MSACL 2016 EU Abstract

The Analysis of C3-Epimers of 25-Hydroxyvitamin D in Serum by LC-MS/MS

Susan Steinike (Presenter)
Restek Corporation

Authorship: Susan Steinike, Frances Carroll, Shun-Hsin Liang,
Restek Corporation, 110 Benner Circle Bellefonte, PA 16823

Short Abstract

Vitamin D exists in two forms, vitamin D2 and vitamin D3; each undergoes metabolism to form 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] which are used as the biomarkers for the assessment of vitamin D status. The epimeric forms of 25(OH)D, 3-epi-25-hydroxyvitamin D2 and D3 may contribute to a large portion of the total 25(OH)D concentration. Since these epimers are isobaric, chromatographic separation is necessary for accurate quantitation. In this study, the Raptor™ FluoroPhenyl column was used for chromatographic separation of 25(OH)D and their C3-epimers.

Long Abstract

Vitamin D analysis has increased dramatically in clinical practice due to its association with multiple human diseases and the prevalence of vitamin D deficiency worldwide. Vitamin D exists in two forms, vitamin D2 and vitamin D3; each undergoes metabolism to form 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] which are used as the biomarkers for the assessment of vitamin D status. The epimeric forms of 25(OH)D, 3-epi-25-hydroxyvitamin D2 and D3, have been identified and may contribute to a large portion of the total 25(OH)D concentration, particularly in infant populations. Studies have shown that the C3 epimers have much lower bioactivity than the primary metabolites; therefore, a specific quantitation of these epimers is necessary for a proper clinical assessment of vitamin D status. Since these epimers are isobaric, chromatographic separation is necessary for accurate quantitation. In this study, the Raptor™ FluoroPhenyl column was used for chromatographic separation of 25(OH)D and their C3-epimers. The established chromatographic method was able to accurately quantitate the 3-epi-25-hydroxyvitamin D2 and D3 metabolites in fortified beagle serum.

Serum was fortified with four analytes, 25(OH)D2, 25(OH)D3, 3-epi-25(OH)D2, and 3-epi-25(OH)D3, and extracted using a liquid-liquid extraction (LLE) method. Serum (400µL) was mixed with 15µL of internal standard solution (1µg/mL of d6-25-OH-D3 in methanol), 0.2 M ZnSO4 (400µL) and methanol (400µL) in a 4-mL glass vial. A 2mL aliquot of hexane was added, mixed for 90 seconds, and then centrifuged for 10 minutes at 4300rpm. The hexane layer was removed and evaporated to dryness under nitrogen at 55°C. The dried extract was reconstituted with 100µL of a 50:50 water:methanol solution and injected (10 µL) for analysis on a Shimadzu Nexera XR UHPLC coupled to a Sciex API 4000™ mass spectrometer.

The calibration standards were prepared in synthetic human serum, SeraFlex LCMSMS, and subjected to the LLE procedure. Good linearity was obtained for all analytes with a concentration range of 1 to 100ng/mL (with 1/x weighting). Standard deviations were ≤10% (the lowest concentration was ≤20%) and R-squared values were 0.996-0.999 for all compounds. The quantitative results of 3 QC levels of fortified synthetic serum samples showed acceptable method accuracy with percent recovery within 10% of the nominal concentration for all QC levels. The %RSD values ranged from 0.9-6.6% and 2.2-4.5% for intra-day and inter-day analyses, respectively, indicating an acceptable method precision. The validated method was used to analyze the 8ng/mL fortified beagle serum which showed acceptable accuracy and precision.

It was demonstrated that the Raptor™ FluoroPhenyl column can provide unique selectivity for accurate and differential quantitation of 25-hydroxyvitamin D and C3-epimers in serum. The chromatographic analysis was performed using 0.1% formic acid in water and methanol as mobile phases with a 7-minute analysis time. The analytical method is applicable to the clinical analysis of total 25-hydorxyvitamin D concentration and provides the option to report the C3-epimer concentrations separately.


References & Acknowledgements:


Financial Disclosure

DescriptionY/NSource
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SalaryyesRestek Corporation
Board Memberno
Stockyes Restek Corporation
Expensesyes

IP Royalty: yes

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