salicylic acid was done by a fluorimetric procedure (8). Under optimized conditions, a chromatogram was resolved in about 10 minutes. At a higher flow rate, the time for an analysis was reduced to about 8 minutes with no loss in resolution. The number of theoretical plates for the column was 1440 (equivalent to a plate height of 0.2 mm), calculated for the most retained peak, p-chloroacetanilide, with a k' of 3.21. The average time required for one complete analysis by this method was about one hour per sample compared to four hours by partition chromatography. In addition, this system has proved extremely reliable. To date, the same column has been in operation for over three months with
(8) G. H. Schenk. F. H. Boyer, C. I. Miles, and D. R. Wirz, Anal. Chem., 44, 1593 (1972).
greater than 1000 injections, and no loss in resolution has been observed.
CONCLUSIONS High-speed, reverse-phase liquid chromatography is an effective dependable, and accurate method for the separation of analgesics. Also, by utilizing an automatic sample injection system and computer interfacing, this method should allow the complete automation of the analysis of these products.
ACKNOWLEDGMENT The author thanks D. Cornish for his help in the preparation of this paper
RECEIVEDfor review April 10, 1974. Accepted August 2, 1974.
Electrochemical Determination of Arylhydroxylamines in Aqueous Solutions and Liver Microsomal Suspensions Larry A. Sternson The Bioanalytical Laboratory, Department of Medicinal Chemistry, School of Pharmacy, The University of Georgia, Athens, Ga. 30602
Certain chemical classes of drugs are converted in the body to highly toxic metabolites. Arylhydroxylamines, isolated as metabolites during enzymatic reduction of nitroaromatic compounds and bio-oxidation of primary arylamines ( I ) , are thought to initiate many toxic responses (2) including carcinogenesis (Y), metagenesis, methemoglobinemia, and hemolytic anemia. Low steady state concentrations of these intermediates and their ease of oxidation has precluded a facile procedure for their detection and quantitation. Several methods for the determination of arylhydroxylamines a t microgram levels have been described in the literature and have recently been reviewed ( 4 ) . These include spectrophotometric ( 4 , 5 ) , fluorometric (6) and gas chromatographic (7, 8) procedures. All of these methods suffer from the common disadvantage that the integrity of the hydroxylamine may be destroyed during the assay. This often prevents the quantitation of hydroxylamine in the presence of large excesses of aromatic nitro compounds and/or amines-compounds that serve as chemical and biochemical precursors to the hydroxylamines. T o detect hydroxylamines in biological fluids, the hydroxylamine must be separated from proteinaceous material by extraction and subjected to one of several rather nonspecific assays. In most cases, this has involved chemical conversion of the hydroxylamine to an amine derivative, making differentiation between hydroxylamine metabolite and amine precursors impossible. In addition, available assays are relatively time consuming. Since arylhydroxylamines are very susceptible to air oxidatlion yielding N - nitroso (1) C. C.Irving, J. Bo/. Chem., 239, 1589 (1964). (2) J. A. Miller, J. W. Cramer, and E. C. Miller, Cancer Res., 20, 950 (1960). (3) J. R. Gillette, J. R. Mitchell, and B. B. Brodie, Ann. Rev. Pharmacol., 14, 271 (1974). (4) R. E. Gammans, J. T. Stewart, and L. A. Sternson, Anal Chem., 46, 620 (1974). (5) E. Boyland and R. Nery, Analyst (London), 89, 95 (1964). (6) L. A. Sternson and J. T. Stewart, Anal. Lett., 6 , 1055 (1973). (7) H. B. Hucker, Drug Metabolism andDisposition, 1, 322 (1973). (8) A. H. Becketl and S. AI-Sarraj, J. Pharm. Pharmacol., 24, 916 (1972).
2228
compounds which are not detected by existing procedures, the accuracy of existing methods may be suspect. We wish to report the development of a rapid chronoamperometric assay for the determination of sub-microgram quantities of arylhydroxylamines directly in aqueous solutions and liver microsomal suspensions. The procedure is based on the anodic oxidation of N- substituted hydroxylamines a t a stationary carbon paste electrode. Although some studies were also conducted in human plasma, results reported herein deal exclusively with sub-fractions of liver suspensions which contain enzymes responsible for generation of transient hydroxylamine metabolites. A chronoamperometric method was chosen because it can be easily automated to accomodate multiple-sample determinations as described by Adams (9) for analysis of serum uric acid. Voltammetric behavior of phenylhydroxylamine a t dropping mercury electrodes (reduction) ( I O ) and a t the graphite electrode (reduction and oxidation) ( 1I ) have been described.
