scan speed, gradients and flushes...

[matthew]

Hi,

My question isn't well formalized, so I'll just dive into it.

Is there any reason to question the sensitivity of the instrument during baseline drift? That is, when more gunk is hitting the detector, is the response still accurate? I often use gradients, and when not using gradients, I still flush after my isocratic runs. Is there any reason to think that there is m/z drift during these 'climbs'?.

I guess my question is: do you ever vary the scan speed during your runs?

[JGK]

My experience (preclinical bioanalysis, LC-MS-MS) is that we employ a switching valve (controlled from the MS software) to send only the area of interest to the MS detector.

the remainder of the chromatography is sent to waste which avoids the worst of the sample matrix ( the gunk) coming into contact with the detector.

[lmh]

A couple of thoughts:

(1) The response of the detector depends on ionising efficiency of the analyte. Since an analyte eluting late in your gradient isn't the same as one eluting early, it would probably have a different response irrespective of what gunk is being washed off by the solvent later in the gradient.

(2) If you have a build-up of gunk in the MS over the course of several runs, yes, you will see a decrease in efficiency. This is where internal standards are valuable. You can also put an external standard later in the sequence to check what's going on. JGK is of course utterly right: better not to let the gunk in at all.

(3) Some of the changed signal and changed sensitivity later in the gradient is due to the solvents. Electrospray generally works better with lots of organic than pure water.

(4) The relationship of sensitivity to coeluting gunk depends on your instrument, and several mechanisms. Coeluting gunk can suppress ionisation in any instrument. In a trap, if there is enough of it, it can also reduce sensitivity by causing the instrument to shorten the time it opens its lenses to fill the trap. The instrument will automatically fill the trap optimally with whatever is in the ion-stream. If most of the ion stream is the wrong thing, the trap will have little room for the right thing. This isn't a problem for Q and QQQ instruments, and it's less serious for big linear traps than older 3d traps of limited capacity.

(5) Personally, when running SIM methods in our simple single quad, I will occasionally vary the SIM dwell time between ions, but this isn't based on the gradient. It's based on which signal is biggest. I've never tried to fiddle with the scan time.

Hope that helps.

[matthew]

Hi guys,

I think that I could have used a better phrase than 'gunk'. Although my work up until now has focused on known analytes (primarily organic acids), I'm getting a bit more free-form in the holiday season and I'm stepping back and using my QQQ for a few metabolomic experiments. With my really clean samples (etoac xn), the baseline is often minimal, even in increasing organic... until I increase the flow rate to shorten analysis time. The peaks that I get as the baseline climbs are still of interest, but I wonder if I should consider using different scan rates as things do co-elute. Perhaps this is an empirical question and I should just do the experiment (with the help of something like mzmine or xcms).

To the points:
JGK,

For targeted approaches, I do like this idea of redirecting the trash to waste. Also, there is apparently an add-on that I can get for my lc that will flip between columns while purging the previously used column... really cutting down on analysis time (are these common tools?). For high-throughput targeted approaches, this is really really getting interesting.

lmh,

(1) The response of the detector depends on ionising efficiency of the analyte. Since an analyte eluting late in your gradient isn't the same as one eluting early, it would probably have a different response irrespective of what gunk is being washed off by the solvent later in the gradient.

...
(3) Some of the changed signal and changed sensitivity later in the gradient is due to the solvents. Electrospray generally works better with lots of organic than pure water.

I have done an experiment with a few analytes in runs of differing %ages of isocratic organic; this really helps one understand ionization efficiency. Prior to doing these injections, I'd had trouble convincing another student that he couldn't say much about the relative abundance of analytes within a run. When one sees the abundance of the same analyte vary so widely, it brings the point home.

(4) The relationship of sensitivity to coeluting gunk depends on your instrument, and several mechanisms. Coeluting gunk can suppress ionisation in any instrument....

To me, this is one of the most interesting challenges for people working in ms. I suspect the answer is to just do the work, but if one is comparing the abundance of an analyte across samples and the composition of the matrix varies between samples, and a coeluting (unseen b/c of MRM approach) compound suppresses/enhances material in some but not other samples, then wow. Bad inference.

This makes me wonder about the influence of RT changes on standards. Hmmm... do you use different standards to differentiate extraction efficiency and RT changes? Is their literature on the effects of RT changes in ionization enhancement/suppression? Is this a bigger problem with MeOH? Do people ignore the issue?

[ryan]

Hi guys,

For targeted approaches, I do like this idea of redirecting the trash to waste. Also, there is apparently an add-on that I can get for my lc that will flip between columns while purging the previously used column... really cutting down on analysis time (are these common tools?)

Yes I believe so! I used to work in high throughput purification by LC/MS purifying compound libraries full of gunk. We used column swithcing valves to switch between two columns, and then used an additional pump (a cheap old one) to wash the column not in use with acetonitrile, then switch back. We had to ensure the column had enough time to equilibrate back to the initial mobile phase conditions, but the lifetime of the column was roughly doubled by this washing process.