Reference standards assay methods

In 1960, at the general assembly of the International Pharmaceutical Federation (FIP), the obsolescence of various national pharmacopeial methods for assaying pharmaceutical enzymes was demonstrated. An international commission on pharmaceutical enzymes was created to deal with this unsatisfactory situation and develop improved assay methods and guidelines for the preparation of pharmaceutical enzyme reference materials. The Center for Standards has a coordination function in organizing collaborative enzyme assays between academic, industrial, and national pharmaceutical control laboratories and in distributing FIP pharmaceutical enzyme standards. Since 1960, many FIP assay methods and standard preparations have been adopted by national and international pharmacopeias, such as the European Pharmacopoeia. The ultimate goal is to provide official, preferentially nonempirical, standardized assay methods by which comparison of commercially available pharmaceutical enzymes is made possible. The most desirable situation would be an international uniformity of enzyme standards and assay methods, which would allow physicians and clinicians to unambiguously compare the potencies of commercially available enzyme products. 

Assay by Reference Standard. It has been realized that the dependence of the turbidimetric assay on a substrate with suitable properties limits the general usefulness of the method to some extent. This shortcoming has been eliminated by the use of a reference standard for hyaluronidase as used by Warren et al. (192) and Alburn and Whitley (1). The enzyme standard is assayed simultaneously with the unknown samples, and the unknown activities are calculated from the turbidity reduction curve of... 

In order to measure the exact amount of a specific protein (analyte) by IHC signal intensity, a critical requirement is the availability of a standard reference material (present in a known amount by weight) that can be used to calibrate the assay (IHC stain). It is then possible to determine the amount of test analyte (protein) by a translation process from the intensity of IHC signals. In this respect it is helpful to consider the IHC stain as a tissue based ELISA assay (Enzyme Linked ImmunoSorbent Assay), noting that ELISA is used in the clinical laboratory as a standard quantitative method for measuring protein by weight in fluids, by reference to a calibrating reference standard. 

Chiral or achiral assay and purity determinations are done according to an external calibration calculation procedure, either with or without internal standardization. The calibration is performed against a 10% w/w (compared to the nominal concentration of the sample solution at 100% w/w) reference standard solution. The sample solution for the purity determination remains at the 100% w/w level, while that of the assay determination is diluted 10 times. The reason for the difference in concentration levels is similar to the purity method. A suggested sample injection sequence can be... 

The assay of stressed samples will usually require the use of some type of external standard. The external standard could be an established reference standard, however, the preferred method is to use the same material/lot as is being stressed. This is easily accomplished by weighing additional samples (that will not be stressed) for use as standards at the same time as the stress test samples are weighed. The standards should then be stored under conditions that will assure that no degradation will occur (e.g., freezer). At the time of analysis, the stressed samples are simply assayed vs. the freshly prepared unstressed standards and the results calculated as percent initial. 

Method robustness was established to show assay consistency with various supplies of the reference standards and two other critical reagents ... 

A fibrin clot containing adsorbed plasmin inhibitors is difficult to define in a chemical or physical sense. Generally, when enzyme reactions occur at surfaces, the porosities and adsorption properties erf which are variable, the reproducibility of enzyme assay methods is questionable. The proteinoses, to which belong the most important pharmaceutical enzymes, may present some difficulties when natural substrates (protein ) are prescribed. Here, the application of a parallel run with a reference standard is recommended. 

The primary reference standard is normally prepared on a laboratory scale using pure starting materials, reagents, and solvents and should be of the highest purity that reasonably can be obtained. The synthetic procedure used to make it and the method(s) used for its purification, also should be provided. (If applicable, the method of manufacture section can be referenced.) The purification procedure is normally performed until little or no change is observed through two consecutive cycles in assay purity and levels of impurities. 

Assay and test results are determined on the basis of comparison of the test sample with the reference standard that has been freed from or corrected for volatile residues or water content as instructed on the reference standard label. If a reference standard is required to be dried before use, transfer a sufficient amount to a clean, dry vessel. Do not use the original container as the drying vessel, and do not dry a reference standard repeatedly at temperatures above 25°. Where the titrimetric determination of water is required at the time a reference standard is to be used, proceed as directed in the Karl Fischer Titrimetric Method under Water Determination, Appendix IIB. 

An interesting example is die assay of clonidine hydrochloride in injections and tablets (British Pharmacopoeia, 1980). In 0.01 Mhydrochloric acid, clonidine exhibits two sharp maxima ne - 272 nm and 279 nm, which are not suitable for precise measurement. However, clonidine forms an ion pair with bromothymol blue, and this can be readily extracted into chloroform for subsequent measurement of the broad maximum near 420 nm. Because of the intiinsic variabihty of reagents used in such methods, a pharmacopoeial reference standard is employed for cahbration. A similar policy is adopted for assays involving chemical modification of the drug, as in the tetrazolium assay for corticosteroids, the assay for folic acid involving hydrolysis, diazotisation, and coupling with N-(l-naphthyl)ethylenediamine, and the reaction of penicillins with imidazole and mercuric salts. 

However, these titration methods can be used in early development when a reference standard is not available. Also, the spectrometric-based assay methods such as ultraviolet (UV) may be nonspecific because most of the drug substance impurities contain a similar chromophore as the parent molecule. If UV is used, UV absorption is measured at one or more wavelengths and the absorbance value is recorded for a particular concentration. Sandor Gorog has critically evaluated the difference between specific and nonspecific assay methods in the European and US Pharmacopoeias [23]. The difference between the mean and the accepted true value with a defined confidence interval should be reported in the acceptance criteria. 

Identity is a general requirement for dosage forms. When determining specificity for identity, the assay and related substances or the content uniformity methods can be used. Assay and content uniformity methods are quantitated by external reference standard. This identity test confirms that the correct active ingredient (s) is present and is present in correct ratio if multiple variants are available. The method could also be used for post-packaging analysis. The general requirements are that the sample and standard chromatograms should correspond in retention time and normalized peak area within 10%. 

The most accurate quantitative results in GC and LC are probably obtained by the use of an internal standard. However, it can be difficult to employ, as the procedure depends upon finding an appropriate substance that will elute in a position on the chromatogram where it will not interfere or merge with any of the natural components of the mixture. Unfortunately, for a multi-component sample, this can become difficult to the extent of being virtually impossible, under which circumstances the external standard method must be used. Having identified an appropriate reference standard, the response factors for each component of interest in the mixture to be analyzed must be determined. It should be noted that this usually does not include all the components. In many instances only certain components of the mixture need to be assayed. A synthetic mixture is then made up containing known concentrations of each of the components of interest together with the standard. 

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