Determination of Benzidine and Its Acetylated. Metabolites in Urine by Liquid Chromatography. John R. Rice and Peter T. Kissinger, Bioanalytical Laboratory, ...
Determination of Benzidine and Its Acetylated Metabolites in Urine by Liquid Chromatography J o h n R. Rice and P e t e r T.
Bioanalytical Laboratory, Department of Chemistry, Purdue
University, West Lafayette, IN 47907
Abstract Methods for the analysis of benzldlne (4,4'-diamlnoblphenyl), or for benzldlne and its acotylated metabolltes, in urine are described. The procedure requires 2.0 ml of urine and involves extraction of the compound(s) followed by quantltetion via liquid chromatography with eloctrochemlcal detection (LCEC). The assay for benzidlne Is linear from 25 ng/ml to 5 /~g/ml, (r = 0.090), with a proclslon of • 0.06% RSD at 250 nglml (n = 6) and • at 25 ng/ml (n = 5).
As part of an ongoing investigation of the oxidative metabolism of toxic aromatic molecules, it was necessary to develop a sensitive assay for benzidine and its acetylated metabolites in complex sample matrices. Reverse-phase liquid chromatography with amperometric detection (LCEC) has proven to be extremely valuable in the determination of trace levels of a host of electroactive compounds (1). Since benzidine and related molecules undergo a facile two-electron oxidation at carbon electrodes, LCEC is a method of choice for its trace determination in very complex mixtures such as urine. The new method is applicable to both metabolism studies and as a means of monitoring the extent of exposure of persons who work with chemical systems in which benzidine might arise as an undesired by-product. Furthermore, a simple modification of this method has recently been applied to the sub-parts-permillion determination of benzidine levels in soil samples taken from an industrial chemical dumping area (2). It is likely that further modifications of the basic procedure outlined here would permit analysis of other biological samples, industrial wastes, azo dye mixtures, or other complex mixtures which may contain benzidine or its congeners at the trace level. Benzidine has long been recognized as a potent bladder carcinogen. Although the intentional industrial production of benzidine per se has now ceased in the United States, and the compound has been placed on OSHA's list of 14 "Regulated Chemical Carcinogens" (3), it was used for many years as the starting material in the manufacture of azo dyes, in the standard clinical test for occult blood, and as a chromatographic spray reagent. Sciarani and Meigs (4) studied the metabolism of benzidine and found that the bulk of an administered dose is excreted as the 3-hydroxy derivative, which may be further conjugated as the sulfate or glucuronide. A small amount (3 to 5%) is excreted unchanged, and the re-
mainder appears as the mono- or diacetylated derivative. In addition, benzidine and monoacetylbenzidine have been found as metabolic products after the feeding of azo dyes to monkeys (5). The majority of previously reported methods for the determination of these compounds involve reaction of the amine with a coupling agent to yield a colored product, which can be determined spectrophotometrically. Chloramine T was particularly popular (4). Aside from being relatively insensitive, these methods lack specificity, since all amine-containing molecules present in solution yield colored products. Other methods available include paper (6) and thin-layer chromatography (7-9), gas chromatography (8,9) and anodic pulsed voltammetry (10). A recent short communication reported the analysis of several aromatic amines, including benzidine, in wastewater by cation-exchange LCEC (11). Experimental
Materials. Benzidine, melting point = 127-129~ (Scientific Products, Allen Park, MI) was used as received. Monoacetylbenzidine, melting point = 199-200~ [literature value = 199~ (12)] was prepared according to the method of Laham (6). Diacetylbenzidine, melting point = 328~ [ l i t e r a t u r e value = 328.30~ (12) or 330-331~ (6)] was obtained as the sole product via the procedure given by Cain (13). Reagent grade chemicals were used in the preparation of all solutions, as was deionized, distilled water. Technical grade methanol was distilled prior to use as an LC mobile phase component. Equipment. Cyclic voltammograms were obtained at a carbon paste electrode versus a Ag/AgCI reference electrode (Model RE-l, Bioanalytical Systems, West Lafayette, IN) using a Bioanalytical Systems Model CV-1A unit and plotted on a Hewlett-Packard (San Diego, CA) Model 7015A X-Y recorder. A Bioanalytical Systems LC-50 Chromatograph was used for final determinations employing a 15-cm stainless steel column slurry-packed with /~-Bondapak C18 material (Waters Associates, Milford, MA), a 20 /~l-sample loop, a carbon paste detector cell (Model TL-3) and a A g / A g C l reference electrode. Isocratic elutions were made using methanol:water (25:75, 0.1 M in ammonium acetate, pH 6.2) mobile phase at ambient temperature with a flow rate of 1.0 ml/minute. Procedure. Urine samples containing benzidine are stable for several hours at room temperature and are stable for at least three weeks when frozen at -35~ Urine (2 ml) was
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delivered to a 12-ml glass centrifuge tube, and 1.0 ml of 2.5 M NH3/NH4CI buffer (pH 9.0) and 1.0 ml of diethyl ether was added. After briefly mixing using a conventional vortex, the tubes were centrifuged to separate the layers. The ether layer was transferred to a second centrifuge tube, and the extraction was repeated with a second 1.0 ml of ether. A 1.0-ml portion of 0.5 M HCIO4 was added to the combined ether layers. The tubes were mixed and centrifuged and 20/~! of the aqueous layer was injected onto the LC column. Quantitation was accomplished by measurement of the peak height and comparison with a linear calibration plot obtained from spiked urine samples.
