Use of mass spectrometry in the identification of in vivo and in vitro metabolites of dihydrotachysterol3 in the rat.

The metabolism of dihydrotachysterol3 (DHT3), a vitamin D analogue, has been investigated in vivo in the rat after intraperitoneal injection, and the metabolism of the 25-hydroxylated metabolite of DHT3 was studied in vitro in the isolated perfused rat kidney. A large number of metabolites have been obtained and some have been identified. The rat plasma or kidney perfusate were extracted and the metabolites separated by high-performance liquid chromatography (HPLC) in straight- and reverse-phase systems and using cyano columns. Metabolites were identified, using a photodiode array assembly which monitored the HPLC eluate, by the characteristic ultraviolet spectrum of DHT compounds. Tentative structures were assigned to some of the metabolites obtained on the basis of their mobility in the various HPLC systems used in comparison to that of known metabolites of vitamin D. Gas chromatography/mass spectrometry (GC/MS) and direct probe mass spectrometry have been used to confirm the identity of seven metabolites formed in vitro, of which only two have been definitely shown also to be formed in vivo. GC/MS was carried out after derivatization forming trimethylsilyl ethers, n-butyl boronate cyclic esters, and N-O-methyl oximes before and after oxidation with sodium periodate and/or reduction with sodium borohydride. Molecular ions of these compounds are usually of low abundance and characteristic mass fragments at m/z 273, 255 and 121 are always seen with metabolites of DHT.