Validation of an LC–HRMS Method for Quantifying Indoxyl Sulfate and p-Cresyl Sulfate in Human Serum

Abstract

Accurate quantification of indoxyl sulfate (IndS) and p-cresyl sulfate (pCS) is essential for understanding their role in chronic kidney disease (CKD) progression and for developing strategies to mitigate their harmful effects, including cardiovascular morbidity and renal fibrosis. Advances in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) enable the integration of powerful diagnostic tools into clinical laboratories. Along with accurate quantification, precise mass measurements allow for untargeted compound identification. Methods: An LC-HRMS was validated for quantifying IndS and pCS in human serum, following EMA guidelines. The method involved protein precipitation with methanol, micro-LC for chromatographic separation, and detection based on accurate mass, with simultaneous high-resolution full-scan acquisition. Clinical samples from patients with varying degrees of renal insufficiency and samples obtained before and after hemodiafiltration were analyzed. Results: The method demonstrated acceptable linearity, precision, and accuracy. The measurement range for both analytes was from 100 to 40,000 ng/mL. Serum levels of IndS and pCS correlated with decreased renal function. After hemodiafiltration, there was a significant reduction of IndS (50%) and pCS (43%). Simultaneous untargeted analysis allowed to identify metabolites significantly underexpressed after hemodiafiltration. Conclusions: An accurate LC-HRMS method was validated for the quantification of IndS and pCS serum levels in patients with CKD, providing insights into toxin dynamics and enabling untargeted metabolic evaluation.