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Sample preparation-specifically, API extraction and dilution-can introduce errors. Use of best practices and automation can reduce variability.
Content uniformity, which ensures that API is present in the right quantity in solid dosage forms, both within and between batches, is crucial to maintaining patient safety. Variability in API loading can become a safety problem when patients split tablets, a trend that has become common a way to reduce the cost of prescription drugs (1).
Content uniformity testing requirements, however, may not always be clear, and the process may be taken for granted, which can result in quality problems. Technology is available to automate more of the sample preparation portions of the test, which tend to be the most error-prone. Ishai Nir, small-molecule products manager at Distek, Inc., describes challenges with Pharmaceutical Technology, and discusses a new automated sample preparation system that was recently introduced to the market.
PharmTech: You have often described content uniformity as ‘the overlooked test.’ Given the predominance of generic drugs today, why would such an important quality-control test be overlooked?
Nir: Content uniformity, which, technically, is part of the United States Pharmacopeia (USP) Chapter <905> Uniformity of Dosage Unit (2), is definitely the red-headed stepchild of solid-dosage form testing. For one thing, it is one of the tests where the least detail or guidance is provided on execution.
For example, the entire section of Chapter <905> that covers the procedure for performing the content uniformity (CU) measurement reads, simply: ‘Assay 10 units individually using an appropriate analytical method.’ That’s it! Compare that with Chapter <711>, the dissolution chapter, which uses more than twice the number of words just to describe the specifications of a paddle, one of seven pieces of equipment that are required for dissolution testing. In 2018 alone, Goggle Scholar currently includes over 5500 references for dissolution testing, yet less than 1300 for content uniformity testing (2).
PharmTech: What impact does this lack of definition most often have on results?
Nir: We haven’t seen the same proliferation of instrumentation dedicated to CU testing that we’ve seen in virtually every other required pharmaceutical test, and this is due to lack of guidance. Instead, companies have developed and validated their own methods based on existing general-purpose laboratory equipment such as stirrers, sonicators, homogenizers, and shakers.
You can think of CU testing as having two components. The first is sample preparation. This entails finding a way of reproducibly extracting 100% of the API out of the dosage form into a solution, and, if necessary, adjusting the concentration of this solution via dilution so that it is suitable for the analytical finish. The solution is then analyzed, typically using ultraviolet (UV) or high-performance liquid chromatography (HPLC), to determine the API concentration. The concentration is then multiplied by the total dilution volume to calculate the original amount of API present in the dosage form.
Most labs have multiple technicians on hand who are highly trained in measuring the concentrations of a given solution using UV analysis or liquid chromatography (LC). As a result, the analytical step has never been the challenging part of the CU test. Problems have generally arisen in sample preparation.
PharmTech: What types of problems typically show up during CU sample preparation?
Nir: There are two major sources of error in CU sample prep. The first is incomplete API extraction, and the second is dilution errors. Incomplete extraction takes several forms. One major source of variation is due to the fact that, as previously mentioned, unlike dissolution or disintegration testing, these procedures are not performed using a specific apparatus. This task is normally accomplished using non-analytical lab equipment such as magnetic stirrers or sonicators. Because the speed, frequency, or power of these units are not rigorously controlled, these parameters can vary over the lifetime of a unit, and between different units in one lab.
Variation can be even greater between units in different labs. So, if a method calls for sonicating the sample for six hours, that may be equivalent to four hours on one sonicator and eight hours on another. Often, method developers ‘solve’ this problem by calling for an overnight run to avoid these issues.
The second source of errors is the dilution steps. Frequently, the 100% extracted solution is too concentrated to be read directly by analytical instruments and requires one or multiple dilutions. Of course, any time a processing step is added, a greater probability of operator error is introduced.
In the case of sonicating samples, mistakes can be even more likely, because prolonged sonicating will normally heat a solution. If the measurements are taken and dilution performed before the solution has returned to room temperature, it can be easy for a lab technician to make a volumetric measurement mistake due to thermal expansion of the liquid.
PharmTech: Why can’t the entire test be automated? Aren’t solutions already available to do this?
Nir: A few systems have attempted to automate the entire CU process, including both sample prep and analysis. However, they are very expensive, starting at over $100,000 and ranging up to several hundred thousand dollars. In addition, they can be complicated to operate, difficult to validate, and expensive to maintain and keep running. In addition, although they address sample preparation concerns, they also attempt to address analysis, which never was much of a problem to begin with.
The PrepEngine, developed by Distek in conjunction with Two Square Sciences, automates only the sample prep portion of the CU test. The goals behind its development were to make the extraction process considerably faster and more reproducible, and, to the extent possible, to eliminate the need for dilution steps entirely. It was also designed to be considerably less expensive than previous solutions.
PharmTech: How does the equipment work?
Nir: The CU sample prep system uses custom plastic vessels, designed for multiple uses, with a built-in blade and filled with appropriate media to extract the API from the dosage form. The main unit comprises 10 positions (to match the 10 samples required for a CU test), the control electronics, and user interface that allows programming the method parameters.
The several-use (typically five runs) tubes are made of polypropylene, and are designed with internal finning, which helps redirect the dosage form down towards the blade as it is being broken apart and blended into the media. They are available in different volumes to match the concentration required for subsequent analysis without further dilution, whenever possible.
As a result, the operator using the equipment must only make one precise volumetric measurement of room temperature media during the entire measurement process. Finally, the tubes are available in amber for light-sensitive products to avoid sensitization before analysis.
Variability in API loading can be a problem, especially with generic formulations. Improving content uniformity testing offers a solution.
PharmTech: Why use disposable sample containers?
Nir: The situation is similar to that of disposable razors. Regardless of what material one uses to make the blades, they will become duller over time and require more agitation time to guarantee complete extraction.
Extending the sample prep time excessively to ensure complete extraction would not be the best solution. Instead, it is better to limit the number of samples that are ground using a given blade.
Some users have taken this concept to the limit, using each tube only once. In this case, the cost of the extra consumables is offset by the elimination of the time and cost associated with cleaning the tubes. This also eliminates the possibility of any product carryover due to inadequate cleaning. Additionally, using each component only once enables pre-sterilization of the tubes for biologics use.
PharmTech: How complicated is method development using the CU Sample Prep Station?
Nir: Method development using the CU Sample Prep Station is simple because analysis and calculation of the results remains largely the same as existing methods. The only exception is generally eliminating the need to correct for dilutions, which become unnecessary.
The sample prep portion of method development involves optimizing two parameters-agitation speed and duration. This requires conducting CU testing at different speeds and durations and extraction efficiency. The goal is to find the lowest agitation speed that will still guarantee complete release in an acceptable test time.
PharmTech: Can you share examples of successful implementations of this technology?
Nir: Distek has published results of some internal proof-of-concept work we have done using an over-the-counter pain medicine (in this case, Tylenol Regular Strength 325-mg tablets from Johnson & Johnson) (3). Tylenol tablets were added to sample preparation vessels containing 500 mL of deionized water. The runs were stopped at different time points, and the extracted API concentration was measured using a fiber optic UV system with a probe that was inserted in the tube.
Figure 1 shows the results of the measurements. For the desired 90-second agitation time, 2500 rpm was clearly a high enough agitation speed to yield complete extraction. Table I shows actual Tylenol CU measurement results obtained from 10 tablets using the sample prep station with 90-second agitation at 3000 rpm. The weight corrected results have a mean of 99.5% and %RSD of 0.12%. USPChapter <905> sets the limit of the CU acceptance value (AV) as less than 15.0 for 10 units. For the data set collected, the corresponding AV value computes to be 0.29.
Pharmaceutical manufacturers that have tested the system have reported similar results and reduced overnight testing times to as little as five minutes. One company that has publicly presented its results is Gilead Sciences, Inc., based in Foster City, CA (4), in work involving a spray-dried dispersion tablet. The original method called for three-hour disintegration of the tablet using a magnetic stir bar.
While using this method, Gilead encountered multiple investigations due to incomplete extraction. With the CU sample prep station, however, the company reported being able to reduce extraction time to 30 minutes. More important than the time savings, the failures and corresponding quality investigations stopped. At this point, dozens of units have been installed globally, and regulatory agencies and authorities such as the US Pharmacopeial Convention have also invested in the technology.
1. A. Ciavarella et al., PDA J. Pharm Sci Technology, 11/12;70(6):523-532 (2016).
2. 2018 U.S. Pharmacopoeia-National Formulary (USP 41 NF 36). Volume 1. Rockville, Md: United States Pharmacopeial Convention, Inc; 2018. <905> Uniformity of Dosage Units; p. 6673.
3. A.Kielt; I.Nir; J. Seely, Dissolution Technologies 25(1):47-48 DOI: 10.14227/DT250118P47
4. A. Wei, et al. “A Fast Sample Preparation Procedure for Spray Dried Dispersion (SDD) Tablets Using Mechanical Assisted Extraction,” presented at the 2016 AAPS Annual Meeting in Denver, Colorado.
Supplement: APIs, Excipients, & Manufacturing 2018
When referring to this article, please cite it as A. Shanley, "Ensuring Consistency in Content Uniformity Testing," APIs, Excipients, & Manufacturing Supplement (September 2018).