Peter Rooney

A pharmaceutical product can become contaminated by metal impurities via a number of ways. There are many inorganic impurities that are deliberately added to the process (e.g., catalysts), while undetected contaminants from starting materials or reagents, or that come from the process, such as leaching from pipes, mixing vessels and other equipment, can raise serious issues. There are also naturally occurring metal ions within plant or mineral sources that are used to produce APIs and herbal medicines. 

Regardless of how they might get into a product, the control of these impurities may be certified by a vendor, but pharmaceutical producers must conduct their own tests to demonstrate the absence of impurities before using these materials in a manufactured article. 

Heavy metal testing is by no means a recent phenomenon. The 

 (Ph. Eur.) 2.4.8 was derived from the heavy metal tests inherited from national pharmacopoeias, many of which had been in existence for years. The 

 (USP) has included a general test for heavy metals since Volume VIII of 1905, which used sulfide precipitation to detect antimony, arsenic, cadmium, copper, iron, lead and zinc. The purpose of the test had more to do with prevention of mislabelling than prevention of contamination as heavy metal salts were often used in therapy, and which salts were present in a treatment had to be known. The detection of residual contamination was introduced in 1942 with Volume XII, in which a lead-containing standard was included in the test. The aim was to detect potentially toxic heavy metal residuals, such as lead and copper, because these were widely used in production equipment at the time. Interestingly, metals such as iron, chromium and nickel were not revealed by the test.  

It is the limitation of the current 'wet chemistry' methods described in USP Chapter <231> that has led to the decision to revise the chapter.  

Current compendial methods were all developed before the introduction of modern analytical instruments. Their advantage is that the methods are easily transferable from one laboratory to another and do not require sophisticated instrumentation or specialist expertise. However, the analysis also involves subjective visual examination and comparison of the sample solution with a lead standard. As with the method of 1905, the compendial methods use a reaction to form the sulfide of any 'so called heavy' metal ions present and the total metal content is reported against the lead standard response as a limit test.  

The validity of this comparison relies on several assumptions, all of which can be questioned. For example, the compendial method assumes that each of the heavy metals in the sample matrix will react in a like manner to lead to form a sulfide species. This assumption applies despite many sulfides being insoluble and some elements having a far more intensely coloured sulfide than lead. Similarly, the compendial method assumes that the reaction kinetics for lead sulfide will be very similar to those for other metal sulfides and that reaction kinetics are not greatly affected by the sample matrix. A final major assumption is that the heating and/or ashing step of the method will have no impact on volatile metals or metal compounds. 

These are not the only reasons to challenge the validity, applicability and reliability of the compendial methods. It is no surprise that additional chapters for the control of specific metals and other inorganic impurities have been added to the pharmacopoeia during the years. Significant amongst these in the USP is Chapter <730> Plasma Spectrochemistry, which was introduced approximately 2 years ago and gives laboratories the opportunity to use techniques such as inductively coupled plasma with either mass spectrometry (ICP-MS) or atomic emission spectroscopy (ICP-AES).  

The advantage of ICP methods is that they provide specific detection and quantification for each of the elements expected to give rise to a positive response in the compendial methods, such as arsenic, selenium, lead, silver and bismuth. The subjectivity of the semiquantitative comparison that is required by the compendial methods is eliminated with ICP. In some cases, ICP is also quicker, requires a smaller sample size and depending on sample preparation may give a higher recovery of some or all of the elements of interest. Additionally, the sample preparation methods that can be used for ICP is less likely to lead to the loss of the volatile elements.  

The proposed revision to Chapter <231> recommends procedures that rely on modern analytical technology and includes limits that are based on toxicity and exposure levels for the selected metals. In its stimuli article for the proposed revision, the USP 

 Advisory Panel on Inorganic Impurities and Heavy Metals and the Expert Committee proposed that "the selection of an instrumental technique and a procedure for the evaluation of the inorganic impurities specified ... requires the evaluation of a large number of variables including, among others, sensitivity, precision, accuracy, compatibility, time, and cost. The method selected may include plasma spectrochemistry, atomic absorption spectroscopy, or any other method that displays requisite accuracy (trueness and uncertainty) and established sensitivity and specificity."

