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.