Results A survey of the electrochemical behavior of benzidine (BD), monoacetylbenzidine (MAB), and diacetylbenzidine (DAB) conducted by cyclic voltammetry showed that benzidine had a significantly lower oxidation potential than its acetylated derivatives. This is clearly illustrated in Figure 1, which is a plot of the peak current response of the three compounds injected into the liquid chromatograph as a function of the potential set on the detector electrode. As this potential surpasses that of the oxidation potential of the compound, the corresponding chromatographic peak height becomes constant for all subsequent potential increases. This value is set equal to unity, and the relative peak heights @) obtained at lower potentials are plotted versus the applied potential. The resulting hydrodynamic voltammograms are very useful in selecting the detector potential to optimize both sensitivity and selectivity. For example, the distinctly higher oxidation potentials of MAB and DAB enable one to selectively "tune out" the MAB and DAB chromatographic peaks by setting the potential of the detector cell to a value adequate for the oxidation of the eluted BD (for example, 500 mV) but insufficient for oxidation MAB and DAB. If desired, the detector can be operated at a higher potential to detect all three compounds. Analysis for benzidine. A detector potential of 450 mV versus Ag/AgCI is adequate for the oxidation of BD alone. Figure 2 shows a chromatogram of an extract from a urine sample containing $0 ng/ml of BD. Determination of BD in this mode was found to give a linear calibration curve over the range 25 ng/ml to 5/zg/ml, with a correlation coefficient of 0.999. A detection limit of 10 pg of BD injected was deter-
POT EN T I A L , vO~.Ts
Figure 2. Example chromatogram of the extract from a 2.0 ml urine sample spiked with 50 nglml of benzidine (BD). Analysis performed with a 15 cm x 4.6 mm column packed with Waters /~-Bondapak C18 material; mobile phase consisted of 25:75 methanol:0.1 M ammonium acetate, pH 6.2, electrochemical detector operated at 450 mV vs. Ag/AgCI.
mined at a signal to noise ratio of 2. At a BD concentration of 250 ng/ml, the precision of the method was :t: 0.69% RSD (n = 6), and at a concentration of 25 ng/ml, the precision was 4.9% RSD (n : 5). The absolute recovery of benzidine (total amount recovered versus the theoretical maximum yield) using this extraction procedure is ~71%, and the relative recovery of BD from urine is ~103% versus a matrix-free soiution. Recovery studies were carried out at 500 ng/ml.
Analysis for benzidine, monoacetylbenzidine, and diacetylbenzidine. In order to detect all three compounds after extraction from urine, a detector potential of 1.0 V versus Ag/AgCl may be used. Figure 3 illustrates a chromatogram of an extract from a urine sample containing 500 ng/ml of each compound. This higher detector potential broadens the void volume response and therefore raises the detection limit of BD over that attained at 450 mV. Further, the relatively large k ' value for DAB and its high oxidation potential limits the detection of this compound. These conditions give a linear calibration curve from 0.5 ~g/ml - 5.0 ~g/ml, with a combined precision for the three compounds of 3.97% RSD (n = 6).
