MSACL 2016 US Abstract

Separating Vitamin D2 D3, their 25-OH Metabolites and C-3 Epimers

Ken Tseng (Presenter)
Nacalai USA Inc.

Authorship: Ken Tseng¹, Toshi Ono¹, Tsunehisa Hirose²
¹Nacalai USA, Inc., San Diego, United States, ²Nacalai Tesque, Inc., Kyoto, Japan

Short Abstract The accuracy of current vitamin D measurements by immunoassays and LCMS has been questioned due to the overlapping LC peaks with identical m/z values epimers and isobars. To solve this problem, we have developed a new HPLC method to achieve baseline separation of vitamin D2/D3, their 25-OH metabolites and C3-epimers in one single run.

Long Abstract

The accuracy of vitamin D measurements by immunoassays and LCMS has been questioned. The amount of its major metabolites measured can differ dramatically for some patients depending on the analytical methods chosen. The overlapping LC peaks with identical m/z values of the vitamin D2/D3 epimers and isobars are likely cause of this inconsistency.

We are proposing a new LCMS method with the baseline separation of all four vitamin metabolites, 25-OH-vitamin D3, 3-epi-25-OH-vitamin D3, 25-OH-vitamin D2, and 3-epi-25-OH-vitamin D2. A novel core-shell type reversed-phase HPLC column with Cholesterol as the functional group (Cosmocore Cholester) is used in this study. The Cosmocore Cholester column has similar hydrophobicity as regular C18 columns, so it is used in the same exact way. The mobile phase is a simple 0.1% formic acid in water and MeOH mixture in isocratic mode. The elution order is 25-OHD3, 3-epi-25-OHD3, 25-OHD2, and 3-epi-25-OHD2.

The baseline separation is so complete that it can be used for quantification by UV detector alone at low concentrations. Gradient condition can be employed to further separate vitamin D2 and vitamin D3 and their four metabolites/epimers, all in one LCMS (or UV) run.

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Financial Disclosure

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