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Anal. Chem.
1981, 53,2258-2263
pected that this compound will also interfere with the ligand exchange procedure, but this has not be verified.
LITERATURE CITED (1) "EPA Methods for Chemical Analysis of Water and Wastes"; Envlronmental Monitoring and Support Laboratory: Cincinnati, OH, 1978. (2) "Standard Methods for the Examination of Water and Wastewater", 14th ed.;American Public Health Association: Washington, DC, 1976; p 361. (3) "1978 Annual Book of ASTM Standards"; American Society for Testing and Materials: Easton, MD, 1978; p 583. (4) Lock, C. J. L.; Wiikinson, G. J. Chem. SOC.1962, 2281. (5) Gilbert, B. L.; Olson, E?. L.; Reuter, W. Anal. Chem. 1974, 46, 1970. (6) Goulden, P. D.; Afghan, P. E.; Brooksbank, P. Anal. Chem. 1972, 44, 1845. (7) Latimer, G. W., Jr.; Payne, L. R.; Smith, M. Anal. Chem. 1974, 46,
311.
(8) Elly, C. T. J.- Water Pollut. Control Fed. 1968, 40, 848. (9) Barton, P. J.; Hammer, C. A,; Kennedy, D. C. J,-Water Pollut. Control Fed. 1978, 50,234.
Williams, H. E.
SOC.Chem. Ind. J . 1912, 31, 468. Ludzack, E. J. Anal. Chem. 1954, 26, 1784. Kruse, J. J.; Melion, M. G. Anal. Chem. 1953, 25,466. Kruse, J. J.; Mellon, M. G. Anal. Chim. Acta 1977, 83, 139. Frant, M. S.; Ross, J. W.; Riseman, J. H. Anal. Chem. 1972, 44, 2227-2230. (15) Schiueter, A. EPA Report 600/4-76-020, June 1976. (16) Luthy, R . G.; Bruce, S. G.; Waiters, R. W.; Nakies, D. V. ,/.-Water Pollut. Control Fed. 1979, 51,2267. (17) Martell, A. E.; Smith, R. M. "Critical Stability Constants"; Plenum: New York, 1974; Vol. IV. (18) L. Lancy, ERC/Lancy, Inc., Zeiienopie, PA, 1980, personal communi-
(10) (11) (12) (13) (14)
cation.
RECEIVED for review April 24,1981. Accepted August 28,1981. The work upon which this publication is based was performed pursuant to Contract No. 68-03-2714 with the U S . Environmental Protection Agency.
Voltammetry of Acetaminophen and Its Metabolites David J. Miner,' John R. Rice, Ralph
M. R i g g h 2 and Peter T.
Kissinger"
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
As background support for a study of acetaminophen metabolism by modern electroanalytlcal end liquid chromatographic technlques, the voltammetric characteristics are reported for acetamlnophen and representatives of its known classes of metabolites. Excellent agreement Is found between cyclic voltammograms of mllllmolar solutions and hydrodynamic voltammograms derived from nanogram quanthles studied by chromatographlcally assisted hydrodynamic voltammetry. This electrochemical information is expected to find application in the qualitative and quantltative determination of acetamhophen metabolites in samples of physlologlcal origin, ultimately revealing new details about the mechanism of acetaminophen toxlclty. Thls work provides a model which can be employed for the study of numerous toxic aromatic amines and phenols.
A common aspect of the metabolism of many cytotoxic agents, including most chemical carcinogens, is the existence of one or more oxidative pathways which convert the native molecular form of the compound into a highly reactive, electrophilic species capable of chemically modifying vital cellular constituents (1). Elucidation of the precise structure of these hypothesized "reactive intermediate" metabolites and investigation of the nature of their reactivity currently stands as one of the greatest challenges to contemporary molecular biochemistry because of the minute amounts of these metabolites apparently formed and because of the enormous complexity of the cellular environment. This laboratory has begun to apply electrochemical techniques to this problem for two important reasons. First of all, a significant number of the parent compounds exhibiting these toxic effects, as well as most of their known metabolites, are substituted aromatic amines, phenols, or aminophenols. Present address: Lilly Research Laboratories, Indianapolis, IN
46285.
Present address: Battelle Columbus Laboratories, Columbus, OH 43201.
