Chromatomass-Spectrometric Method for the Quantitative Determination of Amino- and Carboxylic Acids in Biological Samples

Abstract

A highly sensitive method for the qualitative and quantitative determination of amino- and carboxylic acids, as well as a number of urea and methionine cycle metabolites in the studied solutions, is presented. Derivatives (esterification) were obtained for amino acids by their reaction in a solution of 3 N of hydrochloric acid in n-butanol for 15 min at 65 °C and for carboxylic acids by their reaction with phenol in ethyl acetate with 3 N of hydrochloric acid for 20 min at 65 °C. Experimental work on the determination of individual metabolites was carried out using the HPLC-MS/MS method and included the creation of a library of spectra of the analyzed compounds and their quantitative determination. Multiplex methods have been developed for the quantitative analysis of the desired metabolites in a wide range of concentrations of 3–4 orders of magnitude. The approach to the analysis of metabolites was developed based on the method of the dynamic monitoring of multiple reactions of the formation of fragments for a mass analyzer with a triple quadrupole (QQQ). The effective chromatographic separation of endogenous metabolites was carried out within 13 min. The calibration curves of the analyzed compounds were stable throughout the concentration range and had the potential to fit below empirical levels. The developed methods and obtained experimental data are of interest for a wide range of biomedical studies, as well as for monitoring the content of endogenous metabolites in biological samples under various pathological conditions. The sensitivity limit of the methods for amino acids was about 4.8 nM and about 0.5 μM for carboxylic acids. Up to 19 amino- and up to 12 carboxy acids and about 10 related metabolites can be tested in a single sample.