Assay noise

The limit of tolerable error is generally smallest in a minor component assay. It will have been determined that a particular minor component must be at or below a threshold concentration for the product to be usable. Therefore, the decision to accept or reject an entire production batch may depend on the analytical result. Typical batches may contain the contaminant at a concentration very similar to the specification limit. In a minor component assay, the major component may be overloaded and out of the proper range of detection of the assay. Even so, the minor component may be at such low levels that assay noise interferes. 

HTS assay data is statistically analyzed to identify genuine outliers from systematic or random assay noise, which allows the level of activity for each compound tested in the assay to be determined. The values used in the final analysis are derived from a normalization procedure that converts the raw data to the percent activity relative to the control values (Table 2). Wells that contain controls can be placed in any number of wells on an assay plate, but a common practice is to use < 10% of the plate for controls, often in columns along the left or right edges (Fig. 4a). 

D. Importance of the D. Permeability testing permeability assay noise... 

Operating limits are data driven, with the most critical measure being the process variation. They are established by working inward from the registration limits to provide assurance that we will not fail registration limits because of process or assay noise. At this stage of development the process variation will not be completely known, particularly in the hands of the plant, but the noise observed from the DOEs can be a reasonable starting point. 

A study was conducted to measure the concentration of D-fenfluramine HCl (desired product) and L-fenfluramine HCl (enantiomeric impurity) in the final pharmaceutical product, in the possible presence of its isomeric variants (57). Sensitivity, stabiUty, and specificity were enhanced by derivatizing the analyte with 3,5-dinitrophenylisocyanate using a Pirkle chiral recognition approach. Analysis of the caUbration curve data and quaUty assurance samples showed an overall assay precision of 1.78 and 2.52%, for D-fenfluramine HCl and L-fenfluramine, with an overall intra-assay precision of 4.75 and 3.67%, respectively. The minimum quantitation limit was 50 ng/mL, having a minimum signal-to-noise ratio of 10, with relative standard deviations of 2.39 and 3.62% for D-fenfluramine and L-fenfluramine. 

Essential features of an automated method are the specificity, ie, the assay should be free from interference by other semm or urine constituents, and the sensitivity, ie, the detector response for typical sample concentration of the species measured should be large enough compared to the noise level to ensure assay precision. Also important are the speed, ie, the reaction should occur within a convenient time interval (for fast analysis rates), and adequate range, the result for most samples should fall within the allowable range of the assay. 

When sufficient amounts of sample are available one tries to exploit the central part of the dynamical range because the signal-to-noise ratio is high and saturation effects need not be feared. (Cf. Figures 2.11 and 3.1.) Assays of a major component are mostly done in this manner. 

This approach of combining shape-matching and conformahonal analysis proved a useful complement to HTS. Some of the compounds identified by the computational screen were not detected in the original experimental screen. This was because their relative weak activity was difficult to separate from the noise of the assay. Nonetheless, these compounds had different scaffolds (i.e. were lead-hops ) compared to the previously known inhibitor. The key contribution from conformational analysis was that the newly discovered inhibitors were not found by the corresponding searches based on 2D methods. 

Concentration assays are often the least demanding, since usually the component to be measured is abundant and minor components scarce. Even if resolution is poor or there is detector noise, accurate measurements of concentration can still be obtained. In concentration assays, the principal requirements are stringency in the precision of sample dilution and measurement of column losses of the major component. Detector calibration, another important issue in concentration assays, has been discussed above. 

Couto et al. [11] developed a flow injection system with potentiometric detection for determination of TC, OTC, and CTC in pharmaceutical products. A homogeneous crystalline CuS/Ag2S double membrane tubular electrode was used to monitor the Cu(II) decrease due to its complexation with OTC. The system allows OTC determination within a 49.1 1.9 x 103 ppm and a precision better than 0.4%. A flow injection method for the assay of OTC, TC, and CTC in pharmaceutical formulations was also developed by Wangfuengkanagul et al. [12] using electrochemical detection at anodized boron-doped diamond thin-film electrode. The detection limit was found to be 10 nM (signal-to-noise ratio = 3). 

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