Quantification of free and protein bound uremic toxins in human serum by LC-MS/MS: Comparison of rapid equilibrium dialysis and ultrafiltration

The objectives of this study were (1) to develop a method for the determination of 10 uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, indoxyl sulfate, kynurenic acid, kynurenine, p-cresyl glucuronide, p-cresyl sulfate, phenylacetylglutamine and trimethylamine N-oxide (TMAO)), and 3 precursors (tyrosine, phenylalanine, tryptophan) in serum and (2) to compare two separation methods to determine the free serum fraction: rapid equilibrium dialysis (RED) and ultrafiltration (UF). The method was developed on a liquid chromatography system coupled to a tandem mass spectrometer. Fifty mu L of serum sample were precipitated with methanol after addition of internal standard. The two separation methods were compared using serum samples from patients suffering from renal impairment (n = 30). The method has been validated according to the European Medicines Agency (EMA) guidelines. Calibration curves were linear from 1 to 50 ng/mL up to 10,000-50,000 ng/mL according to the compounds. The comparison between the two separation methods produced similar results for all compounds except kynurenine, tryptophan (around 30% more with UF) and indole-3-acetic acid (around 30% more with RED). This study has allowed the development and validation of a sensitive and robust assay for the quantification of free and total concentrations of 10 uremic toxins and 3 precursors in human serum.