A rapid HPLC method for the extraction and quantification of vitamin B12 in dairy products and cultures of Propionibacterium freudenreichii.

摘要

To overcome nutritional vitamin B12 deficiencies in certain populations in Southern Africa, fortified functional foods have been developed. However, current microbiological methods used to accurately determine vitamin B12 levels in foods, important for quality control and regulatory purposes, are time consuming. Thus, a method was developed for the extraction and detection of vitamin B12 in dairy products and growth media cultured with Propionibacterium freudenreichii. Samples were extracted by mixing in KCN buffer (pH 4.5), autoclaving at 121 degrees C for 25 min, cooling and centrifugation. The resultant supernatant was syringe-filtered prior to RP-HPLC analysis using a methanol-water gradient that was effective in resolving the B12 peak. The method offered excellent linearity with a regression coeff. R > 0.998. The limit of quantification was 0.005 mug ml-1 sample. For samples with vitamin B12 concn. well within the linear range of the assay, good repeatability was demonstrated for the same sample with mean concn. of 2.62 +or- 0.02 and 2.61 +or- 0.02 mug ml-1 detected on day 1 and day 2, respectively. Recovery values ranged from 98.6 to 103.2%, indicating that the extraction method ensured complete dissolution of vitamin B12 from the matrices under study (amasi, skim milk, kefir and culture broth). Sensitivity was enhanced by sample concentration and purification using a series of solid phase extraction steps which resulted in improved peak resolution and removal of interfering peaks. The method was validated by comparison of the HPLC results with those obtained using traditional microbiological methods. The method is a rapid alternative to the more time-consuming microbiological assay.