I hope I am directing this message to the right place. I am presently
> working at the University of Arizona in the College of Pharmacy. At the
> moment I am trying to work out a method for analyzing a metabolite of
> isoniazid, acetylisoniazid. Based on the literature that exists, the
mobile
> phase is a mixture of buffer and organic (MeOH), usually around 90%
buffer.
> Initially there was poor separation between the parent compound and the
> metabolite. I therefore added acetic acid in the hopes of improving the
> separation, which it did. Now I believe I am having problems with carry
> over of the metabolite. This problem especially occurs when I inject my
> samples after they undergo the NAT (N-acetyltransferase) Assay. In this
> assay a mixture of mouse liver cytosol, parent compound, and Acetyl
Coenzyme
> A are incubated at 37 degrees for 10 minutes upon which the reaction is
> stopped with cold Methanol. The reaction mixture is spun and injected as
> is. My injection volume is 20uL and my column is a C18 stationary phase
> 250 x 4.6 mm 5u.
>
> I am assuming that the acetylated metabolite likes the column very much.
I
> have tried increasing the organic to as much as 15% but I seem to lose my
> separation when I do this. Any suggestions as to how I can improve my
> chromatography.
>
> I do realize that decreasing the injection volume would help, but the
> autosampler I am working with does not allow for this.
>
> Thanks for your time.
>
> Melissa A. Bresnahan
> (520) 626-2423

why do you think the problem is in your column? the post logical place to look at when you have carry-over problems would be the autosampler.

Working with ACN:Water system isocratic run, find that a few initial impurity peaks goes on increasing with subsequent injections of the standard. same problem observed with different columns. Sometimes this impurity peak is not at all seen. Could this be due to impurity build up or analyte or impurity carry over from previous injections. Any solutions

What is the nature of the impurities? Are they ionic? if yes, use a buffer...

I think the problem lies with your sample prep. I assume (that word again... doah!)your standards do not contain all the NAT assay components. You state that the reaction is stopped with cold methanol, does the final solution (injected) contain about 10% MeOH, as it should for best results (same as mobile phase). Are the pH and ionic strength of the sample and mobile phase the same, or very similar? I think you have too much of a mis-match. I accept that in the real world matching sample and mobile are sometimes very difficult, but I suggest you look at this carefully; just a word of caution... buffers seldom are!
Cheers
Bryan Wallwork