Endocrine Abstracts

Background: The C-3 epimer of 25 Hydroxyvitamin D₃ (3-Epi-25OHD₃) is produced in the liver by the epimerisation pathway of 25-hydroxy vitamin D₃. It differs from 25OHD₃ in configuration of the hydroxyl group at the third carbon (C-3) position. Despite the fact that little is known regarding its clinical significance, concerns have been raised that isobaric interference may result in over-estimation of total 25OHD when measured by liquid chromatography tandem mass spectrometry (LC–MS/MS).

Objective: The aim of the study was to develop a chromatographic technique to resolve 3-Epi-25OHD₃ from 25OHD₃ and quantify by LC–MS/MS. The method was used to identify the presence of 3-Epi-25OHD₃ in samples received for 25OHD measurement at the Norfolk and Norwich University Hospital.

Method: Serum was precipitated with zinc sulphate and acetonitrile containing hexa-deuterated 25OHD₃. The extract was chromatographed using a 2.6 μm 100×2.1 mm I.D. pentofluorophenyl solid core particle column. Mass detection and quantification were performed by positive electrospray ionization with MS/MS in multiple reaction monitoring mode.

Results: The method was able to fully resolved 3-Epi-25OHD₃ from 25OHD₃. The intraassay CV for the epimer were 6.3 and 4.1% at 25.4 and 62.1 nmol/l respectively; and interassay CV were 8.3 and 6.5% at 27.6 and 63.2 nmol/l, respectively. In the patient samples tested, we found an average of 4.2 nmol/l of 3-Epi-25OHD₃ present. One patient sample had a 25OHD₃ of 187 nmol/l that was shown to contain 141 nmol/l of 25OHD₃ and 44 nmol/l of 3-Epi-25OHD₃.This patient was receiving a high dose of vitamin D supplementation.

Conclusion: We have developed a method for 3-Epi-25OHD₃ that is quick and robust for routine use. The method can be easily adapted into any existing LC–MS/MS system set up for 25OHD measurement. Although the prevalence of 3-Epi-25OHD₃ was found to be low, 3-Epi-25OHD₃ can readily be separated to ensure accurate measurement of total 25OHD₃. This will facilitate further research into the clinical importance of the epimer.