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Anal. Chem. 1987, 59, 2830-2832
AIDS FOR ANALYTICAL CHEMISTS Solid-Phase Extraction Chromatography and Nuclear Magnetic Resonance Spectrometry for the Identification and Isolatlon of Drug Metabolites in Urine Ian D. Wilson*' Drug Development, Hoechst Pharmaceutical Research Laboratories, Walton Manor, Walton, Milton Keynes, Bucks MK7 7AJ, U.K.
J. K.Nicholson Department of Chemistry, Birkbeck College, University of London, London WClE 7HX, U.K. The use of high-resolution 'H nuclear magnetic resonance (NMR) spectrometry for the direct detection, identification, and quantification of drugs, drug metabolites, and endogenous componenta in urine and plasma samples has advantages in that rapid multicomponent metabolite determinations of a complex biological sample can be effected with a minimum of physical or chemical pretreatment (1-13). However, this must be balanced against the relative insensitivity of NMR compared with other methods. Thus for lH NMR to be effective, analytes must be present in relatively high concentrations (>50 pM) and must also possess suitable resonances, usually from CH, CH2, or CH3 protons, which can be readily identified and quantified (1-4). Another problem is the observation of compounds in low concentrations (low millimolar range) in the presence of the very large solvent water signal (4). A number of spectrometer-based water suppression strategies are now available (14-16), which are effective for biological fluids; however, these do not overcome the inherent insensitivity of the technique. The simple expedient of freeze-drying the sample and then reconstitution in a smaller volume of 2H,0, largely overcomes the dynamic range problem and concentrates metabolites but may result in the loss of volatile components and will certainly cause deuteration of exchangeable protons. However, the use of this approach enabled the detection and quantification of the major acidic metabolite of the drug oxpentifylline in urine to be accomplished without difficulty (5). To overcome some of the problems associated with the use of NMR in this area, we have explored the use of sample preparation techniques that enable the rapid and selective removal of interfering endogenous metabolites and allow sample solvent deuteration and concentration of metabolites. On the basis of experience gained with sample preparation for chromatographic analysis, solid-phase extraction methods appeared likely to produce the required results. Thus the results described here show the application of solid-phase extraction chromatography with nuclear magnetic resonance (SPEC-NMR) for the analysis of urine samples exemplified by studies on ibuprofen and its metabolites in human urine. EXPERIMENTAL SECTION NMR Spectroscopy. 'H NMR spectra were measured at ambient probe temperature (25 "C) on a Bruker WM250 spectrometer operating at 250 MHz with quadrature detection. With this spectrometer 350 free induction decays (FJD's) were collected, after 90"pulsea, into 16384 computer points, using a sweep width of 3400-Ehand a data acquisition time of 2.4 s. A further delay Present address: Department of Safety of Medicines, IC1 Pharmaceuticals Division, Mereside, Alderley Park, Macclesfield, Cheshire, U.K. S K l O 4TG.
of 5 s was also added between successive pulses, to allow the nuclear spins to return to equilibrium magnetization via Tl relaxation. The signal from the residual water protons was suppressed by the application of a presaturating secondary irradiation field at the water resonance frequency with the decoupler coils, this field being gated "off' during acquisition of the FID. The 13CNMR spectrum of ibuprofen glucuronide in deuteriated dimethyl sulfoxide (DMSO) was obtained on a Bruker AM200 spectrometer operating at 50.3 MHz 13Cfrequency with a sweep width of 12000 Hz and an acquisition time of 1.4 s. A total of 20 OOO FID's were collected into 32 768 computer points, and an exponential function corresponding to 3 Hz line broadening was applied prior to Fourier transformation. Dosing and Sample Collection. A normal, healthy, male subject (age 32) was dosed orally with a single therapeutic dose acid). Urine of 400 mg of ibuprofen (2-(-isobutylphenyl)propionic samples, including a predose sample, were collected as voided into plastic containers and stored frozen (-20 "C). Sample Preparation. The 250-MHz lH NMR spectra were obtained from all samples by freeze-drying 2 mL of urine and reconstituting in 1mL of 2Hz0. For solid-phase sample preparation: Samples of urine (2 mL) were loaded onto 3-mL ClJ30nd-Elut columns (Analytichem International, purchased from Jones Chromatography, Ltd., Glamorgan, U.K.), which had previously been washed with 5 mL of methanol followed by 5 mL of water. In most experiments the urine samples were acidified with 10 pL/mL of 99% formic acid. At this point the column and was washed with 5 mL of CTH30%Ior mixtures of C2H302H3 2Hz0(as described below). Column eluates were collected into 20-mL scintillation vials. Solvents