Abstract
To study B_6 absorption mechanism, we developed a method for simultaneous determination of pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxine and 4-pyridoxic acid in blood and plasma using high-performance liquid chromatography. Our method employs a reverse-phase column (JASCO Fine SIL C_<18> T), an acidic potassium phosphate mobile phase (0.2% NaH_2PO_4-0.085% H_3PO_4 10:11 pH 2.45) and fluorometric detection (Ex 296nm, Em 391nm). This method is a rapid and simple, and requires smaller amounts of samples (50μl) than other methods. This method was employed to examine luminal absorption of PLP in situ. When PLP (5×10^<-3>M) dissolved in 80mM phosphate buffer was administered into the lumen of a rat ileal segment, 0.68% of PLP administered appeared as PLP and 2.88% as the sum of PLP+PL in the plasma of venous blood collected from the segment for 2 hr.
