Determination of up to twenty carboxylic acid containing compounds in clinically relevant matrices by o-benzylhydroxylamine derivatization and liquid chromatography-tandem mass spectrometry.

Carboxylic acid containing compounds (R-COOH) are involved in a large number of biological processes and they are relevant for several pathological processes such as neurodegeneration or cancer. Comprehensive methodologies for the quantitative determination of R-COOH in biological samples are required. In this study we have developed a LC-MS/MS method for the quantification of 20 endogenous R-COOH belonging to different pathways such as kynurenine metabolism, serotoninergic pathway, glycolysis, tricarboxylic acid cycle, dopaminergic pathway, short chain fatty acids and glycine metabolism. The approach included derivatization with o-benzylhydroxylamine (reaction time 1 h), liquid-liquid extraction with ethyl acetate and LC-MS/MS detection (run time 10 min). The method was optimized and validated in 5 different matrices (urine, plasma, saliva, brain and liver) following two different approaches: (i) using surrogate matrices and (ii) using actual human samples by standard additions. A suitable linearity was obtained in the endogenous range of the analytes. Adequate intra and inter-assay accuracies (80-120%) and intra- and inter-assay precisions (<20%) were achieved for almost all analytes in all studied matrices. The method was applied in several scenarios to confirm (i) human urinary changes produced in glycolysis after exercise, (ii) metabolic changes produced in rat brain and plasma by methamphetamine administration and (iii) metabolic alterations in human plasma caused by vitamin B6 deficiency. Additionally, the application of the method allowed for establishing previously unreported alterations in R-COOH metabolites under these conditions. Due to the comprehensive analyte and matrix coverage and the wide applicability of the developed methodology, it can be considered as a suitable tool for the study of R-COOH status in health and disease by targeted metabolomics.

Improving liquid chromatography-tandem mass spectrometry determination of polycarboxylic acids in human urine by chemical derivatization. Comparison of o-benzyl hydroxylamine and 2-picolyl amine.

Due to its high sensitivity and specificity, liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) could be considered as the gold-standard in targeted metabolomics. Although LC-MS/MS allows for the direct detection of a large number of molecules, the proper quantification of highly polar compounds such as poly-carboxylic acids in complex matrices like urine is still a challenge. Chemical derivatization offers a suitable way to improve chromatographic behavior and sensitivity for these compounds. Several derivatizing agents have been proposed for the LC-MS/MS determination of carboxylic acids but studies dealing with their comparison in challenging scenarios are scarce. Here we present the evaluation of two different derivatization agents; o-benzylhydroxyl amine (oBHA) and 2-picolyl amine (2-PA); for the quantification of the (poly)-carboxylic acids belonging to the tricarboxylic acid cycle in urine. The suitability of both derivatizating agents was compared by validation of the two approaches. Derivatization with oBHA showed important advantages against 2-PA derivatization such as (i) providing better sensitivity, (ii) more stable derivatives and (iii) allowing for the proper validation of a larger number of analytes. Moreover, while 2-PA derivatization failed in the determination of the target analytes in some stored urine samples, oBHA derivatization successfully allowed for their appropriate determination in the same samples. A comparison between the concentrations obtained using oBHA derivatization and those provided by an external laboratory using UV and GC-MS detection revealed a satisfactory agreement between both results. Additionally, the concentrations obtained by the oBHA method for a set of 38 urines are in agreement with those previously reported in the literature. As a conclusion, our results show that the use of oBHA is preferred against 2-PA for the detection and quantification of (poly)-carboxylic acids in urine.