Liquid chromatographic determination with fluorescence detection of B6 vitamers and riboflavin in milk and pharmaceuticals

A simple, reliable and selective high performance liquid chromatographic method with fluorescence detection at different programmed wavelengths has been developed for the simultaneous analysis of B6 vitamers (pyridoxal 5′-phosphate, 4-pyridoxic acid, pyridoxal, pyridoxine and pyridoxamine) and Vitamin B2 in commercial vitaminized milk and in woman milk. The chromatographic separations were performed on a reversed phase octyl column by using a mobile phase consisting of sodium pentanesulfonate in 1% acetic acid-methanol-tetrahydrofuran under gradient elution conditions. The fluorescence intensity of pyridoxal 5′-phosphate was enhanced by post-column photochemical conversion, giving significantly different fluorescence spectra by a on-line photoreactor switched OFF and ON under irradiation at 254 nm. In addition, a simple and rapid method in isocratic conditions without the need of photochemical conversion was proposed for the analysis of Vitamin B6 and Vitamin B2 in pharmaceuticals. Linearity, precision, recovery, selectivity and sensitivity were found satisfactory for each analysed compound. Quantitation limits ranged from 26 to 240 fmol.