Determination of Aminophenol Isomers by High-Speed Liquid Chromatography Hiromu Sakurai and Shunjiro Ogawa, Kyoto College of Pharmacy, Higashiyama-ku, Kyoto 607, Japan

Abstract The simultaneous quantitation of aminophenol isomers (/>, m- and o-aminophenol) and aniline, which with previously reported methods was difficult and/or complicated, has been performed using a high-speed liquid chromatographic method. The resolutions of chromatograms were appropriate for separation and quantitation, when analysis times were 20 minutes. Since the method is applicable in aqueous media, a successful quantitation of p- and o-aminophenol formed by cytochrome P-450 model systems was carried out.*

Reaction Mixtures The reaction mixtures as model systems contained the following reagents; cysteine 10"1 M, ferrous sulfate heptahydrate 10"2 M or 10"3 M, aniline 10"1 M, and sodium hydroxide. The reaction mixtures were adjusted to 10 ml with acetone (80%) and the pH were adjusted to 6. With vigorous shaking in the atmosphere of air, the reactions were carried out at 40° and stopped at various times by addition of 0.5 ml of 2 N hydrochloric acid. Separation of Aminophenols from Reaction Mixtures

Introduction Aminophenols are known as main metabolites of aniline both in vivo and in vitro (1-5). In these reports, aminophenols have been determined by colorimetric, spectrometric, or isotopic methods. However these methods are difficult for determining aminophenol isomers in a sample simultaneously. Recently it was reported that the rapid separation of phenol derivatives including aminophenols was investigated by use of the high-speed liquid chromatograph equipped with a new detector, which is devised to apply the electrolytic flow cell (6). The present paper describes simultaneous quantitation of aminophenol isomers by use of a commercially available high-speed liquid chormatograph. This method was applied in the quantitations of p- and o-aminophenol formed by model systems of cytochrome P-450 (7).

Aminophenols formed by the model systems were extracted from reaction mixtures by the modification of Brodie's method (8). After the evaporation of acetone in the reaction mixture at 40° under reduced pressure, the remaining aqueous solution was saturated with sodium chloride, the pH of the solution was adjusted to 7, and was extracted with ether (25 ml). The ether layer (20 ml) was extracted with 0.1 N hydrochloric acid (3 ml). The aqueous layer (2 ml), extracted with equal volume of chloroform to remove the excess aniline and remaining ether, was used for the analyses. Results and Discussion A chromatographic separation of aminophenol isomers p-, m-, and o-aminophenol, and aniline is shown in Figure 1. The

Experimental High-Speed Liquid Chromatography A Du Pont Model 840 liquid chromatograph equipped with an ultraviolet detector (254 nm) was employed. The column was a Du Pont analytical column packed with Zipax SCX (1 m x 2.1 mm i.d.). The separation of aminophenol isomers was achieved with 0.1 M H3PO4 - 0.1 M KH2PO4 (pH 2.9) as the mobile phase at 25°, and the flow rate of about 0.8 ml/min. Samples of 10 fA were injected directly into the column through the septum. Chemicals All reagents used were of guaranteed grade. Aminophenol isomers, p-, m-, and o-aminophenol, for standards were obtained commercially and purified by sublimation before use. Aniline was redistilled in a N2 stream under reduced pressure. Acetone used for reaction, ether and chloroform for extraction were redistilled. JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 14

Figure 1. Chromatogram for the separation of p-, m-, o-aminophenol and aniline. The separation conditions are given in the experimental section. Peak identity; 1. solvent front, 2. and 3. unknown, 4. p-aminophenol, 5. m-aminophenol, 6. o-aminophenol, 7. aniline. OCTOBER 1976* 499

time required for separation was about 20 minutes. As the resolution was appropriate for the separation of aminophenol isomers in this chromatogram, the separation conditions used in Figure 1 were applied for the quantitation of aminophenols. Figure 2 shows the typical chromatogram obtained from the reaction mixture. It was confirmed that aniline was hydroxylated by model systems, the products were mainly p- and o-aminophenol, and m-aminophenol was detected in no more than trace amounts. The result corresponded to that of twodimensional TLC (plate; cellulosersilicagel G = 5:2, 1st developing solvent; benzene:methanol:acetic acid = 135: 24:12, 2nd developing solvent; chloroform:isopropanol: ammonium hydroxide = 16:3:1, detection; iodine vapor).

