Broad-spectrum analytical method for the simultaneous determination of multiple classes of urinary volatile organic...

Prolonged exposure to volatile organic compounds (VOCs), even at low concentrations, is associated with a range of adverse health effects. The chemical diversity of VOCs results in highly variable biomarker profiles, underscoring the urgent need for comprehensive and efficient analytical strategies to assess urinary VOC metabolites (mVOCs). In this study, we developed a single-run method for the simultaneous quantification of 18 mVOCs with markedly different physicochemical properties, including acidic mercapturic or carboxylic acids, and basic pyrrolidine alkaloids. The extraction workflow was systematically designed based on the acid-base characteristics of the target analytes. Urine samples were spiked with isotope-labeled internal standards, acidified, and loaded onto an Oasis HLB plate. The acidic analytes were collected in eluate, while the basic analytes were collected in the breakthrough, which was later alkalized and re-extracted to recover. Both fractions were combined and analyzed by LC-MS/MS using electrospray ionization (ESI) and multiple reaction monitoring. Notably, carboxyl-containing metabolites were monitored under negative ESI, while pyrrolidine alkaloids were detected under positive ESI within the same chromatographic run. The method exhibited excellent performance, with detection limits ranging from 0.01 μg/L to 2.77 μg/L. Quality control results for ClinChek® reference materials fell within certified ranges, and the method successfully passed the German External Quality Assessment Scheme (G-EQUAS) proficiency testing. Greenness assessments indicated satisfactory eco-friendliness and high applicability. The validated method was applied to the China National Human Biomonitoring Program (CNHBM), revealing significant differences in mVOC concentrations across subgroups stratified by different population types.