I'm trying to develop a method for quantification of drugs (small molecules) in cardiac tissue with LC/MS-MS. The first step is homogenization with ultrapure water with a gentleMAC dissociator (rotor-stator homogenization) followed by a protein precipitation (PP). However, I'm in doubt whether the homogenization and PP adequately disrupts the cells, so I am measuring the total concentration of the drugs. Do anyone have any experience with quantification of drugs in cardiac tissue (or similar tissue)?
Dear Mr. Christian
If you are having problems with homogenisation, you can use liquid Nitrogen to crush the cells.I used this for liver and breast tissue (Mice) samples for extraction of proteins under study and it works well.
Regards
Amand
Dear you can take weighed quantity of cardiac tissue with appropriate amount of water (like. 1 ml/0.25 g tissue). Homogenize it...in comparison to other tissue may cardiac and muscle tissue take more time too dissolve. After homogenization take 100 microL and add upto 2000 ml of ACN. Vortex for 3 minutes and centrifuge it. Take supernatant , dry under nitrogen and reconstitute it with suitable solvent for further analysis.
Cheers
Im using liquid nitrogen to crush my samples, works perfectly and its quick and easy, however what buffers can you help by telling me what buffers I can use to homogenize my samples which preserve the ezyme activity in the samples?
The GentleMACS is a tissue dissociator, not a true homogenizer. It's meant for the dissociation of tissue into viable cells, so I'm not surprised that it wouldn't provide good cell lysis.
For a tough tissue like cardiac tissue, I would recommend a bead mill style homogenizer. The most important things to look for are units that would meet your needs for sample size and throughput. You can find a lot of options here: http://homogenizers.net/collections/bead-mill-homogenizers
http://homogenizers.net/collections/bead-mill-homogenizers
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