Determination of potential vitamin D₂ (the sum of vitamin D₂ and pre-D₂) in UV irradiated products of ergosterol was investigated by a GLC method. Both vitamin D₂ and pre-D₂ were quantitatively isomerized into the thermal cyclized products “pyro- and isopyro-D₂” by GLC. It was previously reported that the separation of pyro-D₂ from lumisterol₂ was difficult (6, 7), but their separation has been found to be successfully achieved by applying the TMS ethers of samples to the GLC using the following apparatus and analytical conditions: apparatus, a Shimadzu GC-4APTF gas chromatograph equipped with a hydrogen flame ionization detector; column, a glass column (0.4×150cm) packed with 1.5% OV-17 on Shimalite W (80-100 mesh); column temperature, 215°; flow rate of nitrogen gas, 80ml/min. Recovery tests of vitamin D₂ in the mixtures of vitamin D₂ isomers by the GLC gave good results, and no special inhibitory peak was observed in the gas chromatogram of irradiated ergosterol products. From these results, the GLC procedure applicating the operating parameters mentioned above after trimethylsilylation of samples and using the peaks of pyro-D₂ TMS ether and stigmasteryl acetate as an internal standard was established as a routine method for the determination of potential vitamin D₂ in irradiated ergosterol products.
The effects of ergosterol concentrations, temperatures, solvents, use of a filter solution and light sources on the yield of potential vitamin D₂ were examined by using this method. The effects of light sources and ergosterol concentrations were large, whereas the other conditions gave little effects.