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Isotope Dilution Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in Human Serum

Isotope Dilution Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Simultaneous Measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in Human Serum

Rosemary L Schleicher, Sara E Encisco, Madhulika Chaudhary-Webb, Ekaterina Paliakov, Leslie F McCoy, Christine M Pfeiffer

Clin Chim Acta. 2011 Aug 17;412(17-18):1594-9.

PMID: 21601563

Abstract:

Background:
An ultra performance liquid chromatography-tandem mass spectrometry method with calibration traceable to NIST SRM was developed and validated to measure concentrations of 25-hydroxyvitamin D(2) (25OHD(2)), 25-hydroxyvitamin D(3) (25OHD(3)) and the C-3 epimer of 25OHD(3) (epi-25OHD(3)) in human serum.
Methods:
Tri- and hexa-deuterated internal standards were added to serum (100 μl) to monitor recovery. Liquid-liquid extraction was used to extract the hexane-soluble materials. Calibration solutions [8-100 nmol/L 25OHD(2,) 12-150 nmol/L 25OHD(3), and 4-50 nmol/L epi-25OHD(3)] prepared in phosphate-buffered saline containing 4% albumin were similarly processed. Using a pentafluorophenyl column (2.1×100 mm) and isocratic methanol/water (72/28, v/v) flowing at 0.4 ml/min, run time was 14 min per sample; 25OHD(3) and epi-25OHD(3) were baseline separated. Atmospheric pressure chemical ionization in the positive ion mode with selected reaction monitoring captured the following transitions: 25OHD(2), m/z 395.3>377.3 (209.1 qualifier); (epi-)25OHD(3), m/z 383.3>365.3 (105.1 qualifier); d(3)-25OHD(2), m/z 398.3>380.3; and d(6)-25OHD(3), m/z 389.3>371.3.
Results:
Recovery averaged ≥98%. Total imprecision was ≤10% when concentrations were ≥20 nmol/l. Bias averaged <5%. Detection limits were <5 nmol/l. Median (nmol/l) 25OHD(2), 25OHD(3) and epi-25OHD(3) were quantitated in 98 blood donors (Conclusions:
This method is highly accurate, precise and specific.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP908126487-A	3-epi-25-Hydroxyvitamin D2		908126-48-7	Price

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