Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices

Abstract

Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20–100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples. Graphical abstract

An Optimized Method for LC–MS-Based Quantification of Endogenous Organic Acids: Metabolic Perturbations in Pancreatic Cancer

“…Methyl malonic acid has been reported as a potential biomarker of vitamin B12 deficiency, while α-ketoglutaric and pyruvic acid levels in urine are considered to be potential biomarkers for type II diabetes and liver cancer [ 5 , 6 ]. Short-chain fatty acids (SCFAs) (fewer than six carbon atoms) are known to play crucial roles in human health and disease [ 7 ]. SCFAs influence systemic metabolism by modulating glucose and lipid metabolism, enhancing insulin sensitivity, and reducing fat accumulation [ 8 ].…”