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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 208; p. 114450
• Main Authors: Gomez-Gomez, Alex, Olesti, Eulàlia, Montero-San-Martin, Blanca, Soldevila, Angie, Deschamps, Tessa, Pizarro, Nieves, de la Torre, Rafael, Pozo, Oscar J.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20-01-2022
• Subjects: Animals; Carboxylic Acids; Chromatography, Liquid; Derivatization; Hydroxylamines; LC–MS/MS; O-benzylhydroxylamine; Rats; Tandem Mass Spectrometry; Targeted metabolomics; Tricarboxylic acid cycle; Tricarboxylic acid cycle; Carboxylic acids; O-benzylhydroxylamine; Targeted metabolomics; Derivatization; LC–MS/MS
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2021.114450

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.
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•A LC-MS/MS method for the determination of 20 carboxylic acids was validated.•The method was successfully validated in urine, plasma, saliva, brain and liver.•The method confirmed metabolic changes previously reported in 3 different scenarios.•Previously unreported metabolic alterations in these scenarios are also described.•The developed method has the potential to be used in targeted metabolomics studies.