Monographs assays

L. Tomatis. "Long-term and Short-term Assays for Carcinogens, lARC Monographs Supplement 2, Lyon, France, 1980. 

A number of handbooks and monographs are available with detailed descriptions of a variety of plant products and their use (Shahidi and Naczk, 1995). From a more practical point of view, an interlaboratory comparison between six university and industry laboratories of 17 extracts of spices, teas, coffees, and grape skin and of tomato peel slurry established within the framework of an EU sponsored programme, would be of interest (Schwarz et al, 2001). In this collaboration, detailed chemical analysis of the content of different phenolic compounds is compared with six antioxidant assays for the 17 extracts including different extraction procedures. 

Non-specific absolute assay methods, e.g. volumetric titration, can be applied to avoid the establishment of a reference substance. This is only appropriate, however, when the monograph describes a separation test for related substances. This approach is certainly valid for the determination of the content of pharmaceutical raw materials but less acceptable for the assay of content of pharmaceutical preparations where the employment of specific assay methods is recommended (ICH Guideline 1994) to take account of decomposition of the active ingredient during the shelf life of the product and to avoid possible interference from excipients. 

The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory, a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in International Units. If an International Standard does not exist, European Pharmacopoeia Units are used. 

Exchanges between pharmacopoeias are co-ordinated by the Pharmacopoeial Discussion Group (PDG) (International Harmonisation 1995) and it is frequent that one pharmacopoeia participates in a collaborative study organized by another pharmacopoeia, or that several pharmacopoeias share the same batch of reference substance to be used in their respective monographs nevertheless, in this case the reference substance can not be considered as harmonized. A new batch of erythromycin was shared between the United States Pharmacopoeia and the European Pharmacopoeia and was established in a common coEaborative study both for the microbiological assay (used in the USP for formulations) and the liquid chromatographic assay (used in the Ph. Eur. and USP for bulk material). 

In contrast to aspirin itself, the U.S.P. monograph for aspirin tablets has undergone considerable changes. For some reason, U.S.P. does not use the ferric salt test for free salicylic acid, as does the British Pharmacopeia of 1973. Apparently, certain excipients such as citric and tartaric acid interfere with this reaction.77 Already in 1913, a double titration method was developed78 which was made an official method in 1926.79 This method was used as the assay method when the aspirin tablets monograph was introduced into U.S.P. XII in 1942. 

Where the test is conducted as a limit test, the specimen is determined to be positive or negative to the test judged against the endotoxin concentration specified in the individual monograph. Where the test is conducted as an assay of the concentration of endotoxin, with calculation of confidence limits of the result obtained, the specimen is judged to comply with the requirements if the result does not exceed (1) the concentration limit specified in the individual monograph and (2) the specified confidence limits for the assay. In either case the determination of the reaction endpoint is made with parallel dilutions of redefined endotoxin units. 

In actual practice, where a test or an assay recommends the usage of a Reference Substance, the spectrophotometric measurements are always performed first with the solution prepared from the Reference Substance by the directions provided in the specific monograph and then with the corresponding solution prepared from the substance under examination. Nevertheless, the second measurement must be done immediately after the first, by employing the same cell and the same instrumental parameters. 

The assay method along with specific experimental parameters are duly stated in the official monograph. Here, two situations arise, namely ... 

It also asked respondents to identify the specifications, tests, and assays in the monographs for these excipients that are most important to be harmonized. 

Equivalence is attributed to monographs, tests, or assays that have arrived at Stage 5B. The nature and reason for divergences are expected to be described in the three pharmacopeial periodicals. Only those stated exceptions are considered nonequivalent. 

If a laboratory was fully confident in a particular assay it might submit it for testing by several laboratories, which would give a measure of how reproducible the assay was in a wider sense with different operators and equipment. For an as.say to succeed in this type of exercise it would have to very robust. For example pharmacopoeial monographs are designed, in theory, to be sufficiently robust to be reproduced relatively easily by many laboratories. Flowever, the tolerances for the precision of such assays might be quite wide. 

Different from foreign impurities, which theoretically are not present and, therefore, are not included when monograph tests and assays are selected, toxic impurities and signal impurities may arise from the synthesis, preparation, or degradation of compendial articles and, differently from ordinary impurities, may present undesirable biological activity, even as minor components. In this context, the following topics should be taken into account ... 

IARC (1980) Long-tenn and short-term screening assays for carcinogens a critical appraisal. Lyon, International Agency for Research on Cancer, 426 pp (IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl 2). 

A modem monograph is devoted to a drug substance and starts with a concise description of the product. This is followed by instructions for packaging and storage, relevant USP reference standards, identification, and procedures for the assay of the drag or the product components. There is sufficient information for those skilled in the arts to be able to analyze the drug and its components, making the compendium... 

Figure 16.5 shows an HPLC chromatogram of the standardized G115 extract. The HPLC method is easy, rapid, relatively inexpensive, specific, precise, and accurate. The robustness has been proved for years in thousands of analyses. The European Pharmacopoeia and the USP recommend the HPLC method for ginsenoside assay in the monograph on P. ginseng they are preparing. 

The subject of impurity analysis of pharmaceutical compounds has been insufficiently addressed in the scientific literature. Many monographs in die U.S. Pharmacopeia have nonspecific assay methods. An attempt has been made to address this problem by focusing on specific methodologies and delineating origination and concentration of impurities found in pharmaceutical compounds. 

Once the low-level radioactive sample has been collected and any chemical procedures performed prior to counting, it is ready for counting. Because of the extremely small disintegration rates encountered, special techniques, called low-level counting, must be used to assay the sample. We shall survey some of the highlights of this area, which has been the subject of many articles and monographs (Knoll, 2000). 

Details of experimental methods are not included. Original papers or appropriate books (2, 3) should be consulted for methods of enzyme preparation and for assays of enzymic activity. For earlier surveys the chapters by G. Schmidt and M. Laskowski, by H. G. Khorana, and by C. F. Anfinsen and T. H. White, Jr., in the second edition of The Enzymes, Vol. 5, and specialized monographs (1, 4) should be consulted. 

---