Journal Article

Determination of the urinary benzene metabolites <i>S</i>-phenylmercapturic acid and <i>trans</i>,<i>trans</i>-muconic acid by liquid chromatography-tandem mass spectrometry

Assieh A. Melikian, Ray O'Connor, Agasanur K. Prahalad, Peifeng Hu, Heyi Li, Mark Kagan and Seth Thompson

in Carcinogenesis

Volume 20, issue 4, pages 719-726
Published in print April 1999 | ISSN: 0143-3334
Published online April 1999 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/20.4.719
Determination of the urinary benzene metabolites S-phenylmercapturic acid and trans,trans-muconic acid by liquid chromatography-tandem mass spectrometry

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To investigate how various levels of exposure affect the metabolic activation pathways of benzene in humans and to examine the relationship between urinary metabolites and other biological markers, we have developed a sensitive and specific liquid chromatographic–tandem mass spectrometric assay for simultaneous quantitation of urinary S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA). The assay involves spiking urine samples with [13C6]S-PMA and [13C6]t,t-MA as internal standards and clean up of samples by solid-phase extraction with subsequent analysis by liquid chromatography coupled with electrospray-tandem mass spectrometry-selected reaction monitoring (LC–ES–MS/MS–SRM) in the negative ionization mode. The efficacy of this assay was evaluated in human urine specimens from smokers and non-smokers as the benzene-exposed and non-exposed groups. The coefficient of variation of runs on different days (n = 8) for S-PMA was 7% for the sample containing 9.4 μg S-PMA/l urine, that for t,t-MA was 10% for samples containing 0.07 mg t,t-MA/l urine. The mean levels of urinary S-PMA and t,t-MA in smokers were 1.9-fold (P = 0.02) and 2.1-fold (P = 0.03) higher than those in non-smokers. The mean urinary concentration (±SE) was 9.1 ± 1.7 μg S-PMA/g creatinine [median 5.8 μg/g, ranging from not detectable (1 out of 28) to 33.4 μg/g] among smokers. In non-smokers' urine the mean concentration was 4.8 ± 1.1 μg S-PMA/g creatinine (median 3.6 μg/g, ranging from 1.0 to 19.6 μg/g). For t,t-MA in smokers' urine the mean (±SE) was 0.15 ± 0.03 mg/g creatinine (median 0.11 mg/g, ranging from 0.005 to 0.34 mg/g); the corresponding mean value for t,t-MA concentration in non-smokers' urine was 0.07 ± 0.02 mg/g creatinine [median 0.03 mg/g, ranging from undetectable (1 out of 18) to 0.48 mg/g]. There was a correlation between S-PMA and t,t-MA after logarithmic transformation (r = 0.41, P = 0.005, n = 46).

Keywords: ES, electrospray; LC, liquid chromatography; MS/MS, tandem mass spectrometry; Q, quadrupole; SAX, strong anion exchanger; S-PMA, S-phenylmercapturic acid; SRM, selected reaction monitoring; t,t-MA, trans,trans-muconic acid.

Journal Article.  5668 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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