Chromatography Forum

Buffer: 1g/L EDTA, Mobiel phase ACN:Buffer = 40:60, 1.5ml/min, luna c18 150x4.6mm , room temperture. the method is validated for both assay(10ul injection) and related compound (50 ul injection). The API is an amide with 8 carbon. This was what happened: 6 related compound standard injection (which were prepared at o.5% of the assay concentration) all gave around 200 K area unit; then 6 finish product giving 80million area unit(with height of 5 au), then a related compound standard check injection giving ONLY around 130K. my overall RSD is bigger than 10, which was a failure. This was repeated for three time in two different LC. Both LC were normal when used in other projects.

any ideas?

Is your related compound standard stable?

JM

very stable . tested for 64 hr <2% change

Tom, Uwe, Mark, Hans or anyone else,
any ideas. I have repeated the forth times but the same result. Like have a curse.
Happy Thanksgiving though

Dear YM°°°,

Your problem seem to be a change related to a possible residue of the API left over after the injection (5AU must be a massive amount). I suspect something left behind has changed the column or injector surfaces and is now adsorbing your related compound. Try to do large blank injections to restore the previous conditions, change the guard column (if any), or try a new "clean column" to run your related compound determinations.

If repeated blank injections eventually restore your original conditions, then it is possible that a large volume injection of a good solvent mix, suchas DMSO/water, in between API and related-compound analyses could prevent this problem.

I wonder how is it that this problem was not detected during the validation.

I hope these comments will help.

josebenjamin

Hi, Jose,
Thank you the input. It approved you are right though I have not straight out yet. I saw the UV spectrum was gone meaning the UV wasnot like what it should be any more. it seems my detector was contaminated.

In validation we were using dilute API concentration but now in the finish product test, we are injectiong at assay level.

Buffer: 1g/L EDTA, Mobiel phase ACN:Buffer = 40:60, 1.5ml/min, luna c18 150x4.6mm , room temperture. the method is validated for both assay(10ul injection) and related compound (50 ul injection). The API is an amide with 8 carbon. any ideas?

What sort of buffer, and concentration?. I suspect that you have deposited some EDTA, or a chelate with a component of your sample, onto the column, and that has grabbed your standards. What solvent is the sample dissolved in?.

Are you certain that the baseline has returned to the original absorbance, and you're not getting a huge displacement offset after the samples?. Is there any trending of the final standards at all, and does the instrument show any pressure changes during the sequence that would indicate deposition?.

Like the previous poster, I believe that a more aggressive flush should clean the column. I'd be concerned at the large sample size ( 50ul ) and high absorbance, and does reducing the size (10 ul ) reduce the problem?. Other alternatives are to change the temperature of the column, concentration of buffer, and see what happens.

I'm surprised that validation didn't pick this up ( was the same sequence fully evaluated? ), and your calibration failure mean that the method will have to modified and revalidated anyway, so you might as well have a play and understand why.

Please keep having fun,

Bruce Hamilton

5AU?!? Guess 1) You have exceeded the linear range of your detector.