Nancy Lewen

The use of atomic spectroscopy techniques and sample preparation procedures is something that is not as routine in the pharmaceutical industry as chromatography-based techniques and sample preparation procedures are. With new requirements being implemented regarding elemental impurities by the United States Pharmacopeia (USP) and International Conference on Harmonization (ICH), analysts in the pharmaceutical industry are, in many cases, working to determine how best to analyze their samples. Sample preparation techniques that can be used for pharmaceutical samples are the same ones that have been used by other industries for many years. This article provides a brief overview of potential techniques.

	As the pharmaceutical industry prepares to implement the 

International Conference on Harmonization

) chapter <232> “Elemental Impurities-Limits” (2), there will be a need for laboratories to perform analyses of drug products, excipients, and active pharmaceutical ingredients (APIs) using 

 <233> “Elemental Impurities-Procedures” (3). 

<233> provides analysts with flexibility in how to perform the analysis on a given sample, while providing two refereed methods for analysis, inductively coupled plasma-optical emission spectroscopy (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS). Although ICP-MS and ICP-OES have been used for metals analysis in the pharmaceutical industry for quite some time (4,5), the techniques have seen limited application for traditional heavy metals testing (6) because of the availability of the traditional wet chemistry 

 <231> “Heavy Metals” (7) test. With the pending retirement of 

 <231>, analysts are transitioning to the use of 

 <233> to perform any analyses that might be required to complete the risk assessment on a product. For some laboratories, the analysis of pharmaceutical samples using plasma spectrochemistry is new and may present challenges.

	Inherent in performing any analysis is the preparation of the sample, which can vary from sample to sample and is a critical and perhaps the most complicated part of the process. One of the frequently used preparation techniques in the field of atomic spectroscopy is acid digestion. Before the advent of modern microwave digestion systems, hot-plate digestions were used as a means to prepare samples for metals analyses. Although they are effective in solubilizing some samples, hot-plate digestions are prone to several potential problems, including loss of potentially volatile analytes such as mercury, sample loss from material spitting out of the digestion container if the sample is rigorously boiled, and simply not providing sufficient heat to adequately dissolve the sample. Studies have been performed comparing the use of hot-plate versus microwave digestion techniques (8–11). As more microwave digestion systems have become commercially available, the use of these systems has become common (12–14), and closed-vessel microwave digestion is the preferred method of sample preparation in 

 <233>. The selection of the acid used to perform the digestion is key to the success of the digestion. Most often, nitric acid (HNO

) is used but other acids, such as hydrochloric acid (HCl), sulfuric acid (H

), or hydrofluoric acid (HF) are also commonly used (15).

	Unfortunately, acid digestions are not always effective in completely solubilizing a sample for metals analysis. Other approaches for preparing samples for metals analysis include fusion, where a flux mixture is added to samples to help lower the melting point of materials in a sample. This approach also helps to achieve a homogeneous fluidity of melted oxide impurities in the sample (16,17). Fusion, performed with fluxes, is a long-used method in the metallurgical industry for determining gold in ore samples, but this old “fire assay” technique is not commonly used for metals analyses, because the majority of samples do not require this rigorous approach to sample preparation.

	When testing is performed to demonstrate that a drug product complies with 

, the manufacturer can elect to test either the drug product or the various components that make up the drug product. In the case of drug product analysis, only the analysis of the drug product is required. Sample preparation procedures may need to be more rigorous, because of the fact that drug products are often mixtures. Additionally, drug products often include capsule shells or tablet coatings that can present challenges in sample preparation and analysis.

	In the case of component analysis, however, each component of a drug product must be analyzed individually, significantly increasing the number of analyses required if the manufacturer opts to test components to demonstrate compliance. Given that the individual components are a single matrix and generally pure materials, their analysis may be easier than drug product analysis. Some manufacturers may choose this option since it minimizes the chance of manufacturing a batch of drug product that fails the elemental impurities specification.

