Chromatography Forum

I have been trying to run HPLC with electrochemical detection for serotonin in serum but have been getting an unknown peak that interferes with serotonin. I have been unable to get rid of this peak and still can't figure out where it's coming from. Details about the protocol and steps are listed below. Any help in getting rid of the unknown peak will be much appreciated!

System:
Electrochemical detector Chromsystems CLC 100
Autosampler Chromsystems CLC 220
Agilent C8 column (100x2.1)

Protocol:
Mobile phase is 150 mM NaH2PO4, 4.8 mM Citric acid, 3mM SDS, 50 uM EDTA, 11 % MeOH, 17% ACN, pH 5.6 with HCL
Electrode set at 550 mV
Flow rate is 0.4 mL/min

Sample preparation:
100 ul Serum + 150 ul ACN, 0.01M HCL
15 min on ice
Centrifuge 13200 rpm, 10 min

Discussion:
The unknown peak occur before peak of serotonin in 0.6 min. (peak of serotonin 4.2 min, unknown peak 3.6 min). Height the unknown peak the same of serotonin peak.

I have taken serum without serotonin. I have got the unknown peak again.
I changed amount of ACN (1/4).

Questions:
What am I doing incorrect? Maybe I must take another precepitation buffer...?

I don't see the problem here, it seems feasible that other compounds in your sample (which is blood, urine,..?) give a response in your detector. Given that the peaks are 0.6min apart and you perform UPLC, they shouldn't interfere unless the peaks are rather wide.

Anyway: does the peak occur in a (water) procedure blank?