Anal. Chem. 1997, 69, 607-612
Directly Coupled 800 MHz HPLC-NMR Spectroscopy of Urine and Its Application to the Identification of the Major Phase II Metabolites of Tolfenamic Acid Ulla Grove Sidelmann,*,† Ulrich Braumann,‡ Martin Hofmann,‡ Manfred Spraul,‡ John C. Lindon,§ Jeremy K. Nicholson,§ and Steen Honore´ Hansen†
Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen, Denmark, Bruker Analytische Messtechnik GmbH, Silberstreifen, D-76287 Rheinstetten, Germany, and Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London WC1H 0PP, U.K.
Tolfenamic acid (N-(2-methyl-3-chlorophenyl)anthranilic acid) is metabolized in vivo to form several oxidized metabolites which are subsequently conjugated with β-Dglucuronic acid. In the present investigation, the metabolism of tolfenamic acid was investigated using 1H NMR spectroscopy of urine samples obtained from a human volunteer after oral administration of 300 mg of the drug. Both one- and two-dimensional NMR experiments were performed in order to identify the phase II metabolites of tolfenamic acid in the stepwise-eluted solid phase extraction fractions of urine. To identify the metabolites unambiguously, directly coupled 800 MHz HPLCNMR spectroscopy was applied to selected samples. The β-1-O-acyl glucuronides of the parent compounds N-(2methyl-3-chlorophenyl)anthranilic acid, N-(2-hydroxymethyl-3-chlorophenyl)anthranilic acid, N-(2-hydroxymethyl3-chloro-4-hydroxyphenyl)anthranilic acid, N-(2-formyl3-chlorophenyl)anthranilic acid, N-(2-methyl-3-chloro-4hydroxyphenyl)anthranilic acid, and N-(2-methyl-3-chloro5-hydroxyphenyl)anthranilic acid were identified. None of these phase II metabolites has previously been identified directly in biofluids. The first practical demonstration of directly coupled 800 MHz HPLC-NMR to a problem in drug metabolism is shown here. By the use of this ultrahigh-field NMR spectrometer, the gain in spectral dispersion allowed the determination of the exact position of hydroxylation on the aromatic rings of tolfenamic acid. High-field NMR spectroscopy is a very powerful tool for the direct investigation of xenobiotic metabolites in complex matrices, i.e., plasma, urine, and bile.1-3 However, in many cases because of the large number of endogenous and exogenous metabolites in biofluids, the signals cannot be fully identified by simple onedimensional (1D) NMR experiments. Different biofluids have different physicochemical and biochemical profiles and thereby †
The Royal Danish School of Pharmacy. Bruker Analytische Messtechnik. § University of London. (1) Nicholson, J. K.; Wilson, I. D. Prog. Drug. Res. 1987, 31, 427-479. (2) Farrant, R. D.; Salman, S. R.; Lindon, J. C.; Cupid, B. C.; Nicholson, J. K. J. Pharm. Biomed. Anal. 1993, 8, 687-692. (3) Holmes, E.; Caddick, S.; Lindon, J. C.; Wilson, J. C.; Kryvawych, S.; Nicholson, J. K. Biochem. Pharmacol. 1995, 49, 1349-1359. ‡
S0003-2700(96)00582-3 CCC: $14.00
© 1997 American Chemical Society