Selection of Sample Preparation Procedure

	Regardless of whether a drug product or its components are analyzed, 

 <233> provides a significant degree of latitude for an analyst performing an elemental impurities determination in a pharmaceutical material, and selecting a sample preparation approach is the first step to performing an analysis with 

 <233>. Analysts can opt to use either traditional sample preparation procedures or more innovative approaches, provided that the validation criteria of <233> are met. With many options available, those analysts who have solid analysis capabilities may use them, but most analysts use solution-based techniques. Figure 1 shows one potential way to approach determining which sample preparation technique to use.

					Figure 1: Sample solubility flow chart.

	When performing direct dilution, the sample should be readily and completely soluble in the given solvent: an aqueous media or an organic solvent. In such instances, analysts simply dilute the sample and perform the analysis. This approach is often appropriate for preparing a sample for elemental impurities analysis, but there are some caveats. Despite the fact that the sample may appear to be completely soluble, dissolved solids may be present, and the analytes may not be completely soluble. In such cases, a digestion technique may be needed to obtain the correct determination of the elemental impurities concentrations in a given sample. Table I illustrates the danger of assuming that a sample is completely dissolved: the same sample run after both direct dilution with dimethyl sulfoxide (DMSO) and microwave digestion using nitric acid can sometimes yield dramatically different results. This point was further highlighted by Lorenz (18), when he compared direct dilution results for various Pd-, Ni-, and Cu-containing catalysts with results obtained using nitric acid digestion, as shown in Table II.

	It is advisable to confirm the accuracy of the results obtained by direct dilution with results obtained on the same samples using a digestion technique to avoid the generation of erroneous results. If results obtained via digestion are consistent with results obtained via direct dilution, then direct dilution is an acceptable approach for sample preparation.

	When digestion is required, there are many potential options available to the analyst. Most often, nitric acid is used, however, other acids such as hydrochloric acid, sulfuric acid, and hydrofluoric acid are viable options. Likewise, combinations of acids or successive digestions may also be necessary to effect full digestion of a sample. Table III provides general information regarding some of the more commonly used different digestion approaches, but is not intended to be an exhaustive listing of digestion options. Table III also provides information on some solid-based techniques and direct dilution approaches for purposes of comparison to digestion.

	Despite best efforts, there are times when a sample cannot be expeditiously dissolved. Analysts may need to weigh efforts expended to completely dissolve a sample with analytical benefits and advantages. In such cases, if possible, an analyst may wish to consider various solid analysis techniques. Laser-induced breakdown spectroscopy (LIBS), laser-ablation ICP-MS (LA-ICP-MS), and X-ray fluorescence (XRF) are the more commonly used techniques, and general information regarding them is provided in Table III. Because these techniques are not available in many laboratories, and because a clear solution is desired, the question then becomes one of whether or not the analytes in question are sufficiently solubilized by the sample preparation procedure used. There has been discussion-especially at conferences (19)-about these so-called “acid leach” procedures. Such procedures should not be undertaken lightly, and require knowledge of the chemistry involved. What is the sample? What are the analytes of interest? What is the solvent being used? Is it reasonable to expect that the analytes of interest would be dissolved in the solvent being used, despite having an undissolved sample? Having a good understanding of the chemistry associated with the sample and analytes is critical in assessing the success of an “unsuccessful” digestion in solubilizing analytes of interest. Even so, it is difficult to compare results obtained from multiple analyses using acid leach procedures, making it difficult to be certain that results are accurate. Consequently, if at all possible, orthogonal, solid-based analytical techniques should be used to help assess the success of an “unsuccessful” digestion to adequately dissolve analytes.

	The sample preparation procedure is key to the success of an elemental impurities analysis, and while

 <233> provides a great deal of latitude in the preparation of a sample, some in the pharmaceutical industry may not be as familiar with various sample preparation options. Pharmaceutical samples can be prepared in a wide variety of ways and analysts can leverage procedures and experiences gained from other industries to perform such analyses on pharmaceutical samples. As the pharmaceutical industry begins to become more accustomed to performing elemental impurities analyses, analysts will be more confident in their ability to perform them.

		http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3D/ Q3D_Step_4.pdf.

