All this glassware was further cleaned by ACN, detergent and pure water after dishwasher cleaning.

I have Waters Acquity UPLC and ZQ 2000 MS, column Water BEH C18 2.1x50x1.7um Mobile A, 0.1% Formic acid , B, ACN; 0.5ml/min, gradient from 25% to 100%B in 3 min; peak at 0.9min, all retention time perfect repeatability. LC MS under SIR positive mode. 1 uL injection

My working standard(100ng/mL) peak area about 1,000,000. Good precision. My 20 ng/mL in ACN solution has peak of 200,000, which is good.

But after many experiments, my 20ng/mL in CIP 200 5ppm solution some times had peak only 100,000(50% Rec) and other times can be as high as 2,500,000.

My analyte is weak base API.

CIP 200 is acidic detergent. 5ppm solution is made by diluting 5ug to 1mL ACN(two steps dilution).

It sounds like that the CIP 200 has tremendous effect on the signal.

Any one has any idea of what's going on? How I can work around ? My boss wants us to validate this method this week. I even can not do this next week if I can not figure out. Please help me.

Thanks a lot.