offer varied analytical challenges in biological NMR spectroscopy. Urine samples are dominated by thousands of signals resulting from endogenous metabolites with low molecular weights, whereas 1H NMR spectra of plasma are dominated by broad unresolved signals from the large concentrations of macromolecules such as lipids and plasma proteins. It is important to take these variations in chemical profile of the sample matrix into account when NMR experiments for biofluids are designed. In the case of urine samples, it is thus often advantageous to simplify the spectra by adding a separation step into the experiment either off-line by combining solid phase extraction with NMR analysis or on-line by directly coupled HPLC-NMR. Solid phase extraction (SPE) has been shown to be very successful for the selective removal of endogenous compounds in biofluids and thereby aid the structure elucidation of unknown drug metabolites.4,5 However, if the metabolic pattern of the drug being investigated is very complex (i.e., many closely related metabolites), it is necessary to separate the metabolites before they can be unambiguously identified. Direct hyphenation of HPLC and NMR has recently proved to be very beneficial for such studies within the field of pharmaceutical chemistry.6-10 With the appropriate chromatographic method, it is possible by use of the HPLC-NMR technique to separate and directly identify compounds in complex mixtures by simple one-dimensional NMR spectroscopic methods. The first study presenting the use of directly coupled 500 MHz HPLC-NMR for analysis of drug metabolites in biological fluids was published by Spraul et al. in 1992.6 The metabolism of the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen was investigated in human urine obtained after ingestion of 400 mg of the drug. Both continuous-flow and stopped-flow HPLC-NMR experiments were performed applying a reversed-phase chromatographic system using gradient elution (4) Wilson I. D.; Nicholson, J. K. J. Pharm. Biomed. Anal. 1988, 6, 151-165. (5) Wilson I. D.; Nicholson, J. K. Anal. Chem. 1987, 59, 2830-2832. (6) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. J. Pharm. Biomed. Anal. 1992, 8, 601-605. (7) Spraul, M.; Hofmann, M.; Lindon, J. C.; Nicholson, J. K.; Wilson, I. D. Anal. Proc. 1993, 3, 390-392. (8) Spraul, M.; Hofmann, M.; Dvortsak, P.; Nicholson, J. K.; Wilson, I. D. Anal. Chem. 1993, 65, 327-330. (9) Wilson, I. D.; Nicholson, J. K.; Spraul, M.; Hofmann, M.; Nicholson, J. K.; Lindon, J. C. J. Chromatogr. 1993, 617, 324-328. (10) Seddon, M. J.; Spraul, M.; Wilson, I. D.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1994, 12, 419-424.
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Figure 1. Metabolic profile of tolfenamic acid (N-(2-methyl-3-chlorophenyl)anthranilic acid (T) and its principal metabolites. All the metabolites shown are believed to form glucuronic acid conjugates in vivo in humans.8 The phase II metabolites identified in the present investigations are the following: T (β-1-O-[N-(2-methyl-3-chlorophenyl)anthraniloyl]-D-glucupyranuronic acid), 1 (β-1-O-[N-(2-hydroxymethyl-3-chlorophenyl)anthraniloyl]-D-glucupyranuronic acid), 2 (β-1-O-[N-(2-hydroxymethyl-3-chlorophenyl)anthraniloyl]-D-glucupyranuronic acid), 3 (β-1-O-[N-(2-formyl3-chlorophenyl)anthraniloyl]-D-glucupyranuronic acid), 4 (β-1-O-[N-(2-methyl-3-chlorophenyl-4-hydroxy)anthraniloyl]-D-glucupyranuronic acid), and 5 (β-1-O-[N-(2-methyl-3-chlorophenyl-5-hydroxy) anthraniloyl]-D-glucupyranuronic acid).