		N. Lewen, M. Schenkenberger, T. Larkin, S. Conder, and H. Brittain, 

		T. Wang, Z. Ge, J. Wu, B. Li, and A. Liang, 

		N. Lewen, S. Mathew, M. Schenkenberger, and T. Raglione, 

		USP38-NF33, General Chapter <231> ”Heavy Metals,” 

		K.M.Dimpe, J.C. Ngila, N. Mabuba, and P.N. Nomngongo, 

Physics and Chemistry of the Earth, Parts A/B/C

		J.M. Cook, M.J. Gardner, A.H. Griffiths, M.A. Jessep, J.E., Ravenscroft, and R. Yates, 

		Z. Kowalewska, E. Bulska, and A. Hulanicki, 

		V. Sandroni, C.M.M. Smith, and A. Donovan, 

		Q. Jin, F. Liang, H. Zhang, L. Zhao, Y. Huan, and D. Song, 

http://www.berghof.com/fileadmin/Dateien-Einpflege/Seitenbaum/Home-Downloads/Produkte/Laborgeraete/Aufschlusstechnik/MW_Theorie_Probenvorbereitung_PT_en.pdf

http://www.mine-engineer.com/mining/assay2.htm

, A. Samchuk adn T. Pilipenko, Eds; translated by S.V. Ponomarenko (VNU Science Press BV, The Netherlands, 1987), pp. 36–39.

		M. Lorenz, “Analysis of Residual Metals in API by Inductively Coupled Plasma- Mass Spectrometry- A Practical Evaluation of Sample Preparation Techniques,” presented at the Eastern Analytical Symposium (EAS), Somerset, New Jersey, 2005.

		D. Seibert, “Analytical Challenges/Round Robin Study,” presetend at the PRI/USP Workshop on Elemental Impurity Requirements in a Global Environment--Next Steps? Rockville, Maryland, 2015.

 is with Bristol-Myers Squibb Analytical and Bioanalytical Development in New Brunswick, New Jersey. Direct correspondence to: 

Articles

The use of atomic spectroscopy techniques and sample preparation procedures is something that is not as routine in the pharmaceutical industry as are chromatography-based techniques and sample preparation procedures. With new requirements being implemented regarding elemental impurities by the United States Pharmacopoeia (USP) and International Conference on Harmonization (ICH), analysts in the pharmaceutical industry are, in many cases, working to determine how best to analyze their samples. Sample preparation techniques that can be used for pharmaceutical samples are the same ones that have been used by other industries for many years. This paper will provide a brief overview of potential techniques.

Preparation of Pharmaceutical Samples for Elemental Impurities Analysis: Some Potential Approaches

The requirements for testing of pharmaceutical products for elemental impurities has been in a state of flux for several years. Impending changes to 

International Conference on Harmonisation

) will have a significant impact on the pharmaceutical industry and its suppliers. Until those changes are adopted as official and implemented, it is difficult to know how to plan or proceed in anticipation of what will come. This article covers the changes so far, impending changes, and options for preparing for those changes.

In the past 10 years, the subject of metals analyses in pharmaceuticals has gained considerable interest. The publication of the 

) "Guideline on the Specification Limits for Residues of Metal Catalysts or Metal Reagents" (1), new 

) chapters <232> "Elemental Impurities: Limits" and <233> "Elemental Impurities: Procedures" (2,3), and 

) 5.20 (4) have forced the industry to begin looking more carefully at the metals content of various materials used in the manufacture of pharmaceuticals. While there are differences between 

 documents, they are, none-the-less, significantly harmonized.  

) recently moved to a step 3 document for 

 completed the public comment period on December 31, 2013. 

 adds many more elements to the listing of elements of interest, and is not well-harmonized with the 

 has indicated that it will harmonize with the 

 document when it is finalized. To that end, the 

 has delayed implementation of the "Guideline on the Specification Limits for Residues of Metal Catalysts or Metal Reagents" for marketed drug products, pending finalization of 

. This will result in significant changes to the 

 has delayed implementation of chapters <232> and <233> and will revisit <232> once 

 is finalized as well, which could also lead to potential changes to <232> in the future. 

To say that the status of elemental impurities in pharmaceuticals is in limbo is an understatement. The word 

 has two widely recognized meanings: "an intermediate state or place" (6); and "a West Indian dance where the players keep bending over backward and passing under a pole that is lowered" (6). Both definitions fit the current state of elemental impurities in pharmaceuticals, where compliance requirements are "on hold," and the position of the "bar" keeps changing, causing those analysts who had hoped to be proactive in meeting compliance requirements to, in some instances, totally revisit their approaches to compliance. As a result of the disparity between the various documents — especially, with 

 not yet finalized and with no projected implementation date for it, the 

with a projected date of December 2015 implementation for the 

 —many in the industry are both frustrated and confused as to how to proceed to prepare their companies and products to comply with what certainly promise to be significant changes in the way metals analysis in the pharmaceutical industry is performed. This article will provide some background and suggestions regarding how to prepare for compliance, as well as ways to leverage the documents currently in limbo to save resources. 

Coming late to the game behind the EMA, EP, and USP, the ICH began looking at the topic of metals analysis in pharmaceuticals and drafted its first pre-step 2 document, 

, in late 2011. This document included elements that were originally proposed for inclusion in, and subsequently not included in, 

 <232>. The Expert Working Group (EWG) indicated a strong desire to achieve step 4 status as quickly as possible, with discussion focusing on mid-2014. A third version of 

 was then finalized as a step 2b document in July 2013. 

 completed the process where the three regulatory sponsors (the United States, European Union, and Japan) submit the document for their own local comment period. When the document reaches step 4, the guideline is then recommended for adoption by the regulatory bodies in the United States, European Union, and Japan. Those regional authorities then establish implementation time lines and guidelines within their own regions. 

 comment period ended on December 31, 2013, followed by a reconvening of the EWG, meeting, a step 4 deadline of June 1, 2014, is quite an aggressive time line.  Even if 

 does not achieve step 4 in June 2014, it will be important for pharmaceutical companies to closely monitor the situation with regard to 

 both regarding its timing and also its potential impact on the already published, but currently on-hold, 

 "Guideline on the Specification Limits for Residues of Metal Catalysts or Metal Reagents" became effective on September 1, 2008, for new materials. At that time, the 

 gave a delayed implementation period of five years for "legacy" products, with the guideline implementation date for those being September 1, 2013. The publication of 

 has caused a delay in that implementation date for marketed products.   

 chapters <232> and <233> was in considerable flux since they were first intended to be official on December 1, 2012. In November 2012, the decision was made to delay making them official. They were reinstated as official in February 2013; however, there were still concerns about the implementation date of May 1, 2014, despite the fact that it already represented a delay in the implementation of <232> and <233>. Consequently, in May 2013, the decision was made to further delay implementation. Concurrently with the decision to delay implementation of <232> and <233>, the decision was also made to delay removing chapter <231> "Heavy Metals from the compendia via General Notices." Additionally, the USP subsequently decided to make changes to <232> and <233>, which would become official in August 2015, and implemented in December 2015, along with removal of <231> from the compendia. With the chapters being official, industry is free to use them, however, it is not required that they be used to demonstrate compliance at this time. Chapter <231> may also still be used. 

Between the EMA, EP, USP, and ICH, there are major differences between the listing of elements and the compliance limits for each element. USP departed from the EMA and EP by including the "Big 4" (As, Cd, Hg, and Pb), potential inadvertent contaminants. Additionally, USP did not include some of the elements listed in the EMA and EP because the limits for those elements were very high, and failure to comply with those limits would represent a GxP failure first. EMA and EP included categories of elements based on toxicities, whereas only an early version (from 2010) of 

 <232> included similar categories of elements. I

 is most like the USP's original listing of elements, and includes Co, Se, Tl, Li, Sb, and Sn, which were all included in the very first draft of 

 also includes Ag, Au, and Ba, which have never been included in 

 also categorizes elements, much like the EMA and EP. Tables I, II, III, and IV provide comparisons between 

Table I: The "Big 4" (1,2,4,5) included in risk assessment			

With different lists of elements, different limits, different elemental categories, unknown implementation dates, and the potential for changes in the future, many analysts feel that it is difficult to prepare for anything regarding metals analysis in pharmaceuticals. Analysts are also concerned that, once the landscape becomes clear, time will be short to achieve compliance. Those who were proactive in attempting to comply with USP, EMA, and EP have now had to regroup after having already spent time and money to comply, as USP has delayed implementation of <232> and <233>, and ICH is now looming.  

