UHPLC Analysis of Vitamin D2 & D3 Metabolites and Epimers on Supel™ Carbon LC Columns

William L. Maule III , Clint Corman , Michael Ye , Cory Muraco , Curtis Frantz, Merck, Bellefonte, PA

Introduction

Analysis of vitamin D metabolites has continued to be a topic of interest in recent publications, primarily as biomarkers for possible disease states and vitamin deficiency. While vitamin D is present in two forms, vitamin D3 and vitamin D2, current ELISA methods demonstrate different cross-reactivities and cannot distinguish between D2 and D3 forms of the vitamin metabolites resulting in erroneous reporting of total 25-hydroxyvitamin D concentrations. Further, there is interest in an analytical method to differentiate the D2 and D3 forms from the D2 and D3 epimers because of their different degrees of bioactivity. This application demonstrates the use of the Supel™ Carbon LC UHPLC column, with its ability to resolve structural isomers, to baseline separate all four analytes.

25-hydroxyvitamin D3

3-epi-25-hydroxyvitamin D3

3-epi-25-hydroxyvitamin D2

25-hydroxyvitamin D2

Chemical Structures of Vitamin D Metabolites

Experimental Conditions for vitamin D2 & D3 Metabolites Analysis

Table 1. Chromatographic Conditions for Analysis of Vitamin D Metabolites on Supel™ Carbon LC

Experimental Conditions
Column	Supel™ Carbon LC, 10 cm x 2.1 mm I.D., 2.7 µm
Detection	UV; 275 nm
Mobile phase	[A] 2-Propanol; [B] Tetrahydrofuran
Gradient	Time (min) 0 15 20 30	%A 100 30 30 100	%B 0 70 70 0
Test Mix	Vitamin D2 & D3 Test Mix, varied concentration, ethanol
Injection Volume	2.0 µL
Flow Rate	0.30 mL/min
Temperature	Column: 25 °C

Table 2. Components of Test Mix Used in Study

Test Mix Analyte/Concentration
1	3-epi-25-hydroxyvitamin D3 (50 µg/mL)
2	25-hydroxyvitamin D3 (25 µg/mL)
3	3-epi-25-hydroxyvitamin D2 (50 µg/mL)
4	25-hydroxyvitamin D2 (100 µg/mL)

Performance Results

Figure 2. Analysis of Vitamin D Metabolites on Supel™ Carbon LC

Table 3. Elution Order of Compounds Seen in Figure 2.

Peak	Compound	Ret. Time (min)
1	3-epi-25-hydroxyvitamin D3 (50 µg/mL)	8.294
2	25-hydroxyvitamin D3 (25 µg/mL)	9.125
3	3-epi-25-hydroxyvitamin D2 (50 µg/mL)	11.126
4	25-hydroxyvitamin D2 (100 µg/mL)	12.052

Conclusion

This application has demonstrated the use of the Supel™ Carbon LC column to resolve vitamin D2 and D3 metabolites and their epimers. Baseline separation of all four analytes was achieved with excellent peak shape and sensitivity.