SIgnal not seen after connecting HPLC to MS/MS (API 4000)

[mdchromatographer]

Hello,

I am working on the method development of Neuropeptide Y 1 36 ( MW is 4272) on API 4000 Mass spectrometer ( Triple Quad).
In the Infusion I am able to see good signal (cps in MRM), optimized the parameters.

When I am connect the HPLC to MS I couldnot see the peak in the chromatogram even at 1o ug/ml (50 ul) injection.
I am using C18 column, solvent is ACN in 0.1% TFA. I have also done the post column infusion ( post column connected T vale ) and signal was good.

Can anyone suggest me why this is happening or anyone has encountered this kind of problem.



Solvent I am using is ACN containing 0.1% Formic ACid.

Thanks
Rao

[Kostas Petritis]

The problem is either in your HPLC instrumentation or your chromatographic conditions. Try to inject your sample through flow injection analysis (FIA). If you see it, it is your chromatographic conditoins if not is your injector/other components of your HPLC.

If you see it, try to analyze your peptide by HPLC-UV and that will give you a good idea if the chromatographic conditions are appropriate. If you get a dinstict peak and you are confident that the peak correponds to your peptide, post again and we will think of something else...