The determination of 25-OHVitamin (D2 D3)‎ in human serum by liquid chromatography tandem mass spectrometry with comparison to IDS enzyme immunoassay

Abstract EN

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.

Hex deuterium 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 with the 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 in good agreement with reasonable correlation (r² = 0.82, P value = 0.000).

Inter-method diagnostic categorization was variable and 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.