The Determination of 25-OHVitamin (D2/D3) in Human Serum by LiquidChromatography Tandem Mass Spectrometry with Comparison to IDS EnzymeImmunoassay

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 quantify25-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 newlyvalidated 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