Chromatography Forum

Hi there, I am working on HPLC assay, the active is ketoconazole. It is dissolved in petrolatum. I heat the formulation to 70C, which is above the melting point for the petrolatum. Then I analyse on the column. The results are variable, and although there is a guard column, the main column is quickly blocked, and washing doesnt help. Could this be due to the nature of the extraction procedure?

Is it an acceptable method of extraction
Thanks for your help
Scio2

You need to give more details. What are your analytical conditions (mobile phase, column, detection, etc.)? From what I can tell, you are not doing any sample preparation. Just melting the sample?

If you are using reverse phase, it is no surprise that you are having major problems. For that compound in petrolatum matrix, I would use normal phase chromatography. Perhaps a hexane/acetone mobile phase with silica column would be a good place to start. With normal phase, you will only have to dilute the sample in your mobile phase and inject.

I'd agree with Sassman's comments except that I'd use a CN bonded phase column instead of bare silica (fewer problems with trace water content in the mobile phase) and start by running a gradient from 1-100% iPrOH/hexane or 0-100% MTBE/hexane to figure out where your compound elutes (then switch to an appropriate isocratic). I'd avoid acetone because of the high UV cutorr.

What's ketoconazole soluble in? If it's soluble in solvents like ACN, methanol, or DMF, I think I'd extract with one of those at higher temperatures to dissolve/disperse the petrolatum, then cool and let the petrolatum kick out of solution, then assay what's soluble in the liquid. Or dissolve in hexane-type solvent and add other orgaic solvent to kick the petrolatum out of solution, chill if necessary.

Thanks everyone,
Much appreciated.
Scio