Such compounds are electroactive in aqueous solution a t modest potentials, an advantageous property for both qualitative and quantitative analytical purposes (2). Even more important is the fact that for some of these compounds it is possible to electrochemically generate the reactive intermediates proposed to be responsible for the toxicity. Initial efforts have focused upon the noncarcinogenic analgesic acetaminophen (N-acetyl-p-aminophenol, Tylenol), a known hepatotoxin in overdose, and have provided strong support for the suggestion ( 3 ) that the reactive metabolite of acetaminophen is N-acetyl-p-quinoneimine (NAPQI) ( 4 ) . In support of these investigations, an examination was made of the fundamental electrochemical properties of acetaminophen, representatives of its known classes of metabolites, several possible metabolites, and several related compounds. Two subsequent papers describe the use of chronoamperometry to study aspects of NAPQI chemistry thought to be relevant to its toxicity (5, 6).
EXPERIMENTAL SECTION All potential values were determined vs. a Model RE-1 Ag/ AgC1/3 M NaCl reference electrode (BioanalyticalSystems, West Lafayette, IN). Melting point values are uncorrected except where noted. Apparatus. The flow-through coulometric cell was used as described previously ( 4 ) . It employs a conventional three-electrode, 25-V potentiostat with a current gain stage and consists of a porous glass tube 18 mm X 4 mm i.d. packed with glassy carbon particles (75-180 wm) which is wrapped with a platinum wire auxiliary electrode, Contact with the glassy carbon working electrode is made with a second piece of platinum wire. The tube is placed in a Plexiglas-capped glass jacket containing electrolyte and a reference electrode. Solutions were pumped through this cell at ca. 1mL/min with a Minipuls I1 peristaltic pump (Gilson Medical Electronics, Middleton, WI). Determinations employing liquid chromatography (LC) and chromatographicallyassisted hydrodynamic voltammetry utilized a Model LC-153 (Bioanalytical Systems) equipped with either a carbon paste or glassy carbon amperometric detector. Work at potentials above + L O V required a Model TL-5A (Bioanalytical Systems) low-resistancedetector cell. The injection volume was 20 gL. A 15 cm X 4 mm stainless steel column was slurry-packed with either 10 pm Lichrosorb RP-2 (E. Merck, Darmstadt, FRG)
0003-2700/81/0353-2258$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981
or 10 pm pBondapak C18(Waters Associates, Milford, MA). For routine analysis of solutions containing the compounds examined in this work, the mobile phase consisted of 1M acetic acid (YO mL), 1 M ammoiiium acetate (35 mL), redistilled methanol (25 mL), and water (400 mL) at a flow rate of 1.0 mL/min. Cyclic svoltammetry was carried out with a Model CV-1A (Bioanalytical Systems) and a Model 7015A X-Y recorder (Hewlett-Packard, San Diego, CA), together with conventional cells. For mnpleu of scarce materials, voltammetry was performed on 100 pL volumes using a previously descrilbed microcell (7).For all other stunples, a cell with a capillary tipped reference electrode positioned ca. 1 mm from the surface of the working electrode and wrapped just below the tip with a platinum wire auxiliary was used. The wlorking electrode for both voltammetry cells was a Teflon sheathed well (diameter ca. 0.16 cm) packed with carbon paste (CP-0, Bioanalytical Systems). Chemicals and Reagents. The best available grades of acetaminophen, phenacetin, potassium 4.nitrosophenolate, 4ethylnitrolbenzene, nitrosobenzene, 4-nitrophenetole, 2-acetaminophenol, and hydroquinone were used as purchased. The hydrochloride salt of 4-minophenol was recrystallizedfrom water prior to use. The potassium salt of acetaminophen-0-sulfate was prepared by the procedure of Burckhardt and Lapworth (8) and was recrystallized from water. Its identity vias confirmed by field desorption mass spectrometry, NMR, and cleavage with sulfahe (Sigma,St. Louis, MO) which yielded acetaminophen, as identified by LC. Acetaminophen-0-glucuronide was isolated by TLC from the urine of a volunteer who had taken a 1.3-g dose of acetaminophen. A 300-pL portion of urine was streaked across the base of a 20 X 20 cm silica gel1 plate (250 pm, Merck silica gel 60, F-254) and the plate vvas developed with ethyl acetate:methanol:water:acetic acid (6030:91) to a height of 11.5cm. The major band (Rf= 0.30) was scraped off mid extracted twice with 4 mL of methanol. The extracts were streaked on a second plate and developed to a height of 11.5 cm using 1-butano1:water:aceticacid (6:l:l). The major band (Rf := 0.14) was excised and washed from the silica with methanol. When this product was examined by LC, the area of the major peak constituted ca. 95% of the tlotal peak areas which were observed at an applied detector potential of +1.1V. Its identity was