Quantifying with MassLynx: are or highest intensity

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

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Hello,

I'm trying to quantify quercetin, i.e. in human plasma. 1) Which is the best way to quantify it? By highest intensity or area? 2) And when it comes to the detection of the area, do you use QuanLynx? Because until now, I measured everything by hand and it takes ages. At the moment I am just investigating quercetin in methanol as a standrad solution. And I always measured the area of my fragments in MRM mode - is this the correct way?

Thanks alot!
Nora
We use targetLynx, not quanLynx, but as I understand it, targetLynx is just the luxury version of quanLynx, the two both being designed to do the same job.

Whether you use height or area depends on the chromatographic context. I prefer area, but some people favour height when there is imperfect separation from a nearby peak. If you do use height, you have to be very careful that your peak shape is consistent and does not vary from sample to sample. If a peak gets wider, it will also get shorter, while its area may be more resistant to change.

Definitely use software rather than picking up areas manually. There are better things to do with life, and it's easier to convince people that your results are not biased by personal expectations. Good luck!
Hey lmh, thanks a lot for your reply. May I send you via direct message or email a chromatogram? Because I couldn't figure out how to upload pictures here.

1) I think it is difficult to define the area because my peaks aren't that beautiful. If I use software, the are will be smaller that what I would have chosen. I think you are totally right, that I am not quite eager to spend my time picking up everything manually....Do you subtract and smooth out your data/peaks before integrate your peaks?

2) "Be slightly cautious about your injection solvent. If you inject in a solvent that is strong enough to elute the analyte, like MeOH, then as your injection volume increases, your chromatography will deteriorate and peak shape will widen. It's best to keep the injection solvent similar to the initial mix for running your gradient, or at least keep it reasonably "weak", consistent with the sample remaining properly dissolved."

At the moment I'm trying different kinds of concentrations of quercetin (0 - 10 uM/ml) for a calibration line but my injection volume (10 uL) and the flow rate (200uL/min) stays always the same. Unfortunately I have peaks until 1 uM/ml but still have lots of noise and they have a strong tailing. I don't know what to do to reduce the noise. I tried different concentratios of acidic acid and formic acid in my eluents (water and acetonitrile), then changed the gradient and changed the column, but my peaks still have a strong tailing. And then I get insecure when it comes to determine the area - with or without the tail?

Unfortunately I do not have a person to help me here or I just question things too much. But I am willing to learn and really want to get this going!

Thanks a lot for your support! I will just write in this post, I'm sorry I posted it in two.
... hm, I don't know how to do pm's here. Others might know better about how to post an image; you might have to put it in a picture hosting site like tinypic. If you can post your chromatogram so everyone can see it, you'll get better answers.

The thing that leaps out is the concentration. Am I right in interpreting what you wrote as micromoles/mL? If so, those look like very, very high concentrations for a triple-quad method. I usually set up our Xevo by infusing 10 micromolar into a flow from the pump, so the instrument is seeing about 0.1 micromolar, and that's for set-up using scan modes. For MRMs once I'm set up, I would expect to use even lower concentrations. If you're injecting far too much, that may give you very strange peak shapes as the mass spec will hit its maximum, and tailing could be serious.
Yes, it's micromol/ml. I know that it's a very high concentration but under 2 uM/ml I just have noise as you can see in this link Image. In the picture you can see the fragment 151 of quercetin with negative ionization (ESI) and 20V collision energy. On the top is a concentration of 0.25 uM/ml, the second last is 10 uM/ml and the last 50 uM/ml.

When you speak from setting up, you mean tuning right? So you tune HPLC and TQ method until you see clear peaks?

Either I have to keep tuning or my TQ ist just not sensitive enough (too old?)..
I'm hampered by not knowing your instrument - I use a XevoTQS.
Yes, by setting up, I mean finding the mass transitions. On my instrument, it would usually be done by the software (Intellistart) while infusing the target compound. The Xevo has built-in fluidics for the purpose of infusing, but you've got a syringe pump on the front, I assume for the same purpose.
Please forgive me if I'm telling you things you already know. What I would have done at the start is this: I'd largely ignore the chromatography, but merely install a clean column and start a flow of typical solvent for about half way through the run (say 500uL/min of 50% acetonitrile with whatever modifiers you might be using, such as 0.1% formic acid). Then, on the Xevo, I would use the internal fluidics, or in your case I'd use the syringe pump, to introduce a small flow of quercetin into the post-column flow. If you haven't got fluidics, put a t-piece post-column, with one leg to the column, one leg to a syringe (leak-tight Hamilton in the range 100-250uL size; gas-tight is good), and the third leg to the spray probe. Close the syringe-driving thingy up to the syringe piston and start it running. This means you should now have a combined flow of typical chromatography solvent, and a little bit of quercetin, running continuously into the spray chamber. On the Xevo, I fill my syringe with 10uM target analyte as standard if I don't know any better, but it's not always right!
Then, on the Xevo, I would use the Intellistart software for developing a method. If you don't have Intellistart (on the Acquity console) then you'd be using the manual tune-page window. It's a good idea, even with Intellistart, to use the manual tune page to check that you have the correct parent ion present, and that the ion disappears when the syringe is turned off (you don't want to develop a method to analyse a background ion!).
If you have to do manual tuning (or what I'd call manual set-up) start with an MS scan and check you've got the precursor. If desired, fiddle with the cone voltage to make sure the signal is as good as it gets. On the Xevo, cone voltage makes little difference, but it might matter a lot more on your instrument. Then change to MS/MS and gradually try out collision energies to look for good, intense fragments, and the corresponding optimum collision energy. Collision energies are often pretty critical; get them wrong by even 5V and the signal will often be much, much less. Once you've done this, you have potential MRMs you can use in your MS method. The actual gas-flows and temperatures in the spray chamber, of course, remain in the tune file. I usually make sure that my mass transitions use cone-voltages from the ms method, not from the tune-file.
Only after completing all this do I start trying any chromatography.
A note on polarity: I notice you're using negative mode. Quercetin will probably work in negative or positive. I don't know if this is true of the Quattro premier, but with the Xevo we find that as it gets dirty, negative mode stops working a bit before positive mode stops working. It's worth trying positive. It's also worth setting up a method that uses positive and negative with polarity switching, because if your instrument is getting dirty, it may be "charging", where something in the system charges up when a constant voltage is applied, and then it all stops working (I have no idea what, physically, is going on behind the scenes when this happens). If so, it ought to be maintained by someone who knows what they're doing (Waters engineer) but you may be able to sneak a result out of it in a polarity-changing method when it fails in a constant negative or constant positive method. (the test for charging is to compare results with and without polarity switching). Hope that makes sense. Good luck.
Thanks for your detailed reply! I set up the system as you described it but it never was perfect and I still kept on measuring in MRM mode. I think that's due to the lack of a contact person and me being an unexperienced (maybe with little patience) student. I'll try it out again from the beginning.

