In my project, I will compare some metabolites in two cell lines with LC-MS. I have two cell lines, their metabolites were extracted, and evaporated, waiting in -20 oC.
I was waiting for my Internal Standard to arrive, so that I can perform LC-MS, but due to some customs problems, I can not proceed. I want to continue my studies as fast as possible. So can you please answer my questions?
An IS is needed for quantitative analysis since the detector response will vary (even if you use a common ion like a CH3 fragment). Without it you will get just a fingerprint of the molecular weight of the fragment which is nice :) But!
Dear Ceren
Since You did not add IS during the first steps of the extraction, it seems like You want to use it only for sensitivity fluctuations correction. [In general, I would not neglect by default a probability of any inconsistency that can occurs at all steps during the extraction and evaporation. But anyway, it is still too late]. So to correct these fluctuations, You can basically use any LC-MS grade compound that can be detected under Your conditions and that is not exist in Your samples. If it is too complicate to determine, another option is to use TIC median value for each of Your chromatograms. For this You will need to analyze the samples in targeted and untargeted modes.
While an SIL IS is essential in quantitative LC-MS, it’s not always readily available, especially in the early exploratory stage of a program. It’s still possible to generate useful quantitative or semi-quantitative data without an SIL IS, as many have been doing. For best possible data, one should optimize the method for robustness/stability in particular and may use an analog of the analyte as an alternative IS.
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