(2-45% acetonitrile over 70 min on a C18 column). Peaks of interest detected by UV absorption were identified by the continuous-flow HPLC-NMR experiment, and stopped-flow 1D single-pulse and 2D TOCSY experiments were performed for unambiguous structure assignment of the individual metabolites of ibuprofen.6,8 The phase II metabolism of paracetamol has similarly been investigated in urine and bile samples from rats and in human urine by directly coupled 500 MHz HPLC-NMR7,10 In the present studies, the metabolites of another nonsteroidal anti-inflammatory drug, tolfenamic acid (N-(2-methyl-3-chlorophenyl)anthranilic acid), were investigated. A combination of SPE and 400 MHz NMR as well as directly coupled 800 MHz HPLC-NMR was applied in order to identify the major phase II metabolites of tolfenamic acid in urine samples obtained 4-6 h after oral intake of 300 mg of tolfenamic acid. The applicability of directly coupled HPLC-NMR, where the chromatographic method developed contained both methanol and acetonitrile in the mobile phase, was additionally demonstrated in the present case. This requires a more complex method for suppression of the NMR resonances of the multiple solvents. The metabolic fate of tolfenamic acid is presented in Figure 1.11,12 The phase I metabolites shown in the figure were found in the present investigations to be conjugated with glucuronic acid (11) Hansen, S. H.; Pedersen, S. B. J. Pharm. Biomed. Anal. 1986, 4, 69-82. (12) Pedersen, S. B.; Alhede, B.; Buchardt, O.; Bock, K. Arzneim. Forsh. Drug. Res. 1981, 31, 1944-1948.
608 Analytical Chemistry, Vol. 69, No. 4, February 15, 1997
(metabolites T, 1-5). It is, however, not known if phase I metabolism takes place before glucuronidation or if the glucuronides are initially formed and then further oxidized. Previous investigations of the metabolites of tolfenamic acid in biofluids have been performed after the samples were treated with β-glucuronidase in order to hydrolyze glucuronic acid conjugates. In the present studies, the phase II metabolites were analyzed directly. EXPERIMENTAL SECTION Chemicals. HPLC solvents were of analytical grade and obtained from Riedel De Hae¨n (Seelze, Germany). All other chemicals were of analytical chemical grade and purchased from Aldrich Chemical Co. Ltd. (Steinheim, Germany). Clotam capsules were kindly donated by GEA A/S (Copenhagen, Denmark). Reference compounds of N-(2-methyl-3-chlorophenyl)anthranilic acid and its phase I metabolites (N-(2-hydroxymethyl-3-chloro-4hydroxyphenyl)anthranilic acid (II) and N-(2-formyl-3-chlorophenyl)anthranilic acid (III)) were also donated by GEA A/S. Solid Phase Extraction (SPE) of Urine Samples Containing Metabolites of Tolfenamic Acid. Urine samples obtained from a human volunteer 4-6 h after oral administration of 300 mg of tolfenamic acid were investigated for metabolites of tolfenamic acid. Two milliliters of urine was applied to a 3 mL solid phase extraction cartridge (Varian, Harbor City, CA) containing 500 mg of C18 sorbent, previously activated by application of
first 5 mL of methanol and then 5 mL of distilled water (acidified with 5 µL of concentrated formic acid). Ten microliters of concentrated formic acid was added to the sample, which was then applied to the activated sorbent. The cartridge was washed with 5 mL of acidified water. A stepwise elution gradient was applied to recover the metabolites, thus 5 mL of each of the eluents consisting of 20:80, 40:60, 60:40, 80:20, and 100:0 methanolacidified water (v/v) was applied to the cartridge. The eluent in the collected fractions was evaporated by rotary evaporation, and the remaining water was removed by freeze-drying. The sample was finally reconstituted in methanol-d4. This SPE method has previously been applied to investigations of the metabolites of oxpentifylline, ibuprofen, naproxen, acetylsalicylic acid, and paracetamol.4,5 Chromatography. The HPLC system consisted of a Bruker LC22C pump (Rheinstetten, Germany) and a Bruker LC33 variable-wavelength UV detector (operated at 200 nm). The outlet of the UV detector was connected to the HPLC-NMR flow probe via an inert poly(ether ether ketone) capillary (0.25 mm i.d.). Data were collected using a Bruker Chromstar HPLC data system. The analytical column was an internal reversed-phase chromatographic column (4.6 mm × 120 mm i.d., 5 µm particles; Supelco HiSep, Supelco Inc., Bellefonte); the flow rate was 1 mL/min. The eluent initially consisted of 5% acetonitrile in phosphoric acid (15 mM). A step gradient was applied after 2 min, resulting in a mobile phase composition of methanol-acetonitrile-15 mM phosphoric acid (28:33:39 v/v/v). UV detection was at 280 nm. NMR and Directly Coupled HPLC-NMR Spectroscopy. The one-dimensional (1D) NMR data of the SPE fractions were acquired using a Bruker AMX-400 spectrometer. 