Examination of structurally selective derivatization of vitamin D3 analogues by electrospray mass spectrometry

The structural specificity of vitamin D derivatization by PTAD (4‐phenyl‐1,2,4‐triazoline‐3,5‐dione) was probed using synthetic analogues and ion trap mass spectrometry. EB 1089, a vitamin D3 analogue which contains a second site for Diels–Alder cycloaddition on its side‐chain, allowed the examination of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD‐vitamin D3 ion fragment, commonly used in metabolite characterization and quantitation of vitamin D3 analogues (m/z 314), were established; ion trap mass spectrometry revealed that the PTAD comprises a portion of this diagnostic fragment, and is not lost by a retro‐Diels–Alder step. Furthermore, the unique structure of the EB 1089 side‐chain also permits facile determination of its side‐chain metabolism. Use of PTAD derivatization and detection of metabolite‐specific ion fragments identify hydroxylation at the end of the EB 1089 sidechain. It is believed that the results from these studies provide a clearer understanding of the mass spectrometry of triazolinedione derivatives, not only in the specific case of EB 1089, but also in their application to other vitamin D compounds. Copyright © 2001 John Wiley & Sons, Ltd.