High-throughput liquid-liquid extraction and LCMSMS assay for determination of circulating 25(OH) vitamin D3 and D2 in the routine clinical laboratory.
BACKGROUND
Increasing focus on vitamin D as essential to health has underscored the need for accurate and precise high-throughput measurement of serum 25(OH)D.


METHODS
Serum was denatured in acetonitrile containing hexadeuterated 25(OH)D3 as internal standard (IS) and automatically applied to filter plates packed with inert diatomous earth material for subsequent heptane extraction. Extracts were chromatographed on a C12 HPLC column, and detected on a triple quadropole mass spectrometer.


RESULTS
The inter-assay precision was 9.4% and 8.8% respectively at 32.0 and 59.7 nmol/l for 25(OH)D3 and 8.6% and 8.0% at 23.4 and 64.4 nmol/l for 25(OH)D2. The detection limit was 10 nmol/l for both metabolites. Three percent of samples contained >50 nmol/l 25(OH)D2. Total run time was 4 min. We have performed more than 200,000 routine samples and the method performs well in external control schemes.


CONCLUSIONS
We describe a robust, high-throughput, LLE-LCMSMS method for accurate and precise quantitation of 25(OH)D3 and 25(OH)D2 in serum. The use of diatomaceous earth material for extraction of vitamin D in 96-well format enables fast, simple and efficient sample preparation. The method offers a cost-effective alternative to immunological methods for use in the routine clinical biochemical laboratory.
Department of Clinical Biochemistry, Arhus Sygehus, Aarhus University Hospital, Denmark.
Carsten S
CS
Højskov
Lene
L
Heickendorff
Holger J
HJ
Møller
D015652:25-Hydroxyvitamin D 2 / Q000097:blood*; D002112:Calcifediol / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015652:25-Hydroxyvitamin D 2; D002112:Calcifediol