Hi Heng,

do you also have UV? It would tell you, that the peptides are eluted off the column in the right amount and disconnect a problem from the HPLC (injector, pumps) from the MS. If UV signals are the same, you have either a supression ( buffer or sample) or a problem with your MS.

Test the system with a "clean" peptide mix like MPDS from Waters. If that is ok, I would suspect some compound in your sampel to supress signals over time. Sample clean up prior to injection could be helpful (ea. Ziptip or detergent removal cartridge).

Another problem could be a dirty MS. Is the  calibration working without problem? Do the signal change if you switch to negative and back to positive? If yes clean the MS including Q1.

Good luck Daniel

Another thing you could try is to monitor all the charge states of your peptide to see if there is any change in charge distribution.

Maybe your peptide of interest is slowly adsorbing to the glass wall of the autosampler vial. Are you using low-adsorption vials? Depending on your injection solvent it could also be precipitating (if the solubility is low). Try agitating the sample before every injection to find out.

Hi, Heng,

I agree with Daniel. From your description, it appeared you had a signal suppression problem. A cleanup with solvents such as IPA between runs could restore the sensitivity. On a separate note, do you notice any increase in the pressure on the column? If it got higher as the runs went on, there might be a precipitation problem which ultimately led to the lower signal.

i think that the problem from pump ( B ) 

see if the pressure normal after 1 inject, if so try to clean only pump ( B ) with 30% water and 70% ACN , :) 

also there is one problem , may be the pressure is above normal try to clean and wash the column ( without MS ) with water about 3 minute after that with solution 10% ACN about 10 minute , 

also be sure from pressure after insert the column because the max is 70 :) 

Thank you for all the answers and it is really helpful.

I forgot to update in these several months.

Things were back to normal after a very thorough Qtrap clean up.