Reference ranges and biological variation of free and total serum indoxyl- and p-cresyl sulphate measured with a rapid UPLC fluorescence detection method.

The uremic toxins indoxyl sulphate (IS) and p-cresyl sulphate (pCS) are absorbed bacterial metabolites of tryptophan and tyrosine respectively and may be predictive of clinical outcome. Long chromatography times, incomplete data on the reference ranges of the free and total fractions and the biological variation limit wider clinical application. An UPLC method with fluorescence detection was developed and reference ranges and biological variation were investigated in healthy volunteers. Chromatography time was 3 min with excellent linearity, precision and low detection limits (IS of 0.02 μmol/L and pCS of 0.05 μmol/L). Both IS and pCS increased with a decrease in renal function and were moderately correlated with eGFR (R(2) 0.65 and 0.33 respectively). The serum reference ranges were (μmol/L): total IS of 0.7-6.3; free IS of 0.0-0.2; total pCS of 0.0-38.4; and free pCS of 0.1-2.4. The intra individual biological variation was estimated at 35.9% and 50.5% with a critical difference of 3.9 μmol/L (100%) and 20.7 μmol/L (141%) for total IS and pCS respectively. We describe a robust analytical method with a short chromatography time that quantifies both IS and pCS. The data on reference ranges and intra-individual biological variation need to be considered in clinical studies that investigate these uremic toxins.