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Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation.
Rapid Commun Mass Spectrom. 2013 Jan 15; 27(1):200-6.RC

Abstract

RATIONALE

The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set.

METHODS

A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems.

RESULTS

Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3.

CONCLUSIONS

Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3.

Authors+Show Affiliations

Clinical Chemistry, University Hospital of Lausanne, CHUV (Centre Hospitalier Universitaire Vaudois), Route du Bugnon 46, 1011, Lausanne, Switzerland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23239334

Citation

Bruce, Stephen J., et al. "Analysis and Quantification of Vitamin D Metabolites in Serum By Ultra-performance Liquid Chromatography Coupled to Tandem Mass Spectrometry and High-resolution Mass Spectrometry--a Method Comparison and Validation." Rapid Communications in Mass Spectrometry : RCM, vol. 27, no. 1, 2013, pp. 200-6.
Bruce SJ, Rochat B, Béguin A, et al. Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation. Rapid Commun Mass Spectrom. 2013;27(1):200-6.
Bruce, S. J., Rochat, B., Béguin, A., Pesse, B., Guessous, I., Boulat, O., & Henry, H. (2013). Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation. Rapid Communications in Mass Spectrometry : RCM, 27(1), 200-6. https://doi.org/10.1002/rcm.6439
Bruce SJ, et al. Analysis and Quantification of Vitamin D Metabolites in Serum By Ultra-performance Liquid Chromatography Coupled to Tandem Mass Spectrometry and High-resolution Mass Spectrometry--a Method Comparison and Validation. Rapid Commun Mass Spectrom. 2013 Jan 15;27(1):200-6. PubMed PMID: 23239334.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry--a method comparison and validation. AU - Bruce,Stephen J, AU - Rochat,Bertrand, AU - Béguin,Alexandre, AU - Pesse,Benoît, AU - Guessous,Idris, AU - Boulat,Olivier, AU - Henry,Hugues, PY - 2012/08/23/received PY - 2012/10/12/revised PY - 2012/10/12/accepted PY - 2012/12/15/entrez PY - 2012/12/15/pubmed PY - 2013/4/30/medline SP - 200 EP - 6 JF - Rapid communications in mass spectrometry : RCM JO - Rapid Commun Mass Spectrom VL - 27 IS - 1 N2 - RATIONALE: The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS: A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS: Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS: Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3. SN - 1097-0231 UR - https://www.unboundmedicine.com/medline/citation/23239334/Analysis_and_quantification_of_vitamin_D_metabolites_in_serum_by_ultra_performance_liquid_chromatography_coupled_to_tandem_mass_spectrometry_and_high_resolution_mass_spectrometry__a_method_comparison_and_validation_ DB - PRIME DP - Unbound Medicine ER -