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;46:226-230
© 2009 Association for Clinical Biochemistry


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Original Articles

Susan Knox1,
John Harris2,
Lisa Calton3 and
A Michael Wallace1

1 Department of Clinical Biochemistry, Macewen Building, Glasgow Royal Infirmary, Glasgow G4 0SF;
2 Presearch, Kingsland Business Park, Basingstoke, Hampshire RG24 8PZ;
3 Waters, Atlas Park, Manchester M22 5PP, UK

Corresponding author: Susan Knox, Department of Clinical Biochemistry, Macewen Building, Glasgow Royal Infirmary, Glasgow G4 0SF, 0141 211 4365, UK. Email: susan.knox{at}ggc.scot.nhs.uk

Background: Measurement of 25-hydroxyvitamin D3 (25OHD3) and D2 (25OHD2)is challenging. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) methods have been described but they are often complexand difficult to automate. We have developed a simplified procedureinvolving an automated solid-phase extraction (SPE).

Methods: Internal standard (hexadeuterated 25-hydroxyvitamin D3) was added to serum or plasma followed by protein precipitation with methanol. Following centrifugation, a robotic instrument (CTC PAL [Presearch] for ITSPTM SPE [MicroLiter Analytical Supplies, Inc]) performed a six-step SPE procedure and the purified samples were injected into the LC-MS/MS. Quantification of 25OHD3 and 25OHD2 was by electrospray ionization MS/MS in the multiple-reactionmonitoring mode.

Results: The lower limit of quantitation was 4.0 nmol/L for 25OHD3 and 7.5 nmol/L for 25OHD2. Within- and between-assay precision was below 10% over the concentration range of 22.5–120 nmol/L for D3 and 17.5–70 nmol/L for D2 (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) withthe IDS-OCTEIA 25-hydroxyvitamin D enzyme immunoassay for samplescontaining less than 125 nmol/L, at higher concentrations theimmunodiagnostic system (IDS) method showed positive bias.

Conclusions: Our simplified sample preparation and automated SPE method is suitable for the measurement of 25OHD3 and D2 in a routine laboratoryenvironment. The system can process up to 300 samples per daywith no cumbersome solvent evaporation step and minimal operatorintervention.

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