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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Greaves, Ronda / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter

IMPACT FACTOR 2018: 3.638

CiteScore 2018: 2.44

SCImago Journal Rank (SJR) 2018: 1.191
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Volume 53, Issue 9


Quantitation of 1α,25-dihydroxyvitamin D by LC-MS/MS using solid-phase extraction and fixed-charge derivitization in comparison to immunoextraction

Nancy Chan / Erin J. Kaleta
Published Online: 2015-01-10 | DOI: https://doi.org/10.1515/cclm-2014-0884


Background: The objective of this study was to present analysis of 1α,25-dihydroxyvitamin D (DHVD) by solid-phase extraction (SPE) using fixed-charge derivitization extraction to enhance ionization for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in comparison to traditional immunoextraction (IE) techniques.

Methods: Full analytical validation of both a SPE and IE LC-MS/MS assay was performed, and included accuracy, intra- and inter-assay precision, limit of detection and limit of quantitation. Performance of these two assays was compared with reference laboratory IE LC-MS/MS testing.

Results: This SPE LC-MS/MS assay demonstrated similar performance to the IE LC-MS/MS assay validated simultaneously. Intra-assay precision for low (12 pg/mL), medium (25 pg/mL) and high (60 pg/mL) control samples was 7.2%, 13.7% and 11.3% for DHVD2, respectively, and 9.1%, 5.9% and 8.9% for DHVD3. The inter-assay precision was 11.6%, 10.3% and 3.9% for DHVD2 and 10.6%, 7.0% and 5.6% for DHVD3. The limit of detection was 1.9 and 2.7 pg/mL for DHVD2 and DHVD3, and limit of quantitation was 4 pg/mL for both DHVD2 and DHVD3. Comparison to a reference LC-MS/MS assay showed excellent correlation (slope 0.936, R2=0.996, –0.2% bias).

Conclusions: The study demonstrated comparability of the SPE-LC-MS/MS assay for analysis of DHVD and offers an attractive option for assessment of vitamin D status as an alternative to traditional IE techniques.

Keywords: 1α,25-dihydroxyvitamin D; liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS); solid-phase extraction


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About the article

Corresponding author: Erin J. Kaleta, PhD, Houston Methodist Hospital, 6565 Fannin Street, Houston, TX 77459, USA, Phone: +1 713 441-4288, Fax: +1 713 441-1565, E-mail:

Received: 2014-09-04

Accepted: 2014-11-21

Published Online: 2015-01-10

Published in Print: 2015-08-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), Volume 53, Issue 9, Pages 1399–1407, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/cclm-2014-0884.

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