Chromatography Forum

Hello All,

I am running a reversed phase HPLC procedure for a drug in animal feed using 85% CH3CN:15% 125 mM aqueous acetate buffer as the mobile phase, with a typical C18 column and fluorescence detection. The sample extract is methanol acidified with 0.5% HCl. I note what appear to be significant carrover peaks in blank feeds at a residue testing level. I do not see such peaks when injecting the extractant or the mobile phase. Is this likely to come from the autosampler (Waters WISP 712) or is something happening on the column? I would appreciate advice on how to identify the source and suggestions to eliminate the problem.

Thanks very much,
CF at NDA

Have you eliminated the possibility that your blank feed may be contaminated with your drug? Have you ever gotten a blank run from your blank feed?

In the past I did not see a response at the retention time of interest, but I have not really done extensive testing. Five other non-medicated feeds from different manufacturers also showed a peak at that retention time during a recent sample run.

Thanks,
CF at NDA

You can test your autosampler in the following way: First inject a high-level standard followed by a solvent blank. If there is carryover originating in the autosampler, it will show in the blank. The WISP injector can give long service, but when the seals go out, it can also give some very strange behavior.

It is also possible that there is some extraneous co-eluting interference that your sample workup is not removing or your column is not resolving. One way to test for that is to use an alternate wavelength and see if you get the same result. A wavelength-scanning fluorescence detector is nice if you can get one.