Annals of Clinical Biochemistry > Volume 46, Number 3 > Pp. 226-230

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Original Articles

A simple automated solid-phase extraction procedure for measurement of 25-hydroxyvitamin D₃ and D₂ by liquid chromatography-tandem mass spectrometry

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 complex and difficult to automate. We have developed a simplified procedure involving 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 ITSP^TM 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-reaction monitoring 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) 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 25OHD₃ and D₂ 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.

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