alexa Simultaneous Quantification of Vitamin D-2, Vitamin D-3, and their 25- Hydroxy Metabolites in Human Plasma by High Performance Liquid Chromatography
ISSN: 0975-0851

Journal of Bioequivalence & Bioavailability
Open Access

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Research Article

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

Syed N Alvi1, Ahmed Yusuf1and Muhammad M Hammami1,2*

1Department of Clinical Studies and Empirical Ethics, King Faisal Specialist Hospital & Research Center, MBC-03, Riyadh 11211, Kingdom of Saudi Arabia

2Alfaisal University College of Medicine, Riyadh, Kingdom of Saudi Arabia

*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.

 

Abstract

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.

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