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Identification of Unknowns – the ultimate analytical chemistry challenge or more than that?

This is my 1st blog post at Maven E&L in 2021, so naturally my thoughts are turning to what will be different this year. For many people 2020 is a year to forget but it did give many people the chance to do or think differently in order to get things done under difficult circumstances.

So perhaps we need to think differently when in comes to perhaps some of the biggest challenges in extractable and leachable (E&L).

E&L sets many challenges but some of the most significant can arise when we are presented with an unknown substance as either an extractable or a leachable. During my time working in this area, I have certainly been presented with a few unknowns so I’d like to share my recommendations for methods and tips for identification.

• Review what you know, unknown substances are never completely without information.

An unknown will have been detected in either a LC or GC analysis, so straight away. You have some information to consider. In a GC analysis, substances which you might detect are either volatile or semi-volatile. Their elution position will tell you something about their volatility. Additionally, in GC-MS you will have a EI mass spectrum. If the unknown was detected using a GC method without MS, I would highly recommend getting the equivalent GC-MS conducted. You might even want to repeat with a high resolution GC-MS if you have available. All that additional information could make the difference. I’m making the assumption here that the mass spectrum doesn’t allow for full identification, but it will have structural information which might infer sub-structure or functional groups. This information can be invaluable. In most cases, you should be able to at least assign a class or structural features which aid in identification. However, beware rushing to declare a substance identified until you have some confirmation of beyond a simple library match particularly if the match is less than perfect. I covered some of the potential problems in my previous blog when discussing the pros and cons of GCMS vs LCMS

For example EI spectra, can be misleading when alcohols and thiols are present as the spectra may resemble the parent hydrocarbon only and long chain alkene and alkanes also can look very similar and hard to assign both a chain length and functional groups without reference materials. Nevertheless, there is a clear reason why GCMS is the most popular method of analysis for extractables and leachables.

In LCMS analysis, the information you have is likely a little different. You might have a suggestion of a molecular ion and hence a tentative molecule weight but less information on substructure. Of course fragmentation can be induced but it is difficult to compare this fragmentation to commercial library in the same manner as GCMS. You will likely have to have a proposed structure in mind to look at the fragmentation data and make sense of it. So its a little different to the GCMS in that regard. I am sure some people will argue you can work from fundamentals to predict the fragments but I have found in on most occasions the fragmentation data is used retrospectively and not prospectively. Hence an unknown detected via LCMS presents different challenges, you may known with some certainty the nominal molecule weight, indeed if you have obtain the high resolution mass spectra you may have the accurate mass, but you are still left with just a number, that number can of course be invaluable and you can suggest a molecular formula (from high resolution data)

GCMS can provide complementary information so consider if you can see the same unknown substance by both GC and LC. That may or may not be easy to establish but don’t dismiss the possibility because the combination of the two approach is very powerful.

Up to now I have been discussing this process as an exercise in analytical chemistry, and as a experienced (old and grey!) analytical chemist I have come to appreciate that these problems are nearly always solved by collective effort. So I would encourage all to embrace this philosophy when looking to identify unknowns.

So stepping away from the beige boxes of GCMS and LCMS for a second.

The second collection of information is the sample itself, what do you know about it? Or what can you find out about it? I appreciate much of today's extractable or leachable testing is done as a outsourced activity in 3rd party laboratories but establishing the source of the unknown is exceptionally useful and could be the key step to identification.

If you see the unknown as a leachable in a drug product, can you see the unknown as an extractable in the inputs for that drug product.? If the unknown is only seen as a leachable that is again a profoundly useful piece of information. Possible reason for that:

a) Unknown is a reaction / degradation product between API and another substance

b) Unknown is from manufacturing system (you have only performed extractable work on container closure materials)

c) Your extractable studies have not been a good simulation for drug product leaching process

Answers to these points is not found in GCMS or LCMS analysis but from collaboration with the wider group.

If you have found the unknown in both drug product and a material, then several more avenues are possible. The material found to be the source has a whole collection of information associated with it:

  • A supplier

  • a manufacturing process

  • a supplier chain of input substances and potential other materials and their inputs

When source of the unknown has been established it allows for the possibility that larger quantities of the unknown can be isolated using techniques such as preparative chromatography. This technique can be very successful in the hands of an expert, so once again I suggest collaboration is king.

With larger quantities and an isolated unknown, analytical techniques such as Nuclear Magnetic Resonance (NMR) and Fourier -transform infra-red (FTIR) can be deployed making elucidation more likely. These spectroscopic techniques might not be found in every laboratory and certainly need expert interpretation for maximum benefit.

Even if you are able to propose tentative structural features that can be useful and deciding what it is not, is sometimes as useful as establishing what it is.

I have already indicated spectral features in the spectrum can be useful, fundamental mass spectral features such as isotope patterns and assignment of probable neutral losses can all add towards proposals for identification. Access to high resolution mass spectra can be very helpful in establishing a proposal for molecule composition although it will most probably require supplementary information to limit the number of different possibilities but without isolation of the unknown the knowledge of source is still of fundamental help and I would encourage the pursuit of this aspect strongly.

Knowledge that the unknown is from a particular plastic or elastomer type can help immensely as it can lead to the types of substances with could be present in typical example. It is also useful to compare the unknown to other known extractables present to see if you can infer any relationships between them which might aid in the identification.This of course means approaching the supplier and producers of the material for ideas and suggestions on what the material could contain and they can prove an essential collaborator.

Another aspect to unknowns sometimes comes from changes, if this unknown has only just been detected why is that? It could be that it the first time someone has looked but if the unknown is a new feature or has grow in intensity that is again a potentially vital clue. Has there been a recent change? Perhaps one which is only apparent on closer questioning of manufacturers or suppliers. The nature of a change is certainly something to understand as in might bring insight e.g. a new cleaning process introduction, has it introduced a new or modified cleaning agent?

Identification of unknowns is very much like a jigsaw puzzle

where each part of the puzzle can help you build up the picture and complete the puzzle. Care is always needed to avoid identification which are a good fit but not actual correct. Except for art work like Tim Klein's below

As ever knowledge and experience can help to guide you plus a bit of good fortune along the way, my last blog in 2020 encouraged people to befriend a toxicologist.

In 2021, I am encouraging all to reach out and work together more perhaps when we are able to shake hands again (with the help of science led vaccines) we will be more likely to stretch and walk to our colleagues and wider collaborators to solve issues such as unknown extractables or leachable together. Since together we can do anything.

On that note, I wish everyone Happy New Year from Maven E&L.

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