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

Abstract

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.

Keywords: 4-Pyridoxic acid; Liquid chromatography; NIST traceable; Pyridoxal 5-phosphate; Vitamin B6.

Fig. 1

Matrix choice for vitamin B6 calibrator solution. PLP derivatized fluorescence product is not stable in saline but remains stable in charcoal stripped plasma.

Fig. 2

Representative chromatograms of vitamin B6 and standard curves. A: Overlay chromatograms PLP and PA from five levels of calibrators. B: A typical standard curve of PLP. C: A typical standard curve of PA. D: Chromatogram of a patient sample with normal level of PLP (130.5 ​nmol/L) and normal concentration of PA (154.5 ​nmol/L). E. Chromatograms of a patient sample with vitamin B6 deficiency (PLP 11.3 ​nmol/L, PA 10.0 ​nmol/L). The yellow diamond indicates the highest calibrator. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Accuracy of the in-house vitamin B6 assay in measuring the NIST standard reference material 3950. The top panel shows that measured results of PLP were about 19% lower than the NIST assigned value using the in-house assay with calibrator values assigned by absorbance measurement. After the calibrators were calibrated against the NIST SRM 3950, measured results of PLP matched the NIST assigned value with difference less than 5%. The Bottom panel shows that measured results of PA matched the NIST assigned value with or without the calibrators calibrating against the NIST reference standards.

Fig. 4

Method comparison between the in-house vitamin B6 assay with/without NIST calibration and a reference laboratory method (n ​= ​26). Left panel: A. Comparison of the in-house HPLC method with a reference lab HPLC method showed equivariant measurement of PLP in plasma specimens using absorbance assigned calibrator values. B. Bias was observed between the in-house HPLC method and the reference lab method after the in-house calibrators were calibrated against the NIST standard reference material 3950. The black dashed line in panel A and B indicate the 1:1 line.