API assay

The API Assay, the Assay of Impurities and Product Stability. The central independent analytical research and quality control unit is responsible for the analytical release of both the API and the formulated drug product for the drug development programs. The central independent QC unit provides all the analytical data needed to build the analytical specification for the IND. It is recognized by those involved that the IND is a relatively raw document compared with the later NDA, which is built on data from a more developed process situation, using more refined analytical techniques. 

TABLE 8-5. Filter Evaluation Results for API Assay-Related Substance Samples... 

The evaluation of the data should begin with an examination of the individual data points that have been collected over the course of the stability study. In a normal data set, some variability is to be expected. The variability can arise from variability in the product, the method, and the analysts performing the testing. The specifications for API assay generally ranges from 98.0% to 102.0% on an anhydrous basis. If the result must be calculated on the anhydrous basis, the variability in the water content will also influence the results. Thus, over the course of the stability study the individual data points will most likely increase and decrease within the specification range. One question to consider is whether the variability in the data can be explained by the expected variability of the determination. The data points for individual impurities and total impurities should be examined as the study continues to ascertain if the specification limits are being... 

Table 1.7 HPLC and UHPLC Method Conditions for API Assay and Impurities...

A crude-oil assay always includes a whole crude API gravity and a TBP curve. As discussed by Nelson (op. cit., pp. 89-90) and as shown in Fig. 13-85, a reasonably consistent correlation (based on more than 350 distillation curves) exists between whole crude API gravity and the TBP distillation curve at 101.3 kPa (760 torr). Exceptions not correlated by Fig. 13-85 are highly paraffinic or naphthenic crude oils. 

Although considered a basic technique, ultraviolet-visible (UV-vis) is perhaps the most widely used spectrophotometric technique for the quantitative analysis of pure chemical substances such as APIs in pharmaceutical analysis. For pharmaceutical dosage forms that do not present significant matrix interference, quantitative UV-vis measurements may also be made directly.114,115 It is estimated that UV-vis-based methods account for 10% of pharmacopoeia assays of drug substances and formulated products.116... 

Borman, P. J., Chatfield, M. J., Crowley, E. L., Eckers, C., Elder, D. P., Francey, S. W., Laures, A. M-F., Wolff, J-C. Development, validation and transfer into a factory environment of a liquid chromatography tandem mass spectrometry assay for the highly neurotoxic impurity FMTP (4-(4-flurophenyl)-l-methyl-l,2,3,6-tetrahydropyridine) in paroxetine active pharmaceutical ingredient (API). J. Pharm. Biomed. Anal., 48, 2008, 1082-1089. 

Direct isolation of sufficient quantities of each metabolite for structural characterization, assay validation and pharmacological or toxicological testing from in vivo studies using biological specimens is, therefore, often impossible, particularly from dmgs with a low therapeutic index. Furthermore, many metabolites have structural modifications which are difficult to replicate by traditional chemical methods. A number of synthetic steps may be required to prepare such metabolites from the API, or, in the worst case, a completely new synthetic route may need to be developed. 

The assay of potency involves the determination of the API to ensure conformance to label claim. For tablets or capsules, a composite assay requires fO-20 units to minimize tablet-to-tablet variation. In assays, it is critical for the quantitative extraction and recovery of the API meet the typical specification limit between 90.0% and 110.0% of label claim. 

This case study illustrates the SP method development of an assay method for a controlled-release analgesic tablet with a single API. Certain considerations were taken into account. First, the analyte within the tablet matrice core had to be extracted quantitatively. Second, the analyte was diluted into a final solution that was compatible with the HPLC mobile phase. Third, short SP time was required (i.e., 30 min) to maximize productivity of the work scheme for processing a large number of samples. 

The method yielded >99% label claim of the API for the composite tablet assay. Table 3 lists the results of a filter validation study showing the quantitative recovery of the API from most membrane media. 

Methods for the quantitation of major components of bulk drug substances or APIs, including preservatives, in finished drug products are classified in Category I. Assay and content uniformity methods fall into this category. These methods demonstrate that the label claim of the drug... 

If reference materials of the suspected impurities are available, the drug substance or finished drug product should be spiked at an appropriate level to demonstrate that the result is unaffected by the addition of the impurity. Figure 2 shows examples of individual chromatograms of the API and three known process impurities. As shown here, none of the three process impurities interfere with the API peak, although peaks for impurities A and C appear to overlap and could co-elute if both were present in the sample. This specificity may be acceptable if the method was designated as an assay method for the quantitation of the API. For a method intended to quantitate process impurities, the overlap of these two components would in most cases be unacceptable due to the inability of the method to accurately measure the two individual components. 

A well-defined, precise, and validated method will help to determine the drug content accurately, whereas an assay method capable of detecting at low levels can help calculate drug losses during the manufacturing process. Different vendors can supply common APIs. The API characterization accompanied by information from the manufacturer on the synthetic route determines the impurities profile and the method used for the active assay. 

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