Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25- Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography
- *Corresponding Author:
- Muhammad M Hammami
Department of Clinical Studies and Empirical Ethics
King Faisal Specialist Hospital & Research Center, MBC-03
Riyadh 11211, Kingdom of Saudi Arabia
E-mail: [email protected]
Received Date: April 17, 2012; Accepted Date: May 28, 2012; Published Date: May 30, 2012
Citation: Alvi SN, Yusuf A, Hammami MM (2012) Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25- Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography. J Bioequiv Availab S14:007. doi: 10.4172/jbb.S14-007
Copyright: © 2012 Alvi SN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
A simple and reliable High Performance Liquid Chromatography (HPLC) method for simultaneous determination of vitamin D-2 (VD-2), vitamin D-3 (VD-3), 25-hydroxyvitamin D-2 [25 (OH) VD-2], and 25-hydroxyvitamin D-3 [25(OH) VD-3] in human plasma was developed and validated. Plasma samples were deproteinized with a mixture of methanol and 2-proponol, and extracted with hexane. After evaporation, the residue was dissolved in methanol:water (9.6:0.4, v/v), centrifuged, and then clear solution was injected onto Zorbax C18 column. The mobile phase (gradient elution mode) consists of methanol, acetonitrile, and water (pH = 3.0); the eluents were monitored by photodiode array detector (wavelength set at 265 nm). 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 5 - 100 ng/mL. The coefficients of variation for inter-day and intra-day assay were all ≤ 9.7% and biases ≤ 13.1%. Mean extraction recoveries of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 from plasma were all over 80%. The method was applied for the determination of vitamin D levels in plasma obtained from healthy subjects. Further, it was used to assess the stability of VD-2, VD-3, 25(OH) VD-2, and 25(OH) VD-3 in plasma under various conditions encountered in the clinical laboratory.