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.

Effects of a 10-day course of a high dose calciferol versus a single mega dose of ergocalciferol in correcting vitamin D deficiency

“…Serum 25(OH)D2, 25(OH)D3, and total 25(OH)D levels were determined using the liquid chromatography-mass spectrometry method developed and validated at King Abdullah University Hospital laboratories. 26 …”

Comparative Analysis of Histamine in Fresh and Processed Fish Sold in Jordanian Market

“…The primary analytical methods for determining histamine in fish and fish products include enzyme-linked immunosorbent assay (ELISA) (19), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) (20), capillary electrophoresis (CE) (4,5,21), and gas chromatography-tandem mass spectrometry (GC/MS) (7). Among these, the ELISA method is most commonly utilized for histamine detection because chromatographic methods involve laborious and time-consuming steps for sample pretreatment such as extraction and concentration, along with the necessity of derivatization in gas chromatography (7,22).…”