Hello everyone.
During my analyses on LC MSMS, the area ratio (Standard Area/Isotopically labelled Standard Area) increases with time for the quality control point.
I'm having trouble explaining this trend.
Also My blanks before and after the quality control are cleaned.
Do you have any hypotheses?
Thank you very much.
Many possible answers and no web forum will provide the specific answer. HPLC systems are very complex and there are many causes for variation in results. All of these are best addressed in-person, on-site, evaluating the specific instrumentation AND the operator (as most problems are the result of poor training). - In your case, you will want to evaluate the condition of the HPLC system and MS settings. If MS peak AREAS alone are increasing (or changing), this may be the result of column fouling, detector overloading (contamination). Sample matrix may be interfering with the detection. Sample degradation over time. Review sample cleanup procedures and all check to make sure that the HPLC method used follow good chromatography fundamentals (e.g. Proper K prime, peak shape, sample is dissolved 100% in mobile phase before injection, column washing with a solvent that is stronger than the mobile phase after each analysis, etc). If your samples have chromophores present, then your HPLC system should have a DAD inline with the MS detector. The combination of detectors is used to collect the most orthogonal data about each sample (UV/VIS, retention times + MS fragmentation data) AND can be very useful to troubleshoot problems.
Speak with a local professional chromatographer, on-site, to find a solution. The LC-MS system is designed to be used by someone with many years of professional training.
in addition to the reasons related to the HPLC system, there is many other reasons for example, the isotopically labeled standard might be degrading faster than the unlabeled standard. This could lead to a decrease in the signal of the labeled standard, causing the area ratio to increase.
another reason, changes in the ionization efficiency of your ms might differentially affect the labeled and unlabeled standards. The isotopically labeled standard might be adsorbing to surfaces within the LC-MS/MS system more than the unlabeled standard, causing a relative decrease in its detected area over time.
also there might be subtle changes in sample preparation or injection volumes that are affecting the labeled and unlabeled standards differently.
another point, the LC column might be aging or accumulating contaminants, which could affect the retention and separation of the labeled and unlabeled standards differently.
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