Use of Fourier transform 1H NMR in the identification of vitamin D2 metabolites.

The use of Fourier transform 1H NMR to characterize vitamin D2 metabolites is described. A 300-MHz spectrometer capable of generating a 6-microseconds pulse and a sweep width of 4000 Hz was used. High-resolution spectra were obtained on 5 micrograms of material using standard 5-mm NMR tubes fitted with glass inserts and isotopically enriched chloroform-d solvent. The data acquisition time under these conditions was 4 h. Application of this technique to a variety of both synthetic and naturally occurring vitamin D2 metabolites, in addition to synthetic delta 22-1,25-dihydroxyvitamin D3, resulted in the reassignment of the chemical shifts for the C-21 and C-28 methyl groups of vitamin D2. The C-21 methyl group resonance is now assigned to the doublet appearing at delta 1.01, whereas the C-28 signal corresponds to the doublet at delta 0.90. An examination of the spectrum of 24 (R),25-dihydroxyvitamin D2 also led to the reassignment of the side-chain methyl group resonances. This technique is an additional means of identifying microgram quantities of vitamin D metabolites.