Dear All, 

I try to measure the concentration of melatonin in blood samples.

For this reason I use the following extraction method:

First, I spin down the blood cells by CF at 1200 rcf / 5 min.

Then collect the serum and add acetonitrile + vortex-mix.

Then CF at 4000 rcf / 20 min, collect the supernatant.

Later I use speed-vac concentrator, and dissolve sample in H2O right before the analysis.

I use d4-melatonin as my internal standard and I aim for 25 nM of ISTD in my samples and standards.

Unfortunately, I always get a little bit higher (i.e. 31 nM) concentraion of d4-mel in my samples. In standards ISTD is about 21 nM but in some of my samples I got higher concentration, and after whole extraction process I would suspect only lower or the same.

WHY?! I checked my ISTD stock solution (its fine), my pipetting techniques (calibration curve is good R=0,9993). What can be wrong?