CE optimization on Agilent QQQ

CE optimization on Agilent QQQ Sarah Michaud  2017-11-06

I getting some strange results when optimizing collision energies for an Agilent 6495 QQQ using Skyline. I am generating the transition list for CE optimization using skyline, then using the method to acquire the data, and importing the data back into Skyline. After importing the data, sometimes the transition results look fine and as expected, but other times the pattern in the peak area graph is really unusual (almost random instead of a curve shape), and other times the results are missing completely. When I check the same data file in the Agilent MassHunter software, the data appears to have been acquired correctly and the peak area pattern is normal. I've attached some slides with examples to try and show what I'm seeing.

I think the problem might be that for some transitions the product m/z value is for some reason different in the acquired data file compared to the method generated by Skyline, and when that data is imported back into Skyline the peak areas associated with each CE step gets jumbled.

Thank you for the help!

Brendan MacLean responded:  2017-11-06
Hi Sarah,
Thanks for the all the effort you put into those PowerPoint slides. I am going to suggest that you first contact Agilent, because this is most likely an issue with the instrument control software you are using. Agilent initially had a problem with rounding method m/z values to the point where they were indistinguishable to Skyline, which they then fixed, but in a way where it was a configurable setting and then eventually that setting was given the wrong default and this sort of random assignment behavior returned.

With any luck, they will simply point you at a settings file and tell you to change a single line it, and your CE optimization will start working.

If that doesn't turn out to be the case, then I will need to get your Skyline document (using File > Share - Complete to create a .sky.zip file) and at least one raw data file (.d folder zipped) to look at. You can always post extra files like these to:


But, I feel confident I would see that all the transitions are coming in with the same m/z.

Hope this helps. Thanks again for all the effort you have clearly put into understanding this issue.

Sarah Michaud responded:  2017-12-06
Thanks for pointing me in the right direction Brendan, this was exactly the problem and Agilent had an update already available to fix it.


m j noga responded:  2020-11-25
Dear Brendan and Sarah,
Could you possibly share the exact version of Agilent software that fixed this problem?
I'm experiencing apparently the same issue on G6490A running MassHunter B.08.02 with Firmware A.00.08.100
Best regards,
Brendan MacLean responded:  2020-11-27
I would recommend contacting Agilent.