Measuring serum total and free indoxyl sulfate and p-cresyl sulfate in chronic kidney disease using UPLC-MS/MS

Measuring serum total and free indoxyl sulfate and p-cresyl sulfate in chronic kidney disease using UPLC-MS/MS

Chronic kidney disease (CKD) is a complex disorder that affects multiple organs and increasesthe risk of cardiovascular complications. CKD affects approximately 12% of thepopulation in Taiwan. Loss of kidney function leads to accumulation of potentially toxiccompounds such as indoxyl sulfate (IS) and p-cresyl sulfate (pCS), two protein-bounduremic solutes that can stimulate the progression of CKD. The aim of this study was toassess whether IS and pCS levels were correlated with CKD stage. We developed andvalidated a method for quantitating total and free IS and pCS in serum by ultraperformanceliquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Serumsamples were pretreated using protein precipitation with acetonitrile containing stableisotope-labeled IS and pCS as internal standards. After centrifugation, the supernatant wasdiluted and injected into a UPLC-MS/MS system. Analyte concentrations were calculatedfrom the calibration curve and ion ratios between the analyte and the internal standard.The calibration curves were linear with a correlation coefficient of >0.999; the analyticalmeasurement range was 0.05e5 mg/L. The limit of quantitation of this assay was 0.05 mg/Lfor both analytes. The reference interval was _0.05e1.15 mg/L for total-form IS, _0.05e5.33 mg/L for total-form pCS, _0.05 mg/L for free-form IS, and _0.12 mg/L for free-formpCS. A positive correlation was observed between analyte concentration and CKD stage.Our sensitive UPLC-MS/MS method for quantifying total and free-form IS and pCS in serumcan be used to monitor the progression of CKD in clinical settings, identify patients at risk,and facilitate development of further therapies for this devastating disease.