Chromatography Forum

Dear Friends,

I need of a method for determination of Isotretinoinand Tretinoin by HPLC. I tried with Methanol as solvent and reverse phase of USP, but the Tretinoin peak are degrading and the Isotretinoin peack are increasing enough.

Thank you.

Carlos Teixeira

Is the peak degrading in the sample solvent? Maybe you need to change that?

Carlos,

These two compounds are known to be photolabile, and will establish an equilibrium unless protected from light. (May also be thermally interconvertible ???).

Uwe suggestion to change diluent away from methanol is appropriate.

Maybe also run the autosampler tray and column at lowered temperatures and store in actinic (amber) vials/ graduated flasks etc.

Please let us know how you resolve the issue.

JMB

In USP 27 ( the latest I have immediate access to ), the related substances method used is normal phase (iC8, IPA, HOAc ), with silica column, but the EP uses a reverse phase with C18 column and methanol-based acidic mobile phase. The EP strongly cautions about heat, light, and oxidation, the USP about light. The compounds are known to be unstable in solution.

You could try the ( older? ) USP normal phase related substance method, but if they have changed to reverse phase, the method may be fragile. I'd be inclined just to follow the standard related substances methods and perform the analysis quickly trying to eliminate the known degradants.

If you want, you could contact the authors of the following study and ask about their methods, more details available at Pubmed.

Pharmaceutical quality of generic isotretinoin products, compared with Roaccutane.
Taylor P.W., Keenan M.H.
Curr Med Res Opin 2006 Mar;22(3):603-15.

Good luck,

Bruce Hamilton

Dear Friends,

Thanks a lot.

Your help were very important to my project.

Bye

Carlos Teixeira

Carlos,

The analysis can be accomplished by using a C18 column with 1 meter of heated stainless steel tubing prior to the column.

HPLC Chemist