HFBA ion pair reagent

[RAD]

We are having problems with our LCMS with rapidly decreasing signal in the middle of a series of injections (99% loss of signal over 4-5 injections). After several more injections the sensitivity returns to normal but the process repeats itself on an irregular basis.
The service techs are questioning the use of HFBA in our chromatography and suggesting this is the cause of the lack of sensitivity.
We use HFBA at 0.2% and 0.4% in bioassays using silica and C18 columns for homocysteine/methionine and for SAM/SAH. We use postcolumn propionic acid addition to minimize inhibition of ionization by the ion pair agents.
Does anyone have any experience with this ion pair reagent and have any comments/suggestions as to periodic loss of signal? We have been told the HFBA can coat the ion optics leading to signal loss. This seems reasonable, but does not explain the signal returning without performing any maintenance or cleaning of the instrument.

[lmh]

all I would say is that you will probably struggle to use your instrument in negative mode as ion pair reagents are notoriously hard to get rid of, and you'll probably have a vast signal of HFBA for ever more in negative electrospray.

[RAD]

Thanks lmh.
I have heard that ion pair reagents are veyr difficult to get rid of.
We are using positive ion and do not see a large signal from the HFBA.
Do you have any suggestions?

Thanks

[lmh]

oh, if you only need to use positive mode, you're fine! In my lab we have to use negative too, and I used nonafluoropentanoic acid for underivatised amino acids once (positive), and regretted it for a couple of months (negative). It had the same mass as one of my analytes.

The way you describe the fluctuating efficiency problem, it doesn't sound like a mobile phase issue. It sounds more sample related. Does the efficiency drop when running a particular sort of sample? Other less likely things might be spray-chamber issues. Is it getting very wet in there? Is the drying gas supply reliable?

Good luck!

[RAD]

Thanks again for your comments.

We currently have 3 assays running on our LCMS. Two of them use HFBA, the third does not. Both assays using HFBA have this problem of periodic loss of sensitivity, the assay which does not use HFBA has not shown this problem although we have not run that particular assay a lot so we may not have seen the problem because we have not run enough samples.

Both assays involving HFBA use plasma only so I cannot comment if we would see it with other samples. I suspect that we would see the problem, as we occasionally have had this issue in the middle of our aqueous standard curves, before any samples have actually been run.
We use a Nitrogen generator and have had no problems with N2 supply, it seems to work very well. We did switch to a tank of N2 for a while just in case this was an issue--the tank did not resolve the problem. We have changed the nebulizer needle and the probe itself recently but the problem is still there. The spray looks appropriate and forms a perfect looking cone so we have ruled out the spray as a possible problem.

We had several problems with this instrument recently and the service engineers have changed the source, cones, hexapole, collison cell, detector and removed and cleaned the quads and lenses. Our sensitivity improved about 10X after all these changes but we still face this dramatic drop in signal periodically. It is possible the HFBA is not the problem, but 3 service engineers have independantly questioned it as a potential problem, especially with the relatively high concentration that we use. I know it is quite sticky and stays around a long time and the engineers are postulating that it coats something and builds up until it causes a problem. I can believe this happens, but have trouble believing that a coating of ion pair reagent can suddenly reverse itself and the problem goes away without any cleaning--which is what happens. We stop running for the day and the sensitivity is back the next day.

[Kostas Petritis]

Did you make sure that the post-column proprionic acid delivery is constant and you do not have "interruptions"?... Although loss of 99% of the signal is really significant... How about the stability of your electrospray (when you observe the loss)?

[RAD]

kostas.
Thanks for your comments.
The nebulizer spray is good. The post column addition is a little more difficult to check. We regularly check for bubbles in the lines and have seen none. We prime the pump each day and it seems to flow properly, although after seeing your comments I have noticed a fluctuation in pressure readings on the pumps which makes me think you might be correct and there is some flow related issue with the post-column.

Thanks again for your help.

[HW Mueller]

I have only done LC + MS, but am keenly interested about modern LCMS methods, such that I am reading almost all the posts with problems in this field. In your case I am confused about what the problem is. Does the signal come back while you are running samples or only after letting the apparatus stand over night? If it is the latter, why are you surprised?

[mreed1272]

If you are seeing a return of performance after letting the instrument sit, it may be due to ion charging. You see this on MALDI-TOFs where matrix material coats the source and develops a charge over time leading to ion deflection. If you let the instrument sit, you get performance back that quickly degrades during use. The only way to fix it is by cleaning the source.

[bbkf]

What instrumentation are you using?

We had something similar (although maybe completely different) with our Thermo Accela and Quantum. The problem ended up being a partially clogged sample transfer line. This line is from where the needle makes the injection to the 6 port valve. Upon replacing this line, we had cured our problem