Assay and Stability Testing

The stability testing requirements for diltiazem hydrochloride include an HPLC assay and dissolution test. The HPLC assay is described in the section for the potency assay (9.2.1.1.) and the dissolution test method is described in Section 9.1. 

Almost all aspects of manual analyses have been automated, ranging from sample handling to measurement with automated calculation and report generation. -96 yhgjjg automated analyses have been applied to many types of tests, including assay, content uniformity, dissolution, and moisture, and are routinely used for product release and stability testing. Some companies have constructed complete robotic laboratories in a centralized or decentralized manner to generate tens of thousands of assay results per year. 

Analytical methods for assay of the toxicology formulations and cleaning validation are developed and validated in preparation for the first GLP studies. Release and stability testing of the toxicology test articles are performed to support the suitability of the materials through their anticipated period of use. Typically, short-term accelerated stability studies are performed on the toxicology batches for at least 3 months to cover the time from date of manufacture through the last dose. 

Yamamura, K. Yamada, J.-L Yotsuyanagi, T. High-performance liquid chromatographic assay of antiinflammatory drugs incorporated in gel ointments. Separation and stability testing, J.Chromatogr., 1985, 331, 383-388. 

For drug substances and drug products, applications for enantiomers and racemates should include a stereochemically specific identity test and/or a stereochemically selective assay. The choice of control tests should be based on the method of manufacture and stability characteristics and, in the case of the finished product, its composition. 

Where antioxidants or antimicrobial preservatives are used, the finished product release specification will need to include identification tests and assays for these two types of excipient. The shelf life specification should also include a specification for assay for antimicrobial preservatives. Stability data will be required for both antioxidants and antimicrobial preservatives in the finished product, and in addition the preservative efficacy of the formulated product should be examined over its shelf life and by means of appropriate in-use stability tests. Preservative efficacy data should also be presented at the lower limit of the preservative assay. 

The application should state the rationale for the design of the in-use stability tests performed. The procedures used should be fully validated. One key factor is that the test should simulate the use of the product as far as practicable. This should include any reconstitution or dilution prior to use. Aliquots should be removed in an appropriate manner following, as far as possible, the usage pattern that will be encountered in practice. Physical (color, clarity, closure integrity, particulate matter, and particulates/particle size), chemical (assays for active ingredient, antioxidants and... 

An important DMPK property of a NCE is oral bioavailability (F) of the compound in various pre-clinical species.3 The oral bioavailability of a compound is dependent on several factors including intestinal permeability (estimated by the Caco-2 assay) and hepatic clearance (estimated with an in vitro metabolic stability assay).3 30 The metabolic stability assay is typically performed by incubating test compounds in liver microsomes or hepatocytes. The results can provide estimates of in vivo stability in terms of metabolic liabilities.3 8 59 62 Several authors described this assay as an important tool for the rapid assessment of the DMPK properties of NCEs.3 6 8111819 26 44 59 62-65... 

In addition to the direct absorbance methods, colorimetric methods are suited for relatively pure proteins as purification progresses. They are accurate if calibrated from a standard curve of the test protein reference sample and fast if automated. However, they are not as simple to perform as direct absorbance methods. Hence they are not as suitable for production as direct absorbance methods. The relative simplicity of colorimetric methods makes them more suited to automated formulation and stability studies and total-protein assays of complex mixtures. Microtiter plate versions of colorimetric assays allow for automation and consumption of relatively small sample sizes while requiring little specialized equipment or training. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

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