Determination of vitamin B12 in food products and in premixes by reversed-phase high performance liquid chromatography and immunoaffinity extraction

A new, faster and simple method to quantify Vitamin B-12, both in foods and in premixes, by reversed-phase liquid chromatography with UV detection has been developed. Vitamin B-12 was extracted from food products with 50 mM sodium acetate buffer pH 4.0 (at 100 degrees C for 35 min) in the presence of sodium cyanide, followed by a purification step on an immunoaffinity column prior to the LC analysis. An enzymatic hydrolysis (pepsin at 37 degrees C and pH 4 for 3 h) prior to the purification step efficiently released the bound Vitamin B-12, and thus, allowed obtaining total Vitamin B I, contend in food products. Vitamin B-12 was monitored by UV at 361 nm after its separation on a reversed-phase narrow-bore column with a gradient of mobile phase made of water/acetonitffle and trifluoroacetic acid (TFA) 0.025%. The specificity of the method was demonstrated by the retention characteristics, UV spectra and by comparing the peak purity with the Vitamin B-12 standard. The calibration Graphs plotted with six concentrations of Vitamin B-12 was linear with a regression coefficient R-2 > 0.9997. The repeatability of the method was evaluated at different levels of concentration on six fortified products and the relative standard deviation (RSDr) was below 3.2%. The value of the relative standard deviation of the intermediate precision was below 5.6% (n = 4). The method was successfully applied to several food products and consistent results were obtained in comparison with microbiological assay (MBA). Our data demonstrate that the immunoaffinity columns are highly efficient for the purification of Vitamin B-12 and that our HPLC could be used as an alternative method to the microbiological assay for the determination of Vitamin B-12 in food products. (c) 2005 Elsevier B.V. All rights reserved.