Bioanalytical assays are widely employed to assess drug products and their metabolites through various stages of drug development projects. Bioanalytical assays measuring analytes in complex biological matrices are foundational components of pharmacokinetics, bioequivalence, and toxicokinetic studies. These studies are performed from the early stages of drug development to human clinical studies. However, what is the drug development process? Drug development aims at screening and identifying potential molecules that can safely and effectively treat diseases and medical conditions.
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The primary steps for each bioanalytical assay method validation are assay development, assay validation, and sample analysis. Sponsors and assay development services should rigorously investigate these steps to ensure that assays produce reliable and reproducible results. The current article focuses on bioanalysis method validation. It highlights the primary parameters and acceptance criteria for assay method development.
Bioanalytical assay development
The primary objective of bioanalytical assay development is to build a procedure for identifying and measuring a novel or unknown compound in a study matrix. Researchers can use several approaches to measure a drug compound. However, the choice will depend on the concentration, sample matrix, speed and time of analysis, measurement, and chemical and physical properties of the analyte of interest. Assay method development includes analyte sampling, separation, analyte detection, and result evaluation. Let us dive deep into these sections to understand the primary parameters and acceptance criteria for bioanalytical assay development.
Method selection and sample information
Before selecting a bioanalytical assay, researchers must conduct literature surveys to gather drug profile data and its physiochemical properties. Based on the physicochemical properties of the drug product, researchers can select an ideal internal standard that is compatible with the analyte of interest. Besides, a literature survey helps discover prior assays that may be used for the analyte of interest.
Initial assay method conditions
Identifying initial method conditions include:
- Internal standard and analyte compatibility with the assay method
- Choosing the mobile phase and its composition
- Identifying diluent based on drug solubility
- Selecting assay column
Running aqueous standards
Before running in biological matrices, researchers first analyze the assay in aqueous standards. The calibration must contain at least four concentrations, including the lowest and highest concentrations. The lowest concentration will depend on preliminary studies, whereas the highest will depend on Cmax values. Moreover, the correlation coefficient must not be less than 0.99.
Sample processing method
The physicochemical data from literature and preliminary studies help select an ideal sample preparation technique. Once the method is compatible with the aqueous standards, researchers next prepare the matrix sample.. The most common sample preparation techniques include solid-phase extraction, liquid-liquid extraction, and protein precipitation.
Running assays in biological matrix.
The final step in bioanalytical assay development is checking the accuracy, precision, and recovery in the sample matrix. This step requires matrix samples with a minimum of three aliquots each of lower quality control, higher quality control, and lower limit of quantification. These samples must be analyzed with one set of extraction calibration standards combined with matrix blank and internal standard plus blank sample. Finally, the result must be compared to quality control samples having similar concentrations.