A HILIC–MS/MS method for the simultaneous determination of seven organic acids in rat urine as biomarkers of exposure to realgar

Realgar (As4S4) is a traditional medicine used in China and Europe for thousands of years. As an arsenical, the toxicity from realgar has raised public concern. Several organic acids in urine are found to be potential biomarkers of realgar exposure, including taurine, citric, glutamic, lactic, pyruvic, succinic and uric acid. In this study, using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), a rapid and sensitive method was developed to separate and quantify these compounds in urine. A ZIC®-HILIC column was used for the separation at an isocratic condition of acetonitrile and 10 mM ammonium acetate in water. Analytes were detected in multiple-reaction monitoring with negative ionization mode, using ibuprofen as internal standard. Good line arities (R2 > 0.996) were obtained for all analytes with the limits of detection from 0.2 to 0.7 μg/mL. The intra-day and inter-day accuracy ranged from 89.1 to 104.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80%with RSD less than 14.0%. The validated method was applied to analyze the urine samples of control and reaglar treated rats, and significant changes of these organic acids were observed.