EXPERIMENTAL Instrumentation. Electrochemical measurements were made with a standard potentiostat ( 1 2 )and electrode configuration ( 1 3 ) that have been previously described. Recordings were made with an Omnigraph Model 2000 X-Y recorder. Potential scales were calibrated with a Biddle student potentiometer. All electrochemical measurements were made in all glass cells at stationary carbon paste (graphite-nujol) electrodes (geometric surface area: 0.64 cm') relative to a saturated calomel electrode with a graphite rod as auxiliary electrode. New carbon paste surfaces were prepared for each determination. Reagents. Arylhydroxylamines were synthesized by reduction (9) G. Park, R. N. Adams, and W. R. White, Anal. Lett., 5,887 (1972). (10) M. Heyrovsky and S. Vavricka, J. €lectroana/. Chem., 43, 311 (1973) and references therein. (11) L. Chuang, I. Fried. and P. J. Elving, Anal. Chem., 36, 2426 (1964). (12) B. A. Feinberg, Ph.D. Thesis, Dept. of Chemistry, University of Kansas, Lawrence, Kansas, 1971. (13) R. N. Adams, "Electrochemistry at Solid Electrodes," Marcel Dekker, New York, N.Y., 1969, p 267.
A N A L Y T I C A L C H E M I S T R Y , V O L . 46, NO. 1 4 , DECEMBER 1974
Table I. Cyclic Voltammetrya Data for Substituted Hydroxylamine Oxidation at Carbon Paste Electrodes Compound
*
P henylhydroxylamine p-Tolylhydroxylamine p-Chlorophenylhydroxylamine a-Naphthylhydroxylamine S-Methylhydroxylamine
Medium
Ep,*'
Buff ere Microsomesf Buffer Microsomes Buffer Microsomes Buffer Microsomes Buffer
+0.11 10.24 -0.05 -0.03 -0.07 -0.03 -0.11 -0.11 +0.62
%ppc*d +0.24 +O. 44 10.15 +O. 34 +0.16 +O. 25
0.00 10.12 +0.81
a Scan rate: 4 V-min 1 at unshielded carbon paste electrode. Final concentration of' all hydroxylamine solutions was 4 X lO-5M V u5 SCE for anodic sweep. d Potential applied in subsequent chronoamperometric determinations. e 0.1M phosphate buffer (pH 7.4). Microsomal suspensions were prepared as described under Methods. Protein concentration of suspensions used in electrochemical measurements was adjusted to 1.5 mg/ml with 0.1M phosphate buffer (pH 7.4).
Table 11. Calibration of Data for Various N-Substituted Hydroxylamines Standard c u ~ y e ~ ~ ~
Compound
Phenylhydroxylamine p-Tolylhydroxylaminc p C hlorophenylhydroxylamine a-Naphthylhydroxylamine
N-Methylhydroxylamine
Medium
Slope
Intercept
Buffer' Microsomesd Buffer Microsomes Buffer Microsomes Buffer Microsomes Buffer
1.801 1.535 1.638 1.319 1.437 1.125 1.043 0.981 1.026
-0.072 -0.091 -0.003 -0.016 0.099 0.008 0.066
0.011 0.082
Graphs obtained by plotting current us. hydroxylamine concentration gave linear relationship for standard curves. Currents were measured after a 9-sec electrolysis a t E,,,,. Such currents were determined for solutions of substituted hydroxylamines present in final concentrations of 1 x 10-4, 4 x 10-5, 2 x 10-5, 1 X 10-5,5 x 10-6, and 2.5 x 10-6M. CO.1M phosphate buffer (pH 7.4). d Microsomal suspensions (1.5 mg protein/ml) were prepared as described under Methods.
