Development and Validation of a High Pressure Liquid Chromatographic Method for the Simultaneous Determination of Four Vitamin D Metabolites in Blood Serum

Abstract

A novel reversed-phase high performance liquid chromatographic method is developed and validated for the simultaneous determination of four vitamin D metabolites: 1,25-dihydroxyD 2 , D 3 and 25-hydroxyD 2 and D 3 in blood serum for the first time. Separation was carried out on an analytical column, Orbit 100 C 18 , 5 I¼m, 250 A— 4 mm, with a mobile phase consisting of ammonium acetate – methanol (MeOH- CH 3 COONH 4 88-12% v/v) delivered isocratically, at a flow rate 0.9 mL/min, at room temperature. Photodiode array detection was performed at 280 nm for the 1I±,25-dihydroxyvitamin D 3 and at 265 nm for the rest compounds. The limit of detection was 0.5 ng/I¼L, while linearity held up to 5 ng/I¼L. Bamifylline (5 ng/I¼L) was used as internal standard. Validation of the method was performed in terms of accuracy and precision: intra-day assay (n=5) and inter-day assay (n=3 A— 5) and was found to be satisfactory, with high accuracy and precision results. Long term stability, short-term stability and stability during various freeze thaw cycles were also studied. The method was successfully applied to blood serum samples of pregnant women and their neonates after an easy preparation procedure. Recovery rates from serum ranged between 88.5 and 103.4 %.