Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25-Hydroxy Metabolites in Human Plasma by High Performance LiquidChromatography

摘要: A simple and reliable High Performance Liquid Chromatography (HPLC) method for simultaneous determination of vitamin D-2 (VD-2), D-3 (VD-3), 25-hydroxyvitamin [25 (OH) VD-2], [25(OH) VD-3] in human plasma was developed validated. Plasma samples were deproteinized with a mixture methanol 2-proponol, extracted hexane. After evaporation, the residue dissolved methanol:water (9.6:0.4, v/v), centrifuged, then clear solution injected onto Zorbax C18 column. The mobile phase (gradient elution mode) consists methanol, acetonitrile, water (pH = 3.0); eluents monitored by photodiode array detector (wavelength set at 265 nm). relationship between concentration VD-2, VD-3, 25(OH) VD-3 their peak area ratio to IS linear over range 5 - 100 ng/mL. coefficients variation inter-day intra-day assay all ≤ 9.7% biases 13.1%. Mean extraction recoveries from 80%. applied D levels obtained healthy subjects. Further, it used assess stability under various conditions encountered clinical laboratory.