0.02

o.oi

o300

100

1*00

Figure 3. Calibration plots of absorbance vs. p- and o-aminophenol concentration on extracted standards. — O —: p-aminophenol, --A--: o-aminophenol.

As an example of quantitation, Table II shows the results of the hydroxylation of aniline by the ferrous ion-cysteine systems. The described method is suitable for the simultaneous quantitations of aminophenol isomers in a sample, which are difficult and/or complicated with previously reported methods. Further, this method may be applied for the detection of aminophenols in biological materials such as liver microsomal fractions, since aqueous samples can be analyzed. Table II. Yields of p- and o-Aminophenol formed by Ferrous lon-Cysteine Model Systems

20

Figure 2. Typical chromatogram of extracted reaction mixture. The reaction mixture contained 10~1 M cysteine, 10~2 M ferrous ion and 10"' M aniline, and was incubated for 60 minutes at pH 6 and 40° in the atmosphere of air. The chromatogram was obtained with injection size of 10 ju. I and sensitivity of 2 x 10~2 absorbance unit full scale. Peak numbers in the chromatogram correspond with those in Figure 1.

To the quantitation of p- and o-aminophenol, a known amount of the standard sample dissolved in phosphate buffer (pH 7) was determined with the same process described in the experimental section. The relation between concentrations and peak heights of p- and o-aminophenol gave a straight line within the investigated concentrations as shown in Figure 3. Both aminophenols were recovered by more than 95% (Table I). Table I. Recoveries of p- and o-Aminophenol Aminophenol p-Aminophenol

o-Aminophenol

Present

Found

Recoveries

(M9)

(M9)

(%)

50 98 195 293 49 98 195 290

100.0 98.0 97.5 97.6 98.0 98.0 97.5 96.6

50 100 200 300 50 100 200 300

500 •OCTOBER 1976

Incubation Time p-Aminophenol

(minutes)

60 120 240

o-Amlnophenol

p-Aminophenol +

if-9)

o-Aminophenol

(^g)

167 ±-25 397 ± 12 749 ± 19

81 241 452

+ + +

8 9 21

248 ± 26 638 ±20 1201 ±27

The reaction mixtures contained 10 -1 M cysteine 10^3 M ferrous ion and 10~1 M aniline. Reactions were carried out at pH 6 and 40° in the atmosphere of air. Each data is the mean + S.O. of four experiments.

Acknowledgment We would like to acknowledge the continual guidance and encouragement of Dr. S. Hirose. We are grateful to Dr. K. Fujitani for helpful suggestions and a critical reading of the manuscript. Manuscript received September 23,1975.

Mean

98.3

97.5

References 1. J.N. Smith and R.T. Williams, Biochem. J., 44, 242 (1949). 2. T. Sato, T. Suzuki, T. Fukuyama, and H. Yoshikawa, Seitai no Kagaku, 6, 225 (1955). (Chem. Abstr., 52, 20534). 3. C. Mitoma, H.S. Posner, H.C. Reitz, and S. Undenfriend, Arch. Biochem. Biophys., 61, 431 (1956). 4. J. Booth and E. Boyland, Biochem. J., 66, 73 (1957). 5. D.V. Parke, Biochem. J., 77, 493 (1960). 6. Y. Takata and G. Muto, Anal. Chem., 45, 1864 (1973). 7. H. Sakurai and S. Ogawa, Biochem. Pharmacol., 24, 1257 (1975). 8. B.B. Brodie, J. Axelrod, P.A. Shore, and S. Undenfriend, J. Biol. Chem., 208, 741 (1954).

JOURNAL OF CHROMATOGRAPHIC SCIENCE • VOL. 14

Determination of aminophenol isomers by high-speed liquid chromatography.

Determination of Aminophenol Isomers by High-Speed Liquid Chromatography Hiromu Sakurai and Shunjiro Ogawa, Kyoto College of Pharmacy, Higashiyama-ku,...
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