Intracellular Vitamin B Analysis in Bacteria

Sample preparation for analysis of intracellular vitamin B content in bacteria was performed using a slightly modified version of the method described for the analysis of bacterial coenzyme a thioesters [21 (link)]. Frozen cell pellets were resuspended in 1 mL of methanol, transferred into 2 mL polypropylene tubes (Sarstedt) containing 0.5 mL glass beads (0.7 and 0.1 mm, Carl Roth), and broken up by homogenization in a mini-bead beater (biospec products). In addition, the extraction was repeated twice with 0.5 mL of methanol, and the solvent was evaporated under a gentle flow of nitrogen gas. All other steps were performed according to Cakić et al. [21 (link)]. The filtered solution was analyzed by liquid chromatography coupled with mass spectrometry. Bacterial vitamin concentrations were calculated by external quantification, as previous tests for recovery and matrix effect have shown that the analytes were all recovered at 98–100% with no ion suppression.