Application of the high-performance liquid chromatography method with coulometric detection for determination of vitamin B6 in human plasma and serum

A reversed-phase high-performance liquid chromatography method (HPLC) with coulometric electrochemical detection has been developed and validated for the simultaneous analysis of pyridoxamine-5′-phosphate (PMP), pyridoxamine (PM), pyridoxal 5′-phosphate (PLP), pyridoxal (PL), pyridoxine (PN) and 4-pyridoxic acid (4-PA) in human plasma and serum. The isocratic separation was achieved on C18 column (250 mm × 4.6 mm, I.D., 5 μm) with a mobile phase consisted of 35 mM sodium phosphate containing 2.5 mM heptanesulfonic acid, adjusted to pH 3.2 with 85% orthophosphoric acid and 12% methanol (v/v). Within-run and between-run precisions expressed by the relative standard deviations were less than 2.7% and 7.7% for all the analysed vitamins and 4-PA, respectively. The limits of detection (LOD) and quantification (LOQ) were: 0.8 and 2.6 nM, 1.1 and 3.8 nM, 1.5 and 4.5 nM, 1.3 and 4.2 nM, 1.1 and 3.7 nM, 2.1 and 6.3 nM for PMP, PM, PLP, PL, PN and 4-PA, respectively. The recoveries ranged from 90.4% to 98.4%. Stability of vitamins was checked under a variety of storage conditions. The developed application demonstrated acceptable sensitivity, precision, accuracy, stability, and linearity over the physiological concentration range. The major advantage of the proposed method is its great simplicity.