Development and validation of a five-channel multiplex LC-ESI-MS/MS method for the quantification of carboxylic acids in urine.

BACKGROUND

Carboxylic acids are vital metabolites widely present in living organisms, playing critical roles in various biological processes. Alterations in carboxylic acid profiles are often associated with inborn errors of metabolism (IEMs), which can lead to severe consequences or even mortality without timely treatment. Monitoring these metabolites through time-series analysis is essential for understanding disease progression. However, achieving high-throughput analysis of carboxylic acids using conventional chromatographic techniques remains a significant challenge.

RESULTS

This study introduces a 5-channel multiplex method using liquid chromatography‒electrospray ionization‒tandem mass spectrometry (LC‒ESI‒MS/MS) to quantify essential carboxylic acids in urine. The method employs butanol isotopes to form butyl derivatives (D0-, D3-, D5-, D7-, and D9-carboxylic acid butyl ester). Validation results demonstrated high accuracy (85.22 %-115.16 %) for mixture estimation, with analyte quantification accuracies ranging from 79.1 % to 116.6 % and precisions below 15 % for most compounds. The validated method was applied to four urine samples in a simulated time-series study, highlighting its effectiveness in tracking metabolic changes.

SIGNIFICANCE: The innovative multiplex strategy significantly enhances the throughput and precision of carboxylic acid quantification. By enabling efficient time-series analyses, this approach contributes to the study of IEMs and accelerates clinical diagnostics, offering promising applications in both research and healthcare.