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 (0.075 ng/5 lL 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 < 5%). A nutritionally relevant amount of active vitamin B12 was produced
by P. freudenreichii in cereal malt matrices (up to 1.9 lg/100 g) in 24 h at 28 C.