I think there are a couple of points to your question. I would regard biological half-life distinct from stability in cell culture media as the two are not necessarily the same. Are the constituents in all cell culture media the exact same in composition as biological fluid such as sinusoidal blood at the liver or interstitial fluid at the lung? The answer is likely no, therefore you need to carefully characterize the stability of your test agent in any media for you to know if the results of your assay is valid. If your assay endpoint is measured in hours, it makes little sense to measure analyte stability in media for days since the two time frames are not equal, yet knowing the stability reasonably beyond the incubation conditions of the assay allows for lag effects that may occur during sample processing. To further answer your last point, you need to have a sense of what effect you want to determine: stability or possible mechanism driving the instability. For one you may need a shorter time, for the other, you may need more drive to prove the mechanism.

As I understood and according to that, I need to establish my drug stability for each new media or conditions I used?.

Your last point is clear, thank you for your answer.

Hi Ibrahim

As point out above by Ron the biological half life is very different from the stability in culture media and is related to whole organism kinetics with typical pharmacokinetic parameters (ADME) having an influence.

Generally we do a stability test of the test drug which is spiked into the culture media, including any additives such as the FCS, at the highest and lowest concentration that will be tested and the media then "cultured" typically for 72 hours for our assays. This endpoint assay is done by LC-MS/MS where we look for the original test compound and any probable breakdown product masses. Generally the biggest problems are binding to the plastic ware and solubility problems where the compounds either float to the surface or precipitate without degrading. The compound recovery is then then very high but the cells would not be in contact with the drug if assayed.

There are some compounds that do break down rapidly in aqueous media and these will need to be added to the assay more regularly during media exchanges that serve to remove the breakdown products that could be toxic or have another effect than the expected outcome.

Thank you Prof. Duncan for your answer, I wonder if there is another way to check for stability in case MS is not available. Also for solubility, what tests could be employed to check for the exact solubility of our drug in certain media or conditions ?

Hi Ibrahim

Any assay that is sensitive to the test compound can be done. The advantage of LC-MS/MS is that it is sensitive enough to be able to use the content of individual wells from a 96 well plate and that the parent drug and breakdown products can be determined from the same sample.

TLC is often a good technique for small samples but you do need a good visualisation technique for your test compounds.

Solubility can be tested by dissolving or adding a solution of the test compound into culture media at the highest concentration to be used. After about two hours in a fridge, this solution is transferred to a centrifuge tube and centrifuged at high RCF at 4 C for about 20 minutes. The concentration of the test compound in the middle zone of the tube is then tested against a reference solution at the concentration generated in the media. Be careful to not take from the top surface or to take from the bottom of the tube where any precipitated compound may have settled.

That's so clear, thank you for your appreciated time and help!

HPLC/UV is another option if you don't have access to a mass spec, though you'll need the compound to be a good chromophore with a lamba max that is separate from that of the solvent.