Repeatability bioanalytical

It is therefore essential that before pivotal (repeat dose) preclinical studies are initiated, bioanalytical assay development must be completed. This has to cover potential test species, normal and diseased humans. The assays must be validated in the sampling matrix of the toxicity test species, and one should also develop suitable assays for antibodies to the test article. 

As with the determination of the intralaboratory repeatability, the intralaboratory reproducibility was determined by analyzing a cleaned sediment extract and a 3-pM 2,3,7,8TCDD standard on 10 separate days and by multiple persons. The reproducibility for the 3-pM 2,3,7,8-TCDD standard was found to be 13.8%, whereas the reproducibility for the cleaned sediment extraet was shown to be 19.9%. Since the observed reproducibilities are in the range of relative standard deviations for two sediment extracts analyzed in 10-fold on the same day (intralaboratory repeatability), the DR CALUX bioassay can be evaluated as a stable and robust bioanalytical tool. 

Table 9. Reanalysis of Study Samples Table 14. Summary of Standard Curve and QC Data for Bioequivalence Sample Analyses Table 15. SOPs Dealing with Bioanalytical Repeats of Study Samples...

Repeatability is obtained when the analysis is performed in one laboratory by one analyst using the same equipment at the same day. Repeatability should be tested by the analysis of a minimum of five determinations at three different concentrations (low, medium and high) in the range of expected concentrations, according to FDA [16], However according to the ICH [79] repeatability could be measured by the analysis of three determinations at three different concentrations or through six determinations at 100 % of the test concentration. The latter one is for analysis when the concentration is supposed to be constant for all samples, e.g., pharmaceutical products. The acceptance criteria for precision depends much on the type of analysis. For compound analysis in pharmaceutical quality control, precision should be better than 2 % [82], For bioanalytical applications the precision values at each concentration level should be better than 15 % except for the lower limit of quantification (LLOQ) where is should not exceed 20 % [16], The intermediate precision shows the variations affected in day-to-day analysis, by different analysts, different instruments etc. Reproducibility, as above, represents the precision obtained between different laboratories. 

Standard Operating Procedures The bioanalytical report should include a brief description of critical SOPs (e.g., acceptance criteria for batches and repeat analysis). As appropriate, SOPs should be included as report appendixes. SOP deviations and their impact on the integrity of the study results should be described. 

Bioanalytical study reports should describe the reason of repetition for each repeated sample. A table that lists each type of repeat is useful and facilitates straightforward and clear reporting. The table should include the original, repeat, and accepted result for use in pharmacokinetic calculations. To facilitate an understanding of the repeats as part of the overall study conduct, it is desirable to include the source of each result by listing the run ID for both the original and repeat results. Samples repeated in error (e.g., the analyst did not intend to repeat the sample) should also be reported. Furthermore, if no samples are repeated, the bioanalytical study report should include a statement in this regard. 

In cases where the study sponsor or pharmacokineticist5 assumes responsibility for selecting samples for repeat analysis, the bioanalytical laboratory should require and maintain written documentation of the request in the study file. The written documentation should identify the samples selected by the sponsor or pharmacokineticist, along with the basis for the selection. Ideally, the selection criteria used by the sponsor or pharmacokineticist should be provided to the bioanalytical laboratory in advance of sample analysis and maintained in the study file. Similarly, if the sponsor or pharmacokineticist assumes responsibility for determining the value reported for pharmacokinetic calculations, the SOP followed by the sponsor or pharmacokineticist in this regard should also be provided to the bioanalytical laboratory to facilitate a complete record for reconstructing the study conduct. 

In addition to analytical and nonanalytical repeats, some samples in certain studies need to be reassayed to demonstrate incurred sample reproducibility (ISR). This type of testing is a critical step for demonstrating the reproducibility of the bioanalytical method with samples from dosed subjects (as distinct from precision demonstrated with QCs, i.e., blank matrix spiked with drug). Written procedures in this regard should include a description of how the samples are selected for reanalysis, the comparison and reporting of the original and repeat results, and the acceptance criteria for variability between results. 

The repeat results from this type of testing are used to confirm the reproducibility of the assay and should be presented in the bioanalytical study report as a separate data table. In cases where repeat testing of incurred samples does not confirm that the method is reproducible, the bioanalytical laboratory should investigate the cause of the nonreproducibility before continuing its use of the method or reporting results from samples previously assayed. 

Bioanalytical method validation guidelines recommend using a minimum of three samples at low, mid, and high concentrations across the calibration curve range for precision testing. Measurements should include the evaluation of precision or repeatability within a single analytical run... 

The precision of a bioanalytical method is a measure of the random error and is defined as the agreement between replicate measurements of the same sample. It is expressed as an RSD. Precision can be evaluated by analyzing replicate samples on the same day (repeatability or within-day precision) or on different days (intermediate precision or between-day precision). The accuracy is defined as the agreement between the measured value and the true value. Accuracy is determined using reference samples, when available, or spiking a... 

Precision of peak areas or heights is important because they are used for calculating amounts during quantification. It is also the most important performance criterion for an LC injection system. Precision should be determined using a minimum of five rephcate chromatograms. For bioanalytical samples, precision is studied at least at low, medium, and high concentrations. This is repeated on separate days to calculate intraday and interday precision. 

It was originally recommended (Matuszewski 2003) that five different batches of blank matrix (biological fluid in their case) should be tested for ME and RE values in order to assure reliable validation of a bioanalytical method. In the later work (Matuszewski 2006) it was further proposed that assay precision and accuracy should be determined in five different lots of a blank matrix (e.g., a biofluid) instead of repeat (n = 5) analyses using a single lot of blank matrix. Then if an isotope-labeled SIS is not available, determination of the slopes of the five calibration curves and their precision, together with use of <3-4 % (CV %) as a criterion, provides a useful guideline for assessment of the presence or otherwise of a significant relative matrix effect. Of course such a procedure is not always possible, especially for environmental samples a recent example of this problem (Stiiber 2004) involved environmental samples (river water). 

Prior to filing an IND, a number of additional studies and tasks must be completed. A formal characterization of the structure and properties of the compound must be completed, methods to analyze the strength, purhy stability of the pure compound and any formulation must be completed and bioanalytical methods to analyze drug in pharmacology and toxicology studies must be developed. Regulatory toxicity in two species, at minimum a single dose toxicity test and a repeat dose toxicity test that matches or exceeds the duration of the planned clinical trial, must be completed. Once a clinical plan is developed, the... 

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