High-throughput UHPLC-MS/MS method for 15 protein-bound uremic toxins in human serum

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a severe complication of chronic kidney disease (CKD), linked to the systemic accumulation of protein-bound uremic toxins (PBUTs). Current methods for PBUT analysis are often constrained by limited analyte coverage and lack of integrated high-throughput quantification of multiple toxins. A high-throughput targeted Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method was developed and validated for the quantitative determination of PBUTs in human serum, including N^δ-(carboxymethyl)lysine (CML) and pentosidine (PE). Reproducible sample preparation and reliable quantification were achieved using 96-well protein/phospholipid removal plates for sample preparation and optimizing chromatographic and mass spectrometric conditions. The method was validated, demonstrating acceptable linearity over the concentration range of 10-2000 ng/mL for all 15 analytes (R² > 0.99), with lower limits of quantification (LLOQ) of 10-20 ng/mL. The limits of detection (LOD) ranged from 5 to 10 ng/mL, and the precision expressed as relative standard deviation (RSD) was below 15%. Application to serum samples from 40 uremic patients and 20 healthy controls revealed significantly elevated levels of 10 PBUTs, with p-cresyl sulfate (PCS) concentrations approximately 400-fold higher in patients. Five PBUTs were undetectable in healthy individuals. This study provides a reliable and accurate method for PBUTs quantification, offering valuable insights into the critical role of PBUTs in the pathogenesis of CKD-MBD, and advancing PBUT-related metabolomic research.