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A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry.
Ann Clin Biochem. 2009 May; 46(Pt 3):226-30.AC

Abstract

BACKGROUND

Measurement of 25-hydroxyvitamin D(3) (25OHD(3)) and D(2) (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE).

METHODS

Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode.

RESULTS

The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D(3) and 17.5-70 nmol/L for D(2) (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias.

CONCLUSIONS

Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) and D(2) in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention.

Authors+Show Affiliations

Department of Clinical Biochemistry, Macewen Building, Glasgow Royal Infirmary, Glasgow G4 0SF, UK. susan.knox@ggc.scot.nhs.ukNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article

Language

eng

PubMed ID

19389886

Citation

Knox, Susan, et al. "A Simple Automated Solid-phase Extraction Procedure for Measurement of 25-hydroxyvitamin D3 and D2 By Liquid Chromatography-tandem Mass Spectrometry." Annals of Clinical Biochemistry, vol. 46, no. Pt 3, 2009, pp. 226-30.
Knox S, Harris J, Calton L, et al. A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry. Ann Clin Biochem. 2009;46(Pt 3):226-30.
Knox, S., Harris, J., Calton, L., & Wallace, A. M. (2009). A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry. Annals of Clinical Biochemistry, 46(Pt 3), 226-30. https://doi.org/10.1258/acb.2009.008206
Knox S, et al. A Simple Automated Solid-phase Extraction Procedure for Measurement of 25-hydroxyvitamin D3 and D2 By Liquid Chromatography-tandem Mass Spectrometry. Ann Clin Biochem. 2009;46(Pt 3):226-30. PubMed PMID: 19389886.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D3 and D2 by liquid chromatography-tandem mass spectrometry. AU - Knox,Susan, AU - Harris,John, AU - Calton,Lisa, AU - Wallace,A Michael, PY - 2009/4/25/entrez PY - 2009/4/25/pubmed PY - 2009/6/24/medline SP - 226 EP - 30 JF - Annals of clinical biochemistry JO - Ann Clin Biochem VL - 46 IS - Pt 3 N2 - BACKGROUND: Measurement of 25-hydroxyvitamin D(3) (25OHD(3)) and D(2) (25OHD(2)) is challenging. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods have been described but they are often complex and difficult to automate. We have developed a simplified procedure involving an automated solid-phase extraction (SPE). METHODS: Internal standard (hexadeuterated 25-hydroxyvitamin D(3)) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSP SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD(3) and 25OHD(2) was by electrospray ionization MS/MS in the multiple-reaction monitoring mode. RESULTS: The lower limit of quantitation was 4.0 nmol/L for 25OHD(3) and 7.5 nmol/L for 25OHD(2). Within- and between-assay precision was below 10% over the concentration range of 22.5-120 nmol/L for D(3) and 17.5-70 nmol/L for D(2) (n = 10). The calibration was linear up to 2500 nmol/L (r = 0.99). Recoveries ranged between 89% and 104% for both metabolites and no ion suppression was observed. The results obtained compared well (r = 0.96) with the IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samples containing less than 125 nmol/L, at higher concentrations the immunodiagnostic system (IDS) method showed positive bias. CONCLUSIONS: Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD(3) and D(2) in a routine laboratory environment. The system can process up to 300 samples per day with no cumbersome solvent evaporation step and minimal operator intervention. SN - 0004-5632 UR - https://www.unboundmedicine.com/medline/citation/19389886/A_simple_automated_solid_phase_extraction_procedure_for_measurement_of_25_hydroxyvitamin_D3_and_D2_by_liquid_chromatography_tandem_mass_spectrometry_ L2 - https://journals.sagepub.com/doi/10.1258/acb.2009.008206?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -