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Quantitative determination of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen by high-pressure liquid chromatography
ANALYTICAL
BIOCHEMISTRY
83, 168-177 (1977)
Quantitative Determination of the Glutathione, Cysteine, and N-Acetyl Cysteine Conjugates of Acetaminophen by High-Pressure Liquid Chromatography A.R.
BUCKPITT,'
Section
D. E. ROLLINS,~ SD. NELSON, R. B. FRANKLIN, AND J. R. MITCHELL~
on Clinical Pharmacology and Metabolism, Ofice of the Director Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20014
of
Received April 18, 1977; accepted June 20, 1977 A quantitative method for the estimation of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen obtained from microsomal incubations has been developed using high-pressure liquid chromatography (HPLC). As little as 5 ng of the thiol conjugates of [ring-Tlacetaminophen can be measured with good precision from microsomal incubations with minimal sample preparation. High-pressure liquid chromatography offers distinct advantages over the standard separation techniques of thin-layer, paper, or gel filtration chromatography for the separation and quantitation of the polar thiol metabolites of acetaminophen.
In recent years there has been considerable interest in the hepatic necrosis produced by acetaminophen (l-4). Acetaminophen is metabolically activated in the liver by the microsomal cytochrome P-450 system to a reactive intermediate which covalently binds to tissue macromolecules (5,6). Significant tissue necrosis and covalent binding occur only after liver glutathione has been depleted to approximately 30% of the control level (7). Thus, glutathione is thought to protect against hepatic necrosis by conjugating with the metabolic intermediate formed from acetaminophen. In addition, the covalent binding of acetaminophen in in vitro microsomal incubations can be reduced significantly by the addition of nucleophiles such as glutathione, cysteine, and N-acetyl cysteine (8). Thus, the electrophilic intermediate produced by the activation of acetaminophen can be trapped as a thiol adduct. 1 Supported by a National Heart, Lung, and Blood Institute Fellowship, No. lF32-HL05236. 2 Staff Fellow, Pharmacology Research Associate Program, National Institute of General Medical Sciences. 3 Address reprint requests to Jerry R. Mitchell, Building 10, Room 8N115, National Institutes of Health, Bethesda, Md. 20014. 168 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN W3-2697
HPLC OF ACETAMINOPHEN
CONJUGATES
169
To facilitate the study of the chemical mechanisms by which acetaminophen is metabolically activated, we have developed a rapid, highly specific, quantitative HPLC method for measuring several of the thiol conjugates of acetaminophen obtained from microsomal incubations. MATERIALS
AND METHODS
Radiochemicals
p-Hydroxy-[ringJ4C]acetanilide (acetaminophen) (1.33 mCi/mmol), purchased from New England Nuclear Corp., Boston, Mass., was shown to be greater than 99.9% pure by the following methods: (1) thin-layer chromatography (silica gel G, 250~pm thickness) with either 100% ethyl acetate (R, 0.60) or 100% diethyl ether (R, 0.28) as the solvent followed by scanning on a Packard Model 7200 radiochromatogram scanner and (2) HPLC on a reverse-phase column with a mobile phase of 12.5% methanoYl% glacial acetic acidf86.5% water as described below. Fractions of the column eluate were collected every 30 set for liquid scintillation counting. [3H]Acetaminophen (generally labeled, 24 mCi/mmol) was obtained from New England Nuclear Corp., Boston, Mass. This material was shown to be greater than 99% pure by the methods described for the ring-14C-labeled material. [Glycine-l-‘4CJgiutathione (reduced form, 25 mCi/mmol) was purchased from AmershanSearle, Arlington Heights, Ill., and was used without further purification. HPLC of an aliquot from a liver microsomal incubation containing only [14C]glutathione showed that no 14C activity eluted in the region of either acetaminophen or its glutathione conjugate. Chemicals
Acetaminophen was purchased from Eastman Kodak, Rochester, N. Y. Glutathione (reduced), N-acetyl cysteine, cysteine-HCl, NADP, glucose 6-phosphate, and MgC& (1 M) were obtained from Sigma Chemical Co., St. Louis, MO. Glucose-6-phosphate dehydrogenase (yeast, 200 IT-J/ml) was obtained from Calbiochem, Gaithersburg, Md. Spectroquality solvents were purchased from Matheson, Coleman, and Bell, Norwood, Ohio, for use in the HPLC. All other reagents were the best available. Microsomal Incubation
Hepatic microsomes were prepared from male NIH general-purpose mice (22-28 g) by previously described methods (8). The thiol conjugates of acetaminophen were formed by incubating [14C]acetaminophen with mouse liver microsomes in the presence of glutathione, cysteine, or N-acetyl cysteine. The incubation mixture contained 75 mM KCl, 20
170
BUCKPITT
ET AL.
mrvr inorganic phosphate, 15 mM MgCl,, 6 mg of microsomal protein, and a NADPH-generating system consisting of 0.83 mM NADP, 20 mM glucose 6-phosphate, and 4.0 IU of glucose-6-phosphate dehydrogenase in a total volume of 3 ml. The incubation mixture was prepared on ice, the NADPH-generating system (cofactor) was added last, and the incubation vessels were transferred to a Dubnoff metabolic shaker. After incubation at 37°C for 10 min, the incubation vessels were placed on ice, and 3 ml of ice-cold spectroquality methanol were added to stop the reaction. The contents of the incubation vessels were centrifuged for 20 min at 1OOOg. The supernatant was filtered through a OS-vrn Millipore filter, and an aliquot was reduced to dryness under a stream of nitrogen. The samples were reconstituted in glass-distilled water for chromatography. HPLC A Waters Associates HPLC (Milford, Mass.) equipped with a M6OOOA pump, a M440 uv detector with a 254-nm filter, a U6K loop injector, and a reverse-phase C,, PBondapak column (30 x 0.39 cm) was used for all chromatographic analysis. Chromatography was performed at room temperature using one of the following solvent mixtures: (i) 12.5% methanol/l% glacial acetic acid/86.5% water, (ii) 10% methanol/l% glacial acetic acid/89% water containing 0.005 M 1-heptane sulfonic acid (Waters Associates, PIC B-7), or (iii) 8% acetonitrile/92% water. One hundred microliters of microsomal supematant was injected onto the HPLC column. For precise quantitative measurements, the injector was backflushed with approximately 10 ml of solvent, and the column was rinsed by the injection of 1 ml of 100% methanol between each sample injection. The eluate from the HPLC was collected directly into scintillation vials at intervals ranging from 15 set to 1 min. Each sample was dissolved in 2 ml of methanol and 15 ml of Aquasol (New England Nuclear, Boston, Mass.) for counting in a Searle Mark III liquid scintillation spectrometer. All samples were counted for 10 min and were corrected for quench using an automatic external standard. Zdentifcation
of Conjugates
The acetaminophen conjugates were collected from the HPLC using 12.5% methanol/l% glacial acetic acid/86.5% water as the mobile phase. The collected eluate was lyophilized, and the conjugate was redissolved in glass-distilled water. To remove trace impurities the sample was then subjected to further HPLC using a C,, PBondapak column with water as the mobile phase. The glutathione conjugate of acetaminophen that was collected from the second purifying injection was characterized in the following manner:
HPLC
OF ACETAMINOPHEN
CONJUGATES
171
(i) Reduction of the conjugate in refluxing ethanol with activated Raney-Ni (ICN/K&K) gave back acetaminophen; (ii) hydrolysis of the conjugate isolated from a dual-label experiment of [3H]acetaminophen and [14C]glutathione with a glutathionase preparation from rat kidney homogenate (9) yielded 3-cysteinyl-[3H]acetaminophen (analyzed by the HPLC method described) and glutamic acid and [14C]glycine as determined by thin-layer chromatography on silica gel GF (Analtech) developed twice in 70:30 n-propanol:NH,OH [glutamic acid (R, 0.35), glycine (R, 0.45)] and (iii) chemical ionization mass spectrometry of the conjugate was performed by a direct probe insertion technique described by Baldwin and McLafferty (10) using a V.G. MicroMass 16F instrument. Conditions were as follows: accelerating voltage, 4 kV; electron energy, 70 eV; ionizing current, 200 PA; ion source pressure using isobutane reactant gas, 0.4 Torr. A small ion occurred at m/e 411 corresponding to the loss of the elements of formic acid from the conjugate. Major fragments were found at rlzle 198 and 184 corresponding to a methylmercapto and a mercapto derivative of acetaminophen which probably arise from thermal degradation of the conjugate. Other major fragments appear at m/e 148, 130, 84, and 76 and probably relate to glutamic acid and glycine degradation products of the peptide backbone. Further mass spectral studies of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen will be reviewed in depth in a separate publication (11). The cysteine and N-acetyl cysteine conjugates were isolated by the HPLC methods described. Electron impact and chemical ionization mass spectrometry of the conjugates gave spectra which were virtually identical to synthetic standards (11). RESULTS
AND DISCUSSION
Glutathione, cysteine, and N-acetyl cysteine have been used to trap reactive drug intermediates in order to gain structural information about the nature of the reactive species. Recently this concept has been used effectively to provide structural information on the reactive acylating agent formed in the microsomal metabolism of acetyl hydrazine (12). Until now the isolation of these conjugates has been a laborious and time-consuming procedure. Previously published methods for the quantitative determination of acetaminophen conjugates include gel filtration on Sephadex G-10 (13), paper chromatography (14), and thinlayer chromatography (15,16). The isolation of the cysteine conjugate of acetaminophen by HPLC on an anion exchange column has been previously reported, but the elution required 4 hr (17). It is now possible to separate and accurately quantify acetaminophen and its glutathione, cysteine, and N-acetyl cysteine conjugates simul-
172
BUCKPITT
ET
AL.
taneously and with a high degree of reproducibility from microsomal incubations. Figure 1 shows both the uv and radioactive elution profiles for three incubations which were identical with the exception that: incubation A contained cofactor and glutathione, B contained cofactor but no glutathione, and C contained no cofactor and no glutathione. The peak eluting at 9.2 min was identified as unchanged acetaminophen by mass spectrometry, and it had an identical retention time when compared with an authentic standard. The possibility that either hydroquinone or paminophenol was formed as a metabolite and was eluted along with acetaminophen could not be eliminated by mass spectrometry since both compounds would yield fragments similar to those for acetaminophen. HPLC of these compounds in 12.5% methanol/l% glacial acetic acid/ 86.5% water yielded peaks at 5.7 min for hydroquinone and at 3.8 min for p-aminophenol. Furthermore, 3,4-dihydroxyacetanilide, which has been suggested by Andrews and co-workers (16) to be an intermediate in the formation of sulfate conjugates isolated from human urine, eluted at 6.0 min. Thus, none of these possible acetaminophen metabolites
a150
5
TIME
(mid
FIG. 1. Chromatographic profile of 100 ~1 of the supematant from a microsomal incubation containing ~O+M [‘YJ]acetaminophen and (A) cofactor plus 10m4tw glutathione, (B) cofactor without glutathione, and (C) no cofactor and no glutathione. Chromatography was performed on a C,, PBondapak column using 12.5% methanol/l.O% glacial acetic acid/86.5% water as the mobile phase at a flow rate of 1.0 mUmin. The solid line is the uv tracing; the arrow indicates a fivefold increase in the uv sensitivity. The dashed line represents the amount of 14C disintegrations per minute eluting from the column. Fractions of 0.5 ml were collected for the first 9.0 mitt, and fractions of 0.25 ml were collected for the remaining 6.0 min.
HPLC OF ACETAMINOPHEN
CONJUGATES
173
was produced in the microsomal incubations. The peak eluting at 12.1 min was identified as an acetaminophen-glutathione conjugate by the following criteria: (i) Omission of glutathione from the incubation reduced the radioactivity in the conjugate fraction to near background levels; (ii) omission of both glutathione and cofactor from the incubation reduced the radioactivity in the conjugate fraction to background; (iii) incubation of [3H]acetaminophen and [14C]glutathione yielded both radiolabels only in the area of the conjugate peak; (iv) reduction of the conjugate with Raney-Ni yielded acetaminophen (see Materials and Methods); (v) glutathionase hydrolysis of the conjugate yielded acetaminophen-cysteine, glutamic acid, and glycine (see Materials and Methods); and (vi) collection of the material eluting at 12.1 min yielded a mass spectrum consistent with an acetaminophen-glutathione conjugate. Furthermore, recycling of the acetaminophen-glutathione conjugate peak on the HPLC in a closed-loop system (six recycles) showed this peak to be only one compound. The amount of acetaminophen-glutathione conjugate formed in incubation A, which contained exogenously added glutathione and cofactor, is approximately lo-fold greater than incubation B, which contained cofactor but no exogenously added glutathione. 6
A
I
h
-2
I
20 0
TIME
I
1
5
I
/
10
,
,
15
20
(mid
FIG. 2. Chromatographic profile of 100 ~1 of the supernatant from a microsomal incubation containing 10m3M acetaminophen and cofactor plus (A) lo-$ M cysteine and (B) no cysteine. Chromatography was done on a C,, FBondapak column using 12.5% methanol/ 1.0% glacial acetic acid/86.5% water as the mobile phase at a flow rate of 0.6 ml/min. The solid line is the uv tracing, and the dashed line is the elution of 14C disintegrations per minute. Fractions of 0.6 ml were collected for the first 11.0 min, after which 0.15-m] fractions were collected for the remaining 9.0 min.
174
BUCKPITT
ET
AL.
Thus, the small amount of acetaminophen-glutathione conjugate formed in incubation B was probably due to the trace quantities of endogenous glutathione which remain with the microsomal fraction of the liver homogenate during its preparation. Figure 2A shows the radioactive and uv elution profiles for a sample prepared from a mouse liver microsomal incubation of acetaminophen, cysteine, and cofactor. The peak eluting at 14.2 min was identified as the cysteine conjugate of acetaminophen by the following criteria: (i) Elimination of cysteine from the microsomal incubation results in no peak at 14.2 min (Fig. 2B); (ii) collection of the material eluting in this peak yields a mass spectrum consistent with that for 3-cysteinylacetaminophen (11). The flow rate for the cysteine-acetaminophen samples was reduced from 1.0 to 0.6 ml/min since adequate separation of acetaminophen and its cysteine conjugate was not obtained at the higher flow rate. Furthermore, reduction of the methanol concentration in the mobile phase to either 5 or 10% had a deleterious effect upon the separation. For precise quantitative measurements, baseline separation of the peaks was achieved by using two C1, PBondapak columns in series with a flow rate of 1.0 mllmin in 12.5% methanol/l% glacial acetic acid186.5% water. A
0
i? a Q
0
5
10
15
20
25
0
TIME
(minl
5
10
15
20
25
FIG. 3. Chromatographic profile of 100 ~1 of the supernatant from a microsomal incubation containing lo-sM acetaminophen and cofactor plus (A) 10e3~ N-acetyl A Cl8 WBondapak column with a mobile cysteine and (B) no N-acetyl cysteine. phase of 12.5% methanol/l.O% glacial acetic acid/86.5% water at a flow rate of 1.0 ml/min was used in the chromatography. The solid line is the uv tracing, while the dashed line is the elution of 14C disintegrations per minute. One-milliliter fractions were collected for 25.0 min.
HPLC OF ACETAMINOPHEN
175
CONJUGATES
Substitution of N-acetyl cysteine for cysteine in the above incubations produced a conjugate eluting at 20.2 min (Fig. 3A) that was identified as 3-(N-acetyl cysteinyl)acetaminophen by the same criteria used to identify the acetaminophen-cysteine conjugate. Omission of N-acetyl cysteine from the incubation eliminated the peak at 20.2 min (Fig. 3B). A mass spectrum of the material eluting at 20.2 min was identical to the one obtained for a synthetic standard of 3-(N-acetyl cysteinyl)acetaminophen (11). Table 1 shows the retention times of acetaminophen and its glutathione, cysteine, and N-acetyl cysteine conjugates in three different solvent systems. The chromatography was very stable; changes in solvent from batch to batch or replacement of a column did not significantly alter the retention times. Routinely, quantitative determinations of acetaminophen and acetaminophen-glutathione were done using 12.5% methanoLI 1% glacial acetic acid/86.5% water as the mobile phase. Paired ion/ion suppression chromatography using 10% methanol/l% glacial acetic acid/ 0.005 M 1-heptane sulfonic acid in 89% water also can be used to separate TABLE
1
RETENTION TIMESOFACETAMINOPHEN AND ITSGLUTATHIONE,CYSTEINE, N-ACETYL~YSTEINE CONJUGATESON A C,,~BONDAPAKCOLUMN WITHTHREE SOLVENT SYSTEMS~
AND
Retention times (min)
Compound 1. Acetaminophen 2. Acetaminophenglutathione 3. Acetaminophencysteine 4. AcetaminophenN-acetyl cysteine
12.5% Methanol/ 1.0% glacial acetic acid/86.5% water
10.0% Methanol/ 1.O% glacial acetic acid/O.005 M l-heptane sulfonic acid/ 89.0% water
8.0% Acetonitrile/ 92.0% water
9.2
9.4
1.5
12.1
19.2
Void volume b
8.2’
14.1
Void volume
20.1
14.8
Void volume
a One hundred microliters of a liver microsomal supernatant, prepared as described in Materials and Methods, was chromatographed in each of the solvent systems at a flow rate of 1.O ml/min. * Compounds eluting in the column void volume are not separated from other uv-absorbing components of the incubation, and they cannot be measured quantitatively. c Acetaminophen-cysteine is not completely separated from acetaminophen. The separation of these two compounds is possible using two C,, PBondapak columns with the same mobile phase and flow rate. Under these conditions, the retention times for acetaminophen and acetaminophen-cysteine are 21.0 and 17.2 min. respectively.
176
BUCKPITT
ET AL.
acetaminophen and its glutathione conjugate from other constituents of a microsomal incubation. However, this solvent system is not suitable for the preparation of compounds for mass spectral studies since lheptane sulfonic acid is not easily removed from the sample. Acetaminophen can be separated from other constituents of a microsomal incubation and can be quantitatively determined in less than 10 min using 8% acetontrile/92% water. However, the polar acetaminophen conjugates eluted in the column void volume and cannot be quantitated using this solvent system. Triplicate aliquots from microsomal incubations of acetaminophen that contained (i) cofactor and glutathione and (ii) no cofactor or glutathione were prepared and analyzed as described in Materials and Methods. The data in Table 2 show that this HPLC method is sufficiently reproducible to permit the quantitation of the acetaminophen-glutathione conjugate and the measurement of total acetaminophen metabolism by the quantitation of cofactor-dependent substrate disappearance. In the lo-min incubation, a total of 13.4 nmol of acetaminophen is metabolized per milligram of microsomal protein, 86% of which (11.6 nmol/mg of microsomal protein) is converted to the acetaminophen-glutathione conjugate. The HPLC method presented here represents a major step in the analysis of the conjugates formed when acetaminophen is metabolized TABLE PRECISION
OF THE HPLC METHOD FOR THE DETERMINATION OF ACETAMINOPHEN AND THE ACETAMINOPHEN-GLUTATHIONE CONIUGATE~ Acetaminophenglutathioneb
(A)
+Cofactor,
k (B)
z%C+ SE
Acetaminophen
b
Recovery (%)
+glutathione
11.44 11.64 11.74 11.61 k 0.88
136.2 138.4 137.6 137.4 k 0.6
102 102 102 102 * 0
-glutathione
0.16 0.30 0.19 0.21 2 0.04
151.4 150.2 150.9 150.8 % 0.3
107 102 99 102 k 2
2 SE
-Cofactor,
2
c
Q Triplicate aliquots were prepared from a microsomal incubation of 2.5 x lo-* M acetaminophen and (A) cofactor and 10m3 M glutathione and (B) no cofactor and no glutathione. b Results are expressed as nanomoles per milligram of microsomal protein per 10 min. c The amount of radioactivity (disintegrations per minute) collected from the HPLC in the acetaminophen and acetaminophen-glutathione fractions was computed as a percentage of the total amount of radioactivity chromatographed. Recoveries from other experiments generally ranged between 98 and 102%.
HPLC OF ACETAMINOPHEN
CONJUGATES
177
to a reactive intermediate in the presence of glutathione, cysteine, or N-acetyl cysteine. It also facilitates studies of the structure of the reactive metabolite of acetaminophen through nucleophilic trapping, isolation of conjugate by HPLC, and nuclear magnetic resonance or mass spectral analysis. In addition, the method can be used to study the role of glutathione in the hepatic detoxification of acetaminophen. Experiments are now in progress to determine whether these methods may be applicable to the analysis of thiol conjugates of other metabolically activated drugs. REFERENCES 1. Davison, D. G. S., and Eastham, W. N. (1966) &it. Med. J. 2, 497. 2. Thomson, J. S., and Prescott, L. F. (1966) grit. Med. J. 2, 506. 3. Prescott, L. F., Newton, R. W., Swainson, C. P., Wright, N., Forest, A. R. W., and Matthew, H. (1974) Lnncet 1, 588. 4. Piperno, E. (1976) Lancer 2, 738. 5. Mitchell, J. R., JolIow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. T&r. 187, 185. 6. Jollow, D. J., Mitchell, J. R., Potter, W. Z., David, D. C., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 175. 7. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Thu. 187, 211. 8. Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 203. 9. Booth, J., Boyland, E., and Sims, P. (1960) Biochem. J. 74, 117. 10. Baldwin, M. A., and McLafferty, F. W. (1973) Org. Mass Spectrom. 7, 1353. 11. Nelson, S. D., Vaishnav, Y., Rollins, D. E., Buckpitt, A. R., and Mitchell, J. R. In preparation. 12. Nelson, S. D., Hinson, J. A., and Mitchell, J. R. (1976) Biochem. Biophys. Res. Commun.
69, 900.
13. Jagenburg, R., Nagy, A., and Rodjer, S. (1968) &and. J. Clin. Lab. Invest. 22, 11. 14. Mitchell, J. R., Thorgeirsson, S. S., Potter, W. Z., and Jollow, D. J. (1974) Clin. Pharmacol.
Ther.
16, 676.
15. Jollow, D. J., Thorgeirsson, (1974)
Pharmacology
16. Andrews,
S. S., Potter, W. Z., Hashimoto, M., and Mitchell, J. R.
12, 25 1.
R. S., Bond, C. C., Burnett, J., Saunders, A., and Watson, K. (1976) Res. 4, Suppl. 4, 34. 17. Mrochek, J. E., Katz, S., Christie, W. H., and Dinsmore, S. R. (1974) Clin. C&m. 20, 1086. Int. Med.
