Simultaneous determination of t,t‐muconic, S‐phenylmercapturic and S‐benzylmercapturic acids in urine by a rapid and sensitive liquid chromatography/electrospray tandem mass spectrometry method

Abstract

We describe a rapid and sensitive high-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC/ESI-MS/MS) method for simultaneous determination of the most relevant metabolites of benzene and toluene, t,t-muconic acid (t,t-MA), S-phenylmercapturic acid (S-PMA), and S-benzylmercapturic acid (S-BMA). Urine samples were purified before analysis by solid-phase microextraction (SPE) on SAX cartridges with 50 mg sorbent mass. The developed method fulfils all the standard requirements of precision and accuracy. Calibration curves were linear within the concentration range of the standards (0-80 microg/L(urine) for t,t-MA, and 0-25 microg/L(urine) for S-PMA and S-BMA), and had correlation coefficients > or =0.997. Limits of detection were 6.0 microg/L for t,t-MA, 0.3 microg/L for S-PMA, and 0.4 microg/L for S-BMA. The method was used to determine t,t-MA, S-PMA and S-BMA levels in urine of 31 gasoline-station workers, with personal monitoring data obtained from radial symmetry passive diffusive samplers. In the context of mean work-shift exposures of 75.9 microg/m(3) (range 9.4-220.2) for benzene and 331.9 microg/m(3) (78.2-932.1) for toluene, metabolite concentrations in end-of-shift urine samples ranged from 23.5-275.3 microg/g(creatinine) for t,t-MA, non-detectable to 0.9 microg/g(creatinine) for S-PMA, and 3.8-74.8 microg/g(creatinine) for S-BMA. No significant correlation was found between the environmental concentrations and urinary metabolites (p > 0.05 for all cases); the ratios of benzene metabolites could be influenced by exposure levels and co-exposure to xylenes and toluene. The high throughput of this procedure should facilitate exploration of the metabolic effects of benzene-related co-exposure to toluene and alkylbenzenes in large populations of subjects exposed to gasoline.

Determination of S‐phenylmercapturic acid in human urine using an automated sample extraction and fast liquid chromatography‐tandem mass spectrometric method

“…The analysis time was 15 min with a lower limit of quantitaion (LLOQ) of 0.4 ng/mL. Another method used 96-well SAX solid-phase extraction and achieved an LLOQ of 0.5 ng/mL but with a long analysis time of 26 min (Barbieri et al, 2004). Pieri et al (2003) reported a method using HPLC purification instead of commonly used solid phase and liquidliquid extraction to clean up the sample.…”

A Concise Review of Chromatographic Methods for the Analysis of Benzene and Its Metabolites

“…The efficiency of this assay was evaluated in human urine specimens from smokers and nonsmokers serving as benzene-exposed and nonexposed groups respectively. Barbieri et al 64 also used LC/MS/MS for determination of t,t-MA and S-PMA in urine samples after SPME based purification. The limit of detection was found to be 6 mg/dm 3 for t,t-MA and 0.3 mg/dm 3 for S-PMA.…”

Method for the Determination of Benzene Metabolite t,t-Muconic Acid in Urine by HPLC-UV with an Ion Exclusion Column

“…On the other hand, there has been widespread use of high performance liquid chromatography (HPLC) with reverse phase columns and ultraviolet and visible spectrophotometry (UV-VIS) or mass spectrometry (MS) detection [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. One way to separate anions by chromatography is to use cation exchange columns where the ions are separated by ion exclusion.…”