Examination of structurally selective derivatization of vitamin D-3 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 Dg analogue which contains a second site for Diels-Alder cycloaddition on its side-chain, allowed the examinati on of derivatization modes and comparisons of ion fragment structures. The origins of a PTAD-vitamin D-3 ion fragment, commonly used in metabolite cha racterization and quantitation of vitamin D-3 analogues (m/z 314), were est ablished; ion trap mass spectrometry revealed that the PTAD comprises a por tion of this diagnostic fragment, and is not lost by a retro-Diels-Alder st ep. Furthermore, the unique structure of the EB 1089 side-chain also permit s facile determination of its side-chain metabolism. Use of PTAD derivatiza tion and detection of metabolite-specific ion fragments identify hydroxylat ion at the end of the EB 1089 sidechain. It is believed that the results fr om these studies provide a clearer understanding of the mass spectrometry o f triazolinedione derivatives, not only in the specific case of EB 1089, bu t also in their application to other vitamin D compounds. Copyright (C) 200 1 John Wiley & Sons, Ltd.