UPLC-MS/MS Determination of Indoxyl Sulfat and p-Cresyl Sulfate in Human Urine

418 urine samples were collected from 295 school-children and 123 pregnant women residing in the central area and at suburbs of Wuhan municipality. 100 μL of the urine sample together with 100 μL of each of the internal standard solution of indoxyl sulfate-d4 and of p-cresyl sulfate-d7, and 700 μL of acetonitrile were added into a centrifuging tube and after swirling for 1 min, the mixed solution was centrifuged at high rate for 10 min, to separate the impurities in the sample by precipitation. 100 μL of the supernatant were taken and used as testing solution for analysis. ACQUITY UPLC^® HSS T3 chromatographic column (100 mm×2.1 mm, 1.8 μm) was used as stationary column, and an aliquot of 10 μL of the testing solution were introduced. Mixtures of 5 mmol·L^-1 NH₄OAc solution and methanol, with their volumic ratio varied in the range of 5%-95% in 4 time intervals within 5 min, were used as mobile phases in the gradient elution, to have the indoxyl sulfate and p-cresyl sulfate well separated. In MS/MS determination, ESI^- with mode of negative ion scanning and the mode of MRM were adopted. As shown by the result that linear relationships between values of ratios of peak areas of indoxyl sulfate and p-cresyl sulfate to the peak areas of their respective internal standards and values of corresponding mass concentration of indoxyl sulfate and p-cresyl sulfate were found in the same range of 5.00-500 μg·L^-1. Values of detection limits (3S/N) were found 0.5 μg·L^-1 (for indoxyl sulfate) and 0.1 μg·L^-1 (for p-cresyl sulfate). Tests for recovery and precision were made by addition of standard solutions of indoxyl sulfate and p-cresyl sulfate at 3 concentration levels to a real sample as matrixes. Values of average recovery were found in the range of 93.0%-98.4% and RSDs (n=6) were found in the range of 4.2%-5.3%. It was shown that the data of urine analysis of the 418 samples provided a reliable basis in judging of function of school-children and pregnant women.