confirmed by cleavage with P-D-glucuronidase (L-11, Sigma, St. Louis, MO) to yield acetaminophen, as identified by LC. The cysteine adduct of acetaminophen was a gift from McNeil Laboratories. The thiomethyl adduct was prepared by reaction of methanethiol with NAPQI generated in the coulometric flow cell. 3-(S)-Thiomethylacetaminophenwas isolated from the reaction mixture by chromatography on a 13 mm X 15 cm column packed with metihed and sieved (106-180 pm) XAD-4 (Mallinckrodt, St. Louis, MO). The compound was eluted with 50:50 methanol/water, the fractions of interest evaporated to dryness and the residue recrystallized from ethyl acetate/hexane. The identity of the product was confirmed by melting point (mp 132 "C, lit. (9) mp 130-131 'C), NMR, and mass spectrometry. N-(4-Hydroxyiphenyl)propionamidewas prepared from 4aminophenol by a standard synthetic procedure (10) and recrystallized from water (mp 170 "C, lit. (11) mp 169-171 "C). 2and 3-Methyl-4-aminophenol were prepared from the corrosponding methyl phenol by nitrosation and subsequent reduction (12). Acetylation yielded the methyl derivativesof acetaminophen. The 2-methyl isomer was recrystallized from ethyl acetatelhexane (mp 179 "C, lit. (13) mp 179 "C). The 3,-methyl isomer was recrystallized twice from water and vacuum dried for 6 h (mp 126 "C, lit. (14) mp 1:?5"C). Electron impact mass spectra of these last three compounds further confirmed their identity. ATHydroxyacetaminophen was synthesized and isolated by the procedure of Mudge et al. (15). N-Hydroxy-N~~(4-ethylphenyl)-3-chloropropionamide was prepared from 4-ethylnitrobenzene (Aldrich Chemical Co., Milwaukee, WI) by established procedures (15,16)(mp 100.2-100.4 "C (corrected)). The identity of the product vvas verified by proton NMR and electron impact mass spectrometry. The latter results were consistent with the anticipated structure and mass spectra of related (compounds(17). Procedures. For each compound to be studied by chromatographically assisted hydrodynamic voltammetry, a potential,
2259
E,, suitable for instantaneous oxidation of the compound is determined by cyclic voltammetry. At this potential, beyond the anodic peak potential, the surface concentration of the analyte is effectively maintained at zero. With the amperometric detector set at E,, chromatographic conditions are found which afford rapid elution of a well-resolved peak for the compound. The applied potential is then sequentially decreased from E,, by 30-50 mV per step. At each potential a constant amount of the compound is injected. A constant quantity of the compound with a reproducible concentration profile passes through the detector for each injection. The peak current, i for the resultant chromatogram is therefore dependent upon tRk voltammetric behavior of the compound at the set potential. At potentials close to E , the peak heights are identical. As the sequence continues i, decreases until a potential is reached where the compound does not react and no peak is seen. For each potential utilized, a ratio 4 is calculated by dividing the peak height (i,) at each potential A plot of 4 vs. E is referred to as by the ip,- recorded at E,,. a normalized hydrodynamic voltammogram. The shape and position of this curve constitute useful qualitative information, which is independent of the chromatographic retention and is normally independent of the amount of compound injected. For the values reported here, the chromatographic mobile phases were chosen to be similar to the supporting electrolyte used in the cyclic voltammetric experiments. Peak heights were recorded manually, and a computer fitting routine was used to obtain the half-wave potential from the raw data. The number of electrons involved in the oxidation of acetaminophen was found by steady-state coulometry. The limiting current produced by the coulometricflow cell was measured when 1.75 mM acetaminophen in 0.1 M citrate, pH 6.8, was passed through it at 1.0 mL/min. Nitrosophenol concentrations were determined by LC employing a C2 column, a mobile phase of 96:4 0.1 M citrate (pH 3.2):methanol,and a detector potential of -253 mV. The response was found to be linear (r > 0.99999) over the range tested (0.5-150 ng injected). The electrode potentials reported here are accurate to & l o mV. RESULTS AND DISCUSSION Voltammetric properties determined via cyclic (CV) and chromatographically assisted hydrodynamic (CAHDV) voltammetry for acetaminophen, a number of its known metabolites, and several related compounds are shown in Figure 1. The method of displaying voltammetric information in this way is defined in the Appendix. The intention is to provide a rapid visual display of information in much the same way as is common for infrared and NMR spectrometry. The comparison of voltammetric information from different techniques is subject to considerable interpretation, especially