Assay and Related Substances

A test method for related substances must be able to separate all known and unidentified components from a given drug product. The phrase that defines this process is called stability-indicating.  

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Owens, P. K., Wikstrdm, H., Nagard, S., and Karlsson, L. (2002). Development and validation of a capillary electrophoresis method for ximelagatran assay and related substance determination in drug substance and tablets.. Pharm. Biomed. Anal. 27, 587-598. 

The utility of CZE for ximelagatran, a direct oral thrombin inhibitor, assay and related substances determination in both drug substance and tablets was confirmed. Separation occurred in a 100 mM sodium phosphate buffer pH 1.9, to which 22% (v/v) ACN and 11 mM... 

Typical testing for stability studies includes appearance, potency, chiral assay, and related substances (impurities, degradation products, and contaminants) by HPLC assay, water content by Karl Fischer, identification by NIR or NMR, melting point by DSC, plus microbial testing. 

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%. 

Suresh Kumag R., Narasimha Naidu, M., Srinivasulu, K., Raja Sekhar, K., Veerender, M. and Srinivasu, M. K. Development and validation of a stability indicating LG method for the assay and related substances determination of Exemestane, an aromatase inhibitor.. Pharm. Biomed. Anal. 50 746-752, 2009. 

Analytical Development of API and Drug Products. Early methods to support synthetic and formulation developments are often developed in the form of potency assay, impurities/related substance assay, dissolution, Karl Fischer, identity, chiral method, and content uniformity. These analytical methods are developed and validated in a fast and timely manner to support all phase II studies. 

Dissolution testing is one of the most common analytical techniques performed in a pharmaceutical analytical laboratory. It is performed primarily on oral dosage forms to determine the in vitro release of a drug from its finished dosage. Dissolution testing complements other analytical tests that are used to characterize the performance of the final dosage form (e.g., potency and related substances assay). 

In this chapter the validation approach will be explained using a simple and complex example. The simple example is a spreadsheet developed and validated for content uniformity determination by UV assay, while the second one is a routine HPLC potency and related substances determination. The simple example is placed directly into the validation section to further illustrate the validation approach, while the second example is located at the end of the chapter for further reading. Different frames will mark descriptions of these examples. 

McGregor, D.B., Riach, C.G., Brown, A.., Edwards, I., Reynolds, D., West, K. Willington, S. (1988) Reactivity of catecholamines and related substances in the mouse lymphoma L5178Y cell assay for mutagens. Environ, mol. Mutag., 11, 523-544... 

More than 220 producers of CRMs throughout the world produce today 12,000 20,000 materials with dif ferent matrixes, analytes and properties [4]. However, many testing (analytical) laboratories cannot find suitable CRMs in the market and develop in-house reference materials (IHRMs) themselves. Often IHRMs are developed in a laboratory to conserve the corresponding expensive CRMs. For example, a pharmaceutical company Chemagis Ltd. produces 30 active pharmaceutical ingredients steroids, benzodiazepines, antihistamines, hipolipidaemics, blood flow reactants, etc. Only for a few of them Mo-metasone Furoate, Fluticasone Propionate and Dobutamine Hydrochloride are of fi-cial reference standards for assay supplied by US, British and European Pharmacopoeias with prices of about 180 per unit (50 200 mg). Thus, to support its customers Chemagis is forced to develop IHRMs for assay as well as for impurities and related substances of each produced compound. Therefore, certification of such IHRMs that leads to traceable values is very important. 

Exp Clin Endocrinol 102 12-22 Spencer CE (2004) Assay of Thyroid Hormones and Related Substances [Internet resource] http //www.thyroidmanager.org/FunctionTests/ assay-frame.htm... 

GC-MS has found wide application in studies of monoamines in both animal models and in human neuropharmacology [452]. Interest has centred on the use of selected ion monitoring in the determination of trace amounts of the amines, their metabolites and related substances with a possible function as neurotransmitters. The SIM approach complements established assay methods such as gas chromatography with electron capture detection (ECD), fluorimetry or enzymic assay. A check on specificity is afforded and in many cases enhancement in sensitivity and precision of measurement can be obtained. Method development, principally relating to estimation of central amine turnover, is noted in this Section and an outline of work on human depression serves to illustrate the potential of GC-MS to the study of CNS dysfunction. 

British Pharmacepoeia (BP) and European Pharmacopoeia (EP) Applications of GC for the Assay Chromatographic Purity Identification Presence of Volatile Matter, Intermediates and Related Substances Organic Volatile Impurities Determination of Water Presence of Isomers and Racemate Ratios Determination of Alcohol and Miscellaneou s Uses of GC in Pharmaceutical Raw Materials and Dosage Forms. 

Sakai A, Suzuki C, Masui Y, Kuramashi A, Takatori K, Tanaka N (2007) The activities of mycotoxins derived frran Fusarium and related substances in a short-term transformation assay using v-Ha-ras-transfected BALB/3T3 cells (Bhas 42 cells). Mutat Res 630 103... 

In this introduction, we do not provide a more detailed review of the classical discoveries in the field of vitamin A research. As already discussed in part, these classical discoveries included the original description of fat-soluble A and the introduction of the term ""vitamin A the recognition of retinol as a substance distinct from its carotenoid precursors the development of quantitative chemical methods for the analysis and assay of retinol and related substances the elucidation of the chemical structure and then the total synthesis of retinol, retinyl esters, and retinoic acid the description of the unique pathology of both hypovitaminosis A and h q)ervitaminosis A in experimental animals and man the elucidation of the fundamental role of retinaldehyde in vision the determination of human and animal needs for retinol or its precursors for adequate nutrition and the development of practical syntheses for the commercial production of retinyl esters to meet those nutritional needs. This historical story has been... 

Udenfriend s development of a spectrophotofluorometric assay for S-HT and related substances has greatly facilitated the study of S-HT biochemistry. S-hydroxy-indoles possess a native and specific fluorescence in acidic conditions. After extraction and isolation, these compounds when excited by U-V light at 27S nm will emit at 330 nm in dilute HCl or at SSO nm in 3N-HCL Generally, less than 1 /ig/ml can be accurately measured. Recently, various procedures have been proposed for converting S-HT into even more fluorescent derivatives. New assay methods based on gas-liquid chromatography or on phosphorescence of indole derivatives should, in the near future, provide further significant improvement (see Hanson in ref. 4). 

Onodera, K., Hirano, S., and Hayashi, H., 1965, Sialic acid and related substances. II. A comparative assay of N-acetylneuraminic acid, Carbohyd. Res. 1 44. 

Assay and analysis of pazufloxacin (8) and its related substances were established by HPLC (98MI89). 

In November 1997, the Department of Health and Human Services along with the International Conference on Harmonisation (ICH) released a draft guidance for the selection of test procedures, which included chiral drugs. For the development of an enantiopure drug substance, acceptable criteria shall include, if possible, an enan-tioselective assay. This assay should be part of the specification for the identification of an enantiopure drug substance and related enantioenriched impurities [16]. 

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