Ultra-Sensitive Quantification of Indoxyl Sulfate and 3-Carboxy-4-Methyl-5-Propyl-2-Furanpropanoic Acid in Plasma Using Ultra-Performance Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry

Abstract

Background: Uremic toxins such as indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) are accumulated in patients with chronic kidney disease (CKD). Recent studies have shown that the accumulation of indoxyl sulfate and CMPF alters the activity of organic anion transporting polypeptide (OATP) 1B and cytochrome P450 (CYP) 3A, respectively. We established and validated a novel ultra-sensitive method for simultaneous quantification of indoxyl sulfate and CMPF using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS).

Method: In this method, plasma samples were prepared by solid phase extraction, then analyzed by UPLC-QTOF/MS to measure concentrations of analytes. Using only 5 μL of human plasma per assay, the calibration range was 0.05-200 μg/mL and the lower limit of quantification was 0.05 μg/mL for both indoxyl sulfate and CMPF.

Results: This assay met the acceptance criteria of the U.S. Food and Drug Administration bioanalytical method validation guidance. The clinical applicability of this assay was evaluated by measuring plasma concentrations in healthy volunteers and CKD patients. All measured concentrations were within the calibration range.

Conclusion: Our novel assay may contribute to the estimation of variation of OATP1B and CYP3A activities in patients, including those with CKD.

Keywords: 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid; chronic kidney disease; indoxyl sulfate; plasma; quadrupole time‐of‐flight mass spectrometry; uremic toxin.