Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices

•A sensitive UHPLC/UV method was developed for the analysis of vitamin B12.•The method was suitable for analysing microbial cells and fermented cereal matrices.•Immunoaffinity purification enabled analysis of vitamin B12 in fermented cereal matrices.•Active vitamin B12 was produced in cells and cereal matrices by P. freudenreichii.•Pseudovitamin B12 could be separated and further identified with UHPLC–MS. A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075ng/5μL injection). As confirmed by UHPLC–MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD