Development and validation of a UHPLC-MS/MS method for measurement of a gut-derived uremic toxin panel in human serum: An application in patients with kidney disease

Gut-derived uremic toxins contribute to the uremic syndrome and are gaining attention as potentially modifiable cardiovascular disease risk factors in patients with underlying chronic kidney disease. A simple, rapid, robust, accurate and precise ultra-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of a panel of four gut-derived uremic toxins in human serum. The panel was comprised of kynurenic acid, hippuric acid, indoxyl sulfate, and p-cresol sulfate. Serum samples were protein precipitated with acetonitrile containing deuterated internal standards. Chromatographic separation of analytes was accomplished with an Acquity BEH C18 (2.1 x 100 mm, 1.7 mu m) column by isocratic elution at a flow rate of 0.3 mL/min with a mobile phase composed of solvent A (10 mM ammonium formate; pH 4.3) and solvent B (acetonitrile) (85:15, v/v). Analytes were detected using heated electrospray ionization and selected reaction monitoring. The total run-time was 4 min. Standard curves were linear and correlation coefficients (r) were >= 0.997 for concentration ranges of 0.01-0.5 mu g/mL for kynurenic acid, 0.25-80 mu g/mL for p-cresol sulfate, and 0.2-80 mu g/mL for hippuric acid and indoxyl sulfate. Intra- and inter-day accuracy and precision were within 19.3% for the LLOQs and <= 10.9% for all other quality controls. Matrix effect from serum was <15% and recovery was >= 81.3% for all analytes. The method utilizes a short run-time, simple/inexpensive sample processing, has passed FDA validation recommendations, and was successfully applied to study patients with kidney disease. (C) 2019 Elsevier B.V. All rights reserved.