Trace determination of the hydrogen sulfide biomarker thiosulfate in human urine by HPLC coupled with element selective ICPMS/MS detection

Hydrogen sulfide is a toxic gas but also established as a naturally occurring gaseous signaling molecule in humans, playing key physiological roles with particular involvement in lung disease including COVID-19. Thiosulfate is the conventional biomarker of hydrogen sulfide and is excreted in human urine at low micromolar levels. Thiosulfate is amenable to detection by the element-selective inductively coupled plasma tandem mass spectrometry (ICPMS/MS), but sulfur speciation in human samples at trace levels is challenging due to the high complexity of human sulfur metabolome and the utility of this detector under such settings has not been demonstrated. We report a method for thiosulfate determination in human urine at trace physiological levels by HPLC-ICPMS/MS. The method involved one-step derivatization to improve chromatographic behavior followed by direct injection. The instrumental limit of detection was 1.4 μg S L−1 (0.02 μM or 0.1 pmol). In a group of samples from volunteers (n = 24), measured thiosulfate concentrations in the diluted urine matrix were down to 8.0 μg S L−1 with a signal-to-noise ratio >10. The method was validated for recovery (80–110%), repeatability (RSD% <5%), and linearity (r2 = 0.9999, at a tested working concentration range of 0.01–1.0 mg S L−1), and the accuracy was assessed by comparing with HPLC-ESIMS/MS which showed agreement within ±20%. This work demonstrates the applicability of HPLC-ICPMS/MS for sulfur speciation at trace levels in a matrix with complex sulfur metabolome as human urine and provides a sensitive method for the determination of the hydrogen sulfide biomarker.