Develop Method "Novel Stable Isotope dilution GC/MS&quo

[louismeme]

I need HELP. I am interested in the level of my drug "X" in rat brain tissue. I then synthesized the stable isotope of that drug "D3-X", generated a standard curve (slope as expected is 1) after dilution of my drug "X" in water spiked with "D3-X".
My question is:
Should I generate my standard curve in brain homogenates from untreated rats? Knowing that after extraction of the brain, I will spike my brain homogenate with my "D3-X". Thanks in advance for all your help.

[Mary Carson]

Some on this forum feel quite strongly that all std curves should be prepared in matrix. I don't. However, during method development you should definitely be testing whether your standard curve needs to be in matrix because of matrix effects, or if you can get away with standard in solvent and just run spiked QCs in matrix. You do need to do experiments to validate your recovery from the tissue, regardless of how you make up your standards. Ideally this is done with radiolabeled dosing and making sure your extraction gets all the radiolabel out. This is not an experiment that can be done with spiking only. However, you can get a reasonable approximation of recovery accuracy (if you are sure there is no protein binding or other major metabolism going on--a big assumption) by doing an exhaustive extraction of tissue from cold dosed rats.

[Peter Apps]

The whole idea behind going to the trouble of synthesising isotope labelled standards is that they mimic the behaviour of the analyte at all stages of the analysis, but can still be distinguished from the analyte. In particular they extract from the sample in the same way as the analyte, and so a calibration against an isotope labelled standard added to the samples does not need a correction for recovery.

The one exception to this happy state of affairs is when the analyte is in a form which does not extract in the same way as labelled standard spiked into the sample. This might happen in your case if the analyte was incorporated by the live rat brain in a way that does not happen to the standard whn you add it to mashed brain. Nonetheless, the best that you can do is to spike your labelled standard into the samples, and calibrate using analyte and standard spiked into brain.

Peter