A simultaneous determination of vitamin B6 group in blood was developed using a high-performance liquid chromatograph (HPLC) equipped with a fluorescence detector. Pyridoxamine, pyridoxal, pyridoxine, deoxypyridoxine (internal standard), and pyridoxic acid were separated on Toyo Soda Gel LS-160 column (8×500mm) using 0.0045 M NaClO4-0.04 M KH2PO4-2 M NaCl (pH 5.5) as an eluent. The blood sample added with citrate buffer and internal standard was deprotenized with trichloroacetic acid (TCA) and shaken with ether and hexane to remove excess TCA. A 100-μl portion of the remaining aqueous solution was injected into HPLC column and the effluent was monitored by fluorescence detector (Ex 325 nm, Em 385 nm). Since B6 5'-phosphates were not separated on this column, they were determined as a free form of B6 after hydrolysis by acid phophatase. The detection limits were 0.1 nmol/ml blood for pyridoxal 0.2 nmol/ml for pyridoxamine and pyridoxine, and 0.3 nmol/ml for pyridoxic acid. The concentration of B6 group in the blood after oral administration of pyridoxal, 5'-phosphate to rabbits and dogs was determined by this method. It was found that pyridoxic acid and pyridoxal were the major metabolites present in rabbit blood, whereas no metabolites other than pyridoxal were detected in dog blood.