BIOCHEMISTRY
83, 168-177 (1977)
Quantitative Determination of the Glutathione, Cysteine, and N-Acetyl Cysteine Conjugates of Acetaminophen by High-Pressure Liquid Chromatography A.R.
BUCKPITT,'
Section
D. E. ROLLINS,~ SD. NELSON, R. B. FRANKLIN, AND J. R. MITCHELL~
on Clinical Pharmacology and Metabolism, Ofice of the Director Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20014
of
Received April 18, 1977; accepted June 20, 1977 A quantitative method for the estimation of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen obtained from microsomal incubations has been developed using high-pressure liquid chromatography (HPLC). As little as 5 ng of the thiol conjugates of [ring-Tlacetaminophen can be measured with good precision from microsomal incubations with minimal sample preparation. High-pressure liquid chromatography offers distinct advantages over the standard separation techniques of thin-layer, paper, or gel filtration chromatography for the separation and quantitation of the polar thiol metabolites of acetaminophen.
In recent years there has been considerable interest in the hepatic necrosis produced by acetaminophen (l-4). Acetaminophen is metabolically activated in the liver by the microsomal cytochrome P-450 system to a reactive intermediate which covalently binds to tissue macromolecules (5,6). Significant tissue necrosis and covalent binding occur only after liver glutathione has been depleted to approximately 30% of the control level (7). Thus, glutathione is thought to protect against hepatic necrosis by conjugating with the metabolic intermediate formed from acetaminophen. In addition, the covalent binding of acetaminophen in in vitro microsomal incubations can be reduced significantly by the addition of nucleophiles such as glutathione, cysteine, and N-acetyl cysteine (8). Thus, the electrophilic intermediate produced by the activation of acetaminophen can be trapped as a thiol adduct. 1 Supported by a National Heart, Lung, and Blood Institute Fellowship, No. lF32-HL05236. 2 Staff Fellow, Pharmacology Research Associate Program, National Institute of General Medical Sciences. 3 Address reprint requests to Jerry R. Mitchell, Building 10, Room 8N115, National Institutes of Health, Bethesda, Md. 20014. 168 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN W3-2697
HPLC OF ACETAMINOPHEN
CONJUGATES
169
To facilitate the study of the chemical mechanisms by which acetaminophen is metabolically activated, we have developed a rapid, highly specific, quantitative HPLC method for measuring several of the thiol conjugates of acetaminophen obtained from microsomal incubations. MATERIALS
AND METHODS
Radiochemicals
p-Hydroxy-[ringJ4C]acetanilide (acetaminophen) (1.33 mCi/mmol), purchased from New England Nuclear Corp., Boston, Mass., was shown to be greater than 99.9% pure by the following methods: (1) thin-layer chromatography (silica gel G, 250~pm thickness) with either 100% ethyl acetate (R, 0.60) or 100% diethyl ether (R, 0.28) as the solvent followed by scanning on a Packard Model 7200 radiochromatogram scanner and (2) HPLC on a reverse-phase column with a mobile phase of 12.5% methanoYl% glacial acetic acidf86.5% water as described below. Fractions of the column eluate were collected every 30 set for liquid scintillation counting. [3H]Acetaminophen (generally labeled, 24 mCi/mmol) was obtained from New England Nuclear Corp., Boston, Mass. This material was shown to be greater than 99% pure by the methods described for the ring-14C-labeled material. [Glycine-l-‘4CJgiutathione (reduced form, 25 mCi/mmol) was purchased from AmershanSearle, Arlington Heights, Ill., and was used without further purification. HPLC of an aliquot from a liver microsomal incubation containing only [14C]glutathione showed that no 14C activity eluted in the region of either acetaminophen or its glutathione conjugate. Chemicals
Acetaminophen was purchased from Eastman Kodak, Rochester, N. Y. Glutathione (reduced), N-acetyl cysteine, cysteine-HCl, NADP, glucose 6-phosphate, and MgC& (1 M) were obtained from Sigma Chemical Co., St. Louis, MO. Glucose-6-phosphate dehydrogenase (yeast, 200 IT-J/ml) was obtained from Calbiochem, Gaithersburg, Md. Spectroquality solvents were purchased from Matheson, Coleman, and Bell, Norwood, Ohio, for use in the HPLC. All other reagents were the best available. Microsomal Incubation
Hepatic microsomes were prepared from male NIH general-purpose mice (22-28 g) by previously described methods (8). The thiol conjugates of acetaminophen were formed by incubating [14C]acetaminophen with mouse liver microsomes in the presence of glutathione, cysteine, or N-acetyl cysteine. The incubation mixture contained 75 mM KCl, 20
170
BUCKPITT
ET AL.