1H NMR spectra were obtained at 400.14 MHz. Free induction decays (FIDs) were collected into 16K computer data points with a spectral width of 5050.5 Hz, 90° pulses were used with an acquisition time of 2.58 s, and the spectra were acquired by accumulation of 64 scans. Prior to Fourier transformation, an exponential apodization function was applied to the FID, corresponding to a line broadening of 0.3 Hz. Two-dimensional COSY and TOCSY experiments were performed on the individual SPE fractions in order to improve signal assignment of the complex aromatic region. The parameters for the COSY experiments were as follows: number of scans per increment, 16; spectral width, 5881.62 Hz; and 512 increments were performed in the F1 dimension. The FIDs were collected into 1K computer data points. The relaxation delay between successive pulses was 1.5 s. The parameters for the TOCSY experiments (where the MLEV pulse sequence was used) were as follows: number of scans per increment, 32; spectral width, 5881.62 Hz; and 512 increments were performed in the F1 dimension. The FIDs were collected into 1K computer data points, and the delay between successive pulses was 2 s. The data were zero-filled by a factor of 2 prior to Fourier transformation. The HPLC-NMR data were acquired using a Bruker DMX800 MHz spectrometer equipped with a 1H flow probe (cell of 3 mm i.d., with a volume of 120 µL). 1H NMR spectra were obtained in the stopped-flow mode at 800.13 MHz. To suppress the solvent signals, the 1D 1H NMR spectra were collected using a NOESYPRESAT pulse sequence.6 This is a one-dimensional version of the nuclear Overhauser effect spectroscopy (NOESY) pulse sequence, and in the present case, triple presaturation for
suppression of the water, the methanol, and the acetonitrile signals was applied. FIDs were collected into 32K computer data points with a spectral width of 16 025.64 Hz, and 90° pulses were used with an acquisition time of 1.01 s. Prior to Fourier transformation, an exponential apodization function was applied to the FID corresponding to a line broadening of 1.0 Hz. Sixty-four scans were accumulated in the three first spectra, whereas the lasteluting peak required 800 scans in order to obtain an appropriate signal-to-noise ratio. RESULTS AND DISCUSSION SPE in Combination with NMR of Urine Containing Tolfenamic Acid Metabolites. SPE was applied to a human urine sample obtained 4-6 h after intake of 300 mg of tolfenamic acid. The individual SPE fractions were then analyzed with respect to tolfenamic acid and its metabolites by one- and twodimensional 1H NMR spectroscopy. It was found that the tolfenamic acid metabolites eluted in the 40, 60, and 100% methanol fractions during the SPE sample preparation step as shown in Figure 2. The metabolites observed along with the parent compound were present as glucuronic acid conjugates, as evidenced by the doublets occurring in the 1H NMR spectra at δ 5.8-6.0; these arise from the 1′ proton on the glucuronic acid ring of β-1-O-acyl conjugates. To identify the metabolites of tolfenamic acid in the SPE fractions, where several metabolites are present in each fraction, 2D COSY and 2D TOCSY NMR experiments were performed. The chemical shift values obtained were compared with the chemical shifts published earlier for tolfenamic acid and its phase I metabolites and from 1H chemical shifts obtained from NMR spectra acquired of the purified phase I metabolites.11 The compounds related to tolfenamic acid can be identified by the doublet of doublets occurring near δ 8, arising from the H3 proton on the aromatic ring of tolfenamic acid and its metabolites (see Figure 1). As can be seen from Figure 2A, the 100% methanol fraction contained primarily a single metabolite, which was identified as the ester glucuronide of tolfenamic acid (T). The 60% methanol fraction (Figure 2B) contained a mixture of at least three phase II metabolites of tolfenamic acid, as can be seen from