by
lmh » Mon Jun 25, 2018 12:59 pm
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.