A one-step NIST traceable HPLC method for quantitation of vitamin B6 and 4-pyridoxic acid in human plasma

OBJECTIVES: Vitamin B6 deficiency is associated with a wide spectrum of clinical syndromes. While vitamin B6 status is primarily assessed by measuring the biologically active form of the vitamin, pyridoxal 5-phosphate (PLP), concurrent measurement of the final metabolite 4-pyridoxic acid (PA) can provide additional information regarding supplement intake and hypophosphatasia. The aim of this study is to develop a simple method traceable to the NIST standard reference material 3950 for simultaneous detection of PLP and PA. DESIGN & METHODS: A one-step reverse phase HPLC method with fluorescence detection was developed by evaluating different derivatization conditions, the use of an internal standard and different calibration strategies. The assay analytical performance was evaluated. RESULTS: Pre-column derivatization with semicarbazide showed the best overall performance in terms of signal to noise ratio, retention time and peak shape when compared to pre- or post-column derivatization with chlorite, pre-column or in-mobile phase derivatization using sodium bisulfite. The final method provided an analytical measurement range from 7.8 to 350 ​nmol/L for PLP and 3.3–339 ​nmol/L for PA, total imprecision <15% and <5% for PLP and PA respectively. Calibration against the NIST standard produced measured values within 3% of NIST assigned PLP values. The use of 4-deoxypyridoxine as internal standard did not improve precision or accuracy when compared to calibration using 5-level external standards. CONCLUSIONS: This method combines derivatization and protein precipitation in one step and is traceable to NIST standard reference material 3950. It is simple and reliable for routine evaluation of vitamin B6 nutrition status.