Chromatography Forum

Does anyone have any suggestions as to the best way to analyze Vitamin D3. It seems like it can be done by reversed phase and normal phase - and probably some other modes as well.

Please let me know of any thought or suggestions.

Much Thanks
Adam

The chromatography is the least of your worries. Even in vitamin supplements, the amounts are low. The sample extraction, concentration, and cleanup is the challenge. I would start by looking at AOAC Official methods, journals of food science or nutrition, and of course Google.

I'd highly recommend starting with :-
" Modern Chromatographic Analysis of Vitamins " by Andre P. De Leenheer, Willy E. Lambert, and Jan F. Van Bocxlaer.
I have access to the 3rd Edition, ISBN 082470316-2 ( 2000 ).
Chpater Two, on Vitamin D and analogues analysis is about 75 pages long, and covers many of the issues.

One of the main issues is light and oxidation stability, especially after the vitamin has been extracted from a sample.

If you are analysising for regulatory purposes, it's especially important to follow the prescibed method completely and carefully.

Good luck,

Bruce Hamilton

We are trying to get the book. But - in the meantime - let me ask the question this way:

"Is there any downside to using a reversed phase method for Analysis of Vitamin D3"

Thank You

Vitamin D2 and D3 were separated on our Cadenza CD-C18 column.
I'd recommend using similar conditions if you are going to use RP:

http://www.imtakt.com/TecInfo/TI121E.pdf

From the suggested book - I just skimmed the chapter...

" The limited use of reversed-phase packings stems from
the aversion of vitamin D analysts to aqueous mobile phases, particularly when competitive binding assay of collected fractions is used for quantitation, since aqueous phases are difficult to evaporate, because they tend to dissolve support material, and, since they contain dissolved oxygen, may give rise to oxidation to the 5,7-diene system. Second, the resolution of the dihydroxylated compounds is not as good as that achieved using silica. Plate counts on bonded-phase columns
are generally lower, pressures are higher, and column lifetimes are shorter than systems using straight-phase silica LC.... "

Bruce Hamilton

Thanks very much guys.

Let me ask one last (most specific) question.

Does anybody see any problems with analyzing Vitamin D3 on a Waters Diol column.

This is what makes the most sense to me - but I don't see any such applications. The one I got from Waters was a reversed phase method - which won't work for our application

We have a normal phase chromatogram using our Unison UK-Amino column - you'll notice Vitamin D2 and Vitamin D3 co-elute - but that can actually be useful depending on your objective:

http://www.imtakt.com/TecInfo/TI330E.pdf