Chromatography Forum

Hi, it seems I always have new problem. This time is about the peak ratio. After the method has been developed and after the instrument have been devoted to other use for some time. I come back to validate my method. Everything is same except the instrument is cleaned and recaliberated. For the standard sample with the same concentration and same amount of internal standard, the peak ratio of internal standard and analyte changed a lot. Anybody has this kind of experience? or anybody know what is the reason? Thanks.

By the way, the instrument is LC/MS-MS (API2000 MS and Agilent HPLC).I am runing a MRM quntitive method for PK study. Let me know if further infromation is need for the analysis of the problem.

I notice my internal standard fluctuates even with time 10-20%. My problem has been that it doesn't always fluctuate at the same ratio versus the analyte(s) of interest.

I usually don't pay a lot of attention to the actual ratio. I am more interested that I have a good linear fit (see FDA guidelines for PK studies). Also, then make sure my QC's fit the guidelines when calculated versu the calibration curve over the time of the anlalysis.

As to what could make it fluctuate. Your calibration might be changing some versus time. If you are not sitting at the top of the peak for the MRM transition for one peak and not the other, etc.

Also, the voltages you enter into the instrument are not always the effective voltages you obtain as the instrument gets dirtier or cleaner...

Good luck.

James, Thank you very much for sharing your experience. Your explaination make sense to explain why I could bring the ratio back last time by recaliberating the instrument. But this time, even after recaliberation the ratio still much off from the original method. The linearity of my method is still good, only for the high concentration analyte, the analyte response is far higher than that of internal standard. looks not well banlanced.