Over the past year, myself and Dr. Christian Zeine of LGC, have been engaging with scientists involved in the analysis of pharmaceutical impurities. During this time, there have been some questions that repeatedly arose, so we thought we would produce this article to answer four of these common questions. If you have any further questions please comment below and we would also be happy to answer these in a follow-up.
1. My API (active pharmaceutical ingredient) is present in the hydrochloride form. Does that mean I can only use impurity reference standards which are also in the hydrochloride form?
No. As far as we can tell, from our own experience and that of our customers, it does not matter in what salt – or even free base – form your impurity reference standards are present. If an API is present as an HCL salt, the impurities in it might be present as an HCL salt or a free base, maybe even as another salt. No matter which, in HPLC the mobile phase determines in what solvated form each impurity passes the column/detector, so retention time, UV spectrum and also mass spectrum will be similar, regardless of the form in which the impurity is present in the tested API sample. Therefore it also does not matter in what form your impurity reference standard is present. But, of course, you do need to know exactly which form this is, in particular if you want to quantify with that impurity standard.
This is where it gets really interesting: We had countless customers in the past switch to our reference standards when their original source for the impurity dried up – for example the API supplier or the pharmacopoeias changed from a neat impurity standard to an impurity mixture. During this switch, it turned out that the customers’ previously used materials wrongly identified the salt form concerned. So the user worked – sometimes for several years – with a misidentified reference standard, which can subsequently cause all kinds of trouble. This issue is also a topic of our recently recorded webinar on impurity reference standards.
2. Is it possible to quantify impurities without the use of reference standards?
This is possible with organic impurities in some situations of HPLC-UV coupling by the setup of so-called relative response factors (RRF), and in some other situations by using Charged Aerosol Detection (CAD). CAD is a near-universal detector that does not suffer the biases that other liquid chromatography detectors incur. The detector works by converting the analyte molecules into dry particles, with the number of particles increasing proportionally with the amount of analyte. A stream of positively charged gas then collides with the analyte particles. The charge is then transferred to the particles—the larger the particles, the greater the charge. The particles are transferred to a collector, where the charge is measured by a highly-sensitive electrometer. This generates a signal in direct proportion to the quantity of analytes present, allowing quantitation without the requirement of reference standards. This recent poster offers an excellent example of using a multi-detector set-up, including CAD, for quantitative sample analysis without reference standards.
Regardless of whether the RRF or CAD approach is utilized, there is still the question of identifying the impurities during the development of the analytical method, and validating it before it gets into routine mode, for which work reference standards are still quite necessary.
3. What are reference standards for impurities, and what are research materials? What is the difference, and can I use research materials for my impurity testing?
Information on what constitutes an impurity reference standard (IRS) is scarce. The European Pharmacopoeia states in text 5.12. that “reference standards are established using suitable procedures and their continued suitability for use is monitored” and further “a CRS (Chemical Reference Substance) corresponding to an impurity is characterized for identity and purity.” Meanwhile the ICH simply requires in impurity guidelines Q3A/Q3B that “reference standards used in the analytical procedures for control of impurities should be evaluated and characterized according to their intended uses.” There is not much further information available, so it is not surprising that approaches to impurity standards are highly variable, from both manufacturers of such standards and end users.
In our opinion, assuring “continued suitability for use” in reference standards, as mentioned above in Ph.Eur. 5.12., requires an ongoing stability or “fit for purpose” monitoring program – an important characteristic which distinguishes reference standards from research materials. Such programs run under the quality accreditation of the RS manufacturer, ideally under ISO 17034:2016 (General requirements for the competence of reference material producers), which represents the highest quality level possible.
Research materials lack this stability-monitoring program. If you are lucky, the manufacturer states that they do in the COA or other documents, but often it is left unclear. Also unclear is how the identity was checked. Often NMR and/or mass spectra are provided, but there is no further explanation of if and how these data were checked. There are even materials on the market calling themselves “reference standards” which are missing this kind of information. A real impurity reference standard should provide identity evidence, i.e. spectra and information on how they were used for proving identity, at least comprising a short formal sentence that on request can be elaborated into a full identity interpretation report.
Can research materials be used for impurity testing? This is a difficult question. Provided the identity was determined correctly and somehow documented, then the material can be used. Users also use research material sometimes for quantitative purposes, hopefully while aware of the following caveats:
Research materials should always be considered 100% pure, even if it is clear they are not. It is not a wise approach to use an analytical statement such as “purity >80%” as an assay value for calculation purposes. The actual assay is normally higher, resulting in underestimation of the research material, and in turn underestimating the impurity in the sample analysis, which cannot be tolerated in any GMP environment.
Overestimation (OE) is an economic risk. OE can trigger false-positive out of specification (OOS) results, leading to unnecessary, time-intensive investigations. When a batch change for the IRS is due, the risk of OOS results is increased. Examination of OOS events is expensive: For simple investigations, financial costs of at least 3000 USD are reported. During the R&D phase of new API/FDF projects, OE may lead to exceeding the qualification thresholds of ICH Q3A/B, triggering lengthy and costly toxicity studies. Any costs incurred by OOS investigations and toxicity studies will almost certainly be several times higher than the costs for a quantitative IRS.
If you are interested in learning more about this topic, we recommend the already mentioned webinar.LGC will also release a white paper on this subject in autumn. Sign up here for the LGC newsletter in order to be notified once it is online.
4. What options are available for the analysis of inorganic impurities and those not amenable to liquid chromatography?
In our various workshops, articles and webinars Christian and I have discussed pharmaceutical impurities and have tended to focus on organic impurities, as my background is liquid chromatography, but the question of analyzing inorganic impurities has usually arisen as well. Organic impurities are broken down into volatile, semi-volatile and non-volatile. Non-volatile impurities are commonly analyzed using liquid chromatography (LC) or hyphenated with mass spectrometry (LC-MS). With volatile and semi-volatile impurities then GC (gas chromatography) / GC-MS is used.
With inorganic or elemental impurities the analysis method of choice is ICP-MS (Inductivity Coupled Plasma Mass Spectrometry). With ICP-MS there are a number of different options available depending on your particular application. A useful ICP-MS selector guide is available on this webpage. In some situations you may be required to use a variety of different techniques to study both organic and inorganic impurities and ensure that you are analyzing all impurities in your sample. For more information on all these techniques for organic and inorganic impurity analysis I would recommend the Extractables and Leachables Learning Centre.
This is just a flavor of some the questions we are regularly asked, but if you have further questions then we would be happy to answer these as well.
For an overview of pharmaceutical impurities and their analysis, we would recommend registering to view this on-demand webinar on the topic.
To take part in a detailed investigation into pharmaceutical impurities visit one of our Pharmaceutical Impurity Workshops, which take place in various locations across Europe and combine both presentations and practical sessions.