Succinic-D4 acid peak issue in HILIC mode

[zokitano]

Dear colleagues,

I hope that someone has more experience and could help me with my issue, which I’m not able to resolve it recently.

I have a LC-MS/MS method for determination of some organic acids in environmental samples, using deuterated internal standards. For the aromatic dicarboxylic acids I use phthalic-D4 acid as IS and for the aliphatic dicarboxylic acids – succinic-D4 acid.

Some of the chromatographic and MS parameters:
- Agilent 1100 LC coupled with AB 4000 QTRAP
- Xbridge Amide 100x3mm, 3.5um column
- Gradient elution using mobile phase containing ACN/H2O with 10 mM ammonium acetate (adjusted to pH=5 in water, before mixing)
- Column thermostating with means of thermostated water bath (25C)
- Negative Single Reaction Monitoring MS/MS method (or (-)MRM, according to the Analyst software)

During FIA-MS optimization of the ion source (Turbospray – ESI source) I can clearly detect and optimize the parameters for both natural and deuterated acids. The problem occurs when I connect the column and run the LC-MS/MS analysis. Phthalic-D4 acid peak shows in the chromatogram (coeluting with the phthalic acid peak) which is ok, but I cannot detect the succinic-D4 acid, although the succinic acid peak is clearly present and symmetric. The SRM (or MRM) transition that I use for succinic-D4 acid is 121.0->59.0 (from syringe injection – MS optimizations of compound dependant parameters).

The stock standard solution of succinic-D4 acid was prepared once in methanol and once in acetonitril with no change/improvement in the chromatogram. The final dilutions of the standard were made in pure ACN. Changing the mobile phase additive from ammonium acetate to ammonium formate and playing with the column temperature didn’t resolve the problem of the missing succinic-D4 peak.
Before I posted this problem, I have searched and read a similar topic on this Forum, where on column degradation of deuterated standard was suggested as possible cause of the lowered peak intensity in chromatogram (the deuterated standard was different from this situation) The link: viewtopic.php?t=3674&highlight=deuterated

Also on the XIC I’m observing higher background intensity for the succinic-D4 transition. I was thinking to try LC-UV to see if this deuterated standard elutes from the column and is detected as the succinic acid is, but its presence during FIA-MS tells me that it is not a MS issue, but chromatographic. And here I'm stuck

Deuterated standards were purchased from Sigma Aldrich.

Did you have similar problems like this. What is your experience? What could be the problem?

I appreciate your time and help.

Best regards

[lmh]

Is there something in the sample runs (absent from the flow injection runs) that has the same parent mass as D4-succinate? If so, is this influencing the sensitivity? I am not a Qtrap person, but as I understand it, the Q-trap isolates trap-style, rather than simple quadrupole style in Q1 when running MRM, in order to enhance sensitivity by pooling ions, so a large dose of irrelevant ions of the same mass might be bad... but I might be writing rubbish.

[zokitano]

Thank you Imh for your reply!

The sample runs during LC-MS/MS have the same compositions as those used during FIA (only for LC-MS/MS the concentration is 10 times larger, which is normal). I am working with standard solutions only.
4000 Qtrap is linear ion-trap, but when it is used in MRM (SRM) mode it behaves and works as triple quad. So I think that MRM mode with this instrument doesn't include any trapping options.
But, I can say something that forgot to tell you previous, that is, the transition m/z 121.0 -> m/z 59.0 is unique among all acids analyzed. So there is no other acid in the standard solution that gives quasimolecular [M-H] ion with m/z 121.0.
But, because i use ammonium acetate and acetate ions have m/z 59.0 which make me to conclude that the high background is partially due to the buffer influence in Q3? But this shouldn't be the case since Q1 and Q3 are fixed to the predetermined m/z values in MRM.

Still confused

[lmh]

it's the higher background with column that bothers me; if the solvent for FIA is the same as the solvent with the column, but putting the column in gives you a higher background, then something is coming out of the column that can be mistaken for your analyte.

Thanks for the explanation of Qtrap workings. What's an enhanced product ion scan in Qtrap-language? I've never quite understood...

[zokitano]

Ok, I didn’t say quite well about the issue of higher background for the succinic-D4 acid SRM transition. The higher background was observed during the FIA also, but I clearly saw the peak during the ion source optimization.

At the end, I think I found the solution of my problem. Namely the most abundant product ion after fragmentation (in the collision cell-Q2) of succinic-D4 quasimolecular ion [M-H] = m/z 121.0 is that with m/z 59.0. Other 2 product ions of m/z 121.0 that were optimized (and were less abundant) were: m/z 61.0 and m/z 77.0 [M-CO2-H]-. Before posting this problem on the Forum I tried to change the SRM transition of succinic-D4 from m/z 121.0 -> m/z 59.0 to m/z 121.0 -> m/z 61.0, but it went without any success during the LC-MS/MS (I didn’t see the peak in the extracted ion chromatogram). I didn’t have any hopes that at least abundant product ion m/z 77.0 (m/z 121.0 -> m/z 77.0) would improve the chromatographic picture. But actually this was the “right moveâ€

[bisnettrj2]

Probably not helpful, but when you ran the optimization injections (direct into source), you stated that you dissolved stock standards in MeOH and MeCN. Maybe you did dissolve in MeOH/H2O/acetate or MeCN/H2O/acetate, but I'm not reading it that way. If you didn't, I would think adding in the acetate might have affected optimization, but I don't know much about MS or MS/MS, so take my 1/2 cent with a giant grain of salt.

[zokitano]

Probably not helpful, but when you ran the optimization injections (direct into source), you stated that you dissolved stock standards in MeOH and MeCN. Maybe you did dissolve in MeOH/H2O/acetate or MeCN/H2O/acetate, but I'm not reading it that way. If you didn't, I would think adding in the acetate might have affected optimization, but I don't know much about MS or MS/MS, so take my 1/2 cent with a giant grain of salt.

I tend to agree with you, because I got the same impression. I did dissolve my standards for syringe infusion optimization (of compound dependent parameters) in the mobile phase that I am actually using (ACN/H2O/Ammonium acetate). And here I also think that it is my mistake. I alter the optimization with the presence of the acetates. I should have dissolved the standard in MeOH/H20 only, but I wanted to be pedantic and to reproduce the condition of the LC-MSMS, which was very wrong. Although my logic said that during syringe infusion you don't need to reproduce the LC-MSMS conditions regarding the solvent/mobile phase. I lost week or so in resolving the problem.

Thank you for pointing that out, because after the problem was resolved I started to check my previous work and concluded that maybe the solvent used for standard solution preparation of the acids for syringe optimization was a bad choice.

Best regards