I was told to just use 10, 20 and 40 V as collision energy because it is easier comparable to literature. But I'll try out different steps.

I didn't notice big differences in RF lens settings in the ESI nor did I notice big effects in Q1/Q3: LM/HM. Do you have literature explaining these components? I couldn't finde any.

The quercetin aglycon with the m/z 301 is always been measured in negative ionization but I'll try positive ionization and polarity switching to see a difference.

One question: "The actual gas-flows and temperatures in the spray chamber, of course, remain in the tune file. I usually make sure that my mass transitions use cone-voltages from the ms method, not from the tune-file."

What do you mean with " gas flows and temperatures stay in the tune file"? Do you just mean that you have different kinds of files? One for tuning for the first time and then another for measuring in MRM mode? Do you need different ones, so that you always have one basic setting?
Not sure how your software works, but when I am doing a setup and tuning I ramp my collision energy from about 5v to 50v and collect a response curve for the daughter ions, where the curve peaks is the optimal voltage for collision energy. I do the same with other lenses to make sure I am getting maximum sensitivity for the analyte I am optimizing for.

Spray temperatures and gas flows need to be optimized with the full mobile phase flow, so that you are getting proper desolvation and ionization, too cold and you are not removing the solvent, too hot and you can cause breakdown of the analyte. You can make quick injections with no column in the system and monitor the sharp peak that elutes within seconds and make adjustments until you reach maximum peak height using the mobile phase and flow rate you will be using for the run, or at least an approximation of the mobile phase mixture when the peak will elute. This just makes it faster than having to make multiple long runs with the column and gradient. Once sensitivity is optimized then run with those source and MS setting and analyze the full chromatographic run and you should have maximum sensitivity on the MS. Last steps are to optimize separation and chromatographic efficiency before calibration and analysis of samples.

It does look that you are making progress :)
The past is there to guide us into the future, not to dwell in.
Good morning James,

I'll try out the CE ramp, thanks for the advice.

Spray temperatures and gas flows: quick injections with the syringe pump, right? So you change i.e. spray temperature, inject a little bit of the sample and then you see if it's the right temperature or not and adjust it again?

You have very good and helpful ideas, thank you!
... all I meant about different sorts of files is that you have the mass spec method and the tune file; mostly they're two different things, but a few parameters (cone voltage, I think) might appear in both.
Does your instrument get any maintenance? Is it reasonably clean?
Yes, you would have differing tune files in MassLynx, depending on the compound. You need to optimize the ESI and it is highly compound dependent. Quercetin will probably require a relatively high ESI temperature and needle voltage. Also, make sure you optimize your collision voltages and gas flow. We use 2E-03 argon for most work but it is compound dependent.
I agree with lmh; uM/mL is WAY too high for routine triple quad work. Literature shows quercetin in the low to mid ng/mL range. High concentrations can radically affect your ionization conditions.
Your RF setting does not have a huge effect, and should typically be below 0.5.
I normally don't use the infusion syringe. Instead I take out the column and do what in essence is flow injection. I'll inject 10 uL into the flowing eluant, which normally I set at 50% buffer and 50% organic. Takes less than a minute to get to the MS and then you see the real effects of the eluant on the ionization of your compound. You can change a lot of parameters in a short time when you have <1 minute runs! Given the broad peak that you are calling quercetin I think taking the column out for optimization will be a big help to you.
Mark Krause
Laboratory Director
Krause Analytical
Austin, TX USA
Hey Mark,

well I know what literature says but I couldn't measure yet concentrations under 2 uM/ml (10 uL injection volume, autoinjection with HPLC) with the TQ. That's why I'm trying to tune it all over again. So today I used the syringe pump and infused 10 uM/ml with a pump flow of 10 uL/min for 2 minutes to change parameters quickly. And my mobile phase consists of 15/85 H2O/ACN because Quercetin elutes at this concentration from the column.

Which concentration do you use when you inject 10 uL and just using the dummy instead of a column?

At the moment my source temperature is 120°C and my desolvation temperature 500°C.
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