Journal of Bioequivalence & Bioavailability

Vitamin D-2, D-3 and their 25-hydroxy metabolites in human plasma: Simultaneous quantification by high performance liquid chromatography

3^rd World Congress Bioavailability & Bioequivalence

March 26-28, 2012 Marriott Hotel & Convention Centre, Hyderabad, India

Syed N. Alvi and Muhammad M. Hammami

Posters: J Bioequiv Availab

Abstract:

A reliable high performance liquid chromatography (HPLC) assay for simultaneous determination of vitamin D-2 (VD-2), vitamin D-3 (VD-3), 25-Hydroxy VD-2 [25 (OH) VD-2], and 25-Hydroxy VD-3 [25 (OH) VD-3] in human plasma was developed and validated. The samples were precipitated with a mixture of methanol and isoproponal (9:1, v:v) and extracted in hexane. After evaporation, the residue was dissolved in acidic methanol and centrifuged. 100 μ l of the clear solution was injected; and separation was achieved on Zorbax C18 column. The mobile phase (gradient elution mode) consists of methanol, acetonitrile and water (pH = 3.0, with acetic acid); the eluents were monitored by photodiode array detector, with the wavelength set at 265 nm. No interference with endogenous components was observed. The relationship between the concentration of VD-2, VD-3, 25(OH)VD-2, 25(OH)VD-3 in plasma and their peak area ratio to the IS was linear over the range of 10 - 100 ng/mL. Mean extraction recoveries of VD-2, VD-3, 25(OH)VD-2, 25(OH)VD-3 from plasma samples were over 80%. The method was applied in assessing the stability of VD-2, VD-3, 25(OH)VD-2, 25(OH)VD-3 in plasma under various conditions generally encountered by clinical laboratory. The assay is suitable to study the bioavailability of vitamin D in humans