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

Chia-Ni Lin ^a,b,*, I-Wen Wu ^c, Yun-Fen Huang ^a, Shu-Yu Peng ^a,b, Ya-Ching Huang ^a,b, Hsiao-Chen Ning ^a,b

^a Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou Medical Center, Taoyuan, Taiwan

^b Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan, Taiwan

^c Department of Nephrology Chang Gung Memorial Hospital, Keelung & Chang Gung University, Taiwan

Chronic kidney disease (CKD) is a complex disorder that affects multiple organs and increases the risk of cardiovascular complications. CKD affects approximately 12% of the population in Taiwan. Loss of kidney function leads to accumulation of potentially toxic compounds such as indoxyl sulfate (IS) and p-cresyl sulfate (pCS), two protein-bound uremic solutes that can stimulate the progression of CKD. The aim of this study was to assess whether IS and pCS levels were correlated with CKD stage. We developed and validated a method for quantitating total and free IS and pCS in serum by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Serum samples were pretreated using protein precipitation with acetonitrile containing stable isotope-labeled IS and pCS as internal standards. After centrifugation, the supernatant was diluted and injected into a UPLC-MS/MS system. Analyte concentrations were calculated from 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 analytical measurement range was 0.05-5 mg/L. The limit of quantitation of this assay was 0.05 mg/L for both analytes. The reference interval was ≦0.05-1.15 mg/L for total-form IS, ≦0.05-5.33 mg/L for total-form pCS, ≦0.05 mg/L for free-form IS, and ≦0.12 mg/L for free-form pCS. 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 serum can 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.