α-Ribazole, a fluorescent marker for the liquid chromatographic determination of vitamin B12 in foodstuffs

A method to determine the contents of free vitamin B12 in various foods by reversed phase liquid chromatography-fluorimetry is reported. It includes a purification of the samples by passage through an immunoaffinity column and a pre-column conversion of vitamin B12 into the fluorescent α-ribazole (successive treatments of the extract with 2.5 M sodium hydroxide (at 100 °C for 15 min) and alkaline phosphatase (7.5 U) at 37 °C and pH 8 for 16 h). An enzymatic hydrolysis prior to the purification step (pepsin at 37 °C and pH 4 for 3 h) made it possible to release the vitamin B12 bound to proteins and thus to obtain the total vitamin B12 contents of these foodstuffs. The method proposed for the determination of free and bound vitamin B12 gives a good recovery rate (95-100%) and a satisfactory repeatability (R.S.D.r between 1.0 and 5.4%). Owing to its low quantification limit (3 ng g−1) and the good resolution of the α-ribazole peak, it could most probably be applied to the determination of this vitamin in any foodstuff.