A LC/UV/Vis method for determination of cyanocobalamin (VB12) in multivitamin dietary supplements with on-line sample clean-up

A HPLC-UV/Vis method using a two-column strategy with a switching valve for on-line sample cleanup was developed for the determination of cyanocobalamin (CN-Cbl/Vitamin 1312) in multivitamin dietary supplement tablets. The method uses two columns: an Agilent Zorbax C8 (150 mm x 4.6 mm, 5 mu m particle size) reversed-phase column and a Waters Symmetry C18 (150 mm x 4.6 mm, 5 mu m particle size) reversed-phase column. Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. Because of the low levels of Vitamin B-12 in the samples, large injection volumes, and thus much interfering material, must be used to exceed the limit of quantitation (LOQ) by UV detection. A switching valve was used to divert most of these early eluting interfering materials to waste, effecting on-line sample clean-up without excessive sample preparation steps. The recovery of CN-Cbl in the method was 99.5% and the LOQ was 10 ng per injection. The method was successfully applied to the analysis of the NIST SRM 3280 multivitamin/multimineral dietary supplement tablet. The method is specific, precise, and accurate for the intended use. Compared to off-line sample clean-up procedures, it offers the advantage of being easier, more economical, and less time-consuming.