Can find analyte in Q1, but not product

[shuku]

I'm having a weird problem with my LCMSMS. I can find an appropriate peak for amphetamine with a Q1 scan, but with a product scan and MRM there are no peaks. The transitions appear in the noise with expected ratios. I've tried tinkering with the declustering potential, electrospray voltage, collision energy.
I have another machine with identical LC and similar MS that works with this analyte just fine.

[JMB]

You need to establish that the electronics to pass ions through Q1/Q2, fragment and detect ions at end of Q3 are all OK.

You might want to lower the resolution to ensure strong signals.

1) Signal at [M + H]+ for full-scan Q1 means that (obviously) the ESI process/lenses/detector for Q1 are OK.

2) Check for [M + H]+ with full-scan Q3 (no collision gas in Q2); if detected, then Q2 and Q3 (and detector for Q3, if different from detector for Q1) are OK; if not detected, then probable issues with Q2 and Q3 (and detector for Q3).

3) If test 2 is OK, then repeat test 2 with gas in Q2; [M + H]+ with full-scan Q3 should then be greatly diminished, and fragment ions should now be present. If not, then excessive collision energy/gas pressure may be causing ion loss by scattering. Optimize collision energy/gas pressure to get good fragment ion spectrum.

4) Once conditions to get signal in test 3 are established, turn gas OFF, select [M + H]+ with Q1
and set Q3 to full-scan; [M + H]+ only should be detected at Q3.

5) Repeat test 4 with gas ON; if OK, set up MRM as required. Check signal; if OK then reset resolution as required. If not OK, then loop to tests 2 and 3.

[shuku]

Thanks for the response JMB.

2) A Q3 scan creates an identical chromatogram

3) With a product ion scan, the noise is reduced by one order of magnitude compared to the Q1 and Q3 scans. When I view the mass spectrum for any given time during the run, I get transitions at about 54, 85, 91, 119, and 136. The noise response from the smaller transitions tend to increase slowly over the duration of the run (1.0 to 8.5 minutes after injection), but form no peaks. I've tried 10 transitions with a few collision energy settings each. I chose them based wide product ion scans (ramping CE and summing signals) with infusions for transitions, followed by ramping CE for each transition to find an optimal CE.

4) I haven't tried that yet, but it will have to wait until next week.

5) I just finished doing a mass and resolution calibration, but the initial results don't show any improvement.

I should add that this is a problem analyte among about 100 other analytes that work fine on this machine.

[bisnettrj2]

Have you infused this compound via a syringe pump teed into the mobile phase at a composition equivalent to the approximate conditions at which the compound elutes, at the flow rate you're using during analysis? I like to infuse my compounds at about 50:50 A:B with a syringe pump teed into the mobile phase at the flow rate I intend to use and at a concentration that gives a significant signal above the noise, just to make sure I'm getting the right peak. If you happen to have a contaminant peak with the same mass present in your system, and you optimize on that mass, you are going to get erroneous declustering potentials, product ions, and collision voltages. Maybe that's what happened here? If all the other compounds work, re-optimizing one compound versus troubleshooting the entire instrument should be easy.

[shuku]

Yeah, to tune it I use 4 ul of 1mg/ml amphetamine standard in 10ml of 50:50 H2O:MeOH, which are the mobile phases. I think I had to dilute it down another 10:1 in that solution to get the peak to resolve clearly. I infuse it with a tee connector at about 10ul/min. I tend to use a lower flow rate on the LC (0.1ml/min), but I can try upping it to injection levels (0.6ml/min). My software has automated compound optimization. I've used information both from that and from manually tuning.
It would be weird for a different contaminate to produce those same transitions. I'm kind of nervous about cleaning the ion source, but I've cleaned and replaced just about everything else that could be contaminating the system.
And like I said, I've tried using parameters that work well on a similar system. I don't think I have bad transitions.

[bisnettrj2]

Can you provide some more details on the software, instrument, mobile phase, and possibly the settings you're getting through the optimization routine?

Have you tuned the instrument and then verified the tune the following day or week, to insure that it is stable and not drifting?