the signals corresponding to the H3 protons on the aromatic ring in the 1H NMR spectrum of this fraction. Also, the spectral region (δ ∼5.7) where the anomeric proton resonances of the β-1-O-acyl glucuronides are found indicates the presence of at least three metabolites and proves that all three metabolites are conjugated with glucuronic acid (i.e., via the carboxylic acid group in the 2-position of tolfenamic acid). The aldehyde 3 is easily identified by the singlet occurring at δ 10.47 (Figure 2B), arising from the CHO group in the 2′-position on tolfenamic acid (see structure in Figure 1). The other two metabolites were identified to be the glucuronic acid conjugates of the phenolic metabolite (4) and the hydroxylated metabolite (1) from the chemical shift pattern of the aromatic region (protons H3, H4, H5, H6), using a combination of the spectra obtained from the COSY and TOCSY experiments (data not shown). Additionally, the metabolites were identified on the basis of the presence of a CH3 group or a CH2OH group in the 2′-position (see Figure 2). The 40% fraction contains one major and one minor metabolite of tolfenamic acid (see Figure 2). The major metabolite was identified to be the glucuronic acid conjugate of the hydroxylated metabolite (1) by a combination of the COSY and the TOCSY spectra of this fraction. The minor metabolite was identified to be the glucuronic acid conjugate of Analytical Chemistry, Vol. 69, No. 4, February 15, 1997
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Figure 2. 1H 400 MHz NMR spectra obtained from the 100% (A), 60% (B), and 40% (C) methanol fractions from a SPE preparation of a urine sample obtained 4-6 h after intake of 300 mg of tolfenamic acid. The stars indicate the expanded signals. The characteristic proton signals of the glucuronic acid conjugates 1, 2, 3, 4, and T (see Figure 1) are indicated in the NMR spectra. 1′β corresponds to the 1H signal of the anomeric protons of the β-1-O-acyl glucuronides.
the hydroxylated phenolic metabolite (2) by use of the 2D COSY and 2D TOCSY experiments. In the 60% methanol SPE fraction, where at least three glucuronic acid conjugates were observed, confirmation of the structure of the metabolites could not be achieved directly because the presence of methyl group oxidation (see Figure 1) and position of hydroxylation on the aromatic ring in the individual metabolites were not obtained, i.e., metabolites 4 and 5 as opposed to metabolites 1 and 2 (see Figure 1). To obtain the exact identification of the metabolites in this fraction, further separation before NMR analysis was therefore necessary. Directly Coupled 800 MHz HPLC-NMR Spectroscopy. In the present investigation, the glucuronides of tolfenamic acid found in the 60% methanol SPE fraction of a urine sample were additionally separated and identified by a directly coupled 800 MHz 1H HPLC-NMR experiment. A reversed-phase chromatographic system was developed that separated four glucuronides of tolfenamic acid metabolites in the 60% methanol fraction. The chromatogram obtained using UV detection is presented in Figure 3. The structures of all four glucuronides could be elucidated using this technique, contrary to what was possible by the 1H 400 MHz analysis performed directly on the SPE fractions. The peaks in the chromatogram shown in Figure 3 were identified by the directly coupled 800 MHz stopped-flow HPLCNMR experiment. The individual 1D 1H NMR spectra obtained from these investigations are presented in Figure 4, where only the spectral region corresponding to δ 2-11 is shown. The 610 Analytical Chemistry, Vol. 69, No. 4, February 15, 1997
Figure 3. Chromatogram obtained when 50 µL of the 60% methanol SPE fraction of a urine sample obtained 4-6 h after intake of 300 mg of tolfenamic acid was injected into the chromatographic system. The analytical column was an internal reversed-phase chromatographic column (Supelco HiSep), and the flow rate was 1 mL/min. The eluent was 5% acetonitrile in phosphoric acid (15 mM), and then a step gradient was applied, resulting in a mobile phase composition of methanol-acetonitrile-15 mM phosphoric acid (28:33:39 v/v/v). UV detection was at 280 nm. 1, 3, 4, and 5 refer to the metabolites of tolfenamic acid, as was shown in Figure 1. All the metabolites seen are conjugated with glucuronic acid via the carboxylate group in the 2-position (see Figure 1).
