How to set an MS2 Scan method in Triple Quad 6460 to get proper MS2 scan spectra

Hello MS scientist, 

I am facing a problem to set an MS2 Scan method in Triple Quad 6460 instrument. I am trying to analyze caffeine at the MS2 level but I am not getting a proper MS2 scan peak. 

Please guide me with the proper method.  

  • Hi,

    Got to the Method Editor in MassHunter - on the QQQ tab go to the Time Segments table and in the 'Scan Type' drop down choose MS2 Scan. Choose 'To MS' in the Div Valve column. Set the Delta EMV to 0 for both positive and negative. Then in the Acquisition tab set your start and end masses according to the mass of the analyte you are measuring. So for caffeine in positive ion mode, you should expect an ion at 195.1m/z so a start mass of 50 and an end mass of 250 should be fine. A scan time of 500ms should be ok too. Set the Polarity to 'Positive'

    What are you observing when you open the data file?

  • The below screenshot might help - it didn't attach to my previous reply!

  • Thank you professor for your reply, 

    I tried these parameters, unfortunately still not getting them.

    Please find the method image that I was used for the data acquisition.  


  • Are you just seeing noise? You have quite a large injection volume - what is the concentration? What are the mobile phases you are using?

    I would also set the Sheath Gas Temp to 400oC and the Drying Gas Temp to 200oC and try again. The other parameters will need optimizing once you have your MRM setup.

  • Hi, 

    Thank you for your suggestions. Concentration- 100ug/ml, 15ul injection volume, Mobile phase- A-0.1% FA in water, B-0.1% FA in 100% methanol. 

    Please find the Mass Hunter Quantitative software image. This image is showing just the MS spectrum.  

  • So you have a peak in the TIC at approx 14.5 minutes. What does the spectrum look like? Can you share the peak apex spectrum? The monoisotopic mass of caffeine when protonated (M+H+) is 195.1m/z but the molecule can fragment to give a product ion with a monoisotopic mass of 138.1m/z. You should look for those two masses in the spectrum. Anyway it looks like you may have your caffeine peak there - good news Smiley. Caffeine is relatively polar - on our Eclipse Plus C18 columns it typically elutes at approx 10-15% Acetonitrile. If you want an earlier elution time, simply increase the percentage of methanol you are delivering from the pump or shorten the gradient time - its not clear if you are using an isocratic or gradient method. 

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