Content uniformity assays

Since its creation around 1973, modern high-pressure liquid chromatography (HPLC) has played a dominant role in the analysis of pharmaceuticals. It is used in many different applications for example, in content uniformity assays and stability-indicating methods, for the purity profiles of drug substances, or in the analysis of drug metabolism in animals and humans. The heart of all of these assays is the HPLC column. In this chapter, we will describe the fundamental properties of HPLC columns as well as how these properties influence column performance and separation characteristics in pharmaceutical assays. 

ICH Q2A suggested validation of the characteristics of accuracy, precision, specificity, linearity, and range for potency and content uniformity assay. A detailed discussion of each of these parameters is presented later in this chapter. Some examples of validation data are presented along with a brief critical discussion of the data. 

As recommended by the ICH, the usual range for the potency assay of a drug substance or a drug product should be 20% of the target or nominal concentration and 30% for a content uniformity assay. At least five concentration levels should be used. Under normal circumstances, linearity is achieved when the coefficient of determination (r2) is >0.997. The slope, residual sum of squares, and... 

For content uniformity, assay, and dissolution assays, a filtered aliquot is acceptable if the difference between the response of the filtered aliquot is not greater than 1.0% of the response of the reference sample. For impurities methods, the response of each impurity after filtration should be within 20% of the... 

Repeatability. Repeatability expresses the precision under the same operating conditions over a short interval of time. The recommended testing for automated content uniformity, assays, degradation and impurity methods, and dissolution methods are listed in Table 5.1. 

Equivalency. This test compares the results of the automated procedure with the results of the validated manual method. If accuracy of the automated procedure has been proven, it may not be necessary to perform the equivalency study. However, if the manual method does not exist, then accuracy and reproducibility data should be used to assess the suitability of the automated method. The recommended testing for content uniformity, assays, degradation and impurity methods and dissolution methods are listed in Table 5.4. 

An automated HPLC tablet content uniformity assay for a multipotency range of tablets (the active constituent not identified being at 0.1,0.2, or 0.3 mg/tablet) has been described [66]. In this application, a further degree of communication was introduced between the robotic and HPLC systems, with the robot being able to alter its actions, dependent on the result of the HPLC analysis. 

Another piece of evidence to suggest that content uniformity data is not normally distributed can also be seen in the content uniformity data of Rohrs et al.4 They report the content uniformity assay results for 11 batches of tablets. In every case except one, the actual mean potency is below the target value. If the content uniformity data were normally distributed, as assumed by the Yalkowsky and Bolton approach,2 one might expect to see a more even distribution of mean values above and below the target. The one exception was for the lowest potency batch for which one tablet was assayed to be 292% of intented value, as mentioned above. This characteristic is... 

Finished product test results to include content uniformity, assay, hardness, friability, etc. 

Content Uniformity/Assay. The question most often asked is when NIR will be able to be used as a release test. In the earliest NIR assays, tablets and capsules were not analyzed intact. Prior to scanning, the active was extracted and scanned in a clear liquid. The first use of NIR for tablets was reported in 1968. Sherken assayed the meprobamate content in tablet mixtures and commercial preparations. Allen used NIR to analyze a three-component mix carisoprodol, phenacetin, and caffeine. The powder was extracted with chloroform and scanned in the NIR. Several other publications took advantage of the dissolve and scan approach.t ... 

F.g.2 Flow diagram of basic robotic procedure for content uniformity assay. 

Tablets must meet the U.S.P. requirement for content uniformity testing (62). The content uniformity assay is performed by assaying ten tabiets individually. The samples are...

The content uniformity assay is performed by assaying ten tablets individually. Each tablet is ground to a fine powder and transferred quantitatively to a 50-mL volumetric flask. About 40-mL of water/acetonitrile (25/75 v/v) diluent are added to the flask and the solution is sonicated for 20 minutes. The solution is then treated as described in the potency assay procedure. 

Before the performance of method transfer activities involving protocols and acceptance criteria, it was customary for a receiving laboratory to repeat some or all of the validation experiments. This laboratory was thereby deemed to be qualified as described above. The choice of validation parameter(s) depends highly on the type of method being transferred. For example, content uniformity assays to determine consistency of product potency depend heavily on the method and system precision. As a second example, a determination of trace impurities in an API could not be reproduced between two sites if their instruments did not yield similar limits of detection and limits of quantitation. A detailed discussion on the rational choice of validation parameters that would need to be repeated by the receiving laboratory is beyond the scope of this chapter. The reader is referred to the method validation chapter by Crowther et al. for additional information on this subject. 

Figure 6.4 shows an example of an FIPLC assay of a single API product (capsule) with the content uniformity assay results summarized in Table 6.4. 

Table 6.4. An Example of Content Uniformity Assay Report...

Many of the techniques described above, excipient compatibility, blend uniformity by HPLC dissolution, and content uniformity/assay by HPLC can be effectively automated by robotic sample preparation. Each of these techniques requires that the sample under study be dissolved in an appropriate solvent and fully extracted from any excipients. There are a number of commercially available products that have proven to be effective and robust in this sample preparation role. This robotic process can reduce both the analyst hours required to prepare a number of samples, and turnaround time on the sample analysis, since the robotic systems will operate unattended over night and on weekends. There is of course a cost to pay for laboratory automation. There is a significant capital cost, and then an ongoing maintenance cost for the continued operation of the system. Also it is critical that a specialist be available in-house to care for the system, develop the methods, and troubleshoot any issues with the system. The cost of the system and specialist must be weighed against the advantages of speed and lab capacity enhancement realized with a successful automation implementation. 

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