if the electron transfer rate constants are not fast. Factors such as the scan rate in CV and the flow rate in CAHDV become important experimental variables in such cases (18). Nevertheless, when the intent is t o use the information for analytical rather than physical purposes, there is far less need for caution, as long as the experiments are carried out under reproducible conditions. The close agreement of the CV and CAHDV data illustrated in Figure 1 demonstrates the unique ability of the CAHDV technique to derive voltammetric information from nanogram quantities of individual chromatographically resolved mixture components. Such information frequently gives insights helpful to the identity of unknown components in complex mixtures. The compounds examined in this study may be broadly classified in two groups on the basis of their structural, and consequently voltammetric, characteristics. Those in the first group are oxidized between +500 and +700 mV and have a free 4-hydroxyl group. The second group consists of those in which the 4-hydroxy group is conjugated with sulfate or glucuronide and which are oxidized in the region from +900 to +1100 mV. In general, primary aromatic amines are more easily oxidized than phenols (compare 4-aminophenol and hydroquinone), but the acetamide substituent makes acet-
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981 I
I
I
I
I
I
I
I
I
, Flgure 2. Cyclic voltammetry of acetaminophen. Conditions: 1.O mM acetaminophen, 1.6 mm diameter carbon paste electrode, 150 mV/s, E vs. (AgIAgCV3 M NaCI). Scans initiated in positive direction at 0.0 V.
I 1 1
I
I
09
I
I 07
I
I 05
I
I
03
POTENTIAL IvoLYsI
Flgure 1. Voltammetric properties of acetaminophen and related compounds obtained by cyclic voltammetry and chromatographically
assisted hydrodynamic voltammetry. The conventions used in expressing the data are given in the Appendix. Cyclic voltammetry conditions: 1.0 mM analytes in pH 4.0 citrate, 1.6 mm diameter carbon paste electrode, 150 mV/s. Hydrodynamic conditions: analytes in pH 4.0 citratdmethanol (86:14), except acetaminophen-0-sulfate and glucuronide (97:3),3 mm diameter carbon paste electrode, linear velocity 7 c m k
aminophen more difficult to oxidize than hydroquinone. This effect of acetylation of aromatic amines has been further demonstrated by CAHDV (19). Also of note is the relative ease of oxidation of the 4-isomer as contrasted with N acetyl-2-aminophenol. A more detailed discussion of the individual classifications of the compounds examined is given below: Acetaminophen a n d the Thioether Metabolites. The coulometrically determined number of electrons involved in the oxidation of acetaminophen was found to be 2.1 f 0.1. On the basis of this evidence and previous work on the chemical oxidation of acetaminophen and the two-electron electrochemical oxidation of related aminophenols (20,21),it is clear that the product is N-acetyl-p-quinoneimine. Over the pH range 0-8, acetaminophen and NAPQI are largely uncharged. The involvement of other protonated or unprotonated forms is unlikely. Koshy and Lach, in studying the acid catalyzed hydrolysis of acetaminophen, found a linear relationship of In (rate) vs. pH from neutral pH down to pH 0.1 (22). This plot would not have been linear throughout this range had the acetamido group been significantly protonated. A pK, less than zero for this group is also consistent with measurements of the pK, for the protonated form of acetanilide (23). The pK, for the phenolic proton has been determined to be 9.5 (24). Protonation of the nitrogen of NAPQI doubtless occurs well below p H 0. A comparison of the acidities of the protonated forms of several N-substituted p-quinoneimines and the corresponding primary amines (e.g.,
p-benzoquinoneimine vs. ammonia) shows that the protonated quinoneimines are much stronger acids (23). The pK, for the protonated form of acetamide is -0, thus NAPQI can be expected to have a pK below 0. Consequently, over the pH range k 8 , the interconversions of acetaminophen and NAPQI apparently involve a total of two protons and two electrons. The cyclic voltammetric peak potential for acetaminophen at a scan rate of 150 mV/s and at carbon paste was determined over a range of pHs. Representative voltammograms are shown in Figure 2. As the pH is increased above 10 the oxidation become kinetically less favorable, presumably due to the presence of the phenoxide form. Over the pH range 0-9, the peak potential is a linear function (correlation coefficient = 0.99) of pH: EP,,(V vs. Ag/AgCl) = +0.751 0.037 [pH]. For a Nernstian two-electron, two-proton process the slope would be expected to be -59 mV/pH unit. The -37 mV/pH unit slope indicates that the process is more complex. The thioether metabolites of acetaminophen are believed to result from the detoxification of NAPQI in vivo ( 4 ) . As ring-substituted