High-Performance Liquid Chromatographic Determination of Urinary Metabolites of 2, 4-Dinitrotoluene in Wistar Rats

Abstract

Urinary metabolites of 2, 4-dinitrotoluene (2, 4-DNT) were quantitated by high-performance liquid chromatography (HPLC) after administration to male Wistar rats. The urine was extracted with ether after adjusting the pH to 11 and 2. The ether extracts were evaporated to dryness, and each residue was dissolved in aqueous methanol and subjected to HPLC analysis. A 54 μl portion of aqueous methanol solution was analyzed by HPLC on a reversed-phase column. The mobile phases were 40% methanol, 40% methanol in 0.0018 M tetra-n-butylammonium chloride (TBACl), 20% methanol in 0.0018 M TBACl, pH 7.4 phosphate buffer and 20% methanol in 0.0027 M sodium 1-hexanesulfonate (HSANa), pH 3.7 acetate buffer, at a flow rate of 0.6 ml/min. 2, 4-Dinitrobenzoic acid (2, 4-DNBA) was excreted most abundantly, followed by 2, 4-dinitrobenzyl alcohol glucuronide (2, 4-DNBG), 2-amino-4-acetylaminobenzoic acid (2A4AABA), 2, 4-dinitrobenzyl alcohol (2, 4-DNB), 4-amino-2-nitro (2-amino-4-nitro) benzyl alcohol glucuronides (4A2N (2A4N) BG), 4-amino-2-nitro (2-amino-4-nitro) benzyl alcohols (4A2N (2A4N) B) and 4-amino-2-nitrotoluene (4A2NT). Though 2, 4-dinitrobenzaldehyde (2, 4-DNAl), a potent mutagen, was not detected in this study, the oxidative conversion of 2, 4-DNB to 2, 4-DNBA in vivo might be correlated to the carcinogenicity of 2, 4-DNT, since 2, 4-DNBA are the major metabolites of 2, 4-DNT.