Separation and quantitation of B-6 vitamers in rat plasma by high performance liquid chromatography

A high performance liquid chromatographic method was developed for the separation and quantitation of the B-6 vitamers in rat plasma. The separation time was under 20 minutes. The analyses were accomplished using a reverse phase C₁₈ column; a binary system of methanol and heptane sulfonic acid, the ion pair; and a fluorescence detector using 300 nm excitation and 375 nm emission filters. The minimum detectable quantity was determined to be 5 ng for PL and PN and 4 ng for PM. Samples were extracted using potato acid phosphatase; phosphorylated forms of the B-6 vitamers were converted to nonphosphorylated. The B-6 vitamers were measured in plasma from rats whjch had intakes of vitamin B-6 ranging from 7% - 551% of the basal requirement for the vitamin. PL was found to be the predominate B-6 vitamer in plasma from animals consuming <50% B-6 basal requirement and PM for the lowest intake group. PL and total B-6 values were significantly correlated (r = 0.99, p < 0.0001). Significant differences (p < 0.01) in plasma PL and total B-6 concentrations were observed for rats with inadequate and adequate B-6 intakes. The method seemed to be a sensitive indicator of vitamin B-6 status in rats thus indicating potential for use with humans.