analogues of acetaminophen, these compounds will exhibit electrochemical properties qualitatively similar to those seen for acetaminophen itself. However, it appears from examination of the voltammetric behavior of the two thioethers in Figure 1 that the addition of the sulfur-linked substituent in the 3-position lowers the peak potential by 140 mV. The peak potential for oxidation of 3(S)-thiomethylacetaminophen is somewhat lower than that of the cysteine adduct, due to near-ideal reversibility of the former and less reversibility of the latter. It is expected that the remainder of the unconjugated thioether metabolites are oxidized similarly. Those that have been conjugated with sulfate or glucuronide are expected to be oxidized near +950 mV, similarly shifted in contrast to the major acetaminophen metabolites, which are discussed below. A similar negative shift is observed for the methyl analogue, 3-methylacetaminophen. A reversal of this trend is seen with methyl substitution in the 2-position, which apparently increases the peak potential. The Major Metabolites of Acetaminophen. These molecules arise upon enzymatic conjugation of free acetaminophen at the 4-hydroxyl group with sulfate or glucuronic acid. The acetaminophen-related analgesic phenacetin ( p ethoxyacetanilide) is the 4-ethoxy analogue. These three molecules are more difficult to oxidize, exhibiting cyclic voltammetric peak potentials around +1.0 V, as seen in Figure 1. Their relative ease of oxidation parallels the electron donating ability of the substituted group (phenacetin > glucuronide > sulfate). Although the sulfate and the glucuronide
ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981
2261
-CH,COOH
R
Figure 4. Mechanisms of oxidation of (A) N-hydroxy-Narylacetamides
and (B) N-hydroxyacetaminophen.
il
I
OCH,CH,
i
IC
r
A
l6
Io 9pA
Flgure 3. Cyclic voltammogram of phenacetin. Conditions: 1.O mM phenacetiri in 0.1 M citrate, pH 4.0, 1.6 mm diameter carbon paste electrode, 150 m'V/s, E 11s. (Ag/AgC1/3 M NaCI). Scan initiated in positive direction at 0.0 W.
are the most difficult to oxidize of the known metabolites of acetaminophen. the potentials are sufficiently low to be analytically useful. The mechanism O P oxidation of these derivatives also involves formation of a quinoneimine. but the oxygen remains substituted and acquires a positive charge. Water (or hydroxide) attacks rapidly, liberating sulfate, glucuronide, or ethtmol and producing NAPQI. This mechanism is illustrated by Ihe cyclic voltammogram of phenacetin shown in Figure 3. Following the initial oxidation, a corresponding reduction wave IS not observed. Instead, a redox couple attributable to the reduction of NAPQI to acetaminophen and the subsequent reoxidation of acetaminophen are observed. The cyclic voltammetric behavior of acetaminophen sulfate or glucuronide is very similar. A rate constant of 2500 s-' for the decomposition of the charged intermediate of the phenacetin two-electron oxidation at pH 6.7 was found by double potential step chronoarnperometry (5). The Acetaminophen-3-(0)Metabolites. A second class of ring-substituted metabolites have been reported (25) in which the ring has been substituted with a sulfate ester or a methoxy group in the %position. Free 3.hydroxyacetaminophen has not yet been identified. Although these species were not examined in this study, their voltammetric properties at pdl 4 may be estimated from shifts previously reported for methoxy substitution of quinones, anilines, and phenols (26, 27) and those given in Figure 1 for other acetaminophen metabolites. Thus, both 3-methoxyacetaminophen and the unknown 3-hydroxyacetaminophen would be expected to oxidize near +500 mV, while the sulfate and glucuronide conjugates of 3- inethoxyacetaminophen would be expected to oxidize a t approximately +900 mV The 3-sulfoester conjugate of acetaminophen is assumed to oxidize at a slightly higher potential than acetaminophen, due to the influence of the sulfabe substituent. M-Hydroxyacetaminophen. The role of N-hydroxyacetaminophen as a proximate toxic metalbolite has been both suggested (15) and refuted (28, 29) recently, and thus the properties of this molecule are of particular interest. Gemborys et al. (30) found that N-hydroxyacetaminophen quantitatively decomposed to an equimolar mixture of 4-nitrosophenol arid acetaminophen. The oxidlation of other N hydroxy-Ai-arylamides by chemical, electirochemical,and enzymatic means have been reported (31, 33). All involve one-electron oxidation to yield a nitroxide radical which undergoes further reactions, as shown in Figure 4. In the case of the electrochemical oxidation, two one-electron waves were
I'
j2
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ict/ / B. E (+)--+
OJ
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1
0.1 I
1
1
1
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.
