The Determination of 25-OH Vitamin ( D2 / D3 ) in Human Serum by Liquid Chromatography Tandem Mass Spectrometry with Comparison to IDS Enzyme Immunoassay

Abstract

The proper assessment of the status of vitamin D requires the accurate measurement of both 25-OH vitamin D2 and 25-OH vitamin D3, which collectively constitute 25-OH vitamin D, the best indicator of vitamin D status. Currently, numerous assay methods are available for 25-OH vitamin D measurement but their comparability is uncertain. We employed isotope dilution liquid chromatography coupled with tandem mass spectrometry (ID-LC-MS/MS) to quantify 25-OH vitamin D2 and 25-OH vitamin D3 in human serum. Hexadeuterium labeled 25-OH vitamin D3 internal standard was added to calibrators, controls prepared in 6% bovine serum albumin in phosphate buffered saline, and patients’ sera. Zinc sulphate was added to release 25-OH vitamin D metabolites for vitamin D binding protein, followed by a precipitation step withthe addition of acetonitrile. Subsequent online phase extraction by trap column followed by chromatographic separation on a C-8 column using a water/acetonitrile gradient was employed. Detection was performed using Atmospheric Pressure Chemical Ionization (AP-CI) in a Multiple Reaction Monitoring (MRM) mode. The method was linear from 4 to 70 ng/mL. The intra and inter-day CV% were ≤ 10 for both 25-OH vitamin D2 and 25-OH vitamin D3. Recoveries ranged between 39.09 % to 64.31 % for 25-OH vitamin D2 and 30.44 % to 58.66 % for 25-OH vitamin D3, while recoveries from hexadeuterium 25-OH vitamin D3 ranged from 44.11 % to 67.5%.We compared the newly validated LC-MS/MS with a commercial Enzyme Immunoassay from Immunodiagnostic Systems (IDS EIA) in terms of inter-method agreement, correlation, and impact of assay variation on the diagnostic categorization of vitamin D status through the measurement of 182 subjects’ sera. The mean bias % of the IDS EIA relative to the LC-MS/MS was -34.28 ± 10.15 (mean ± std) with 95% CI [-24.13 to 44.43]. The two methods were ingood agreement with reasonable correlation (r²=0.82, P value = 0.000). Inter-method diagnostic categorization was variableand depended on the type of assay method and the applied cut offs used. Cross calibration and standardization of vitamin D assay methods is crucial for proper clinical assessment of vitamin D status. This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D2 and 25-OH vitamin D3 in human serum.