In enabling heavy metal limit testing to be conducted using ICP-MS and other plasma spectrochemistry, the revised USP <231> will not entirely rid itself of problems as the stimuli article raised several questions, which any revision will need to address.  

Any standard method always runs the risk of being too general and unable to deal with particular circumstances. The proposed revisions to Chapter <231>, as outlined in the stimuli article, do raise some concerns; for example, certain elements have not been included in its Table 1, which sets out the proposed element limits for oral and parenteral materials. 

Silver is not in the list of elements in the table, which is surprising given that silver would have been detected by the methods given in Chapter <231>. With the current changes to water purification processes, many industries use sacrificial copper/silver anodes to kill microbial contamination and so one might have expected silver to be included in a list of elements having toxicological properties.  

Other elements have also been omitted from Table 1, such as titanium, barium and zirconium, that have toxic forms. It is feasible that the USP intends that the individual limit tests for barium will remain as they are now, rather than being incorporated into this revision. However, given that rarely used elements such as iridium and thallium have been included, it is curious that other elements such as those listed above, as well as germanium and gallium, both of which are toxic, have not been included.  

From a practical perspective, there is also room for debate regarding the reliability of the proposed methods; for example, it would appear that spiking of samples is being proposed as a means of confirming recovery. However, where the concentration of certain metals is already high, the spiked amount may be too small an increase to be detected above the level already found in the sample. In extreme cases, the contribution of the spike could be within the tolerances of normal result variability. If, for example, the method prescribed in the stimuli article were to be applied to ferrous fumarate to determine the levels of mercury, arsenic and lead, the system suitability would not be achievable for iron. This observation means there will have to be specific exclusions in the chapter or individual monographs to cope with such occurrences. 

The stimuli article illustrates a flow chart for determining the sample preparation method. However, no acknowledgement is made that with a water soluble sample, solution viscosity and salt strength must also be taken into account, as both of these properties will have severe effects on the performance and life of the nebulizer in the ICP instrumentation.  

In the case of water insoluble samples, decisions must also be made about which solvents are appropriate to use. For the time being, the stimuli article makes no recommendations and proposes no list of acceptable/unacceptable solvents. Again, this choice will have major impacts on the cost of the analysis and lifetime of equipment. Should the method force the use of analysis in an organic solvent, it may oblige testing laboratories to buy solvent system nebulizers and plasma torches as standard torches are often unsuitable where organic solvents are involved. There is also an assumption that the elements of interest will be soluble in the solvent of choice. This may lead to instances where known sample solubility data are used to select a solvent, but the trace metals will not be solubilized and because of the low concentrations this will not be apparent as out of solution. Interestingly, the option of microwave digestion is only offered when a sample is deemed to be insoluble in water or 'another' solvent. In many cases, microwave digestion may be preferable because it destroys the sample matrix and allows a solution of sufficient sample concentration to be produced, enabling the laboratory to achieve the Method Reporting Limit and ensuring the maximum lifetimes from the plasma components that are exposed to the sample solution. In our view as a testing laboratory, it would be preferable to allow microwave digestion to be used as a first resort preparation technique, (where appropriate) rather than as a last resort when all other methods have proved ineffective.   

These critical observations of the stimuli article should only serve to illustrate the enormous complexity involved in creating pharmacopoeial methods that are required, in effect, to cover every current and foreseeable circumstance arising from pharmaceutical production. That it has taken some time for the revision of Chapter <231> to get to this stage should come as no surprise to anyone, and it is to be hoped that the final revision brings to an end what has been a lengthy and sometimes faltering process. An earlier attempt to revise the chapter had to be reversed and it would be a shame if the current process met with a similar end.  

www.usp.org/pdf/EN/USPNF/2008-04-10InorganicImpuritiesStim.pdf

Articles

The closing date for comments to be received by the US Pharmacopeia (USP) for proposed revisions to Chapter <231>, which deals with analysis of heavy metals, is 15 December 2008. The USP has been working towards for approximately 4 years and the task has not been easy.