lower field strengths are overlapped. This is illustrated in Figure 5, where the stacked plot of the aromatic spectral region (δ 6.38.5) of the four glucuronides is shown. The third chromatographic peak (Figure 4C) contains the glucuronide of the phenolic metabolite (4), which is the metabolite that is hydroxylated in the 4′-position and where the 2′-methyl group is intact (see Figures 4C and 5C). Surprisingly, the signal corresponding to H6 shifts 0.81 ppm upfield as a consequence of the hydroxylation in the 4′-position of tolfenamic acid. However, the 2D TOCSY and 2D COSY results on the nonseparated SPE fraction proved that the assignment made was correct. The 1D 1H NMR spectrum of the last-eluting peak (Figures 4D and 5D) indicates that this compound is the glucuronide of the metabolite of tolfenamic acid that is hydroxylated in the 5′-position on the aromatic ring (5), and, as in the previous compound, the 2′-methyl group is intact (see Figures 4 and 5). This metabolite was not identified in the 1H 400 MHz NMR spectra of the whole 60% methanol SPE fraction because of spectral overlap and because of the relatively low concentration of this metabolite. The chemical shifts of the glucuronides of tolfenamic acid and the glucuronides of its oxidative phase I metabolites obtained in the investigation described here are summarized in Table 1. CONCLUSIONS SPE in combination with NMR analysis is an efficient means of rapidly obtaining information on the structure of unknown drug metabolites in biofluids. This is true especially if the drug under investigation possesses hydrophobic properties (as for metabolites of tolfenamic acid) and thus can easily be separated from the endogenous metabolites using C8 or C18 solid phase extraction columns. However, if the metabolic pattern is complex, as was the case here, where the different positions of hydroxylation on tolfenamic acid give rise to a highly complex pattern in the aromatic spectral region (δ 6.3-8.5), then 2D NMR experiments must be used in order to identify the metabolites. Alternatively, directly coupled high-field HPLC-NMR can be used, and four glucuronic acid conjugates of metabolites of tolfenamic acid were separated and identified by 1H NMR in the 60% methanol SPE fraction. By use of directly coupled stopped-flow 800 MHz HPLCNMR, it was further possible to identify a minor compound, the glucuronic acid conjugate of V (5). This metabolite was, due to spectral overlap, not seen during the 2D NMR experiments performed directly on the 60% methanol SPE fraction at the lower field strength. Because of the improved dispersion obtained with the ultrahigh-field (i.e., 800 MHz) NMR spectrometer, it was
Figure 4. 800 MHz 1D 1H NMR spectra obtained from a stoppedflow HPLC-NMR experiment on the 60% methanol SPE fraction of a urine sample containing metabolites of tolfenamic acid. The spectral region (δ 2-11) is shown. CHO indicates the aldehyde proton in the 2′-position on the aromatic ring of metabolite 3, as was denoted in Figure 1, 1′β is the 1′ proton of the β-anomer of glucuronic acid conjugates. CH3 is the methyl group in the 2′-position on the aromatic ring, as was denoted in Figure 1. Spectrum A corresponds to metabolite 1, B corresponds to metabolites 1 and 3, C corresponds to metabolite 4, and D corresponds to metabolite 5.
