Compound Stability

Ever wondered just how stable those compounds are that you receive from your local medicinal chemist? Well we have, and thought it would be good to dive into the subject a little deeper. To this end, there are two published studies we have relied on over the years that directly evaluate compound stability. Please see the adjacent sidebar for references and links – we encourage our readers to download both publications and review the complete data sets.

The first study by Kozikowski and colleagues (Procter & Gamble Pharmaceuticals) selected a query set of 9,280 compounds for their analyses. Within this set were 2,041 samples that could not be used. The authors made this statement regarding this revelation:



This is of course a rather substantial number of compounds (and kind of scary, don’t you think?). Regardless, there were a total of 7,236 that could be evaluated. This evaluation consisted of resuspending samples in DMSO to a concentration of 20 mM and storing them at room temperature. A subset of the samples was then randomly evaluated by flow injection analysis–mass spectrometry (FIA-MS) at specific time points following resuspension. It is important to note that this technique facilitates detection of the anticipated compound as a function of its molecular mass, but does not address how much of that mass may be present. In other words, the compound may still be there, but most it may already be degraded. The actual data are shown below:


Compound stability as a function of time at room temperature


These data tell us that after six months at room temperature, 83% of the compounds could still be “detected”, but after one year, half of the samples were no longer detectable and were presumably completely degraded. This is helpful, but what we want to know is how much of the sample has been degraded at earlier time points. Information addressing this question can be found in the second study of Cheng and colleagues (Abbott Labs).

For their study, compounds were resuspended in DMSO to a concentration of 10 mM and integrity was monitored using quantitative liquid chromatography/ultraviolet spectroscopy/mass spectrometry (LC/UV/MS) measurements. An accelerated stability study was conducted at 40 °C, with analyzed variables including atmospheric (specifically the presence and absence of humidity and oxygen), repeated freezing and thawing, and different container materials. Before reviewing their data, let me first present what the authors said regarding tolerable compound stability: “We set the criterion of 80% of the test compounds having 80% or more of the initial concentration remaining as acceptable compound retention”. Seems reasonable to us…that means a sample could have an initial IC50 value of 500 nM, and after “acceptable” degradation, might have an IC50 of 600 nM. Whatever the outcome, we’d be happy with that.

So, let’s have a look at those data (presented below). First and most telling is the “pilot” data set composed of 644 compounds stored at 40C for 26 weeks: These data tell us that after six months – regardless of how the compounds were handled – slightly more than half of the compounds had more than 80% of their initial mass remaining. The remaining data relate to how the compounds compared relative to an internal reference standard – the important observation being that they’re all the same.


Compound degradation versus time at 40C


Next, let’s look at the outcome of comparing “dry” versus “wet” samples:


Compound stability over time - wet versus dry DMSO

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Please don’t be misled – dry is not a powder form, but rather DMSO free of water. “Wet” on the other hand is DMSO with water in it – something you’re likely to have after repeated freeze/thaw cycles…or in compounds exposed to a normal laboratory environment found somewhere like Seattle (I’m kidding). Seriously, most of us probably have “wet” DMSO (unless you’re lucky enough to have an inert gas system readily available). Bottom line – don’t worry too much about that water that accumulates in your DMSO.


Read the paper and evaluate the data for individual compounds, we think they accurately reflect the summarized data above. Here is an example of one of the more unstable compounds and its degradation over time:


Accumulation of degradation products over time


What do we learn from this? Well, half of the compounds we study are quite stable over an extended period – and the other half?


Not so much…


Over time then, compounds will absolutely degrade and of course we want to retard that process to the best of our ability. So, the best thing to do is freeze them – right? Certainly for a large corporate library this is advisable. However, freezing compounds means they must cycle through at least one, and likely repeated cycles in and out of the freezer. What impact does that have on our compounds? Well, here are those data:


Compound stability versus freeze/thaw cycles

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Perhaps what you might expect. First, some compounds crash out of solution after freezing them only once, more so with repeated cycles. Second, whether we vortex or repeatedly aspirate/dispense (the authors call this “suck and spit”), those insoluble compounds often don’t go back into solution. We suspect many of you have traveled this road before. Despite these issues with solubility, it is reassuring that most compounds remain soluble throughout multiple trips through the freezer – as demonstrated by these data representing the population:


Effects of freeze/thaw cycles on average compound stability


To summarize, most compounds stand up well to repeated cycles in and out of the freezer. But those that don’t often remain insoluble.

Finally, the authors examined the effects of the vessels the compounds are stored in (glass versus polypropylene plastic):


Stability as a function of container composition

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The room temperature condition is provided for comparison, with the outcome being that – if anything – plastic is a better container than glass.

What then have we learned? Over the short term (let’s just call that one to two months), most compounds are very stable – even at room temperature – and can be expected to give similar results to compounds that have been freshly solvated in DMSO (particularly considering the “accelerated” 40C conditions of this study – see the paper). However, there is a risk of compounds crashing out of solution once frozen – and then remaining that way for the duration. So, our advice to those clients that are sending us compounds to evaluate; ship them at room temperature and we’ll keep them that way during the study. Both powder and liquid form are acceptable, though we recommend provision of solids – then you don’t have to worry about the compound leaking out of the tube. This is not only better for the compound; it will do wonders for your shipping budget! After it leaves your building, don’t worry; you can rely on the data generated by ACD. Want us to hang on to the compound for a longer period – just in case you want to perform one or more follow-on studies? No problem, we’ll keep it in our freezer until your ready (the limit for such storage is six months unless prior arrangements are made).

In closing, please let us provide this word of warning for everyone – if you pull a vial that’s been tucked away in a lab drawer for the last couple of years, then don’t be surprised if it no longer “works”.