Characterizing the In Vitro Conversion of Benzene Exposure Urinary Biomarker 6-Hydroxy-2,4-cyclohexadienyl...

Urinary phenyl mercapturic acid (PhMA) is a specific biomarker of benzene exposure that has been widely used in biomonitoring of the general population and in occupational exposure studies. However, previous research has identified significant interlaboratory variation in urinary PhMA concentrations due to differences in the acidity of the sample treatment conditions. This variation arises from the need to convert the benzene's precursor metabolite 6-hydroxy-2,4-cyclohexadienyl mercapturic acid (pre-PhMA) to PhMA. In this study, we systematically examined the influence of sample treatment pH on this reaction across various acidic treatment conditions representative of the reported PhMA assays. The resulting pre-PhMA and PhMA levels were quantified using an established liquid chromatography-tandem mass spectrometry assay. PhMA levels increased with more acidic treatment conditions until pH -0.6 when PhMA formation was the greatest at 53.1% formation of the total pre-PhMA. The formation of PhMA was dependent on sample treatment pH. Thus, a quadratic regression was modeled on PhMA formation vs pH across all acid types. The resulting regression model (y = 0.874 × pH² - 12.146 × pH + 41.99, R² = 0.978) can be used to determine the extent of PhMA formation for a specific treatment pH, improving the ability to compare PhMA results among studies with different analytical methods or to absolute health-based cutoffs such as the biological exposure index. To explain the incomplete PhMA formation, we utilized gas chromatography-mass spectrometry to identify and quantify the formation of benzene as a major byproduct of the acid-derived dehydration of pre-PhMA. Further, base-derived dehydration of pre-PhMA to PhMA was performed to validate the benzene formation mechanism observed with acid dehydration.