It is still possible, however, to begin to plan, despite the differences between ICH, USP, EMA, and EP. The EMA and EP have indicated that they will harmonize 100% with the finalized ICH document. It is possible that the current 

 document will remain largely unchanged as it moves to step 4. It is also possible that additional changes may be made to the document.  

Although the USP has a strong desire to harmonize wherever possible, 100% harmonization cannot be absolutely assured. While not desirable, it is possible that toxicologists working with the USP will not agree with the ICH regarding the listing of elements or their corresponding limits, or vice versa. Certainly, it is clear that toxicologists are not in agreement regarding elements of potential interest or their limits, or there would not be differences between the ICH, USP, EMA, or EP listings of elements and corresponding limits.  

Table IV: ICH Class 3 metals (1,2,4,5)			

One approach to determining how to prepare for compliance with 

, EMA, EP, and USP requirements is to wait. The EMA and EP have already indicated that once it is finalized, they will fully harmonize with 

. Whether the USP harmonizes 100% may or may not happen, but even with the ever-changing landscape it is possible that waiting until everything is finalized, with due dates firmly set in stone, is a valid option for companies. The drawback to waiting is that, depending on time lines and the number of materials a company may have to evaluate, they may be rushed to accomplish the full assessments in whatever time is finally allotted before implementation of ICH, EMA, EP, and USP requirements. The advantage of waiting is that all requirements and deadlines will be known.  

For companies with a large number of materials to assess, or for those who wish to proceed for other reasons, it is still possible to begin to prepare for compliance, despite the fact that the requirements are in a state of flux. It is unlikely that the listing of elements and limits in 

 will change, but changes are possible. It is also possible that the USP will change. It is most likely that USP, EMA, and EP will harmonize either fully or in part with 

. If total harmonization is not achieved, it will likely be the result of differences in regional acceptance of limits. Should limits for a given element diverge, the simplest option is to work with the lower of the two limits. In that way, compliance with the lowest limit ensures compliance with the highest, as well. Should the listing of elements of interest differ between 

 and USP, companies can choose to assess the combined listing of elements, or they may use the risk-based approach to rule out some elements completely. By using a combined listing of elements and the lowest of limits between the various listings, companies can then begin to prepare to meet compliance requirements before deadlines are established. With this in mind, a conservative approach to developing a plan in advance of finalization of ICH, USP, EMA, and EP would be to adopt the required elements from each listing, and adopt the lowest limits for all analytes. Consequently, the listing of elements and limits might look something like that found in Table V.  

Table V: Hypothetical listing of elements and limits (2,5)			

Armed now with a listing of elements and limits, it is possible to begin to prepare for compliance with unharmonized documents — especially when the concept of a risk-based approach is encouraged by all parties. First and foremost, it is absolutely essential that a company have a thorough understanding of their product's process, suppliers processes, raw materials, and equipment being used. A sound risk-based approach that will comply with all guidelines and compendia simultaneously must be based on full knowledge of the process by which a drug is manufactured. Vendor qualifications; understanding of how raw materials, intermediates, and excipients are made or derived; and knowledge of whether any materials included in the drug product are naturally sourced or plant-based are all critical in determining which elements must be monitored. A careful evaluation of that information can help to narrow the listing of analytes to a more manageable one than that provided in Table V. Figure 1 provides an example on how to determine if a metal (other than one of the big 4 or the ICH mandatory metals) must be monitored or not.  

Figure 1: Sample flowchart for assessing the need to test.			

What About As, Cd,  Mo, Pb, Hg, Se, V, and Co?

