Targeted mass spectrometry method for the determination of multiple gut-microbiota metabolites in human plasma

The gut microbiota profoundly impacts human health by producing metabolites that can act as biomarkers for disease diagnosis and therapy. However, accurately measuring these metabolites in biomatrices is challenging due to their low concentrations, high molecular diversity, and interference from matrix components, demanding advanced and precise analytical methodologies. Hence, an ultra-high-performance liquid chromatography method coupled to triple quadrupole-tandem mass spectrometry detection, combined with a chemical derivatization procedure, was developed and validated to quantify seven gut metabolites, namely acetic acid, propionic acid, butyric acid, p-cresol sulfate, 3-indoxyl sulfate, indole-3-acetic acid, and L-tryptophan, in human plasma. Samples were prepared by protein precipitation with acetonitrile and subsequently derivatized using 3-nitrophenylhydrazine. Chromatographic separation was achieved using a BEH C18 column, with elution performed at a flow rate of 0.2 mL min⁻¹ and in gradient mode using formic acid-water (1:1000, v/v) and formic acid-acetonitrile (1:1000, v/v) as mobile phase components. The mass spectrometer was operated in negative ionization mode and data was acquired in selected reaction monitoring. Good linearity was achieved (r² > 0.997) for all the target gut metabolites in the evaluated concentration ranges, with low LLOQ values (0.4–8 μM). The method proved to be accurate (87.0–114 %) and precise (CV ≤ 13.5 %), achieving a score of 65 in the Blue Applicability Grade Index (BAGI) metric, which confirmed its practicality. The developed method was ultimately employed to the analysis of plasma samples from children and adults involved in clinical studies, demonstrating its usefulness in medical research.