mrvr inorganic phosphate, 15 mM MgCl,, 6 mg of microsomal protein, and a NADPH-generating system consisting of 0.83 mM NADP, 20 mM glucose 6-phosphate, and 4.0 IU of glucose-6-phosphate dehydrogenase in a total volume of 3 ml. The incubation mixture was prepared on ice, the NADPH-generating system (cofactor) was added last, and the incubation vessels were transferred to a Dubnoff metabolic shaker. After incubation at 37°C for 10 min, the incubation vessels were placed on ice, and 3 ml of ice-cold spectroquality methanol were added to stop the reaction. The contents of the incubation vessels were centrifuged for 20 min at 1OOOg. The supernatant was filtered through a OS-vrn Millipore filter, and an aliquot was reduced to dryness under a stream of nitrogen. The samples were reconstituted in glass-distilled water for chromatography. HPLC A Waters Associates HPLC (Milford, Mass.) equipped with a M6OOOA pump, a M440 uv detector with a 254-nm filter, a U6K loop injector, and a reverse-phase C,, PBondapak column (30 x 0.39 cm) was used for all chromatographic analysis. Chromatography was performed at room temperature using one of the following solvent mixtures: (i) 12.5% methanol/l% glacial acetic acid/86.5% water, (ii) 10% methanol/l% glacial acetic acid/89% water containing 0.005 M 1-heptane sulfonic acid (Waters Associates, PIC B-7), or (iii) 8% acetonitrile/92% water. One hundred microliters of microsomal supematant was injected onto the HPLC column. For precise quantitative measurements, the injector was backflushed with approximately 10 ml of solvent, and the column was rinsed by the injection of 1 ml of 100% methanol between each sample injection. The eluate from the HPLC was collected directly into scintillation vials at intervals ranging from 15 set to 1 min. Each sample was dissolved in 2 ml of methanol and 15 ml of Aquasol (New England Nuclear, Boston, Mass.) for counting in a Searle Mark III liquid scintillation spectrometer. All samples were counted for 10 min and were corrected for quench using an automatic external standard. Zdentifcation
of Conjugates
The acetaminophen conjugates were collected from the HPLC using 12.5% methanol/l% glacial acetic acid/86.5% water as the mobile phase. The collected eluate was lyophilized, and the conjugate was redissolved in glass-distilled water. To remove trace impurities the sample was then subjected to further HPLC using a C,, PBondapak column with water as the mobile phase. The glutathione conjugate of acetaminophen that was collected from the second purifying injection was characterized in the following manner:
HPLC
OF ACETAMINOPHEN
CONJUGATES
171
(i) Reduction of the conjugate in refluxing ethanol with activated Raney-Ni (ICN/K&K) gave back acetaminophen; (ii) hydrolysis of the conjugate isolated from a dual-label experiment of [3H]acetaminophen and [14C]glutathione with a glutathionase preparation from rat kidney homogenate (9) yielded 3-cysteinyl-[3H]acetaminophen (analyzed by the HPLC method described) and glutamic acid and [14C]glycine as determined by thin-layer chromatography on silica gel GF (Analtech) developed twice in 70:30 n-propanol:NH,OH [glutamic acid (R, 0.35), glycine (R, 0.45)] and (iii) chemical ionization mass spectrometry of the conjugate was performed by a direct probe insertion technique described by Baldwin and McLafferty (10) using a V.G. MicroMass 16F instrument. Conditions were as follows: accelerating voltage, 4 kV; electron energy, 70 eV; ionizing current, 200 PA; ion source pressure using isobutane reactant gas, 0.4 Torr. A small ion occurred at m/e 411 corresponding to the loss of the elements of formic acid from the conjugate. Major fragments were found at rlzle 198 and 184 corresponding to a methylmercapto and a mercapto derivative of acetaminophen which probably arise from thermal degradation of the conjugate. Other major fragments appear at m/e 148, 130, 84, and 76 and probably relate to glutamic acid and glycine degradation products of the peptide backbone. Further mass spectral studies of the glutathione, cysteine, and N-acetyl cysteine conjugates of acetaminophen will be reviewed in depth in a separate publication (11). The cysteine and N-acetyl cysteine conjugates were isolated by the HPLC methods described. Electron impact and chemical ionization mass spectrometry of the conjugates gave spectra which were virtually identical to synthetic standards (11). RESULTS
AND DISCUSSION
Glutathione, cysteine, and N-acetyl cysteine have been used to trap reactive drug intermediates in order to gain structural information about the nature of the reactive species. Recently this concept has been used effectively to provide structural information on the reactive acylating agent formed in the microsomal metabolism of acetyl hydrazine (12). Until now the isolation of these conjugates has been a laborious and time-consuming procedure. Previously published methods for the quantitative determination of acetaminophen conjugates include gel filtration on Sephadex G-10 (13), paper chromatography (14), and thinlayer chromatography (15,16). The isolation of the cysteine conjugate of acetaminophen by HPLC on an anion exchange column has been previously reported, but the elution required 4 hr (17). It is now possible to separate and accurately quantify acetaminophen and its glutathione, cysteine, and N-acetyl cysteine conjugates simul-
172
BUCKPITT
ET
AL.
taneously and with a high degree of reproducibility from microsomal incubations. Figure 1 shows both the uv and radioactive elution profiles for three incubations which were identical with the exception that: incubation A contained cofactor and glutathione, B contained cofactor but no glutathione, and C contained no cofactor and no glutathione. The peak eluting at 9.2 min was identified as unchanged acetaminophen by mass spectrometry, and it had an identical retention time when compared with an authentic standard. The possibility that either hydroquinone or paminophenol was formed as a metabolite and was eluted along with acetaminophen could not be eliminated by mass spectrometry since both compounds would yield fragments similar to those for acetaminophen. HPLC of these compounds in 12.5% methanol/l% glacial acetic acid/ 86.5% water yielded peaks at 5.7 min for hydroquinone and at 3.8 min for p-aminophenol. Furthermore, 3,4-dihydroxyacetanilide, which has been suggested by Andrews and co-workers (16) to be an intermediate in the formation of sulfate conjugates isolated from human urine, eluted at 6.0 min. Thus, none of these possible acetaminophen metabolites
a150
5
TIME
(mid
FIG. 1. Chromatographic profile of 100 ~1 of the supematant from a microsomal incubation containing ~O+M [‘YJ]acetaminophen and (A) cofactor plus 10m4tw glutathione, (B) cofactor without glutathione, and (C) no cofactor and no glutathione. Chromatography was performed on a C,, PBondapak column using 12.5% methanol/l.O% glacial acetic acid/86.5% water as the mobile phase at a flow rate of 1.0 mUmin. The solid line is the uv tracing; the arrow indicates a fivefold increase in the uv sensitivity. The dashed line represents the amount of 14C disintegrations per minute eluting from the column. Fractions of 0.5 ml were collected for the first 9.0 mitt, and fractions of 0.25 ml were collected for the remaining 6.0 min.
