The mercapturic acids are critical urinary biomarkers for evaluating the short-term exposure to typical food processing contaminants in vivo. However, limited studies have focused on methodological supports for biomonitoring the internal exposure to these multiple contaminants. Here we developed a reliable method for simultaneous determination of five mercapturic acids originating from acrylamide, 3-monochloropropane-1,2-diol (3-MCPD), and glycidol by ultra-high performance liquid chromatography tandem mass spectrometry. The limits of quantification of the five analytes ranged 0.3‒0.9 ng/mL, while the recovery rates were measured as 102%‒119%, 107%‒118%, and 105%‒111% at low, intermediate, and high spiking levels of analyte standards, respectively. Chromatographic separation at baseline level and rapid run time (7 min per sample) for all mercapturic acids also supported the current effective analysis. We enrolled 408 Chinese participants (203 male and 205 female) who had not been served with special diets before and collected their urine samples in the morning after fasting overnight. The urinary concentrations of mercapturic acids were associated with typical demographic characteristics of the participants, including sex, age, body mass index, and smoking status. The current method was successfully used for biomonitoring urinary metabolites from daily exposure to acrylamide, 3-MCPD and glycidol among Chinese participants.

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