Chromatography Forum

Hello everybody

Here's the new round of the fight: Q.M. vs me (Let's get ready to ruuuumble)
So what I faced
I was optimizing a method for mycotoxins determination. First I dealt with the ones which ionize in positive mode (14 compounds) and it seemed quite OK, ending with rather acceptable signal in MRM mode. Then I started with negative mode (7 compounds) and this is where the problems begun.
I performed the optimization by syringe infusion, then started with HPLC injection and got plain MRM chromatograms, no peaks at all! Only one of 7 compounds gave some peak only at giant concentration of 1 microgram per ml.

Then I tried the SIR mode - all 7 were present!
I made record of some separate in daughter scan mode (for example, compound DON has a precursor 355 and daughter 295 - then I record "Daughters of 355" , scan window 295+/-0.5) - again I get the intensive peak. But if I record MRM - no way!

After all, to exclude possible compound-dependant issues, I decided to make test on chloramphenicol (CAP), which normally inonizes well in negative mode. The same data - great intensity in SIR mode (321) , complete fail in MRM (321->152, 321->157).

I checked the mass calibration - it is OK, both mass accuracy and scan speed compensation.
Certainly the coliision gas is on and the pressure is enough. The collision energy shows the appropriate readback.
Now there's a lack of ideas, I just get lost.

Later I'm gonna attach the chromnatograms and screenshots of Tune page or MS Method window, if that will be of use for anyone who has any ideas.

cheak the collision gas(argon) pressure it is near 0.5 bar(7-8psi).
In mix mode having positive and negative ionisation cheak Tune page the desolvation gas,cone gas,source temperature,desolvation temperature will be same in positive and negative ionisation tune page.

Hello David, thanks for advice
the collision gas pressure is allright - I wrote in 1st post
mix mode - you mean introduce both POS and NEG acquisition periods in one injection? - that's an idea, I'll try. When running in Neg only, the readbacks seem allright

I had a similar problem some two years ago: I left a sequence of cortiscoreroids running overnight and the next morning there were no peaks; either for internal standards or spiked samples. That method had been used for a couple of years at least and was fully validated and accredited. (By the way these compounds are analyzed in ESI-) . After much trying this and that I only got signal in MS1 but nothing in MS2 scan or MRM. So I ended running my samples in SIR (they was some urgency for the results).

Next day I infused directly a 10 ug/ml std of dexamethasone and got mass peaks both in MS1 and in daughter scan. Incidentally the masses were exactly the same used in the (MRM) MS File of the sequence with no peaks. Then I injected cloranphenicol in MRM and got a analyte peak with the usual intensity. And to end the tale I injected the failed samples using the original method and lo and behold! everything was OK.

Apparently the QMicro mended itself or came out of its tamtrum or whatever.

I don´t know if this is important: the failed sequence had the shutdown active, so, perhaps, when it went into standby the Qmicro reinitialized itself. Try to reboot the Qmicro from the Tune page or more drastically to shut it off (and I mean completely off ) and turn it back on. Since vacuum is not broken it should take a very little time to reach operating conditions.

Hope it helps