Simultaneous quantification of seven B vitamins in human faeces by stable isotope label-based high-performance liquid...

B vitamins in the human distal gut are primarily derived from the gut microbiota because daily dietary vitamins are fully absorbed in the small intestine under normal dietary and physiological conditions. Quantitative determination of B vitamins in the distal gut and faecal samples is crucial for understanding the intricate relationship between gut B vitamins, gut microbiota, and host health. In this study, we developed a rapid, robust, and reliable method with a simple extraction procedure for the simultaneous analysis of seven B vitamins in human faeces using high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (HPLC-ESI-MS/MS) with stable isotope-labelled internal standards. A protein precipitation approach using methanol as the precipitant was employed to extract vitamin B from human faecal samples. Seven B vitamins were adequately separated and quantified within 9 min by HPLC-ESI-MS/MS with a Pursuit PFP column (2.0 ×150 mm, 3.0 µm), including vitamins B1, B2, B3, B5, pyridoxic acid, pyridoxine, and B7. The lower limits of quantification were within the range of 0.1-25 ng mL^-1. The intra-day and inter-day precision and accuracy were both within 15 %. The validated method was successfully applied to 55 faecal samples collected from healthy individuals, patients with type 2 diabetes, and obese patients. Compared with healthy controls, obese patients had lower faecal concentrations of vitamins B1 and B3 and pyridoxic acid, whereas patients with type 2 diabetes had lower faecal concentrations of vitamins B1 and B5.