756) project for partial support of this work and a Cefic LRi Innovative Science Award. T.J.A. acknowledges the Royal Society for provision of an International Travel Grant (2010/R4) that contributed to the completion of this work.

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CONCLUSIONS We have illustrated how the addition of a chiral solvating agent to intact human urine samples can be used to differentiate by 1 H NMR spectroscopy the enantiomers of a common drug (ibuprofen) and the diastereoisomers of one of its main metabolites alongside observations of prochiral selectivity in other metabolites. The relative simplicity and minimal sample preparation required by this approach is attractive when compared with techniques that utilize chromatographic separation (that may suffer from deleterious matrix effects) and/or derivatization (that may suffer from differential reactivity). Future work will focus on the current limitations relating to chemical shift referencing and the additional spectral complexity that results from enantiodifferentiation (e.g., overlap). We believe that this approach opens up a wide range of possibilities for application in the field of metabonomics, where enantiodifferentiation is barely ever conducted, yet could have a huge influence on the hypotheses generated by such experiments. Using CSA-NMR analysis directly in body fluids could reveal hitherto unreported interindividual variability in the chiral fate of drugs and endogenous metabolites. This might lead to novel predictors of efficacy and toxicity and provide new avenues for personalized medicine. 2873



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Chiral Metabonomics: 1H NMR-Based Enantiospecific Differentiation

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