Significant changes in response when flow and gradient are changed

I am trying to optimize a method for narcotics to include buprenorphine and norbuprenorphine.  Why is there a significant change in response (AUC) for these two analytes when the flow and gradient are changed?  Specifically, response is greater when flow (0.8 mL/min) and gradient are increased and less when flow (0.35 mL/min) and gradient are decreased.  None of the other 11 analytes in the same run perform this way.  The same mobile phases (0.1%FA in Water and 0.1%FA in Methanol) and instrument conditions (other than flow and gradient) are being used and baseline resolution is achieved in both methods.  I'm using a 1260 Infinity LC and 6420 MS/MS.

Was this helpful?