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I am trying to analyse samples of Rosuvastatin in rat plasma using LC-MS/MS and ran into the problem that I get two peaks for Rosuvastatin, one after a retention time of around 7 mins and one around 10. Both peaks are existent if only the plasma that does not contain any Rosuvastatin is measured (blankplasma; see the first Figure) and further regardless of concentration, however the higher the concentration, the smaller the relative size of the second peak (example in the second Figure).

If I measure samples containing Rosuvastatin solved in water instead of Plasma, no second peak is present. Two Peaks when sampling only Plasma

Two Peaks when sampling Plasma mixed with Rosuvastatin

I redid the analysis with newly created samples but the second peak persists.

Since the samples containing rosuvastatin solved in only water ran fine after the plasma based samples, I assume that the LC-MS is clean, and that every component works as intended.

My thoughts were that there is a compound inside of rat plasma that has the same m/z value as Rosuvastatin, but my search has not yet yielded any results.

Does anyone have an idea what the reasons could be or how to interpret this result?

Also, I am a newbie in this forum, if this is more fit for the BiologyExchange feel free to tell me.

Thanks in advance.

Edit below: Clarification of experimental details.

I'm showing multiple reaction monitoring on a Waters instrument (Micromass Quattro Ultima PT & a Waters 2695 HPLC).

In the shown MRM method the sample gets analyzed for the mass transition of rosuvastatin (482 > 258) and the internal standard carbamazepin (237>194).

The chromatograms shown are results of the processing with QuanLynx for quantification. In the QuanLynx method the retention time of rosuvastatin is set to ca. 7 minutes, such that the shaded peak is recognized as rosuvastatin and therefore integrated by QuanLynx.

The non shaded-peak is the unknown peak which appears when measuring with rosuvastatin spiked plasma for the calibration curve, rat plasma samples after rosuvastatin injection in the rat, and in the blank plasma that did not contain any Rosuvastatin.

When I inject only acetontrile however, I only see a small peak at 7.18 min rosuvastatin trace without the unknown second peak.

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  • $\begingroup$ I think SE Chemistry is a good home for this question. Are you showing multiple reaction monitoring on a Waters instrument? A bit more experimental detail would be helpful. Also, what is shaded vs not shaded on the chromatogram? $\endgroup$
    – Karsten
    Commented Sep 10, 2023 at 1:59
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    $\begingroup$ @Karsten I have added the additional information regarding MRM, as well as the devices and quantification method used to the post. Thanks for your feedback. $\endgroup$
    – Ratpricker
    Commented Sep 10, 2023 at 7:19
  • $\begingroup$ Can you explain better this "Both peaks are existent if only the plasma that does not contain any Rosuvastatin is measured (blankplasma; see the first Figure)" so you mean that the blank has two peaks, but there is presence of Rosuvastatin you should only see one of them? $\endgroup$
    – Jojostack
    Commented Sep 10, 2023 at 9:24
  • $\begingroup$ @Jojostack I analyzed plasma containing no rosuvastatin as a "blank" to get an idea what is wrong with my measurements. I performed (as with every sample) a protein precipitation with an acetonitril-carbamazepin-soultion and therefore would only expect the carbamazepin peak but expect none of the two peaks of the rosuvastatin trace which can be observed in the first figure. $\endgroup$
    – Ratpricker
    Commented Sep 10, 2023 at 10:12
  • $\begingroup$ I think you can easily find some other MRM transitions that are specific for rosuvastatin, and then see if the mysterious peaks from the blank plasma show up at that time. For example, the MS2 spectrum of rosuvastatin leads me to believe you could try 482 -> 446 or 482 -> 376. If you don't see peaks with those MRMs instead of your original one, then you've identified that the problem is an unrelated contaminant that just happens to be active on your chosen MRM. $\endgroup$
    – Curt F.
    Commented Sep 11, 2023 at 18:53

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Analysis of biomolecules chromatography can be a challenging exercise because these biomolecules can stick to any fluidic path of the chromatograph if they want to. Just to make sure, is every connection (autosampler needle, connection tubings etc) biocompatible? Now you have three separate problems:

  1. Seeing two peaks for a single analyte "rosuvastatin": In a chromatographic experiment a single molecular species will not have two retention times. Two peaks at different retention times means two different molecular species, perhaps same molecule but a different shape. A different conformation of the same molecule, if it is formed for whatever reason might have a different retention time. It could have formed due to the solvent conditions/pH. Check the quality control sheet that came with the compound. Usually they will mention related impurities which might undergo the same transitions in MS. For example, N-desmethyl rosuvastatin might be present.

  2. Both peaks are observed even when measuring plasma that does not contain any Rosuvastatin, referred to as "blank plasma."

Seeing peaks in a blank plasma means "carry over" from previous injections. Even high-end instruments have carry over. Do you have an autosampler needle wash protocol? Before injection a blank, make large volume injections (three times the volume you are injecting for the sample) of several wash solvents in which rosuvastatin is highly soluble. Does the peak intensity go down by repeating this protocol? It should.

  1. As the concentration of Rosuvastatin increases, the relative size of the second peak decreases. This behavior is exemplified in the second figure.

Again it indicates, either a very closely related impurity or conformation change of a certain fraction of your analyte under your solvent system.

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