There is considerable confusion regarding the requirements to test for As, Cd, Pb, and Hg to comply with the USP, and there will undoubtedly be some confusion regarding the ICH requirements to test for As, Cd, Pb, Hg, Se, Co, V, and Mo. Many believe that testing for these elements in every material (drug product or components) is mandatory for every batch. Although that certainly is one approach to meeting the requirements and demonstrating compliance, it is generally not considered the most effective use of a risk-based approach. While it is necessary to make certain that none of those elements is present at levels exceeding their limits for both the USP and the ICH, it is also important to realize that a risk-based approach has the potential to minimize or eliminate testing for some or all of those elements in any given product or component. Although the ICH requires that these elements be included in a risk assessment, it is important to note that the ICH does not require that these elements, necessarily, be included in all analyses. The risk assessment is part of a risk-based approach. For example, if a component is a mined material or if a drug product contains a mined material, a risk-based approach might require the testing for some or all of those elements. Mined materials may vary considerably from batch to batch, given the fact that different locations in mines may have varying concentrations of potential elements of interest. On the other hand, if the material is not mined it may not be necessary to test every batch for those elements. The decision regarding whether or not to test for those elements must be based on a thorough knowledge of the entire process and sourced materials. Lacking that knowledge, testing is the safest way to ensure compliance.  

It may also be possible to use skip-lot testing or eliminate the need for testing if results for pilot scale or production scale batches demonstrate that a metal does not exceed 30% of the guideline limit. The decision on eliminating elements or performing skip-lot testing is something each company will have to carefully consider and assess within the context of the company's policies and procedures, as well as the level of acceptable risk a company is willing to assume. 

It must be said that although risk-based approaches are highly encouraged by the USP, EMA, EP, and ICH, it remains to be seen how regulatory agencies will respond to using a risk-based strategy for metals testing. For this reason, it is important for a manufacturer to work closely with local authorities to ensure that whatever approach is taken is acceptable.  

 chapter <233> may be used for the analytical procedure for the determination of elemental impurities in pharmaceuticals. The ICH, EMA, and EP do not specify an analytical procedure, so <233> would be appropriate for those organizations, as well as USP. In deciding how to develop the method, it is important to first determine if the drug product will be analyzed or if components of the drug product will be analyzed. Either practice is acceptable for USP, ICH, EMA, or EP. After that decision is made, it is then necessary to determine if the maximum daily dosage is different than 10 g/day, and if so, what impact that might have on the limits for each analyte of interest. Using the following calculation, analysts may determine their analytical target range for a given analyte: 

Concentration (ppm) = PDE/daily dose [1]  

where PDE is the permitted daily exposure from the EMA guideline shown in Table I, and is in micrograms per day; and daily dose is the maximum daily dosage, and is in grams per day. The maximum daily dosage can have a significant impact on the final compliance limits. Table VI illustrates the impact of a few different maximum daily dosages on limits for a few selected analytes.  

Table VI: Impact of maximum daily dosages 			

Clearly, the selection of the analytical technique to be used for the analyses may be impacted by the maximum daily dosage. Although inductively coupled plasma–optical emission spectrometry (ICP-OES) and inductively coupled plasma–mass spectrometry (ICP-MS) are the methods of choice listed in 

 <233>, analysts may use any other technique that meets the validation requirements of that chapter. Consequently, depending on the maximum daily dosage, analysts may not need the sensitivity that ICP-MS, or even ICP-OES, affords.   

 <233>, it is important for the analyst to remember some general guidelines: 					

 = the term used to represent the concentration (by weight) of any element of interest at the target limit for that element. It may be necessary to dilute to the working range of the instrument being used for the analysis.

Target limit (or target concentration): This is the value used, for any elemental impurity under evaluation, that provides the acceptance value for that elemental impurity.   

When using direct dilution of a sample, it is important to be certain that the elements in question are fully soluble in that solvent. If this is not the case, then microwave digestion may be required.

Alternate procedures may be used, provided they meet the requirements of the 

 <1225> differs from the validation requirements of <233>, the validation requirements of <233> take precedence.

Requirements in a monograph trump the requirements in 

 <232>, which provides the basis for limits being tested via 

This article covers the changes so far to United States Pharmacopeia (USP) chapters <232> and <233>, the International Conference on Harmonisation (ICH), the European Medicines Agency (EMA), and the European Pharmacopoeia (EP), impending changes, and options for preparing for those changes.

Nancy Lewen

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