resolve as Duplicate of 51
Hi Thomas,
In order to go to 6 steps, you will need to expand the "Method match tolerance m/z" in the Transition Settings - Instrument tab. I think 0.065 should work. This is a duplicate of the long-standing issue 51. It happens because Skyline introduces a slight shift in precursor m/z for each CE step, and when that shift is great enough it will move the chromatogram precursor m/z outside the range that Skyline will accept as matching the target.
Thankfully, still very few people have tried to expand the number of steps beyond 5, probably because of the impact on the number of transitions that can be measured. More often we have seen people decreasing the step count (e.g. to 3) and increasing the step size. I believe this is what Agilent recommends.
Hope this helps. I agree on showing the voltage and wish that we could in all cases record the actual voltage used by the instrument. If we could do that reliably, then we wouldn't need the m/z shift. Unfortunately, not all instruments report the true CE voltage used for each transition, which makes it difficult to confidently declare anything other than that a chromatogram matches a certain "step", and even then, as you have seen, the matching is somewhat delicate. Wish we could do better of course.
--Brendan
151012_OptCE_SkyReport.pptx