ai3 -
--.&
of
-02
-03
04
VOLTS
E
vs
SCE
a
of the corresponding nitro compounds with zinc and ammonium chloride ( 2 4 ) . Physical constants of products were in agreement with literature values (25, 16). N- Methylhydroxylamine was purchased from the Aldrich Chemical Company. Compounds used as potentially interfering substances were secured from commercial sources and purified by either recrystallization or distillation before use. All solutions were prepared with triply distilled water and thoroughly deaerated before use by passing argon through and over the solutions. Microsomal Suspensions. Sprague-Dawley rats (100-150 grams) were sacrificed by decapitation and their livers immediately excised. Livers were subsequently homogenized with four volume of cold 0.1M phosphate buffer (pH 7.4) and centrifuged a t 9,000 X g to sediment undesired cellular components. Microsomal fractions were obtained as previously described ( I 7). Protein levels were determined by the method of Lowry ( 1 8 ) using bovine serum albumin as protein standard and protein content of microsomal suspensions was adjusted to 1.5 mg/ml with 0.1M phosphate buff(14) E. E . Smissman and M. D. Corbett, J. Org. Chem., 37, 1847 (1972). (15) A . Lapworth and L. Pearson, J. Chem. Soc.. London, 119, 765 (1921). (16) R. D. Haworth and A. Lapworth. J. Chem. Soc., London, 119, 768 (1921). (17) R . A. Wiley. L. A. Sternson. H. A. Sasame, and J. R. Gillette, Biochem. Pharmacol., 21, 3235 (1972). (18) 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R . J. Randall, J. Bid. Chem., 193, 265 (1951).
Figure 1. Cyclic voltammograms for 4 X lOP5Mp-tolylhydroxylamine in ( A ) 0.1M phosphate buffer (pH 7.4) and ( B ) hepatic microsomal (protein concentration: 1.5 mg/ml) suspensions at a carbon paste electrode. Scan rate, 4 V/min. Two traces are shown to show reproducibility in successive scans
er (pH 7.4) for subsequent electrochemical studies. Analytical Procedure. The 4 X lOW5Msolutions of each of the investigated hydroxylamines were prepared in both 0.1M phosphate buffer and microsomal suspensions. Cyclic voltammograms were recorded (sweep rate: 4 V/min) for all solutions at 2 5 O , and the potential (Eapp)for subsequent chronoamperometric (potentiostatic) measurements determined. Stock solutions of the various hydroxylamines (1 X 10-3M) were prepared in deaerated phosphate buffer. Standards for the electrochemical chronoamperometric determinations were prepared by diluting 1000, 400, 200, 100, 50 and 25 fil of the stock hydroxylamine solutions to 10 ml with 0.1M (pH 7.4) phosphate buffer or microsomal suspension. These solutions corresponded to final concentrations of 1 X 10-4, 4 x 2 X 1X 5 X and 2.5 X 10WM hydroxyl amine. Solutions must be prepared immediately before use because of the proclivity of the substrate to undergo oxidation upon standing. These solutions were placed in electrochemical cells and current was determined for each solution after a 9-sec controlled electrolysis at E,,, from recorded current-time curves. Seven replicate determinations were made for each solution. Standard curves were generated for all studied substituted hydroxylamines in buffer and microsomal suspensions by plotting current (after 9sec electrolysis) levels us. hydroxylamine concentration.
RESULTS AND DISCUSSION Arylhydroxylamines are very easily oxidized a t carbon paste electrodes (Table I) a n d y i e l d well-defined cyclic vol-
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, NO. 14, DECEMBER 1974
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Table 111. Analysis of Known Arylhydroxylamine Mixtures for Arylhydroxylamine in Microsomal Suspensionsa Arylhydruxylam ine c oncn In mlxmes,
Mixture
Components
1
p -Tolylhydroxylaminec
$1
Foundb .+I ~ 1 0 - 5
Recovery, $4.
1 x 10-5 0.94 i 0.01 94 i Id 1 x 10" Nitrobenzene 2 1 x 10-5 p -Tolylhydroxylamine 1.00 f 0.02 100 i 2 Acetanilide 5 x 10-2 1 x 10-5 p -To ly lhy d roxy la m ine 0.96 i 0.01 96 i 1 1 x 10-2 12-Butylamine 1 x lo-; p -Tolylhydroxylamine 1.03 & 0.02 103 i 2 Acetamide 1 x lo+ 1 x 10-5 p-Tolylhydroxylamine 1.01 * 0.03 101 i 3 Aniline 2 x lo-'? 6 p-Tolylhydroxylamine 1 x lo-5 1.03 i 0.04 103 * 4 Nitrobenzene 5 x 10-2 Aniline 1 x lo+ 7 p-Tolylhydroxylamine 1x 1.01 f 0.02 101 f 2 NADPHe 2 x 10-4 a All mixtures were prepared in microsomal suspensions, made up in 0.1M phosphate (pH 7.4) buffer. Protein concentration was adjusted to 1.5 mg/ml. b Based on 5 replicate determinations of each solution. E,,,,, = 0.30 V us. SCE. Confidence limits at p = 0.05. e NADPH (Sigma);or formed enzymatically by reduction of NADPH with glucose-6-phosphate dehydrogenase ( 1 4 ) .
