Assay methodology

Stability of a drug substance and product is monitored throughout the development and clinical phases. This monitoring requires stability-indicating assay methodology, and this is a subject that is separate from performulation per se. In most instances, the major, feasible decomposition products are identified early [51], and as such it is known if the pathways are hydrolytic, oxidative, or photochemical. 

Disparate effects on T-cell proliferative responses have been reported following exposure to JP-8. Significant suppression of T-cell proliferation is reported following either inhalation or dermal exposure to JP-8 [ 18,20,36], while the response is unaffected following either the oral or dermal exposure routes in other studies [66,71,72], These differences may be explained by variations in exposure routes and in assay methodology, as agents used to evaluate T-lymphocyte activation and proliferation were diverse and included Con A plus IL-2 [18,20], anti-CD3 [36], or Con A only [66,71,72],... 

Skin tape stripping can be used to determine the concentration of chemical in the stratum comeum at the end of a short application period (30 min) and by linear extrapolation predict the percutaneous absorption of that chemical for longer application periods. The chemical is applied to skin of animals or humans, and after a 30-minute skin contact application time, the stratum comeum is blotted and then removed by successive tape applications. The tape strippings are assayed for chemical content. There is a linear relationship between this stratum comeum reservoir content and percutaneous absorption. The major advantages of this method are (1) the elimination of urinary and fecal excretion to determine absorption and (2) the applicability to nonradiolabeled determination of percutaneous absorption, because the skin strippings contain adequate chemical concentrations for nonlabeled assay methodology. 

Lastly, every research project presents a unique combination of parameters (target, assay methodology, hit rate, throughput limit, and so forth) and project team requirements for interesting actives (physiochemical properties, novelty, and so forth). This suggests that an optimal selection scheme should be easily tailored to meet these unique needs, which is certainly not true of a simple Top X approach. 

In addition to the pharmacokinetics of the drug, the first study in man can provide important information about its metabolites. If assay methodology has been developed, metabolites in plasma can be detected and the AUCs and half-lives determined. Further information can be obtained from assaying urine for drug and, if possible, metabolites. Renal clearance can be calculated over time intervals and the ratio of... 

Plasma concentrations, pharmacokinetics, any difficulties with assay methodology... 

The assay methodology selected should ensure specificity, accuracy, interday and intraday precision, linearity of standard curves, and adequate sensitivity, recovery, and stability of the samples under the storage and handling conditions associated with the analytical method. 

The concentration of active ingredient D1 for batch to batch is shown in Figure 13. The mean potency of all batches is 0.1 mg/5 ml above target. The control chart did not respond to tests for unnatural patterns and trends. It is noteworthy that the calculated UCL (16.7 mg/5 mL) for the 20 batches in this study exceeds the release specification for the product (15.5 to 16.5 mg/5 ml. A probability thus exists that a batch may eventually fail to meet the release criteria. Raw material purity is not a factor in the potency of an individual batch because it is taken into consideration at the time of manufacture. A possible explanation for the wide historical control limits is the assay methodology for... 

Steroid hormones assay standardization Immunoassay vs. GC-MS and LC-MS/MS, validation of assay methodologies, establishing standard pools of steroid hormones in women and man, utilizing the pools for cross comparison of various methodologies. [18]... 

Since radioimmunoassay has permitted the quantitation of substances present in concentrations as low as a few pg/ml of a biological fluid, and since the inherent selectivity of antibodies often make sample preparation requirements minimal and assay methodology simple, RIA has naturally been considered as a means for measuring blood levels of cannabinoid compounds. 

Samples should not be submitted to the FDA with the application. The reviewing chemist will contact the applicant and provide the laboratory addresses) where the samples should be sent. The applicant should prepare four representative samples in sufficient quantity to permit the FDA to perform each test described in the application three times to determine whether the drug substance and the drug product meet the specifications given in the application and whether the assay methodologies work in the FDA s hands. The four samples are ... 

Detail the regulatory specifications and test methods used to assure the identity, strength, quality, purity, and bioavailability of the drug product. The emphasis lies in the assay methodology(ies) used to quantitate the presence of degradation products that assure stability of the drug product. 

Aspeslet LJ, LeGatt DF, Murphy G, Yatscoff RW. Effect of assay methodology on pharmacokinetic differences between cyclosporine Neoral and Sandimmune formulations. Clin Chem 1997 43 104-8. 

Radioactivity, however, is still a very sensitive means of measuring the presence or absence of a given material. Assay methodology has now come full circle, to the development of an ultrasensitive enzyme RIA. In this technique, an antigen is bound to a solid phase. Antibody will bind to the antigen, which could be a drug-protein conjugate, and the presence of bound antibody is detected by means of a second antibody coupled to alkaline phosphatase. So far this is the standard enzyme-linked immunosorbent assay (ELISA). However, if the substrate is tritium-labeled adenosine monophosphate, it is converted by the enzyme to tritium-labeled adenosine, which may be readily separated and measured. The detection limit for this assay for cholera toxin is approximately 600 molecules of the toxin (22). 

In spite of the demonstration that at least the receptor assays using receptor from bovine lung measure very specifically /-propranolol (62), most other workers appear to have used the racemic material as a standard and to have recorded assay results in terms of racemic substance. That this procedure can give usable results is supported by the generally good correlation between the receptor assay methodology and other nonenan-tioselective techniques such as GLC or fluorometry. However, it would seem that given the enantioselective properties of the receptor system, it would behoove researchers to analyze and report /-propranolol or /-propranolol equivalents whenever possible. 

The reagents used for the Opus system are totally self-contained in the test modules. There are two types of assay methodologies used with the analyzer multilayer film assays and fluorogenic ELISA assays. 

In implementing the infra- and inter- laboratory trials an extensive assessment of specific components of the assay, methodology and pertinent issues of operation, application and production have been examined. 

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