were removed by using a stream of nitrogen and freeze-drying if necessary. Stepwise Elutions. Stepwise elution was employed for some samples in order to obtain selective recovery of metabolites. Thus, following the application of urine samples to the CIScolumns as described above the cartridges were washed with acidified water and then eluted with methanol-water mixtures (pH 2, 5 mL washes of increasing eluotropic strength, i.e. 20:80,40:60,60:40, 80:20, and 100% methanol. Methanol was removed from the eluates with a stream of nitrogen and then the samples were freeze-dried to remove the residual water. The residues were then for spectrometry. redissolved in 2Hz0 RESULTS AND DISCUSSION The 250-MHz 'H NMR spectrum of the freeze-dried predose urine dissolved in 2Hz0is shown in Figure 1. It should be noted that the spectrum contains several frequency "windows" (e.g. 0-1,1.5-1.9,5-7, and 7.8-12 ppm, ref 5 ) that contain few resonances from endogenous compounds and which would allow signals due to drug metabolites to be observed without difficulty. Solid-phase extraction of the urine sample shown in Figure 1without prior acidification resulted in only urinary pigments being retained. Following acidification however (formic acid, pH 2) the bulk of the urinary aromatic acids (e.g. hippurate, indoxyl sulfate, etc.) were se-
0003-2700/87/0359-2830$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
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Flgure 2. (A) A 25oMHr 'H NMR spe3n.m of a sample of freezedrkl utine (2 mL) obtained from a subject following oral dosing with 400 mg of Ibuprofen. The sample was redissolved in H ' ,O for spectroscopy. Structures of the metabolites present are shown above the figure with provisional assignments on the metabolite signals as Indlcated. (B) A 250-Mk 'H NMR spectrum of the sample shown in Figure 3A following solld-phase extraction on a CIS Bond Eiut cartridge and elution with methanol. Methanol was removed and the residue dissolved in 2H20 for spectrometry. The key Is the same as that given for Figure 1.
lectively retained on the column, while most of the nonaromatic amino acids and certain neutral molecules, such as glucose, passed through the column into the eluate. As would be expected, washing the column with methanol resulted in the recovery of the retained metabolites. Subsequent experiments showed that as little as 20% methanol in the eluting solvent was sufficient to wash off all of these components.
Solid-phase extraction chromatography was then applied to acidified urine samples containing ibuprofen metabolites (see below). The NMR spectrum shown in Figure 2A was obtained from a urine sample collected 2-4 h after a single oral dose of 400 mg of ibuprofen. The predose urine sample is shown in Figure 1. After dosing, many new resonances were observed including a broad envelope of overlapping aromatic resonances at about 7.25ppm, together with a prominent doublet at 5.4 ppm. This signal is due to the B-anomeric protons of glucuronides of ibuprofen and certain of ita metabolites. The region from 0.8 to 1.5 ppm contained several signals of drug origin including a large singlet at 1.18 ppm (methyl group from a metabolite with a hydroxylated isobutyl side chain, i.e. 2-[4-(2hydroxy-2-methylpropyl)phenyl]propionicacid, HMPPP). Solid-phase extraction of 2 mL of this 2-4 h urine, following acidification, on a C18 column, followed by elution of the retained material with methanol gave the result shown in Figure 4B. In this spectrum the signals from the ibuprofen metabolites were much more prominent. When processed by using the stepwise gradient elution te&nique, the ibuprofen metabolites were fractionated into two groups essentially free of endogenous compounds. The first group, recovered by eluting with 40% methanol, contained a glucuronide of HMPPP, and the side chain oxidized metabolite 2-[4-(2-carboxy-2-methylpropyl)phenyl]propionic acid (CMPPP). Elution with 60% methanol led to the recovery of further material consisting predominantly of ibuprofen glucuronide (Figure 3). A small amount of contamination of the 60% fraction with metabolites predominantly eluting in the 40% methanol wash was also observed. On the basis of these results a further extraction was performed by incorporating a 50% wash in between the 40% and 60% washes. This resulted in ibuprofen glucuronide being eluted essentially free of contaminants. This metabolite was thus obtained in a fraction of the time that would have been required for the development of a preparative scale HPLC or TLC method. Indeed it proved feasible by scaling up the
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 23, DECEMBER 1, 1987
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Flgure 4 A 250-MHz 'H NMR spectrum of the concentrate obtained following the solid-phase extraction of 12 mL of the sample shown in Figure 2A on a C,B Bond Eiut cartridge. The ibuprofen metabolites were eluted with methanol. After removal of the solvent the residue was dissolved in H ' ,O for spectrometry.