The long awaited revision

If the scientists responsible for drug discovery are the 'star-performers' in pharmaceutical science, then those performing stability studies deserve the nomination for 'best supporting actors'. They don't attract much attention, but without reliable stability studies and shelf-life testing, there would be no drugs on the market and no viable pharmaceutical industry to speak of. 

That said, a stability study is something of a misnomer as its purpose is to detect signs of instability, and sometimes to create the conditions whereby any instability in the product or its packaging might be encouraged, accelerated and otherwise brought to light.  

As such, stability studies are not only concerned with the shelf life of the end product, but accompany and support all phases of drug development. This includes specific studies required to support progress through to the various phases of clinical trials. After all, if the drug can't be produced in a form that can be effectively packaged, distributed, stored and administered, its efficacy is largely immaterial. That being the case, stability studies play a much greater role in drug development than many people realize. Though largely routine and time consuming, and, therefore, an ideal candidate for contracting out, stability studies are integral to the drug development process. Therefore, the choice of partner laboratory is a significant decision for a pharmaceutical manufacturer, not least because it has implications for the speed and cost of bringing drugs to market. 

There are essentially two aspects to the stability study, at least in terms of facilities and expertise. The first of these is appropriate storage of the drugs for the period of the study and the second is the testing for degradation, spoilage, contamination and so on. While it is possible to separate the two by choosing different laboratories to store and test, there are clear advantages in having both disciplines under the same roof. 

Appropriate storage facilities will include all rooms, chambers, ovens, fridges and freezers that can provide controlled environments of temperature, humidity and light as appropriate to the specific drug substance or product being challenged. In accordance with the International Conference on Harmonisation (ICH) guidelines, these facilities must be validated initially to confirm operation to the required tolerances and thereafter monitored continuously during use. Revalidation is performed as an assurance of continued performance. However, equipment failure can occur and as such back-up facilities are recommended to prevent long-term excursions from the specified conditions. In the case of short-term excursions, an investigation into the effects on the stability results will be required. 

Despite the ICH requirement for validation there is little actual guidance on how the validation should be performed. The validation protocol is, therefore, one of the key areas for assessing the quality of the laboratory entrusted with sample storage. Indeed, the rigour of the initial validation process, the robustness of in-use monitoring and the frequency of revalidation will all impact on the reliability of a stability study.  

The specific conditions chosen for storage will depend on the aspect of stability that is being assessed. Long-term storage conditions will differ from accelerated testing conditions, or stress testing conditions. Depending on the market and manner in which the drug is likely to be used, it may make sense to perform storage at the more extreme conditions of temperature and humidity, 30?°C and 65% relative humidity (RH), as specified in Q1F for long-term storage. 

For multidose products that have an extended use period and will have the pack integrity broken on opening, it may be advisable to also conduct in-use stability testing from which an in-use product shelf life can be established. While it is not always necessary to set an in-use shelf life, the decision not to do so must be justified and fully substantiated. 

An example of the type of product where in-use testing is sensible is one where a moisture-proof seal is used on a pack containing several days' treatment. CPMP/QWP/2934/99 gives guidance on the in-use stability testing of human medical products.

  It refers specifically to products that: "Due to repeated opening and closing, may pose a risk to its contents with regard to microbiological contamination, proliferation and/or physicochemical degradation once the closure system has been breached."  

The storage regime should, as far as possible, simulate the actual use of the product and include testing at intermediate time points throughout the proposed shelf life period. Such tests might be physical (assessing changes to colour, clarity, particle size, etc.), chemical (assaying the active substance, degradation products, pH, etc.) and microbiological (total viable count, sterility, spoilage). 

