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

• Rapid UHPLC method for the determination of six B6 and two B2 vitamers in cow’s milk.
• Separation of eight analytes was achieved within 6.0 min.
• Sample preparation sustains the native composition of possible vitamins.
• First comprehensive investigation of 4-pyridoxic acid in cow’s milk.

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 6.0 min 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 2.69 μmol L−1. Vitamin B2 was present as riboflavin and its phosphate ester up to 12.86 μmol L−1.