extraction to obtain not only 'H NMR data on this metabolite but also a 13Cspectrum. By use of repeated SPE with a much shallower gradient (5% increments) and reextraction of the fractions, it was also possible to obtain the hydroxyglucuronide metabolite of ibuprofen HMPPP and the side chain oxidized metabolite CMPPP as individual components. However, these two metabolites are relatively similar in their properties on SPE and therefore difficult to obtain in the pure form. In such cases, it would invariably be more efficient to use the SPE step to obtain a concentrate, purified of endogenous contaminanta, and then separate them by using HF'LC or TLC to resolve them into individual components. Alternatively two-dimensional NMR methods could be used for structure elucidation with metabolite mixtures. We also investigated the capacity of the C18 cartridge to retain ibuprofen metabolites by gradually increasing the amounts of urine, which contained 1-2 mg/mL of drug related material, loaded onto the column until no further retention of metabolites was obtained. Until about 15 mL of urine had been loaded onto the 3-mL C18 cartridge there was little evidence of breakthrough of metabolites. With larger amounts (e.g. 20 mL) some breakthrough of drug-related material did take place. However, elution of the material from the cartridge with methanol gave concentrated samples that contained primarily drug metabolites. These concentrated samples enabled high-quality NMR spectra to be obtained, with ex-
cellent signal-to-noise ratios, after the collection of 16-32 FID's. A typical spectrum is shown in Figure 4, which was the result of the application of 12 mL of urine (i.e. insufficient to cause metabolite breakthrough). In this instance the CI8 phase clearly had a greater capacity or affinity for the acidic drug metabolites of ibuprofen than the endogenous components such as hippurate. Thus few signals from endogenous components were observed, even though no attempt was made to remove them by the use of stepwise elution procedures. The high column loading was thus beneficial in terms of both purification and ease of NMR detection, although this should not be expected in all cases.
ACKNOWLEDGMENT We are grateful to P. J. Sadler and I. M. Ismail for their advice and R. Pickford for technical assistance. Registry No. HMPPP, 51146-55-5; CMPPP, 110512-90-8.
LITERATURE CITED (1) Nlcholson, J. K.; Buckingham, M. J.; Sadler, P. J. Biochem. J . 1983, 277(3), 605-615. (2) Nlcholson, J. K.; O'Flynn, M.; Sadler, P. J.; Macleod, A.; Juul, S. M.; Sonksen, P. H. Blochem. J. 1984. 217, 365-375. (3) Nlcholson, J. K.; Bales, J. R.; Sadler, P. J.; Juul, S. M.; Macled, A.; Sonksen, P. H. Lancet 1884, ii, 751-752. (4) Bales, J. R.; Higham, D. P.; Howe, I.; Nlcholson, J. K.; Sadler, P. J. Clin. Chem. (Whston-Salem, N . C . ) 1984, 30, 426-432. (5) Wilson, I. D.; Fromson, J.; Ismail, I . M.; Nicholson, J. K. J . Pharm. Biomed. Anal. 1887, 5(2), 157-163. (6) Wilson, I. D.; Ismail, I. M. J. Pharm. Biomed. Anal. 1986, 4(5), 663-665. (7) Bales, J. R.; Sadler, P. J.; Nicholson, J. K.; Timbrell, J. A. Clin. Chem. (Winston-Salem, N . C . ) 1984, 30(10),1631-1636. (8) Nlcholson, J. K.; Tlmbrell, J.; Sadler, P. J. Mol. Pharmacol. 1985, 2 7 , 644-65 1. (9) Nlcholson, J. K.; Timbrell, J.; Higham, D. P.; Sadler, P. J. Hum. Toxicol. 1984, 3(4), 334-335. (10) Bales, J. R.; Nicholson, J. K.; Sadler, P. J. Clin. Chem. (Winston&/em, N . C . ) 1885, 31, 757-762. (11) Tullp, K.; Nlcholson, J. K.; Timbrell, J. A. Biological Reactive Intermediates; Kocsls, J. J., Ed.; Plenum: New York, 1986; Vol. 3. pp 941-950. (12) Everett, J. R.; Jennings, K.; Woodnut, G. J. Pharm. Pharmacol. 1985, 3 7 , 869-873. (13) Tullp, K.; Tlmbrell, J. A.; Nlchdson, J. K.; Wllson, I. D.; Troke, J. Drug Meteb. Dispos. 1988, 14(6), 746-749. (14) Rabensteln, D. L.; Fan, S.; Nakashlma, T. T. J. Magn. Reson. 1985, 64, 541-546. (15) Rabenstein, D. L.; Fan, S. Anal. Chem. 1986, 5 8 , 33718-3184. (16) Connor, S.; Everett, J.; Nlcholson, J. K. Magn. Reson. M e d . 1887, 4 , 461-470.
RECEIVED for review January 21, 1987. Accepted August 10, 1987. We thank the National Kidney Research Fund for supporting this and related work.