HPLC OF ACETAMINOPHEN
CONJUGATES
173
was produced in the microsomal incubations. The peak eluting at 12.1 min was identified as an acetaminophen-glutathione conjugate by the following criteria: (i) Omission of glutathione from the incubation reduced the radioactivity in the conjugate fraction to near background levels; (ii) omission of both glutathione and cofactor from the incubation reduced the radioactivity in the conjugate fraction to background; (iii) incubation of [3H]acetaminophen and [14C]glutathione yielded both radiolabels only in the area of the conjugate peak; (iv) reduction of the conjugate with Raney-Ni yielded acetaminophen (see Materials and Methods); (v) glutathionase hydrolysis of the conjugate yielded acetaminophen-cysteine, glutamic acid, and glycine (see Materials and Methods); and (vi) collection of the material eluting at 12.1 min yielded a mass spectrum consistent with an acetaminophen-glutathione conjugate. Furthermore, recycling of the acetaminophen-glutathione conjugate peak on the HPLC in a closed-loop system (six recycles) showed this peak to be only one compound. The amount of acetaminophen-glutathione conjugate formed in incubation A, which contained exogenously added glutathione and cofactor, is approximately lo-fold greater than incubation B, which contained cofactor but no exogenously added glutathione. 6
A
I
h
-2
I
20 0
TIME
I
1
5
I
/
10
,
,
15
20
(mid
FIG. 2. Chromatographic profile of 100 ~1 of the supernatant from a microsomal incubation containing 10m3M acetaminophen and cofactor plus (A) lo-$ M cysteine and (B) no cysteine. Chromatography was done on a C,, FBondapak column using 12.5% methanol/ 1.0% glacial acetic acid/86.5% water as the mobile phase at a flow rate of 0.6 ml/min. The solid line is the uv tracing, and the dashed line is the elution of 14C disintegrations per minute. Fractions of 0.6 ml were collected for the first 11.0 min, after which 0.15-m] fractions were collected for the remaining 9.0 min.
174
BUCKPITT
ET
AL.
Thus, the small amount of acetaminophen-glutathione conjugate formed in incubation B was probably due to the trace quantities of endogenous glutathione which remain with the microsomal fraction of the liver homogenate during its preparation. Figure 2A shows the radioactive and uv elution profiles for a sample prepared from a mouse liver microsomal incubation of acetaminophen, cysteine, and cofactor. The peak eluting at 14.2 min was identified as the cysteine conjugate of acetaminophen by the following criteria: (i) Elimination of cysteine from the microsomal incubation results in no peak at 14.2 min (Fig. 2B); (ii) collection of the material eluting in this peak yields a mass spectrum consistent with that for 3-cysteinylacetaminophen (11). The flow rate for the cysteine-acetaminophen samples was reduced from 1.0 to 0.6 ml/min since adequate separation of acetaminophen and its cysteine conjugate was not obtained at the higher flow rate. Furthermore, reduction of the methanol concentration in the mobile phase to either 5 or 10% had a deleterious effect upon the separation. For precise quantitative measurements, baseline separation of the peaks was achieved by using two C1, PBondapak columns in series with a flow rate of 1.0 mllmin in 12.5% methanol/l% glacial acetic acid186.5% water. A
0
i? a Q
0
5
10
15
20
25
0
TIME
(minl
5
10
15
20
25
FIG. 3. Chromatographic profile of 100 ~1 of the supernatant from a microsomal incubation containing lo-sM acetaminophen and cofactor plus (A) 10e3~ N-acetyl A Cl8 WBondapak column with a mobile cysteine and (B) no N-acetyl cysteine. phase of 12.5% methanol/l.O% glacial acetic acid/86.5% water at a flow rate of 1.0 ml/min was used in the chromatography. The solid line is the uv tracing, while the dashed line is the elution of 14C disintegrations per minute. One-milliliter fractions were collected for 25.0 min.
HPLC OF ACETAMINOPHEN
175
CONJUGATES
Substitution of N-acetyl cysteine for cysteine in the above incubations produced a conjugate eluting at 20.2 min (Fig. 3A) that was identified as 3-(N-acetyl cysteinyl)acetaminophen by the same criteria used to identify the acetaminophen-cysteine conjugate. Omission of N-acetyl cysteine from the incubation eliminated the peak at 20.2 min (Fig. 3B). A mass spectrum of the material eluting at 20.2 min was identical to the one obtained for a synthetic standard of 3-(N-acetyl cysteinyl)acetaminophen (11). Table 1 shows the retention times of acetaminophen and its glutathione, cysteine, and N-acetyl cysteine conjugates in three different solvent systems. The chromatography was very stable; changes in solvent from batch to batch or replacement of a column did not significantly alter the retention times. Routinely, quantitative determinations of acetaminophen and acetaminophen-glutathione were done using 12.5% methanoLI 1% glacial acetic acid/86.5% water as the mobile phase. Paired ion/ion suppression chromatography using 10% methanol/l% glacial acetic acid/ 0.005 M 1-heptane sulfonic acid in 89% water also can be used to separate TABLE
1
RETENTION TIMESOFACETAMINOPHEN AND ITSGLUTATHIONE,CYSTEINE, N-ACETYL~YSTEINE CONJUGATESON A C,,~BONDAPAKCOLUMN WITHTHREE SOLVENT SYSTEMS~
AND
Retention times (min)
Compound 1. Acetaminophen 2. Acetaminophenglutathione 3. Acetaminophencysteine 4. AcetaminophenN-acetyl cysteine
12.5% Methanol/ 1.0% glacial acetic acid/86.5% water
10.0% Methanol/ 1.O% glacial acetic acid/O.005 M l-heptane sulfonic acid/ 89.0% water
8.0% Acetonitrile/ 92.0% water
9.2
9.4
1.5
12.1
19.2
Void volume b
8.2’
14.1
Void volume
20.1
14.8
Void volume
a One hundred microliters of a liver microsomal supernatant, prepared as described in Materials and Methods, was chromatographed in each of the solvent systems at a flow rate of 1.O ml/min. * Compounds eluting in the column void volume are not separated from other uv-absorbing components of the incubation, and they cannot be measured quantitatively. c Acetaminophen-cysteine is not completely separated from acetaminophen. The separation of these two compounds is possible using two C,, PBondapak columns with the same mobile phase and flow rate. Under these conditions, the retention times for acetaminophen and acetaminophen-cysteine are 21.0 and 17.2 min. respectively.
