Two polar metabolites of vitamin D3 have been isolated from the plasma of a hypoparathyroid patient that had been treated with 1.5 mg of vitamin D3 per day. One of the metabolites (peak IV) has been conclusively identified as 25-hydroxycholecalciferol (25-HCC) by means of mass spectrometry, ultraviolet absorption spectrophotometry, and gas-liquid chromatography. Furthermore, this metabolite has the same potency as 25-HCC in the cure of rickets in rats. The second metabolite (peak V) was identified as 21,25-dihydroxycholecalciferol (21,25-DHCC) on the basis of its ultraviolet absorption spectrum and cochromatography with authentic 21,25-DHCC in three different chromatographic systems.
Michael F. Holick, Hector F. DeLuca, Louis V. Avioli. Isolation and Identification of 25-Hydroxycholecalciferol From Human Plasma. Arch Intern Med. 1972;129(1):56–61. doi:10.1001/archinte.1972.00320010060005