I am having trouble with my internal standard reading about 3-4 times higher than the amount that was put in during the extraction procedure. I am adding 50 pg of DHBA to the sample and eluting with 250 ul of perchloric acid. I am injecting 20 ul of eluent into the HPLC, so the most internal standard that could possibly be on column is 4 pg of DHBA, yet I am getting 3-4 times this amount.

Also, it does not happen everytime. Sometimes I get a good DHBA response and other times it is a large spike in DHBA.

My thoughts are that:
1) There is some component in plasma that is binding to DHBA and causing the ECD to read it at higher than what it should be.

2) I need to switch the internal standard to something else.

Are there alternate internal standards that could be used for plasma catecholamine extractions that may not give erroneous results?

Thanks!