Simultaneous quantification of total and free protein-bound uremic toxins in serum by LC-MS/MS: method development, validation, and clinical application in hemodialysis patients

Protein-bound uremic toxins (PBUTs), including indoxyl sulfate (IxS), p-cresol sulfate (pCS), indole-3-acetic acid (IAA), and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF), accumulate in patients with End-Stage Kidney Disease (ESKD) due to their resistance to conventional dialysis clearance and have been implicated in cardiovascular complications, oxidative stress, and disease progression. Accurate quantification of both total and free fractions of these toxins is essential for understanding their clinical significance, yet validated methods with population-specific reference intervals remain scarce. In this study, a rapid, sensitive, and fully validated LC-MS/MS method was developed for the simultaneous quantification of total and free serum concentrations of four PBUTs using a 4-minute chromatographic run and a single-step protein precipitation with an acetonitrile/acetone (1:1, v/v) solvent system. For the first time, indole-3-carboxylic acid was employed as an internal standard for IAA and CMPF quantification, while dihydrochlorothiazide (DHCT) was used for IxS and pCS. Method validation was performed in accordance with European Medicine Agency (EMA) guidelines, demonstrating excellent linearity (R² > 0.99), recovery (90-110%), minimal matrix effects (95-105%), and intra- and inter-day precision below 10% coefficient of variation (CV) for all analytes. Reference intervals for total PBUT concentrations were established using data from 120 healthy volunteers from a Turkish population cohort, representing the largest reference dataset reported to date for these analytes. The validated method was applied to serum samples from 118 maintenance hemodialysis patients, revealing significantly elevated total PBUT concentrations compared to healthy controls (p < 0.001 for all analytes), with IxS and pCS showing the most pronounced accumulation (approximately 29- and 23-fold higher, respectively). ROC curve analysis demonstrated excellent diagnostic performance for IxS (AUC = 0.986), pCS (AUC = 0.980), and CMPF (AUC = 0.943) in discriminating hemodialysis patients from healthy controls, while IAA showed limited discriminatory ability (AUC = 0.633), likely reflecting its distinct protein binding dynamics in uremic conditions. Spearman correlation analysis revealed significant associations between total PBUT levels and biochemical markers of renal function, further supporting the clinical relevance of the developed method. This validated LC-MS/MS approach offers a practical and reliable tool for simultaneous PBUT monitoring in clinical and research settings.