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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 (R A s) 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 R A s of 13 C-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., (© 2024. The Author(s).)

Deoxynivalenol (DON) is a trichothecene mycotoxin regularly occurring in cereals. Rats are often used to study toxicokinetics of DON and related compounds, yet only about 30 % of the administered dose is typically recovered. Recently, it was reported that DON is partly metabolised to previously undetected DON- and deepoxy-DON (DOM) sulfonate in rats and tentative structures were proposed. The present work describes the production and characterisation of DON-, DOM- and DON-3-glucoside (D3G) sulfonates of three different series; the development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for determination of DON, DOM, D3G and their sulfonates in rat faeces and urine; and application of the methods to samples from a DON and D3G feeding trial with rats. In addition to previously produced DON sulfonates (DONS) 1, 2 and 3, D3G sulfonates 1, 2 and 3; and DOM sulfonates (DOMS) 2 and 3 were synthesised, purified and characterised. The developed methods showed apparent recoveries of all investigated compounds between 68 and 151 % in faeces and between 48 and 113 % in urine. The recovery of DON, D3G and their metabolites from faeces and urine of rats (n = 6) administered in a single dose of 2.0 mg/kg b.w. DON or the equimolar amount of D3G was 75 ± 9 % for the DON group and 68 ± 8 % for the D3G group. DON-, DOM- and D3G sulfonates excreted in faeces accounted for 48 and 47 % of the total amount of administered DON and D3G. Urinary excretion of sulfonates was <1 %. In both treatment groups, DONS 2 was the major metabolite 0-24 h after treatment, whereas DOMS 2 was predominant thereafter. The developed methods can also be used for investigation of DON (conjugate) sulfonate formation in other animal species.