Determination of monobromobimane derivatives of phenylmercapturic and benzylmercapturic acids in urine by high-performance liquid chromatography and fluorimetry

A method was developed for the determination in human urine of S-phenylmerc apturic (PMA) and S-benzylmercapturic (BMA) acids, metabolites respectively of benzene and toluene. PMA and BMA were determined, after alkaline hydrol ysis, to give respectively thiophenol and benzylmercaptan, and coupling of the thiol-containing compounds with monobromobimane (MB), by reversed-phase HPLC on a diphenyl-silica bonded cartridge (100x4.6 mm I.D., 5 mum particl e size) with fluorimetric detection. Wavelengths for excitation and emissio n were 375 and 480 nm, respectively. The recovery of PMA and BMA from spike d urines was >90% in the 10-500 mug/l range; the quantification limits were respectively 1 and 0.5 mug/l; day-to-day precision at 42 mug/l was C.V. <7 %. The suitability of the proposed procedure for the biological monitoring of exposure to low-level airborne concentrations of benzene and toluene, wa s evaluated by analyzing the urinary excretion of PMA and BMA in subjects e xposed to different sources of aromatic hydrocarbons, namely occupationally -unexposed referents (non-smokers, n=15; moderate smokers, n=8; mean number of cigarettes smoked per-day=17 cig/day) and non-smoker workers occupation ally exposed to toluene in maintenance operations of rotogravure machines ( non-smokers, n=17). Among referents, non-smokers showed values of PMA rangi ng from <1 to 4.6 mug/l and BMA from 1.0 to 10.4 mug/l; in smokers, PMA val ues ranging from 1.2 to 6.7 mug/l and BMA from 9.3 to 39.9 mug/l, were obse rved. In occupationally exposed non-smoker subjects, BMA median excretion v alue (23.6 mug/l) was higher than in non-smoker referents (3.5 mug/l) (P<0. 001) and individual BMA values (y, <mu>g/l) were associated and increased w ith airborne toluene concentration Gs, mg/m(3)) according to the equation y =6.5+0.65x (r=0.69, P<0.01, n=17). The proposed analytical method appears t o be a sensitive and specific tool for biological monitoring of low-level e xposure to benzene and toluene mixtures in occupational and environmental t oxicology laboratory. (C) 2001 Elsevier Science B.V. All rights reserved.