The specifics of the testing regime, in terms of frequency and nature of tests required, are governed by a variety of factors. These include, but are not limited to, the particular characteristics of the product and whether it is a new drug or existing product. In the latter case, the use of existing stability data may be permissible.  

Needless to say, there is a vast range of chemical, microbiological, physical and microscopy tests that may need to be evaluated to assess the stability of a pharmaceutical product and its packaging. Any contract research company (CRO) performing such studies will need to be equipped with considerable analytical resources and expertise. 

Key to the success of outsourcing stability studies is the relationship/partnership between the client and CRO. It involves a high degree of trust and mutual support. The client must be willing to share potentially commercially sensitive information with the CRO, including technical transfer of analytical methods. The CRO must be fully communicative throughout the course of the trial and particularly vigilant to identify and communicate any out of trend/out of specification (OOT/OOS) observations.  

From the CRO's perspective, openness may simply be a matter of regular communication and a readiness to respond to client questions. However, modern IT systems permit greater transparency through a web-enabled LIMS, which may allow clients to access and assess the progress of their projects. 

In the case where progress is halted, perhaps by the reporting of OOT/OOS results, there are potentially serious cost implications for the drug manufacturer — another area where the choice of CRO could prove significant. A CRO with investigative abilities as well as routine testing capability will certainly be able to save a client time and money, especially when investigating OOS results. Investigative analysis will provide critical information to the client and help determine the course of action to be taken. 

The CRO may also be able to save money for the client by careful bracketing and/or matrixing of the stability study. Bracketing describes the design of a stability schedule such that at any time point only the samples on the extremes are tested, for example, the minimum and maximum dosage forms, or smallest and largest pack sizes. Clearly, bracketing has an inherent assumption that intermediate conditions are covered by the extremes.  

Matrixing is a statistical design of a stability schedule so that only a subset of the total number of samples is tested at any specified time. At subsequent set times, a different subset of the samples will be tested, with the aim that all samples are tested at some point during the lifetime of the study. Clearly, where an OOT/OOS situation occurs, samples not scheduled for testing can be tested to provide supplementary data to support or negate the continuance of the stability trial. In any event, matrixing can be applied to samples where there already exists a high degree of certainty concerning their stability. In all cases, scientific and often statistical justification of the matrix design is required. 

Both bracketing and matrixing seek to reduce the resource required for a stability study without compromising data quality and patient safety.  

Stress testing is typically targeted at determining the stability of the drug substance. According to the ICH definition, stress testing "helps to determine the intrinsic stability of the drug substance by establishing degradation pathways to identify the likely degradation products and to validate the stability indicating power of the analytical procedure used". Photostability testing is seen as an integral part of stress testing and may be pertinent to the testing of drug substances and drug products. Indeed, the ICH gives specific guidance (in Q1B) for photostability testing.

An experienced CRO can help to define the specific stress parameters to be applied to the test material. Such variables may include temperature, humidity, pH, oxidation and light. The CRO may also be able to suggest appropriate tests for identifying the degradation products, which may be unanticipated and difficult to isolate/identify. 

There are many more aspects to stability studies than appear at first sight. Their duration and the long periods of inactivity between each round of testing can suggest that stability testing is more about the waiting than the testing. The name too can also create the expectation of stability and the absence of anything to report, whereas as noted above, the study is principally concerned with detecting instability.  

As such, stability studies play a key role in drug development and in getting new drugs to market. The scientists playing the role of 'best supporting actors' are not mere walk-ons, but an essential part of the drug development team, a fact which both the client and CRO should not take for granted when stability studies are being outsourced. 

is a technical manager (pharmaceuticals) at RSSL Pharma (UK).

www.emea.eu.int/pdfs/human/qwp/293499en.pdf

Key to the success of outsourcing stability studies is the relationship/partnership between the client and CRO. It involves a high degree of trust and mutual support