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25-Hydroxyvitamin D Isomerizes to pre-25-hydroxyvitamin D in Solution: Considerations for Calibration in Clinical Measurements

25-Hydroxyvitamin D Isomerizes to pre-25-hydroxyvitamin D in Solution: Considerations for Calibration in Clinical Measurements

Mary Bedner, Katrice A Lippa

Anal Bioanal Chem. 2015 Oct;407(26):8079-86.

PMID: 26359234

Abstract:

Reference standards for the vitamin D metabolites 25-hydroxyvitamin D3, 25-hydroxyvitamin D2, and 3-epi-25-hydroxyvitamin D3 were evaluated using liquid chromatography (LC) with ultraviolet (UV) absorbance and mass spectrometric (MS) detection to assess purity. The chromatograms for solutions of all three 25(OH)D compounds, obtained using a pentafluorophenyl (PFP) stationary phase, revealed peaks that increased in area over time and had MS spectra that were nearly identical to the parent compound, indicating isomers had formed in solution that were unrelated to the reference standard purity. However, when the purity evaluations were completed with a cyanopropyl stationary phase, the isomeric products coeluted with the parent compounds and were not observable. The rates of formation of the isomeric products were found to increase when heated and were confirmed to be pre-25-hydroxyvitamin D compounds using spectral information from both MS detection and nuclear magnetic resonance (NMR) spectroscopy. The rates of conversion of 25(OH)D3 to pre-25(OH)D3 was studied in solutions of ethanol and bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The solutions prepared with BSA/PBS were found to form twice as much pre-25(OH)D3 as the solutions in ethanol. The isomerization of 25(OH)D in solution has implications for calibration of 25(OH)D in clinical measurements, which are discussed.

Chemicals Related in the Paper:

Catalog Number	Product Name	CAS Number	Price
AP63283363	25-Hydroxyvitamin D3 solution	63283-36-3	Price
AP73809059	3-epi-25-Hydroxyvitamin D3 solution	73809-05-9	Price
AP908126487	3-epi-25-Hydroxyvitamin D2 solution	908126-48-7	Price
IAR42410580	Vitamin D3-25,26,27-13C3 solution		Price

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