Anal. Chem. 1996, 68, 106-110
750 MHz HPLC-NMR Spectroscopic Studies on the Separation and Characterization of the Positional Isomers of the Glucuronides of 6,11-Dihydro-11oxodibenz[b,e]oxepin-2-acetic Acid Ulla G. Sidelmann,†,∇ Eva M. Lenz,† Manfred Spraul,‡ Martin Hofmann,‡ Jeff Troke,§ Paul N. Sanderson,⊥ John C. Lindon,⊥ Ian D. Wilson,| and Jeremy K. Nicholson*,†
Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London WC1H 0PP, U.K., Bruker Analytische, Gmbh, Rheinstetten, Silberstreifen, Germany, Drug Metabolism, Hoechst Marion Roussel, Walton Manor, Walton, Milton Keynes MK7 7HL, U.K., Department of Physical Sciences, Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, U.K., and Department of Safety of Medicines, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, U.K.
Ester glucuronides (β-1-O-acyl-D-glucopyranuronates) of many drugs can undergo a series of acyl migration reactions, resulting in positional isomers and anomers which can react with serum proteins with possible toxicological consequences. We have investigated the acyl migration of the ester glucuronides of the model drug 6,11-dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid in pH 7.4 buffer using directly coupled 750 MHz stopped-flow HPLC-NMR spectroscopy. Using a reversed phase isocratic HPLC method with 21% acetonitrile and 79% D2O in the mobile phase, it was possible to separate and hence identify the individual positional isomers of the model drug glucuronide by 750 MHz HPLC-NMR. The order of elution of the isomers from the C18 column was 4r-, 4β-, aglycon, 1β-, 3β-, 3r-, 2r-, 2β- (r- and β- referring to the anomerization state at C1 on the glucuronide ring and the numbers referring to the carbon number on the glucuronide ring to which the drug moiety has migrated). It is shown that directly coupled ultra-high-field HPLCNMR spectroscopy offers a unique analytical advantage for obtaining structural information of interconverting compounds in equilibrium mixtures, and this method will be of value in the study of reactive drug glucuronides of toxicological importance. 6,11-Dihydro-11-oxodibenz[b,e]oxepin-2-acetic acid (I) is an experimental non-steroidal antiinflammatory drug (NSAID) that is excreted mainly in the urine (>99%).1 The major metabolite of I in humans is the β-1-O-acylglucuronide.2 Several other glucuronide-forming NSAIDs (Zomiperac, Benoxaprofen, Indopro†
Birbeck College, University of London. Bruker Analytische. § Hoechst Marion Roussel. ⊥ Wellcome Research Laboratories. | Zeneca Pharmaceuticals. ∇ Current address: Department of Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, Universitetesparken 2, DK-2100 Copenhagen, Denmark. (1) Illing, H. A.; Wilson, I. D. Biochem. Pharmacol. 1981, 30, 3381-3384. (2) Hucker, H. B.; Quaint, K. C.; Dugan, D. E. Prog. Drug Met. 1978, 5, 199204. ‡
106 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
I fen, and Alclofenac) have been withdrawn from clinical use because of a high incidence of toxicological reactions associated with drug therapy.3,4 The toxicological reactions observed are thought to be mediated via covalent drug-protein adducts which act as haptens and can cause immune responses.5-7 The ability of NSAIDs to undergo rapid acyl migration appears to be related to the incidence of allergic reactions.3 Ester glucuronides have been shown to undergo hydrolysis (regeneration of parent compound),8 intramolecular rearrangement (isomerization by acyl migration)9-11 and covalent adduct formation with nucleophilic groups on proteins.10,12-14 The ester linkage of the acyl glucuronides is susceptible to nucleophilic reactions, and this allows the acyl moiety to migrate from one hydroxyl group to an adjacent hydroxyl group on the glucuronic acid ring via an ortho ester intermediate.15 Both the R- and β-anomers of 2-, 3-, and 4-O-acyl positional isomers are formed (3) Spahn-Langguth, H.; Benet, L. Z. Drug Metab. Rev. 1992, 24, 5-48. (4) Clive, D. M.; Stoff, J. F. N. Engl. J. Med. 1984, 310, 563-572. (5) Pohl, L. R.; Satoh, H.; Christ, D. D.; Kenna, J. G. Annu. Rev. Pharmacol. 