Rapid determination of the various native forms of vitamin B₆ and B₂ in cow's milk using ultra-high performance...

As the formation of pyridoxal phosphate, the active cofactor of vitamin B₆, is dependent on riboflavin 5-phosphate, we propose a fast and simple ultra-high performance liquid chromatography method for the simultaneous determination of the native B₆ vitamers pyridoxal, pyridoxine, pyridoxamine, their mono phosphorus esters and 4-pyridoxic acid as well as vitamin B₂ as riboflavin and its phosphorus ester riboflavin 5-phosphate in milk. Separation was achieved under 6.0min by reversed-phase and pH gradient elution. Sample preparation was optimized regarding various acids and pH levels. Changes in those parameters led to significant deviations of sample matrix breakdown efficiency. The optimized method was then validated regarding specificity, accuracy, precision, linearity, range, detection and quantification limits. As the method performed satisfactory, is was used to study commercial liquid cow's milk (n=31), regarding effects of the employed preservation technique (pasteurization, extended shelf-life, ultra-high temperature) on the composition and content of B₆ and B₂ vitamers. In cow's milk, vitamin B₆ mostly consists of pyridoxal and its phosphate ester, with pyridoxal phosphate being the bulk component. The catabolite of the vitamin B₆ metabolism, 4-pyridoxic acid was present in significant amounts in all studied samples, with up to 2.69μmolL^-1. Vitamin B₂ was present as riboflavin and its phosphate ester up to 12.86μmolL^-1.