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Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.

Abstract

BACKGROUND

An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum.

METHODS

Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples.

RESULTS

Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3.

CONCLUSION

This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.

Authors+Show Affiliations

Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands. Electronic address: j.v.d.ouweland@cwz.nl.Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.Department of Clinical Chemistry, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

25125396

Citation

van den Ouweland, Johannes M W., et al. "Overestimation of 25-hydroxyvitamin D3 By Increased Ionisation Efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS Methods Not Separating Both Metabolites as Determined By an LC-MS/MS Method for Separate Quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 967, 2014, pp. 195-202.
van den Ouweland JM, Beijers AM, van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;967:195-202.
van den Ouweland, J. M., Beijers, A. M., & van Daal, H. (2014). Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 967, 195-202. https://doi.org/10.1016/j.jchromb.2014.07.021
van den Ouweland JM, Beijers AM, van Daal H. Overestimation of 25-hydroxyvitamin D3 By Increased Ionisation Efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS Methods Not Separating Both Metabolites as Determined By an LC-MS/MS Method for Separate Quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in Human Serum. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Sep 15;967:195-202. PubMed PMID: 25125396.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. AU - van den Ouweland,Johannes M W, AU - Beijers,Antonius M, AU - van Daal,Henny, Y1 - 2014/07/31/ PY - 2014/04/18/received PY - 2014/07/10/revised PY - 2014/07/14/accepted PY - 2014/8/16/entrez PY - 2014/8/16/pubmed PY - 2015/5/12/medline KW - 25-hydroxyvitamin D KW - 3-epi-25-hydroxyvitamin D(3) KW - C(3)-epimer KW - Ionisation behaviour KW - LC–MS/MS KW - MS response factor SP - 195 EP - 202 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J Chromatogr B Analyt Technol Biomed Life Sci VL - 967 N2 - BACKGROUND: An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. METHODS: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. RESULTS: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. CONCLUSION: This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3. SN - 1873-376X UR - https://www.unboundmedicine.com/medline/citation/25125396/Overestimation_of_25_hydroxyvitamin_D3_by_increased_ionisation_efficiency_of_3_epi_25_hydroxyvitamin_D3_in_LC_MS/MS_methods_not_separating_both_metabolites_as_determined_by_an_LC_MS/MS_method_for_separate_quantification_of_25_hydroxyvitamin_D3_3_epi_25_hydroxyvitamin_D3_and_25_hydroxyvitamin_D2_in_human_serum_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(14)00478-4 DB - PRIME DP - Unbound Medicine ER -