Simultaneous quantitative analysis of uremic toxins by LC MS/MS with a reversed-phase/cation-exchange/anion-exchange tri-modal mixed-mode column

Column choice is crucial to the development of liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods because analyte selectivity is dependent on the nature of the stationary phase. Recently, mixed-mode chromatography, which employs a combination of two or more stationary phases and solvent systems, has emerged as an alternative to multiple, complementary, single-column systems. This report describes the development and validation of a novel analytical method based on LC-MS/MS employing a reversed-phase/cation-exchange/anion-exchange tri-modal column (Scherzo SS-C18; Imtakt) for the simultaneous quantification of various uremic toxins (UTx), including creatinine, 1-methyladenosine, trimethylamine-N-oxide, indoxyl sulfate, p-cresyl sulfate, phenyl sulfate and 4-ethylphenyl sulfate. Stable isotope-labeled compounds were prepared as internal standards (ISs) for each analyte. Mobile phase optimization and appropriate gradient conditions resulted in satisfactory retention and peak resolution that could not have been attained with a single stationary phase LC system. The essential validation parameters, including intra- and inter-assay precision and accuracy, were adequate. The validated method was applied to measure serum levels of the aforementioned compounds in 19 patients with chronic kidney disease. This is the first report detailing the simultaneous quantification of these analytes using stable isotopes as ISs. Our results suggest that Scherzo SS-C18 columns will be considered breakthrough tools in the development of analytical methods for compounds that are difficult to quantify simultaneously in traditional LC systems.