1988, 28, 367-387. (6) Williams, A. M.; Worall, S.; Jersey, J.; Dickinson, R. G. Biomed. Pharmacol. 1992, 43, 745-755. (7) Smith, P. C.; McDonagh, A. F.; Benet, L. Z. J. Clin. Invest. 1986, 77, 934939. (8) Smith, P. C.; Song, W. Q.; Rodriguez, R. J. Drug Metab. Dispos. 1992, 20, 962-965. (9) Bradow, G.; Kan, L. S.; Fenselau, C. Chem. Res. Toxicol. 1989, 2, 316-324. (10) King, A. R.; Dickinson, R. G. Biochem. Pharmacol. 1991, 42, 2289-2299. (11) Smith, P. C.; McDonagh, A. F.; Benet, L. Z. Drug Metab. Dispos. 1990, 18, 634-644. (12) Feneselau, C. In Conjugation-Deconjugation Reactions in Drug Metabolism and Toxicity; Kauffman, F. C., Ed.; Springer-Verlag: Berlin, 1994; pp 367389. (13) Weiss, J. S.; Guatam, A.; Lauff, J. J.; Sundberg, M. W.; Jatlow, P.; Boyer, J.L.; Saligson, D. N. Engl. J. Med. 1983, 309, 147-150. (14) Ruelius, H. W.; Young, E. M.; Kirkman, S. K.; Schillings, R. T.; Sisenwine, S. F.; Janssen, F. W. Biochem. Pharmacol. 1985, 34, 451-452. 0003-2700/96/0368-0106$12.00/0
© 1995 American Chemical Society
Scheme 1. Acyl Migration and Hydrolysis Reactions of the Glucuronic Acid Conjugate of I
during the acyl migration reactions (Scheme 1). The acyl migration kinetics of drug glucuronides are very complex, and we have previously presented a theoretical kinetic model involving 16 first-order rate constants describing all of the acyl migration and anomerization reactions of the ester glucuronides as well as hydrolysis of the β-1-O-acyl isomer.16 The acyl migration reactions occur spontaneously in aqueous solution, and it has been shown for the β-1-O-acylglucuronide of I that the migration rates are highly dependent upon pH.1 Acyl migration reactions are inhibited in acidic solution, but above pH 7, the migration rates increase markedly.1,16,17 There are also relationships between the chemical structure (i.e., steric and electronic properties) of the aglycon and the observed acyl migration rates.17 Bulky groups in the vicinity of the carbonyl group can obstruct the attack from an adjacent hydroxyl group on the carbonyl carbon, and inductive effects can make the carbonyl carbon more susceptible to nucleophilic attack. As acyl migration is closely associated with drug-protein adduct formation and possible consequent immunogenic reactions,10 it is important to study the factors controlling the rates of the intramolecular acyl migration reactions. NMR spectroscopy allows the direct identification of such isomeric compounds, and it is therefore possible to characterize the novel positional isomers and anomers of drug ester glucuronides in an equilibrium mixture resulting from acyl migration reactions by the use of the twodimensional NMR experiment TOCSY.18 We have previously shown that directly coupled HPLC-1H NMR can be highly effective for the assignment and identification of acyl-migrated positional glucuronide isomers of fluorobenzoic acid glucuronides.19 Directly coupled 750 MHz HPLC-NMR (15) Fischer, E. Chem. Ber.1920, 53, 1621-1633. (16) Sidelmann, U.; Gavaghan, C.; Carless, H. A. J.; Farrant, R. D.; Lindon, J. C.; Hansen, S. H.; Wilson, I. D.; Nicholson, J. K. J. Am. Chem. Soc., submitted. (17) Spannh-Langguth, H.; Benet, L. Z. Drug Metab. Rev. 1992, 24, 5-48. (18) Farrant, R. D.; Spraul, M.; Wilson, I. D.; Nicholls. A. W.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1995, 13, 971-977.
