I have done a lot of homogenization of striatal tissue for analysis of catecholamines and have a few recommendations. Avik's suggestions are great but I have one thing to add.

I would highly recommend using a hand-held sonicator rather than a piston or other mechanical homogenizer. Anything that relies on pulverizing the tissue will likely leave small fragments that will get stuck in your HPLC. We used to use a tissue homogenizing pestle and the pressure in the HPLC would increase steadily over the course of running samples, leading to a change in analyte elution time. Our best guess was this was due to microscopic tissue chunks getting stuck in the HPLC and clogging things. A hand-held sonicator does a great job of fully lysing the tissue and yields more consistent results.

Hope that helps!

I want to add few points:

1. After homogenizing the brain samples, homogenates should allowed to stand for 15 minutes at 2-8 °C in refrigerator to aid the precipitation of proteins, cell debris, microscopic tissues etc.

2. Then proceed for centrifugation and collect supernatant

3. Before injection in to HPLC, clear supernatant must be passed through syringe filters.

I think, in this you can get rid of the problems of clogging or stuck of HPLC column. 

Hello to everyone!!!

           Still i have a problem with neurotransmitter estimation by using HPLC-ECD system.

I am using the following method for for the the sample preparation:

I am using 1 ml of  17% perchloric acid in deionised water for 100 mg of tissue and homogenized by hand held homoginizer by keeping homogenized sample in ice bucket, then incubated the sample in 2-8 C for 15 minutes then centrifuged 12000 rpm for 15 mins.

The supernatant was then filtered through 0.22 micron filter then this was injected to the pump.  

Dear Govindaraj!

Do you have reference/article for "using 1 ml of 17% perchloric acid in deionised water for 100 mg of tissue"?

If yes its ok. Otherwise I refer you another method.

Hello Cheema!!!

        Yeah is there.. could you please tell other method??

Hello Govindaraj,

First of all 1 ml volume is too big to homogenize properly by a hand held homogenizer. Even the fat contents of the rat brain tissue will not be homogenized efficiently by this method. It is recommended to use sonication for this purpose. Moreover you can take less amount of tissue and solubilize it 10 times volume of 0.1 or 0.2 M perchloricacid.