Improved quantitation of short-chain carboxylic acids in human biofluids using 3-nitrophenylhydrazine derivatization and liquid chromatography with tandem mass spectrometry (LC-MS/MS)

Short-chain carboxylic acids (SCCAs) produced by gut microbial fermentation may reflect gastrointestinal health. Their concentrations in serum and urine are indicative of specific metabolic pathway activity; therefore, accurate quantitation of SCCAs in different biofluids is desirable. However, it is often challenging to quantitate SCCAs since matrix effects, induced by the presence of a vast variety of other compounds other than SCCAs in complex biofluids, can suppress or enhance signals. Materials used for sample preparation may introduce further analytical challenges. This study reports for the first time a LC-MS/MS-based method to quantitate ten SCCAs (lactate, acetate, 2-hydroxybutyrate, propionate, isobutyrate, butyrate, 2-methylbutyrate, isovalerate, valerate and hexanoate) and evaluates the matrix effects in five human biofluids: serum, urine, stool, and contents from the duodenum and intestinal stoma bags. The optimized method, using 3-Nitrophenylhydrazone as a derivatization agent and a Charge Surface Hybrid reverse phase column, showed clear separation for all SCCAs at a concentration range of 0.1−100 µM, in a 10.5 min run without carry-over effects. The validation of the method showed a good linearity (R2 > 0.99), repeatability (CV ≤ 15%) assessed by intra- and inter-day monitoring. The lowest limit of detection (LLOD) was 25 nM and lowest limit of quantitation (LLOQ) was 50 nM for nine SCCA except acetate at 0.5 and 1 µM, respectively. Quantitative accuracy in all biofluids for most compounds was