Chromatography Forum

I am undertaking an HPLC method validation on a product with 3 compounds of interest. I am having injection to injection variability from the same sample vial but for only one out or the 3 compounds. This indicates to me it isn't an injection issue. There aren't any integration issues. Can any one help?

OK, so what's different about the one misbehaving peak? For example:
- Is it degrading (plot response versus injection number and see if there's a downward trend)?
- Is your detection wavelength parked on a steep slope of the absorbance spectrum for that compound?

It isn't degradation. The system suitability and bracketing standard injections are consistent on a 60 injection long sequence it is only duplicate injections from the same vial of sample preparations. The problematic peak of interest isn't on the Gradient the base line looks good (flat and level with very little noise). It is very confusing because the %RSD in the system suitability and bracketing standards id <0.3% for each peak of interest the injection to injection variability in the sample preparations for 2 of the peaks of interest is less than 0.15% but for the other peak approximately 4% and yet when 6 sample preparations are made the RSD of the average assay of the six sample preparations is approximately 0.5% even though duplicate injections from the same sample vial are so varied.

You did not describe what the "injection variability" actually is. How much does the area vary by? Perhaps the integration events are not appropriate for the peak? Perhaps their is a solubility issue with the sample or the injection solution is not appropriate? More info is needed to suggest cause(s).

The injection to injection variability is the peak height/area is changing by upto 5% between injections from same vial but only for one out of three peaks of interest (this is only happening for the sample solutions NOT the standard solution). It isn't consistent either sometimes it is the first injection gives the higher area and the second injection gives the lower result and then the next sample vial gives lower result on the first inject and then higher result on the second injection. The second checker always checks the integration before approving the analysis. There is no issue with the integration events.

Could you further describe the sample matrix and preparation of the sample? If it's only the sample you are seeing variability in, then more information about it might help.