New fully automated gas chromatographic analysis of urinary S-phenylmercapturic acid in isotopic dilution using negative chemical ionization with isobutane as reagent gas

The determination of urinary S-phenylmercapturic acid (S-PMA) represents the most reliable biomarker to monitor the intake risk of airborne benzene. Recently, the European Chemical Agency deliberated new occupational exposure limits for benzene and recommended an S-PMA biological limit value of 2-μg/g creatinine. This limit is an order of magnitude lower than the previous one, and its determination constitutes a challenge in the analytical field. We developed and validated a method that allows the fully automated and sensitive determination of S-PMA by the use of gas-chromatography negative chemical ionization tandem mass spectrometry in isotopic dilution. For negative chemical ionization, we selected a mixture of 1% isobutane in argon as reactive gas, by studying its chemical ionization mechanism and optimal parameters compared with pure isobutane or pure methane. This gas mixture produces a more abundant signal of the target analyte than isobutane or methane, and it extended the operative lifetime of the ion source, enabling us to start a high-throughput approach of the S-PMA analysis. Moreover, energy-resolved mass spectrometry experiments were carried out to refine the MS/MS analysis conditions, testing nitrogen and argon as collision gases. The method optimization was pursued by a chemometric model by using the experimental design. The quantification limit for S-PMA was 0.10 μg/L. Accuracy (between 98.3% and 99.6%) and precision (ranging from 1.6% to 6.4%) were also evaluated. In conclusion, the newly developed assay represents a powerful tool for the robust, reliable, and sensitive quantification of urinary S-PMA, and because of its automation, it is well suited for application in large environmental and biological monitoring.