Development and Validation of a Comprehensive LC-MS/MS Targeted Assay for Uremic Solute Profiling in Chronic Kidney Disease

Uremic solutes are toxic metabolites derived from endogenous and gut microbial metabolism normally excreted by the kidneys. Their diverse chemical properties present challenges for analytical analysis. A comprehensive uremic solute assay was developed to optimize a workflow to increase coverage of clinically relevant uremic solutes in pediatric Chronic Kidney Disease (CKD). From an initial database of 130 candidates, 25 uremic solutes were selected based on high fold-change significance, etiology, clinical reporting in uremia, and cost. A targeted metabolomics assay was developed using Multiple Reaction Monitoring (MRM) on a Waters TQ-S Micro triple quadrupole mass spectrometer, utilizing both ionization modes. ¬-Chromatographic separation was achieved via a Waters ACQUITY UPLC with a BEH C18 column. Absolute quantification was processed through TargetLynx software using a 15-point calibration curve. Validation was performed in charcoal-stripped human plasma and normalized against internal isotopic standards. Finally, the assay was applied to 22 pediatric CKD samples (healthy to kidney failure). A rapid LC-MS/MS assay using a 7- minute MRM method was developed and validated for the quantification of 18 uremic solutes, including creatinine, hippuric acid, and indoxyl sulfate. This assay validated LOQ utilizing internal standards and charcoal-stripped plasma calibration. Most targeted solute levels of uremic solutes strongly correlate with CKD progression with hypoxanthine as an exception. In conclusion, this assay provides a robust tool to monitor pediatric CKD progression at a mechanistic level.