I am currently working on the Vit D3 Method 1 from the USP. Here are the analytical conditions:
Column: Spherisorb NH2 (150 mm x 4.6 mm, 3 um particle size), Packing L8
Mobile Phase: Hexane, IPA (99:1)
Flow rate: 1.0 ml/min
Column temp: 25 deg C
Detection: UV; 265 nm
Sample volume: 100 ul
When I inject my working standard (2 ug/ml) of Vit D3, I get two peaks. One smaller peak with an RT of ~3.2 (7-dehydrocholestrol) and a much larger peak with an RT ~4.2 (cholecalciferol~Vit D3). The resolution solution is made by heating a portion of the working standard for 1 hour at 60 deg C in a waterbath. Once injected, the peak area of the Vit D3 decreases and the peak area of it's precursor, 7-dehydrocholestrol increases. I used Vit D2 as an internal standard but with the analytical conditions set forth, the Vit D2 only added to the area of Vit D3. So I will have to find another internal standard
Here are a few questions I have:
Has anyone tried this analytical condition? If so , does this retention time seem too quick off the column? I see other posts mention 15-20 min retention time but that is with different packings I think. I let each sample run time go for 6 minutes.
The method calls for n-hexane but the HPLC hexane is only 60% n-hexane, but is >99% total hexanes. This is still of high enough purity to use since the HPLC column will not seperate these isomers unless using chiral HPLC..Is this correct?