operating in the stopped-flow mode was used here to identify the various positional glucuronides of I in the complex mixture of interconverting compounds. EXPERIMENTAL SECTION Chemicals. HPLC solvents were analytical grade, obtained from Riedel-de Hae¨n (Germany). All other chemicals were of analytical chemical grade, purchased from Aldrich Chemical Co. Ltd. (Gillingham, Dorset, U.K.). I was kindly donated by Hoecsht Pharmaceuticals (Milton Keynes, U.K.) Purification of I-β-D-Glucopyranuronic Acid from Human Urine. The β-1-O-acylglucuronide of I was isolated and purified by preparative HPLC from 280 mL of urine obtained 0-3 h after oral administration of 250 mg of I to a human subject. The HPLC system comprised a Milton Roy Constametric III pump with a Milton Roy spectrometer III UV/vis variable wavelength detector (Stone, Staffs, U.K.) and a Rheodyne injector. The chromatographic column used was a Spherisorb ODS-2 (Phase Separations Ltd., Deeside, Clwyd, U.K.), 5 µm (25 cm × 4.6 mm i.d.). The eluent consisted of 34% acetonitrile in water, acidified with formic acid to pH 2.5. The flow rate was 1 mL/min. The UV detector was operated at 280 nm. The purity of the β-1-O-acylglucuronide of I, measured from the 1H NMR spectrum of the purified product in D2O, was 84%. The 1H NMR spectral data for the isolated β-1-O-acylglucuronide were as follows: δH1 8.21, δHR 4.00, δH3 7.71, δH4 7.29, δH6 5.45, δH7 7.69, δH8 7.72, δH9 7.85, δH10 7.99, δH1′ 5.70, δH2′ 3.68, δH3′ 3.65, δH4′ 3.65, δH5′ 3.80. Establishment of the Equilibrium Mixture of the Glucuronide Isomers. A 1.5 mg/mL sample of the purified product of the β-1-O-acylglucuronide of I was incubated in potassium phosphate buffer (20 mM) at pH 7.4, 25 °C, for up to 24 h. The (19) Sidelmann, U. G.; Gavaghan, C.; Carless, H. A. J.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. Anal. Chem. 1995, 67, 3401-3404.
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Figure 1. Chromatogram obtained when 5 µL of a 1.5 mg/mL solution of the partly equilibrated mixture of positional isomers of the ester glucuronide of I was injected onto the column. 4R/β are the R- and β-4-O-acyl isomers, A is the aglycon, 1β is the β-1-O-acyl isomer, 3R/β are the R- and β-3-O-acyl isomers, 2R is the R-2-O-acyl iosomer, and 2β is the β-2-O-acyl isomer. Chromatography was performed on a Knauer column (120 mm × 4.6 mm i.d.) packed with Spherisorb ODS-2, 5 µm. Mobile phase was acetonitrile/0.2 M potassium phosphate (pH 7.4)/ deuterium oxide (21:10:69 v/v/v), with a flow rate of 1 mL/min. Identification of the HPLC peaks in the chromatogram was based on results obtained using directly coupled HPLC-NMR.
equilibration was monitored by HPLC (UV detection at 280 nm), and when the relative amounts of the positional isomers had stabilized, the sample was stored at -20 °C until analysis, in order to prevent hydrolysis of the glucuronide isomers. Analytical 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 ketone) capillary (0.25 mm i.d.). Data were collected using a Bruker Chromstar HPLC data system. Analytical chromatography was performed on a Knauer column (120 mm × 4.6 mm i.d.) packed with Spherisorb ODS-2, 5 µm. The mobile phase was acetonitrile/0.2 M potassium phosphate in D2O (pH 7.4)/deuterium oxide (21:10:69 v/v/v), with a flow rate of 1 mL/min, and the injection volume was 50 µL. Identification of the compounds corresponding to the HPLC peaks in the chromatograms was based on results obtained using directly coupled HPLC-NMR. NMR Spectroscopy. The HPLC-NMR data were acquired using a Bruker DMX 750 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” mode20 at 750.13 MHz. To suppress the solvent signals, the 1D 1H NMR spectra were collected using a modification of the NOESYPRESAT pulse sequence,21 with double presaturation for suppression of the water and the acetonitrile signals. Free induction decays (FIDs) were collected into 32K computer data points, with a spectral width of 12 019 Hz; 90° pulses were used with an acquisition time of 1.36 s, and the spectra were acquired by accumulation of 200 scans. Prior to Fourier transformation, an exponential apodization func(20) Wilson, I.; Nicholson, J. K.; Spraul, M.; Hofmann, M.; Nicholson, J. K.; Lindon, J. C. J. Chromatogr. 1993, 617, 324-328. (21) Nicholson, J. K.; Foxall, P. J. D.; Spraul, M.; Farrant, R. D.; Lindon, J. C. Anal. Chem. 1995, 34, 793-811.