1 4
!" ia
Flgure 5. Cyclic voltammograms of (A) N-hydroxyacetaminophen and (B) 4-nitrosophenol. Conditions: 1.0 mM analytes in pH 4.0, 0.1 M citrate/4% 2-propanol, 1.6 mm diameter carbon paste, 150 mV/s, E vs. (Ag/AgC1/3 M NaCI). Scan initiated In positive direction at 0.0 V.
observed at a graphitic membrane electrode (32). Cyclic voltammetry on N-hydroxy-N-(4-ethylphenyl)-3-chloropropionamide in pH 4.0 citrate with 33% methanol revealed similar behavior. The initial positive scan shows two oneelectron waves. If the scan is reversed after the first wave, the coupled reduction is seen and at a more negative potential a small reduction wave which matches that for 4-ethylnitrosobenzene. This presumably is formed by dismutation of the nitroxide radical. If on the initial scan the direction is reversed after the second oxidation wave, the same reductions are observed, but the wave due to the nitroso derivative is much larger than before. The cyclic voltammogram obtained for N-hydroxyacetaminophen is considerably different, as seen in Figure 5A. No corresponding reduction is seen for the first wave and a highly reversible reduction for the second wave is seen near +800 mV. The nature of this oxidation was explained when cyclic voltammetry was performed on nitrosophenol (Figure 5B). The reduction of 4-nitrosophenol at a mercury electrode is considered as the classical "ECE' mechanism (34). Chemical oxidation of 4-nitrosophenol which yielded 4-nitrophenol has been reported (35), but electrochemical oxidation of 4nitrosophenol a t carbon apparently has not been reported. The oxidation wave at +790 mV was determined to be a one-electron wave by comparing the peak height to that of a reference compound using the same carbon paste surface. This is consistent, since a two-electron oxidation would yield
2262
ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981 260 mV/,ec
/I
Over the pH range 6.5-9, the reduction peak potential Ep,c observed for NAPQI at 150 mV/s was a linear function of p H (mV vs. Ag/AgCl) = $479 - 59[pH], with a correlation coefficient of 0.99. This range is limited on the low end because of the kinetic shift just discussed and on the high end because of the slow oxidation kinetics of acetaminophen. The slope in this region is -59 mV/pH unit, consistent with a 2e-, 2H+ process.
65 mV/,e,
ii \I
\I U
ia
Voltammograms of acetaminophen as a function of a scan rate at pH 4.0. Conditions: 1.0 mM acetaminophen in 0.1 M citrate, same 1.8 mm diameter carbon paste electrode used for both, Evs. Ag/AgCI/3 M NaCI. Scan initiated in positive direction at 0.0 V. Flgure 6.
