DETERMINATION OF TOTAL PYRIDOXAL IN HUMAN PLASMA FOLLOWING ORAL-ADMINISTRATION OF VITAMIN-B6 BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITHPOSTCOLUMN DERIVATIZATION

An HPLC method for determining total pyridoxal from plasma was develop ed for a relative bioavailability comparison of two oral vitamin B-6 ( pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosph ate with the acid phosphatase enzyme, the total pyridoxal was determin ed by HPLC. Pyridoxal was separated on a reversed-phase column, post-c olumn derivatized to pyridoxal-semicarbazide, and then detected by flu orescence and quantitated. The limit of detection was 2 ng/mL and inte rday 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 B-6 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 e ndogenous pyridoxal detected on the 2nd day varied in plasma between 1 3 and 17 ng/mL. Pharmacokinetic parameters corrected for background ar e reported for two vitamin B-6 (40 mg) preparations. Briefly, the phar macokinetic results for the Ratiopharm preparation compared with the H offmann-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 C(max), 193.0 and 197.1 ng/mL; t(max), 1.25 and 1.44 h; and relative bioavailabilit y, 97.9% (West-lake, 88-112%).