. . . . . . . .o o o
Figure 1. Hydrodynamic voltammogram of benzidine (BD), monoacetylbenzidine (MAB) and diacetylbenzidine (DAB) showing the relative ease of oxidation of the three compounds. ~ represents the relative chromatographic peak height obtained at various detector potentials upon injection of a constant amount of each compound. J O U R N A L OF A N A L Y T I C A L T O X I C O L O G Y * V O L . 3
Discussion This assay was developed in part to fill the obvious need for a modern chromatographic method for trace determination of benzidine and structurally related molecules, many of which are known or suspected of being highly toxic. Haley (14) has reviewed the occurrence, chemistry, and pharmacology of benzidine and its congeners and has called for a thorough and statistically sound study of the quantitative biotransformation of such compounds at subacute levels, as well as studies of M A R C H / A P R I L 1979e65
This work was s u p p o r t e d by t h e National I n s t i t u t e of General Medical Sciences a n d t h e National Science Foundation. The a u t h o r s wish to t h a n k D.J. M i n e r a n d R.E. S h o u p for helpful discussions, a n d P.C. Conrad a n d R.W. Pariza for technical advice a n d assistance.
M a n u s c r i p t received N o v e m b e r 15, 1978
O.S n A
Figure 3. Example chromatogram of the extract from a 2.0 ml urine sample spiked with 500 ng/ml each of benzidine (BD), monoacetylbenzidine (MAB), and diacetylbenzidine (DAB). Chromatographic conditions used were identical to those of Figure 2, but with a detector potential of 1 .O V vs. Ag/AgCI.
epidemiologicai a n d p u l m o n a r y e x p o s u r e of industrial workers, a m o n g o t h e r n e c e s s a r y l o n g - r a n g e studies. The p r e s e n t m e t h o d is well suited for much of this work. The specificity of t h e m e t h o d arises from a simple extraction, r e v e r s e - p h a s e liquid c h r o m a t o g r a p h y , a n d electrochemical detection. F u r t h e r work is currently in p r o g r e s s to e x t e n d this m e t h o d to t h e detection of o t h e r m e t a b o l i t e s of b e n z i d i n e which m a y arise by m i n o r metabolic pathways.
6 6 e M A R C H I A P R I L 1979
1. P.T. Kissinger. Amperometric and coulometric detectors for high-performance liquid chromatography. Anal. Chem. 49: 447A456A (1977). 2. J.R. Rice. Unpublished results. Details of this method are available upon request. 3. Fed. Regist. 39:3756 (1974). 4. L.J. Sciarini and J.W. Meigs. The biotransformation of benzidine. II. studies in mouse and man, A.M.A. Arch. Environ. Health 55: 423-428 (1961). 5. E. Rinde and W. Troll. Metabolic reduction of benzidine azo dyes to benzidine in the rhesus monkey. J. Nat. Cancer Inst. 55: 181182 (1975). 6. S. Laham, J.P. Farant, and M. Potvin. Biochemical determination of urinary bladder carcinogens in human urine. Occup. Health Rev. 21:14-23 (1970). 7. E. Rinde and W. Troll. Colorimetric assay for aromatic amines. Anal. Chem. 48:542-544 (1976). 8. R.L. Jenkins and R.B. Baird. The determination of benzidine in wastewaters. Bull. Environ. Contain. Toxicol. 13:436-442 (1975). 9. J. Schulze, C. Ganz, and D. Parkes. Determination of trace quantities of aromatic amines in dyestuffs. Anal. Chem. 50: 171-174 (1978). 10. W.M. Chey, R.N. Adams, and M.S. Yllo. Anodic differential pulse voltammetry of aromatic amines and phenols at trace levels. J. Electroanal. Chem. 75:731-738 (1977). 11. I. Mefford, R.W. Keller, R.N. Adams, et al. Liquid chromatographic determination of picomole quantities of aromatic amine carcinogens. Anal. Chem. 49:683 (1977). 12. R.C. Weast, Ed. Handbook of Chemistry and Physics, 58th Ed., CRC Press, Cleveland, OH, 1977, p. C-204. 13. J.C. Cain. Nitrosoacetylamino-derivatives of the benzene and diphenyl series. J. Chem. Soc. 95:714-730 (1909). 14. T.J. Haley. Benzidine revisited: A review of the literature and problems associated with the use of benzidine and its congeners. Clin. Toxicol. 8:13-42 (1975).
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