4-nitrophenol, the reduction of which was not observed. The ability of 4-nitrosophenol to undergo electrochemical oxidation is apparently due to the hydroxy group. Nitrosobenzene, 4-ethylnitrosobenzene, and 4-ethoxynitrosobenzene showed so such oxidation. Contrasting Figure 5A and Figure 5B,it appears that because of the availability of the quinone-type structure, N-hydroxyacetaminophen undergoes a two-electron oxidation to yield an intermediate which rapidly loses acetate to leave 4-nitrosophenol (see Figure 4B). It is likely that hydroxide ion is principally responsible for attacking the charged intermediate, since in citrate buffer a t pH 6.0 the formation of nitrosophenol appears greater, while in 0.1 M HCIOI the decomposition is slow enough that a wave for reduction of the intermediate is seen. A significant amount of 4-nitrosophenol exists in each of its two tautomeric forms (36). On the reverse scan the reduction of 4-nitrosophenol to N-hydroxy-4-aminophenol is seen at -140 mV. This wave is still seen if the scan is reversed between the two oxidation waves and not seen if the scan is initiated in a negative direction. The formation of 4-nitrosophenol upon oxidation of Nhydroxyacetaminophen Cas confirmed by using the coulometric flow cell. N-Hydroxyacetaminophen (0.1 mM) in 0.1 M citrate, pH 4.0, was passed through the cell with the potential set at 0.0, +0.70, +0.75, and +0.8 V, and the nitrosophenol concentration in the cell effluent was determined with LCEC. At 0.0 V no product was seen, while at +0.70 V nearly quantitative formation of nitrosophenol was observed. The yield decreased a t the higher potentials, probably due to further oxidation of nitrosophenol. Reverse-ScanReduction of N-Acetyl-p-quinoneimine. At p H 0 reduction of NAPQI is not observed, due to rapid hydration of NAPQI (5). The reduction waves observed for NAPQI at pH 2 and 6 appear chemically reversible, but the position of the wave shifts toward a more negative potential (see Figure 2). At intermediate pH a flat, apparently “mixed” wave is observed, the shape of which is dependent upon the scan rate, as shown in Figure 6. At low scan rates the wave is less shifted than at higher scan rates. This behavior suggests that a competition exists between two forms of NAPQI. It is postulated that the protonated form of NAPQI is the species being reduced at low pH. At neutral pH the unprotonated form is reduced, but with a greater overpotential at the carbon electrode. Around pH 4 a mixed mechanism occurs. The pH a t which such transitions occur is dependent upon the proton transfer rate constant and is frequently several p H units separated from the pK for the species involved (37). This appears to be the case here, since the pK, of protonated NAPQI is less than zero.
APPENDIX Voltammetric information may be presented in graphical format, such as Figure 1 in the present paper, according to the following considerations: (1)For cyclic voltammetry (CV), one end of a rectangle is placed at the potential where the current value is equal to half that of the peak current (EPjz)and the other end a t the potential of the peak maximum (E,) for the first peak obtained during the initial forward scan. Consequently, the width of the rectangle is indicative of the relative rate of electron transfer for the process represented. If the data had been estimated or calculated, the rectangle is composed of dashed lines. For experimentally obtained data, the interior of the rectangle is indicative of subsequent processes, as follows: (a) A single solid rectangle indicates that one peak is observed during the initial scan and a second, single peak is seen during the reverse scan, which together indicate a system which is both chemically and electrochemicallyreversible, and in which the electron transfer is sufficiently fast so that both foreward and reverse processes occur on the time scale of the CV experiment. (b) A single rectangle containing diagonal lines indicates that one peak is observed during the intital scan and that one or more peaks arising from the opposite electron transfer process are seen when the scan is reversed, such that the system is electrochemically reversible but chemically irreversible. (c) A single, empty rectangle indicates that only one forward peak is observed and no other peak is obtained on the reverse scan between limiting potentials of the medium. (d) A rectangle in which an end boundary is absent indicates that a forward peak was obtained as a poorly defined shoulder on a second peak, and consequently the E p j zvalue was estimated as the potential where the current value is equal to half that a t the maximum of the shoulder peak. (e) Solid bar(s) placed adjacent to a rectangle indicate the peak potential(s) of additional peaks(s) observed during the initial forward scan but for which EPpvalues cannot be determined due to poor resolution between adjacent peaks. (2) For sigmoidal-shaped voltammetric techniques, an empty rectangle containing a single vertical line is placed such that the center line indicates the E l p potential and the ends and E3I4potentials. Here, also, the width represent the of the rectangle reflects the relative rate of the electron transfer process. (3) For peak-shaped voltammetric techniques such as differential pulse methods, an empty rectangle containing a single vertical line is also used. In this case, the center line is placed at the peak potential and the ends of the rectangle a t the potentials where the current value is half the peak current. Note Added in Proof. Hinson et al. recently reported the identification of 3-hydroxyacetaminophen as a microsomal metabolite of acetaminophen (38). ACKNOWLEDGMENT The authors wish to thank L. J. Felice, C. R. Preddy, and D. A. Meinsma for their important contributions. LITERATURE CITED (1) Miller, E. C.; Miller, J. A. I n “Chemical Carcinogens”; Searle, C. E., Ed.;American Chemical Society: Washington, DC, 1976; Chapter 16.
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