Vitamin D assay
Shah et al. Nutrition Journal 2011, 10:46 http://www.nutritionj.com/content/10/1/46 RESEARCH
Open Access
Misleading measures in Vitamin D analysis:
A novel LC-MS/MS assay to account for epimers and isobars
Iltaf Shah1, Ricky James1, James Barker2, Andrea Petroczi1 and Declan P Naughton1*
Abstract
Background: Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars.
Methods: A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction
Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified.
Results: The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with
25OHD3 epimer 3-epi-25OHD3 and isobars 1-a-hydroxyvitamin-D3 (1aOHD3), and 7-a-hydroxy-4-cholesten-3-one
(7aC4). The new assay was capable of
Open Access
Misleading measures in Vitamin D analysis:
A novel LC-MS/MS assay to account for epimers and isobars
Iltaf Shah1, Ricky James1, James Barker2, Andrea Petroczi1 and Declan P Naughton1*
Abstract
Background: Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars.
Methods: A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction
Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified.
Results: The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with
25OHD3 epimer 3-epi-25OHD3 and isobars 1-a-hydroxyvitamin-D3 (1aOHD3), and 7-a-hydroxy-4-cholesten-3-one
(7aC4). The new assay was capable of