assignments were based on the chemical shift values for the nonconjugated metabolites found in the literature11 as well as on the coupling patterns and the coupling constants. The first-eluting peak (Figure 4A) corresponded to the glucuronic acid conjugate of the hydroxylated metabolite 1, as was also found from the 1D 1H 400 MHz NMR experiment to be the major metabolite in the 60% methanol fraction. The second chromatographic peak (Figure 4B) consists of the glucuronides 1 as well as 3. The fact that this peak contains both glucuronides was caused by chromatographic peak overlap. The increased dispersion obtained with the 800 MHz NMR spectrometer aids the assignment of the aromatic protons, especially the H5, H5′, and H6′ protons that at Table 1.
T 1 2 3 4 5b
1H
NMR Chemical Shifts for the Glucuronic Acid Conjugates of Tolfenamic Acid and Its Metabolitesa
δH3
δH4
δH5
δH6
δH4′
δH5′
δH6′
δ-CH3
8.12 dd 8.08 dd 7.90 dd 8.12 dd 8.05 dd 8.09 dd
6.82 t 6.85 t 6.55 t 7.20 t 6.70 t 6.80 t
6.82 t 7.40 t 7.40 t 7.65 t 7.28 t 6.85 t
7.26 d 7.20 d 6.85 d 7.11 d 6.45 d 7.39 d
7.21 d 7.15 d
7.21 t 7.28 t 6.88 d 7.41 t 6.86 d
7.21 d 7.37 d 7.04 d 7.60 d 7.05 d 6.75 d
2.25 s
7.28 d 6.70 d
δ-CH2-OH
δ-CHO
4.65 s 4.63 s 10.47 s 2.30 s 2.12 s
δ1′β 5.80 d 5.78 d 5.71 d 5.78 d 5.69 d 5.69 d
a Metabolite key, see Figure 1. b Measured only in the 800 MHz HPLC-NMR experiment. Coupling pattern: d, doublet; dd, double doublet; t, triplet; s, singlet.
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Figure 5. 800 MHz 1D 1H NMR spectra obtained from an HPLC-NMR experiment on the 60% methanol SPE fraction of a urine sample containing metabolites of tolfenamic acid. The aromatic spectral region (δ 6.3-8.5) is shown for clarity. All the metabolites seen were present in the form of glucuronic acid conjugates, identified on the basis of the resonance of the β-1′-proton at δ 5.8 (not shown in the figure). Additionally, the metabolic state of the CH3 side chain was identified on the basis of the presence of a singlet at δ 2.3 (shown in Figure 4). H3-H6 and H3′-H6′ are the protons on the aromatic rings of tolfenamic acid and its metabolites, as was shown in Figure 1. Spectrum A corresponds to metabolite 1, B corresponds to metabolites 1 and 3, C corresponds to metabolite 4, and D corresponds to metabolite 5.
possible to assign all the aromatic protons by 1D NMR spectroscopy and thereby determine the exact point of hydroxylation on tolfenamic acid. The experiments described here show the first application of directly coupled 800 MHz HPLC-NMR and show the applicability of the HPLC-NMR technique within the field of pharmaceutical and biological chemistry. Large amounts of structural information can be obtained very rapidly, because of the saving in analysis time compared to off-line analysis, where the individual metabolites would have to be separately isolated and purified before they could be identified by NMR analysis. Furthermore, this study describes the identification of the intact glucuronic acid conjugates of tolfenamic acid in biofluids. In previous investigations, only the
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phase I metabolites of tolfenamic acid have been identified after β-glucuronidase treatment of the biological samples. Finally, the position of hydroxylation on the aromatic rings of tolfenamic acid can be easily assigned in the metabolites identified because of the increased dispersion obtained in the spectra by using a ultrahigh-field NMR spectrometer, i.e., directly coupled 800 MHz HPLC-NMR. Received for review June 12, 1996. Accepted October 17, 1996.X AC960582W X
Abstract published in Advance ACS Abstracts, December 15, 1996.
