Don't see product-ions of steroides.

[dRima]

Good afternoon, colleagues!
Sorry for my bad English.

Once again I need your help!
Now examine the steroids. I'm working on optimizing the parameters of the detection mass spectrometer Agilent tripleQuad LC/MS/MS 6410 (MRM, ESI(+))

The problem is that I see the parent ion of steroid (M + Na+ = 503), still see a peak with a mass of 525 (assuming that (M-H +2 Na).
But when I start to investigate product-ions, there is absolutely no fragments.
The fragments are not formed at low collision energy, and high energy.

What do you think about this situation?
Thank you very much!

[ian51]

The most similar situation I experienced to this was the analysis of cortisol and 6-B-hydroxycortisol. Precursor ions for both could be seen in positive mode ESI, however, for the hydroxycortisol I could generate no useful product ions. Switching to negative ion, formate adducts were produced in Q1 and these ions both produced useful product ions to use in SRM mode. I have seen other manuscripts, however, using APCI in positive ion mode and emploting SRM.

[trozen]

Good afternoon, colleagues!
Sorry for my bad English.

Once again I need your help!
Now examine the steroids. I'm working on optimizing the parameters of the detection mass spectrometer Agilent tripleQuad LC/MS/MS 6410 (MRM, ESI(+))

The problem is that I see the parent ion of steroid (M + Na+ = 503), still see a peak with a mass of 525 (assuming that (M-H +2 Na).
But when I start to investigate product-ions, there is absolutely no fragments.
The fragments are not formed at low collision energy, and high energy.

What do you think about this situation?
Thank you very much!

sodium clusters are very stable. that's why you see no reasonable fragmentation.
switch to negative mode (if this is a corticosteroid), formate adducts should work well
or change the eluent composition to have MH+ instead of M+Na (this could be tricky..)

[carras]

I completely agree with trozen, in my experience you can't fragment a Na adduct no matter what you do. Also it is very difficult to get rid of Na, it seems to be everywhere (it leaches out of the glass in tubes, pippetes, mobile phase bottles...).
You best chance is switching to ESI- and use formic or acetic acid to get the corresponding adducts. It works great with corticosteroids.

[kerri]

You also could try adding 1-2mM ammonium formate buffer with 0.1% formic acid to see if that helps to lessen the M+Na adduct.

Kerri

[aalbre]

Hi everyone,

I have a similar problem and I really hope someone can help me out. I am using a QQQ MS (APCI) and I am trying to fragment lutein. I see M+ and M- in pos and neg APCI modes beautifully, but when I try to fragment these precursor ions I get zero product ions (m/z range 10 - 1200)! And it doesnt matter what kind of CID energy I use, it is always the same - nothing. Only at really high energies I eventually get just lots of low abundant noise with no discernable MS signals.

Could anyone have any idea what could be wrong? Is perhaps something wrong with the MS? According to the multiple reports in the literature, lutein should produce nice product MS spectra in both APCI+ and APCI-...

Any help is much obliged...

Best
A

[bcd_GCLCMSMS]

Has the instrument be tuned recently?

First, I'd run a known compound that fragments and confirm the instrument is performing correctly.

I suggest checking that the largest parent is either [M+H]+ or [M-H]-. If not, work with mobile phase additive composition, try to reduce inorganic ions (as suggested above) in your mobile phase, and adjust source settings. Ammonium formate (+ (optionally) formic acid) mixtures seems fairly universal.

Next purge and check your collision cell gas lines and check the delivery pressure at the instrument (and confirm CAD gas it is 'on' for the QQQ experiment).

There is always the possibility that the instrument settings/tuning for the ion path after Q1 are very far from optimal and need correction/optimization to see fragment signals.

[aalbre]

Thanks for the quick reply. The MS system hasn't been tuned for a while, that is true, but I am not sure this is the problem here. Let me elaborate...

When I use the same source (and housing) and solutions on an ion trap MS that we also have in the lab, I get the expected fragment ions in both APCI+ and APCI- (tune parameters between QQQ and IT were more or less the same). I use only acetic acid as an additive in the mobile phase, however, I don't see any adduct ions in the full scan MS (not from the additive, solvent or from other possible contaminants such as sodium, etc.). Moreover, when I run Q1 full scan and Q3 full scan, the resulting spectra look more or less the same (abundant precursor ion). The collision gas readout equals the set value of 1.5 mtorr (Ar), so I am running out of ideas what I could be doing wrong (or what is wrong with the MS system).

Oh, and another thing, when I use ESI+,- MS2 or SRM with other compounds, I don't seem to have any problems with observing product ions, so could this by some strange coincidence also be ion source-related?

If there are any additional ideas I would be happy to try them out!

Thanks...
A

[bcd_GCLCMSMS]

I recommended autotuning just to see if the instrument behaves normally with the tuning solution and if the instrument gives a successful autotune.

I wouldn't expect the same fragments/ratio with IT vs. triple-quad.

If the Q1 and Q3 scans both show the parent at a reasonable intensity, then I doubt it is a source problem, unless you were relying on in-source fragmentation previously.

The next step after seeing a consistent parent ion is to immediately switch to run Q3 scans at various collision energies (or set up a couple known SRM's from literature (with one of them being the parent mass) and run a collision energy scan from 0 to moderately-high values).

I assume you are seeing only parent ion mass after varying the collision energy? In that case, something is likely wrong with the collision cell.

If the parent mass goes away, even at near 0 CE and there are no fragments at any collision energy, then there is likely something bigger wrong (or you have a multiply charged

Either way, the parent mass ion signal should decrease at some point as collision energy is raised.

[aalbre]

Thank you so much for the comprehensive reply.

I also suspect there is something wrong with the colission cell, however, the colission gas pressure readback (1.5 mtorr) coresponds to the set value and I already performed the CE ramping you suggested. At 0 CE I can see the precursor ion as expected and as the energy increases the precursor ion decreases somewhat until there is only noise left. In other words, no fragment ions (other than the precursor ion) are observed at any of the CE levels.

I know that MS2 spectra from IT and QQQ cannot be directly compared, but I have never experienced a total lack of fragment ions during MS2 analysis before, and this is what is keeping me up at night. Especially since this is a well known compound and there are already several reports in the literature regarding its fragmentation pattern (either ESI or APCI).

I will try to run some calibration tests and see what I come up with... any further suggestions are more than welcome.

Many thanks!
A