Determination of total pyridoxal in human plasma following oral administration of vitamin B6 by high-performance liquid chromatography with post-column derivatization.

An HPLC method for determining total pyridoxal from plasma was developed for a relative bioavailability comparison of two oral vitamin B6 (pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosphate with the acid phosphatase enzyme, the total pyridoxal was determined by HPLC. Pyridoxal was separated on a reversed-phase column, post-column derivatized to pyridoxal-semicarbazide, and then detected by fluorescence and quantitated. The limit of detection was 2 ng/mL and interday variation (3 days) over the whole concentration range (13-215 ng/mL spiked) was < 4.1%. In the relative bioavailability study, 16 human subjects were put on a low vitamin B6 diet for a period of 3 days. On the 2nd and 3rd days, 14 blood samples were taken per subject at the same times each day. The drug was administered on the 3rd day. Total endogenous pyridoxal detected on the 2nd day varied in plasma between 13 and 17 ng/mL. Pharmacokinetic parameters corrected for background are reported for two vitamin B6 (40 mg) preparations. Briefly, the pharmacokinetic results for the Ratiopharm preparation compared with the Hoffmann-La Roche preparation are, respectively: AUC0-24, 369.2 and 352.6 ng.h/mL; AUC24-48, 1638.2 and 1662.3 ng.h/mL; net Cmax, 193.0 and 197.1 ng/mL; tmax, 1.25 and 1.44 h; and relative bioavailability, 97.9% (Westlake, 88-112%).