Objective To establish a rapid ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous quantitative analysis of perchlorate (ClO₄^-), thiocyanate (SCN^-), and nitrate (NO₃^-) in human serum, and to apply this method to sample analysis in a real-world cohort.

Methods Serum samples were directly injected for analysis after vortex mixing, centrifugation, and filtration using an Amicon Ultra-0.5 centrifugal filter. The chromatographic column was a Waters Torus DEA Column, 130 Å (100 mm×2.1 mm, 1.7 μm), and the mobile phases were 20 mmol/L aqueous ammonium formate (containing 0.5% formic acid) (A) and acetonitrile (B), at a flow rate of 0.5 mL/min. Mass spectrometry was performed using an electrospray ionization source (ESI) in negative ion mode with multiple reaction monitoring (MRM). Perchlorate-¹⁸O₄ (ClO₄^--¹⁸O₄), thiocyanate-¹³C (SCN^--¹³C), and nitrate-¹⁵N (NO₃^--¹⁵N) were used as isotopic internal standards to investigate the specificity, linear range, precision, recovery, matrix effect, and stability of the method.

Results The linear ranges for ClO₄^-, SCN^-, and NO₃^- were 1.0-150.0 μg/L, 3.0-450.0 μg/L, and 1.0-150.0 mg/L, respectively, with lower limits of quantification of 1.0 μg/L, 3.0 μg/L, and 1.0 mg/L, respectively. The intra- and inter-batch precision were ≤10%. The average recovery rates at three mass concentrations ranged from 98.46% to 103.06%, while the RSDs of matrix effect values for serum quality controls at low, medium, and high concentrations ranged from 2.33% to 7.35%. The stability of the samples was good when stored at -80 ℃ for 30 days and subjected to three freeze-thaw cycles.

Conclusion This method is operationally simple, highly sensitive, and interference-resistant, enabling precise determination of ClO₄^-, SCN^-, and NO₃^- in the serum of environmentally exposed populations.