title: Determination of vitamin B6 vitamers and pyridoxic acid in plasma: development and evaluation of a high-performance liquid chromatographic assay.
abstract: Marginal deficiency of vitamin B6 has recently been related to cardiovascular diseases. Because of that there is an increasing interest in a suitable and reliable method for quantifying this vitamin in routine laboratory medicine. We have developed a HPLC-based method able to quantify the B6 vitamers pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxamine 5'-phosphate (PMP), pyridoxine (PN), and pyridoxamine (PM) and the degradation product 4-pyridoxic acid (4-PA). The separation was accomplished using a C18 (ODS) analytical column and an ion-pair reversed-phase chromatography. B6 vitamers were eluted with a gradient of acetonitrile (0.5-15%) in a potassium phosphate buffer with 1-octanesulfonic acid and triethylamine, pH 2.16. The concentration of the vitamers was determined with fluorescence detector (328 nm excitation, 393 nm emission) after postcolumn derivatization with phosphate buffer containing 1 g/L sodium bisulfite. The performance of the assay was evaluated by analyzing six plasma samples with interrelated concentration and two control samples (unspiked and vitamer spiked) over a 3-months period. The HPLC method was able to identify PLP, 4-PA, PM, PL, PN, and PMP from all other compounds in plasma in an analytical run of 46 min. The imprecisions and mean values (presented in parenthesis in nmol/L) were (unspiked and spiked sample) 9-8% (41-65) for PLP, 12-7% (18-40) for 4-PA, 67-28% (4-19) for PL, 15% (21) for PN, 10% (27) for PM, and 27% (17) for PMP. All three B6 vitamers (PLP, 4-PA, and PL) present in unspiked plasma showed an excellent linearity within the range of (nM) 8-60 (4-PA), 1-19 (PL), and 11-99 (PLP). In conclusion, we report a HPLC-based method that separates and detects nanomolar quantities of six B6 vitamers and demonstrate that the method will be suitable for routine quantitation of PLP and 4-PA in human plasma.
authors.affiliation: Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK-8000 Aarhus C, DK-8000, Denmark.
authors.forename: Marianne R
authors.initials: MR
authors.lastname: Bisp
authors.forename: Mustafa Vakur
authors.initials: MV
authors.lastname: Bor
authors.forename: Else-Marie
authors.initials: EM
authors.lastname: Heinsvig
authors.forename: Morten A
authors.initials: MA
authors.lastname: Kall
authors.forename: Ebba
authors.initials: E
authors.lastname: Nexø
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004492:Edetic Acid / Q000097:blood; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry; D006801:Humans; D011735:Pyridoxic Acid / Q000097:blood*; D012107:Research Design / Q000706:statistics & numerical data*; D013997:Time Factors; D025101:Vitamin B 6 / Q000031:analogs & derivatives / Q000097:blood*
chemical_list: D005456:Fluorescent Dyes; D011735:Pyridoxic Acid; D025101:Vitamin B 6; D004492:Edetic Acid