Accuracy and clinical implications of seven 25-hydroxyvitamin D methods compared with liquid chromatography–tandem mass spectrometry as a reference

Ann Clin Biochem 2008;45:153-159
© 2008 Association for Clinical Biochemistry


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Original Article

Heinz Jürgen Roth 1 ,
Heinrich Schmidt-Gayk 1,
Holger Weber 2 and
Christoph Niederau 3

1 Limbach Laboratory, Department of Endocrinology and Oncology, Im Breitspiel 15, 69126 Heidelberg, Germany;
2 Labor Prof G Enders und Partner, Rosenbergstraße 85, 70193 Stuttgart, Germany;
3 Labor Dr Niederau, Leopoldstr 10, 44147 Dortmund, Germany

Corresponding author: Heinz Jürgen Roth. Email: pablolim{at}

Background: The most reliable assessment of vitamin D status is measurementof plasma 25-hydroxyvitamin D (25[OH]D) concentration. Highvariability in 25(OH)D measurements due to utilized test andassay technologies and the lack of standardization against referencematerials and reference method often confounds proper assessmentof vitamin D status.

Methods: We evaluated the accuracy of six routinely available methodologies: high-performance liquid chromatography (HPLC), the IDS-radioimmunoassay (IDS-RIA) and enzyme immunoassay (IDS-EIA), the Nichols Advantage automated protein-binding assay (Advantage), two versions of the DiaSorin automated immunoassay (Liaison 1 and Liaison 2) – and one prototype automated immunoassay (Elecsys) for assessment of the 25(OH)D3 status in a cohort of 300 randomlyselected patients’ samples compared with the reference methodliquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results: Passing-Bablok regression analysis demonstrated a slope for each method compared with LC-MS/MS that varied from 0.62 (IDS-EIA) to 1.0 (HPLC). The Advantage and the Liaison 1 showed significant deviation from linearity with highly variable individual results vs. the LC-MS/MS. Difference plots revealed a considerable persistent proportional bias for the IDS-RIA and IDS-EIA. All evaluated methods except HPLC demonstrated a more or less considerable deviation of individual 25(OH)D3 values compared with LC-MS/MSdefined target concentrations.

Conclusions: Standardization of methods for the quantification of 25(OH)Don a human-based sample panel by means of LC-MS/MS would helpto reduce the inter-method variability with respect to accuracyexisting in 25(OH)D measurement considerably. However, therewill still remain differences in the accuracy of methods utilizingsample purification before final quantification or immunologicalreaction when compared with those methods without separate samplepurification.

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