title: Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay.
abstract: The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum. Hexadeuterium labelled 25-OH vitamin D3 internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's < or = 6% for both 25-OH vitamin D3 and 25-OH vitamin D2. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D3 and 82.7 and 100.3% for 25-OH vitamin D2. Our results for the DEQAS serum pools averaged -7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r(2)=0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r(2)=0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum.
authors.affiliation: Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. j.v.d.ouweland@cwz.nl
authors.forename: Johannes M W
authors.initials: JM
authors.lastname: van den Ouweland
authors.forename: Antonius M
authors.initials: AM
authors.lastname: Beijers
authors.forename: Pierre N M
authors.initials: PN
authors.lastname: Demacker
authors.forename: Henny
authors.initials: H
authors.lastname: van Daal
mesh_terms: D001331:Automation; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D016018:Least-Squares Analysis; D008163:Luminescent Measurements / Q000379:methods*; D011863:Radioimmunoassay / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000097:blood
chemical_list: D014807:Vitamin D; C104450:25-hydroxyvitamin D