Challenging Pharmaceutical Impurity Analyses Part 1: Derivatization

With pharmaceutical impurities under increasing scrutiny, recently so with the nitrosamine issues in Ranitidine and prior to that Valsartan, the question of ‘What’s in our medicines?’ has never been more pertinent.

Chromatography is at the heart of impurity testing, with the typical pharmaceutical testing lab looking similar wherever you go. You will be greeted with rows and rows of HPLC and GC systems as the stock standard tool for analysing pharmaceutical impurities with some LC-MS, GC-MS and perhaps one or two Ion Chromatographs. But what happens if the analyte of interest isn’t suitable for analysis by HPLC or GC?

Nitrosamines are notoriously difficult to analyse and the approved method following ICH Q2(R1) requires HPLC coupled to high-resolution mass spectrometry to enable detection, which is not your standard laboratory equipment. Labs are constantly being challenged with difficult compounds to analyse and high-end equipment is entering into the routine sector. Are there other ways to meet these challenges? Amines are just one topical example; think also of small organic acids often used at starting materials and require tracking through the chemical process. Can chromatographically challenging compounds be converted into a more ready form and what factors should be considered with this?

Is derivatization the answer?

When an analyte of interest isn’t volatile, has no chromophore or cannot be retained on reverse phase columns, one option is to derivatize it into a form we can analyze. Sounds simple! But for anyone that has worked in a lab developing these methods, this is not always the easy option.

Not only do you need excellent knowledge of the chemistry involved and the conditions required to carry out a full derivatization to achieve 95-100% conversion, but you need to consider the purity of the chemicals used and what sort of recovery will be achieved? Whilst this usually works beautifully with standards, how will the derivatization reagent react with other compounds in the sample matrix?

Then will the derivative give you the sensitivity you require and is it fully resolved from interfering peaks and finally, is the derivative stable? Or will you start to see a loss of sensitivity over time? In the case of organic acids, derivatization is not so successful.

Derivatization can mean a lot of work in developing and validating the method, then there is the increased sample preparation time when it comes to running samples, and how will this method transfer to a manufacturing environment?

There is also the risk of opening up a whole new line of troubleshooting.

From experience, the more steps in a method, the higher chance of issues occurring. Some examples may be:

- peaks going missing

- low detection response

- extra peaks

- a new batch of reagent not yielding the same conversion

In a GxP environment these issues, not only take time, expertise and valuable resources but can soon escalate into a full-blown quality investigation.

With all this in mind, should derivatization be the first choice? In reality, derivatization is perhaps the last resort, but does this reflect the practices in the current pharma laboratories?

Perhaps there are other options not previously considered which offer a direct approach. Is it uncomfortable when GC and HPLC are not the answer? We shall discuss more in the next part of this series.