108 Analytical Chemistry, Vol. 68, No. 1, January 1, 1996
tion was applied to the FID, corresponding to a line broadening of 1.0 Hz. A single pulse 1H NMR spectrum was obtained for the equilibrium mixture of the glucuronide isomers of I on a Bruker AMX-600 spectrometer at 600.14 MHz; 256 FIDs were collected in 32K data points. An exponential apodization function of 0.3 Hz was applied to the FID before Fourier transformation. RESULTS AND DISCUSSION The β-1-O-acylglucuronide of I was purified from the urine samples by HPLC, and an equilibrium mixture of the glucuronide isomers was obtained by incubation of the purified compound at pH 7.4 and 25 °C. A HPLC method was then developed which enabled separation of the positional isomers and the aglycon from the equilibrated mixture of the β-1-O-acylglucuronide (containing the β-1-O-acyl isomer, the R- and β-2-O-acyl isomers, the R- and β-3-O-acyl isomers the R- and β-4-O-acyl isomers and the aglycon). The chromatographic conditions were designed to facilitate direct NMR detection in a dedicated flow probe. In particular, a simple isocratic solvent elution system was needed to minimize solvent suppression problems in the NMR spectrometer, and to facilitate later direct detection of the kinetics of the acyl migration reactions on-line, the pH of the mobile phase was titrated to 7.4. The mobile phase consisted of a 20 mM, pH 7.4 phosphate buffer and 21% acetonitrile, which gave good separation of the positional glucuronide isomers and partial separation of the anomers. The resulting chromatogram of a partly equilibrated mixture of glucuronide isomers is shown in Figure 1. Complete separation of the R- and β-anomers was only obtained for the last eluting isomers (the R- and β-2-O-acyl isomers). However, this did not preclude NMR assignment of the anomers of the other positional isomers (see below). All the positional glucuronide isomers as well as the aglycon corresponding to the UV chromatogram in Figure 1 were identi-
Table 1. 1H NMR Resonance Assignment for the Positional Isomers of the Ester Glucuronide of 6,11-Dihydro-11-oxodibenz[b,e]oxepin-2-acetic Acid (I), Obtained by Stopped-Flow 750 MHz HPLC-NMR R group (I) 1H NMR signals (δ, ppm)
glucuronide ring 1H NMR signals (δ, ppm)
isomer
tRa
R
1
3
4
6
7
8
9
10
1′
2′
3′
4′
5′
R-4-O-acyl β-4-O-acyl aglycon R-3-O-acyl β-3-O-acyl R-2-O-acyl β-2-O-acyl β-1-O-acyl
2.69 2.69 3.50 4.76 4.76 5.21 5.63 b
3.98 3.98 3.62 4.01 4.01 4.00 4.00 4.00
8.17 8.17 8.12 8.21 8.21 8.21 8.21 8.21
7.70 7.70 7.67 7.71 7.71 7.72 7.72 7.71
7.28 7.28 7.23 7.29 7.29 7.29 7.29 7.29
5.44 5.44 5.43 5.44 5.44 5.45 5.45 5.45
7.69 7.69 7.68 7.69 7.69 7.69 7.69 7.69
7.72 7.72 7.72 7.73 7.73 7.72 7.72 7.72
7.85 7.85 7.85 7.86 7.86 7.86 7.86 7.85
7.99 7.99 7.99 7.99 7.99 7.99 7.99 7.99
5.37 4.76
3.75 3.46
4.00 3.80
5.02 5.02
4.31 3.95
5.37 4.77 5.48 4.77 5.70
3.84 3.55 4.88 4.88 3.68
5.31 5.13 4.06 3.80 3.65
3.81 3.78 3.71 3.71 3.65
4.25 3.89 4.22 3.83 3.80
a Retention time in minutes. b Determined from NMR measurement on the starting material (not present in significant amounts in the equilibrium mixture).
