Indirect spectrophotometric determination of vitamin B12 using 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-cobalt complex formation.

Abstract

A sensitive spectrophotometric determination of vitamin B12 (V. B12) in complexing vitamin preparations is proposed. Since V. B12 contained cobalt, it is possible to use cobalt for the V. B12 assay. Cobalt reacts with 3-(2-pyridy1)-5, 6-dipheny1-1, 2, 4-triazine (PDT) to form a cationic chelate ion and the complex can be extracted into 1, 2-dichloroethane with tetrabromophenolphthalein ethyl ester (TBPE) at pH 9.0. The composition of the ternary complex is assumed to be Co(PDT)3(TBPE)2. A predetermined amount of V. B12 (0.1-3mg) was heated at 200°C for 2h with 20ml of nitric acid. After adding 10ml of nitric acid and 2ml of perchloric acid, the mixture was evaporated to dryness. The residue was dissolved in 30ml of 1M hydrochloric acid by heating and diluted to 50ml with distilled water. The required volume of the sample solution containing 0.5-2.5μg cobalt (10-60μg V. B12) was transferred to a 50-ml volumetric flask. To this, 1ml of 10-4 M potassium periodate, 2% Tiron, 7.5×10-3M PDT, 10ml of the borate-phosphate buffer (pH 9), and 1ml of 1.35×10-3M TBPE, were added. It was then diluted to 50ml with distilled water. The mixture was transferred to a separatory funnel and shaken for 5 min with 10 ml of 1, 2-dichloroethane. The organic phase was centrifuged and the absorbance was measured at 610 nm against a reagent blank. Molar absorptivity was 134000mol-1 cm -1 l, and relative standard deviation, 1.7% for ten determinations of 30 ng ml-1 cobalt. Fe(II) was tolerated up to 0.1μg ml-1 (5-fold for cobalt) by addition of 2×10-6 M potassium periodate and 0.04% Tiron and also Cu(II), 0.1μg ml-1 by adding 0.2M thiourea. This method can be applied to the indirect assay of V. B12 compounds such as cianocobalamin, hydroxocobalamin and mecobalamin in commercial preparations containing other vitamins.