Accuracy bioanalytical

For high-throughput analysis, it is important to increase the specihcity of each bioanalytical method. The enhancement of chromatographic resolution presents various limitations. Better selectivity can be obtained with TOF mass analyzers that routinely provide more than 5000 resolution (full width at half-mass or FWHM). The enhanced selectivity of a TOF MS is very attractive for problems such as matrix suppression and metabolite interference. In one report of quantitative analysis using SRM, TOF appeared less sensitive than triple quadrupole methods but exhibited comparable dynamic range with acceptable precision and accuracy.102... 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

Partial Validation. Partial validations are modifications of already validated bioanalytical methods. Partial validation can range from as little as one intraassay accuracy and precision determination to a nearly full validation. Typical bioanalytical method changes that fall into this category include but are not limited to ... 

The magnitude of the uncertainty associated with a measurement should always be evaluated even if method development is able to generate the best, error-free standard curve possible in order to obtain the best, error-free concentration values for the unknown. Precision and accuracy values define the compromise between the demand for certainty in reported results and the inherent uncertainty in bioanalytical measurement. Precision and Accuracy are the most important parameters used to set the performance standards of bioanalytical methods. They define if the zone of uncertainty connected with the data produced by the method is or is not acceptable for the specific task [13,21]. 

Internal Standard. The use of internal standard is critical in bioanalytical methods to improve precision and accuracy. The role of internal standard is to mimic the analyte of interest. It should be added before sample preparation/extraction to account for losses and errors introduced during the process. The more sample handling steps there are, the greater the error becomes, because errors are additive. In this case, the use of internal standard minimizes errors significantly. 

Even if a reconstitution solvent blank is normally used to evaluate carryover during validation and sample analysis, it is suggested to use all three types of blank as mentioned earlier for carryover evaluation during the method development. As considered previously (see Section 8.3.2) in the quantitative evaluation of carryover, linearity of detection together with sufficient accuracy and precision below LLOQ should be assumed. This assumption is questionable, and for this reason some bioanalytical laboratories have chosen different criteria to evaluate the carryover. 

Quality Control (QC) QC samples are used to check the performance of the bioanalytical method as well as to assess the precision and accuracy of the results of postdose samples. QC samples are prepared by spiking the analyte of interest and the IS into a blank/control matrix and processing similar to the postdose samples. QC samples cover the low (3 x LLOQ LLOQ = lower limit of quantitation), medium, and high (70-85% of ULOQ ULOQ = upper limit of quantitation) concentration ranges of the standard curve and are spaced across the standard curve and the postdose sample batch. 

Characterize bioanalytical method, including sample preparation procedure, for linearity, sensitivity, specificity, precision, and accuracy. 

One of the critical steps in qualitative and quantitative analysis is the sample preparation procedure. Sample preparation step can affect specificity, sensitivity, accuracy, precision, and throughput of a bioanalytical procedure. In addition to development and optimization of the chemistry involved in sample processing, the use of semiautomated or fully automated protocols has been... 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

For HPLC methods, significant changes could occur, and the recommended parameters for revalidation are summarized in Table 2. For bioanalytical methods, precision, accuracy, and limit of quantitation are considered to be the minimum revalidation tests. 

The internal standard (I.S.) method is a more accurate method. The I.S. technique can compensate for both instrumental and sample preparation errors and variations (e.g., dilution and extraction) [45, 46], Sample pretreatment steps such as extraction often result in sample losses, and a proper I.S. standard should be chosen to mimic the variations in these steps. Thus, both the accuracy and precision of quantitative data increase if an I.S. is included in the procedure. The I.S. should be similar but not identical to the analyte, and the two should be well resolved in the chromatographic step. The standard curves are obtained from standards of blank samples spiked with different known concentrations of the analyte of interest and addition of an I.S. at constant concentration. Also to the unknown samples the same constant concentration of the I.S. is added. The standard samples are processed in parallel with the unknown samples. In the calibration curve, the ratios of analyte to I.S. peak area (or height) are plotted versus the concentration of the analyte. A proper I.S. in a bioanalytical chromatographic method should fulfill the following requirements [44] ... 

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