While attempting to validate a method forced degradation was performed on the sample. The analysis was performed on a PDA which showed that the product had two maximums. One at 200 and another at 220. When the sample was degraded the maximum at 220 increased. This method was developed by another analysist that is no longer with the company. He had the nm analyized in the valley between the two maximums. So when we compared the degraded sample with the neat the area counts went up for the degraded sample.

We have performed peak purity on the analyt and it passed. The nm at 200 did decrease some but not in the same proportion that the max at 220 increased. What would cause one nm to increase as the other decreases? Any help in this matter would be greatly appreciated.

The degradation product you are seeing has a higher max at 220. It's just the chemistry of your degradents.

I'm sorry I did not say this was on HPLC and the analyte is resolved of all degradents. The peak purity program even says that it is a pure peak. So I again ask how would the sample degrade and change the ratio of the maximums?

If it is not on LC what is it? What purity program are you running?

Obviously I am not communicating well today. My second comment was to assure you I was using HPLC. The degradation product is not coming out at the same time as the component. We only degrade our samples from 5 to 20%. So the answer you first gave doesn’t seem likely.

Ok, let me try this again. I apologize for the confusion as well. From rereading your original post it sounds like you have a coeluting peak whether you want to believe it or not. That is the only explanation that makes sense. When you degrade your sample, you normally see a decrease in area counts for your peak of interest. You are seeing an increase as well as a shift in the peak apex spectrum.

I ask the question agian, what software package are you using. I suggest you rerun the purity test. It is possible to have a peak perfectly co-elute and not show up in the purity analysis. Although it is rare.

Here is another possibility, not knowing your LC conditions or how you are stressing your samples. It is possible that your stressed sample has changed the properties of your sample solution. If, for example, you added base to degrade and didn't adjust the sample pH back to mobile phase or sample prep pH. The pH difference could effect the response of your peak at 220. This, of course, may not be applicable to your situation.

Dan