176
BUCKPITT
ET AL.
acetaminophen and its glutathione conjugate from other constituents of a microsomal incubation. However, this solvent system is not suitable for the preparation of compounds for mass spectral studies since lheptane sulfonic acid is not easily removed from the sample. Acetaminophen can be separated from other constituents of a microsomal incubation and can be quantitatively determined in less than 10 min using 8% acetontrile/92% water. However, the polar acetaminophen conjugates eluted in the column void volume and cannot be quantitated using this solvent system. Triplicate aliquots from microsomal incubations of acetaminophen that contained (i) cofactor and glutathione and (ii) no cofactor or glutathione were prepared and analyzed as described in Materials and Methods. The data in Table 2 show that this HPLC method is sufficiently reproducible to permit the quantitation of the acetaminophen-glutathione conjugate and the measurement of total acetaminophen metabolism by the quantitation of cofactor-dependent substrate disappearance. In the lo-min incubation, a total of 13.4 nmol of acetaminophen is metabolized per milligram of microsomal protein, 86% of which (11.6 nmol/mg of microsomal protein) is converted to the acetaminophen-glutathione conjugate. The HPLC method presented here represents a major step in the analysis of the conjugates formed when acetaminophen is metabolized TABLE PRECISION
OF THE HPLC METHOD FOR THE DETERMINATION OF ACETAMINOPHEN AND THE ACETAMINOPHEN-GLUTATHIONE CONIUGATE~ Acetaminophenglutathioneb
(A)
+Cofactor,
k (B)
z%C+ SE
Acetaminophen
b
Recovery (%)
+glutathione
11.44 11.64 11.74 11.61 k 0.88
136.2 138.4 137.6 137.4 k 0.6
102 102 102 102 * 0
-glutathione
0.16 0.30 0.19 0.21 2 0.04
151.4 150.2 150.9 150.8 % 0.3
107 102 99 102 k 2
2 SE
-Cofactor,
2
c
Q Triplicate aliquots were prepared from a microsomal incubation of 2.5 x lo-* M acetaminophen and (A) cofactor and 10m3 M glutathione and (B) no cofactor and no glutathione. b Results are expressed as nanomoles per milligram of microsomal protein per 10 min. c The amount of radioactivity (disintegrations per minute) collected from the HPLC in the acetaminophen and acetaminophen-glutathione fractions was computed as a percentage of the total amount of radioactivity chromatographed. Recoveries from other experiments generally ranged between 98 and 102%.
HPLC OF ACETAMINOPHEN
CONJUGATES
177
to a reactive intermediate in the presence of glutathione, cysteine, or N-acetyl cysteine. It also facilitates studies of the structure of the reactive metabolite of acetaminophen through nucleophilic trapping, isolation of conjugate by HPLC, and nuclear magnetic resonance or mass spectral analysis. In addition, the method can be used to study the role of glutathione in the hepatic detoxification of acetaminophen. Experiments are now in progress to determine whether these methods may be applicable to the analysis of thiol conjugates of other metabolically activated drugs. REFERENCES 1. Davison, D. G. S., and Eastham, W. N. (1966) &it. Med. J. 2, 497. 2. Thomson, J. S., and Prescott, L. F. (1966) grit. Med. J. 2, 506. 3. Prescott, L. F., Newton, R. W., Swainson, C. P., Wright, N., Forest, A. R. W., and Matthew, H. (1974) Lnncet 1, 588. 4. Piperno, E. (1976) Lancer 2, 738. 5. Mitchell, J. R., JolIow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. T&r. 187, 185. 6. Jollow, D. J., Mitchell, J. R., Potter, W. Z., David, D. C., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 175. 7. Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Thu. 187, 211. 8. Potter, W. Z., Davis, D. C., Mitchell, J. R., Jollow, D. J., Gillette, J. R., and Brodie, B. B. (1973) J. Pharmacol. Exp. Ther. 187, 203. 9. Booth, J., Boyland, E., and Sims, P. (1960) Biochem. J. 74, 117. 10. Baldwin, M. A., and McLafferty, F. W. (1973) Org. Mass Spectrom. 7, 1353. 11. Nelson, S. D., Vaishnav, Y., Rollins, D. E., Buckpitt, A. R., and Mitchell, J. R. In preparation. 12. Nelson, S. D., Hinson, J. A., and Mitchell, J. R. (1976) Biochem. Biophys. Res. Commun.
69, 900.
13. Jagenburg, R., Nagy, A., and Rodjer, S. (1968) &and. J. Clin. Lab. Invest. 22, 11. 14. Mitchell, J. R., Thorgeirsson, S. S., Potter, W. Z., and Jollow, D. J. (1974) Clin. Pharmacol.
Ther.
16, 676.
15. Jollow, D. J., Thorgeirsson, (1974)
Pharmacology
16. Andrews,
S. S., Potter, W. Z., Hashimoto, M., and Mitchell, J. R.
12, 25 1.
R. S., Bond, C. C., Burnett, J., Saunders, A., and Watson, K. (1976) Res. 4, Suppl. 4, 34. 17. Mrochek, J. E., Katz, S., Christie, W. H., and Dinsmore, S. R. (1974) Clin. C&m. 20, 1086. Int. Med.