A Validated LC-MS/MS Assay for the Quantification of Cholate Isotopes in Human Serum.

ABSTRACT

BACKGROUND: Current methods for evaluating liver health rely on nonspecific blood tests, elastography surrogates for fibrosis, and invasive procedures, none of which directly measure liver function and physiology. Herein we present the analytical validation of a unique, highly sensitive LC-MS/MS assay and dual-sample oral (DuO) cholate challenge test to reliably quantify serial serum concentrations of cholate isotopes administered to patients with liver diseases. The clearance of administered cholate isotopes measured by the assay provides information about liver function and physiology. METHODS: Analytical method validation of the cholate assay analytes (endogenous unlabeled cholic acid, 24-13C-cholic acid, and 2,2,4,4-D4-cholic acid) in terms of accuracy, precision, analytical sensitivity, analytical specificity, and range of reliable response was completed in human serum samples spiked with quality controls and calibrators in accordance with applicable guidelines. DuO test parameters were validated using samples from 48 subjects representing various liver disease etiologies. RESULTS: Accuracy (mean biases) for all analytes ranged from 0.1% to 3.7%. Using a nested components-of-variance design (20 days, 2 runs per day, 2 replicates per sample), total imprecision for all analytes ranged from 2.3% to 8.4%. Lower and upper limits of quantitation were established and validated at 0.1 to 10.0 µM. Matrix effects and potential interferents did not affect assay performance. DuO test validation met all prespecified acceptance criteria. CONCLUSIONS: The method validation studies described herein established the performance characteristics in terms of accuracy, precision, analytical sensitivity, analytical specificity, reportable ranges, and reference intervals of the LC-MS/MS cholate assay and DuO test.