Figure 2. 1H NMR spectra obtained from the chromatographic peaks in the chromatogram of the glucuronide isomers of I in their equilibrium mixture: (A) Peak corresponding to the R- and β-4-O-acyl isomers, (B) peak corresponding to the aglycon, (C) peak corresponding to the Rand β-3-O-acyl isomers, and (D) peak corresponding to the R- and β-2-O-acyl isomer.
fied using stopped-flow HPLC-NMR. Directly coupled 750 MHz HPLC-NMR was used to ensure maximum signal dispersion and sensitivity needed for this complex mixture of highly similar isomers. The assignment of the 1D 1H NMR spectra was based on the characteristic chemical shift values and coupling constants of acyl glucuronides.19,22 The NMR signal assignments are summarized in Table 1. In Figure 2, the spectra corresponding to the various positional glucuronides of I in anomeric pairs are shown. For comparison, the 1D 1H NMR spectrum of the equilibrium mixture of the isomeric glucuronides of I is shown in Figure 3. The directly coupled HPLC-NMR technique makes it possible to identify the isomers in the equilibrium mixture by (22) Kaspersen, F. M.; Van Boeckel, C. A. A. Xenobiotica 1987, 17, 1451-1471.
use of simple 1D 1H NMR acquisitions, where it would otherwise take more complex and time-consuming two-dimensional correlation experiments to assign all the resonances in the mixture, as we have shown previously.23 It was, in the present study, possible to identify the three positional glucuronide isomers of the β-1-Oacylglucuronide of I, as well as their R- and β-anomers and the aglycon. The elution order of the positional isomers observed in the chromatographic run is the same as the elution order observed for the 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers20 and the 2- and 3-trifluoromethylbenzoic acid glucuronide isomers at pH 7.4 which was determined earlier.24 The structure of the β-1(23) Farrant, R. D.; Spraul, M.; Wilson, I. D.; Nicholls, A. W.; Nicholson, J. K.; Lindon, J. C. J. Pharm. Biomed. Anal. 1995, 13, 971-977.
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Figure 3. Single-pulse 600 MHz 1H NMR spectrum of the equilibrium mixture of the glucuronide positional isomers of I. The assignments presented in the figure are based on the HPLC-NMR spectral data. The 2,1′R is the 1′-proton on the glucuronide ring of the R-2-O-acylglucuronide of I, the 3,3′β is the 3′-proton on the glucuronide ring of the β-3-O-acylglucuronide of I, etc.
O-acyl isomer of I is very different from those of the other model ester glucuronides that we have studied.24,25 This indicates that the structure-chromatography relationship that determines the elution order of these glucuronide isomers and anomers is consistent, even though the parent compounds are retained to very different extents on the C18 column used. This is exemplified by the fact that the fluorobenzoic acid glucuronides are eluted within 10 min from the column with only 1% acetonitrile,19 whereas the glucuronides of I are eluted with 21% acetonitrile in the mobile phase, and the trifluoromethylbenzoic acid glucuronides are eluted within 10 min with 10% acetonitrile in the eluent.24 HPLC-NMR studies on drug glucuronide isomerization reactions necessitate NMR detection at very high observation frequencies, as high sensitivity and maximal signal dispersion are needed to accurately assign the signals. This is because of the relatively low column loading possible if chromatographic separations of the glucuronide anomers and isomers are to be obtained and because of the complexity and narrow frequency range of the glucuronide ring 1H NMR signals in the chemical shift region δ3.5-4.0. We have previously shown that 600 MHz HPLC-NMR (24) Sidelmann, U. G.; Nicholls, A. W.; Meadows, P.; Gilbert, J.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. J. Chromatogr., in press. (25) Sidelmann, U. G.; Gavaghan, C.; Carless, H. A. J.; Spraul, M.; Hoffman, M.; Lindon, J. C.; Wilson, I. D.; Nicholson, J. K. Anal. Chem., in press.
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is adequate for “simple” model drug glucuronides, such as the fluorobenzoic acid glucuronides.19 Even the fluorobenzoic acid glucuronides require 750 MHz HPLC-NMR when operating in a continuous-flow mode to give adequate NMR sensitivity for the HPLC loading possible.25 However, more complex model drugs, such as I and its glucuronides, pose more significant chromatographic and spectroscopic analytical challenges. Directly coupled 750 MHz HPLC-NMR spectroscopy has proved to be highly effective, allowing full characterization of the glucuronide isomers and anomers, even though complete separation of the R- and β-anomers was not achieved. The method presented here should be of value in studying the structural physicochemical properties that determine drug glucuronide reactivity and will be applicable to study of the reactivity of ester glucuronides of NSAIDs and related compounds. ACKNOWLEDGMENT We thank The Danish Research Council and the EPSRC for supporting this project. Received for review July 31, 1995. Accepted October 16, 1995.X AC950752P X
Abstract published in Advance ACS Abstracts, November 15, 1995.
