Chromatography Forum

I validated analysis method using LC-MS/MS. Now I am evaluating matrix effect of analytes.
The method is this
1. 99 uL of beagle plasma + 1 uL working solution (in ACN) + 200uL MEOH
2. 14,000rpm 20 min centrifuge
3. 5uL injection to LC-MS/MS

However in my case matrix effect value using Matuszewski method is over than 150%. I use ACN, MEOH, DW as neat solution.
And then I tested 50mM potassium phosphate buffer instead of plasma 99 uL. In this result, peak height is similar as plasma result. So I think phosphate will enhance my analytes' ion response.
In my case what can I do for removing ion enhancement.
Same phenomenon is observed at addition of ethyl acetate 1mL to upper method and evaporation under nitrogen gas and reconstituted with meoh : dw (1:1) solution.

I can't find this case in other studies.

Let me know what can I do please....

There are plenty of things you can try to lower matrix effects. The question is, what are you allowed to change to a method that is already validated?

I do not think that phosphate will cause a matrix effect on your analyte. For this to happen, it has to elute at the same retention time of the analyte - which is very unlikely. But we don't know any details about the LC run and analyte itself.

There is a lot of literature on LC-MS matrix effects in serum samples that are prepared by a protein-precipitation protocol.