Assay Validation: The Critical Step for Ensuring Accurate and Reliable Results!

Assay Validation: The Critical Step for Ensuring Accurate and Reliable Results!

Bioanalytical method development and validation are crucial for generating accurate and reliable results. The assay validation component establishes reference methods and assessments for demonstrating the competencies of assay development services to deliver reliable bioanalytical results. Assay validation and good laboratory practice studies such as GLP toxicity studies require maximum relevant confirmation of ideal parameters. The current article discusses these parameters that are crucial for demonstrating assay validity. 

Bioanalytical assay validation

Assay validation is critical for biochemical evaluations. It confirms that the bioanalytical method is suitable for its intended applications. Assay validations protocol includes assessing multiple features of an assay and determining its usefulness and validity. These parameters include selectivity and specificity, precision, linearity, accuracy, stability, range, and limit of detection and quantification. Let us explore each of these parameters in detail. 

Selectivity is the ability of a bioanalytical assay to measure the analyte of interest. On the other hand, specificity is concerned with identifying the analyte among different sample components. 

Precision is a measure of agreement among different test results when employed repeatedly. Precision can be determined through standard deviation and mean values or using the Horwitz equation. 

Accuracy is concerned with test results matching the true value.

Researchers spike the sample matrix with the known concentrations of analytes to measure assay accuracy. Generally, this process varies from matrix to matrix and depends on the study plan. 

The linearity of a bioanalytical assay is proportional to analyte concentrations within a specified range. Generally, linearity assessments include a series of standards in the range of 50% to 150% of the desired working range. 

The assay range includes the upper and lower levels of analyte concentrations determined with accuracy, linearity, and precision. 

Often analytes may decompose before the investigation

For example, during extraction or sample clean-up. Hence demonstrating stability is a crucial component of assay validations studies. 

The limit of detection is the lowest concentration at which an analyte is detected. On the other hand, the limit of quantitation is the lowest limit at which an analyte is detected and quantified. 

Today most assay validations studies employ a fit-for-purpose concept. This concept mentions that the level of assay validation should depend on the intended purpose of a study. Therefore, assay validations can be full, partial, or cross-validation. 

Full assay validation is required for a new bioanalytical method or revision of an existing validated assay. 

Partial validation is necessary during modifications of an already validated assay. So, Partial assay validation can range from one single parameter to nearly complete assay validations. Partial method validation is generally required during transfer between laboratories or changes in instruments, procedures, methodology, assay matrix, or critical reagents. 

Cross-validation is required to compare various parameters between two or more bioanalytical assays used within the same study or among separate studies. Besides, cross-validation is necessary to conduct experiments at different sites. 

In Conclusion

Assay validation is crucial to fulfill regulatory requirements and to ensure that the assay generates accurate, reliable, and reproducible results. Moreover, laboratories should follow the US FDA and GLP guidelines during bioanalytical method development and validation. 

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