Clinical Quantitative Method Development of serum indoxyl sulfate (IS) assay using LC-MS/MS

Indoxyl sulfate (IS) accumulates in the blood with decreases in the voided volume due to a decline in renal function and promotes the progression of renal failure. Assay reagents for IS are marketed by several companies, but they have not been standardized because of the absence of a standard assay method. In this study, the performance of the LC-MS/MS (liquid chromatography tandem mass spectrometry) was evaluated for the establishment of a standard assay method.

The same analytical procedure was performed by two examiners using the AB SCIEX QTRAP® 4500 LC-MS/MS System. A calibration curve was prepared according to the target analyte (IS)/internal standard: 3-Indoxyl sulfate-d4 potassium salt (IS-d4) peak area ratio, and the linearity, reproducibility, selectivity tests, interferent tests, limit of blank (LOB), limit of detection (LOD), lower limit of quantification (LLOQ), and correlations were evaluated.

By dilution tests of IS standard solution, linearity (R² = 0.9995) passing through the origin was observed up to 440 μmol/L. Repeatability was coefficient of variation 2.6-4.7 % at 8.3-124.6 μmol/L, and the intermediate precision was 7.9-9.2 % at 12.9-171.2 μmol/L. In the selectivity testing, the recovery rate was 101.0-104.3 %. Regarding the effects of interferents, the interference rate was about 5.0 % at the maximum. LOB was 0.004 μmol/L, LOD was 1.248 μmol/L, and LOQ was 3.23 μmol/L. The correlation between this method (y) and conventional enzymatic analysis (x) was R = 0.996, y = 1.13x - 2.89.

The basic performance of this method was satisfactory. Also, because excellent precision was obtained in both examiners, the stability of the assay method was also suggested to be guaranteed. This method is considered useful as a reference method for IS assay.