Hello,

I am developing a quantitative method using HPLC-ESI-MS (ion trap mass spectrometry) in the negative ionization mode. I face an issue but I cannot tell why it is happening. I inject the same pure pool standards that consist of 4 molecules (>98% purity) prepared in HPLC water, e.g. I prepare 25µg/ml, and I repeat this injection 8 times from the same injection vial, with the same instrumental method, and I do not get reproducible results. The relative standard deviation was more than 95% to one of the standards (M/Z-->419), and I have only one molecule that is reproducible (m/z-->659) (However, this molecule happens before being poorly detected for the method that used to work for like 2 years, what changed Idk). For example, I did a calibration curve two days ago, and it worked fine with the M/Z-> 659. The same calibration curve was repeated yesterday, and it was detected poorly.

In another test, I prepared a calibration curve, and I gave the order to inject the one vial in triplicate. I still had a huge variation from one injection to another that was injected from the same injection vial with the same concentration.

DOES anyone have an idea what I can do to solve this issue? Or why is that happening? I am confused! I have been working on this repeatability test for more than 3 weeks! We also did a deep cleaning and still have faced unexplained results.