tammograms in phosphate buffer. A typical curve is illustrated in Figure l a and indicates a quasi-reversible redox couple. Electrochemical oxidation of these hydroxylamines in microsomal suspensions occurs a t slightly more anodic potentials and oxidation becomes more irreversible in protein solution (Figure l b ). Aliphatic hydroxylamines are oxidized at considerably more anodic potentials than aromatic analogs precluding their analysis in proteinaceous solutions. Potentials approximately 100-200 mV more positive than were chosen as applied potentials (Eapp)for subsequent chronoamperometric determinations (Table I). The instantaneous current ( i t ) at a planar electrode under semi-infinite linear diffusion conditions is given by Equation 1 and indicates that the current is directly proportional to the concentration (C b, of electroactive species in the bulk of the solution, where the remaining symbols have their ususal significance and subscript '(0''indicates oxidant species.
Current-time curves were recorded for aqueous buffer and microsomal solutions of hydroxylamine a t concentrations ranging from 1 X 10-4M to 2.5 X 10-6M. Current levels were determined after a 9-second controlled (potentiostatic) electrolysis at E app and plotted us. hydroxylamine concentration. Selection of electrolysis time was not critical, so long as it was chosen to be less than 11seconds. Currents from 0.6 to 35 HA were observed for solutions over this range of hydroxylamine concentrations. Currentconcentration calibration plots for the tested arylhydroxylamines were linear and are shown in Table 11. These graphs served as standard curves for analysis of unknown concentrations of hydroxylamines in the range from 1 X lop4to 2.5 X 10-6M with an accuracy of 3-5%. The accuracy of the procedure is quite high especially in light of the fact that the system is operating far from linear diffusion control. A t the potentials utilized for chronoamperometric measurements, essentially no residual currents were detected in phosphate buffer while in microsomal suspension small background currents (0.2-0.3 PA) were observed. 2230
Considerable difficulty was experienced in attempting to monitor plasma levels of arylhydroxylamines. High concentrations of uric acid in serum (9) limit the practical anodic range which can be scanned. In addition, high levels of soluble plasma proteins have hampered the reproducibility of measurements at carbon paste electrodes. Plasma proteins are emulsifying agents and may destroy the integrity of the graphite-nujol electrode surface resulting in decreased precision of measurements. Because arylhydroxylamines are normally present in biological systems at steady state levels far below the concentrations of their metabolic precursors, a useful and practical assay must allow for the analysis of hydroxylamine in the presence of large excesses of substrates known to generate N - hydroxylated intermediates. As shown in Table 111, arylhydroxylamines (1 X 10-5M) can be quantitated in the presence of a 10,000-fold excess of aromatic nitro compound and 5,000-fold excess of arylamine. In addition, the procedure allows the determination of arylhydroxylamines in the presence of relatively high concentrations of aromatic and aliphatic amides and amines. Enzymatic formation of arylhydroxylamines by either reduction of nitro compounds or oxidation of primary amines requires NADPH as cofactor. As shown in Table 111, no interference with arylhydroxylamine determinations was observed in the presence of 2 X 10-4M NADPH. The method is as accurate and precise as previously reported methods. However, it has the advantage over existing methods that no reagents or sample preparation are required. In addition, arylhydroxylamines can be determined rapidly and directly in biological fluids in concentrations ranging from 1 X 10-4M to 2.5 X 10-6M in the presence of high concentrations of their chemical and biochemical precursors. ACKNOWLEDGMENT The author gratefully acknowledges the technical assistance of S. J. Bannister.
RECEIVED for review May 6, 1974. Accepted August 5 , 1974. This investigation was supported in part by NIH grant CA-14158-02 from the National Cancer Institute.
A N A L Y T I C A L C H E M I S T R Y , VOL. 46, N O . 14, DECEMBER 1974
