Achieving greater selectivity for the analysis of o-, m-, p-methylhippuric acids in workers’ urine by ultra performance liquid chromatography coupled with tandem mass spectrometry

•A selective method for o-, m- and p-methylhippuric acid analysis has been developed.•Chromatographic separation has been optimized to resolve the three isomers at baseline.•Simultaneous acquisition of analyte chemical structure information and quantitative data.•Simultaneous full scan acquisitions did not prevent the robustness of the quantitative data.•The method includes a full validation.

A selective method analyzing separately o-, m- and p-methylhippuric acid isomers in workers’ urine samples has been developed using ultra performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation has been optimized to resolve the three isomers at baseline. Combined with this optimal separation, the mass spectrometer allowed rapid switching from MRM scan to full scan and product ion scan within the chromatographic peak. This feature allowed the retention of analyte chemical structure information for the three methylhippuric acid isomers in parallel with the simultaneous acquisition of quantitative data. Such an approach is unequaled for the reliability of the data generated and it can be applied to each isomer separately. The method was adjusted to a dynamic range between 0.2 mM and 8.12 mM for o-methylhippuric acid and p-methylhippuric acid, and between 0.41 mM and 16.23 mM for m-methylhippuric acid in order to cover the biological exposure index. A negligible matrix effect was observed with the conditions used. Also, intra-day and inter-day precisions were both <6% for all the concentration levels tested and the accuracy was evaluated at 97 ± 4%. The inclusion of simultaneous full scan acquisitions did not prevent the robustness of the quantitative data. The method applied to the determination of inter-laboratory proficient test samples led to results in the tolerated range. Moreover, urine samples from workers were robustly quantified and the MHA levels were all below the biological exposure index reference value.