**6.2 Specific recommendations for bioanalytical method validation**


**161**

*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions*

curve. By using a least five samples independent of standards and its coefficient of variation, the LLOQ can be established. The LLOQ should not be confused with the limit of detection and/or the low-Quality Control (QC) samples. The upper limit of quantification will be defined by highest

• The accuracy and precision should be determined by using minimum of five determinations per concentration level excluding blank samples. The average value should be within ±15% of the theoretical value. The LLOQ should be up to ±20%. The coefficient of variance of precision should not exceed 15% and for LLOQ should not exceed 20%. The methods which give the results of accuracy and precision with these above-mentioned values should be

• There should be proper demonstration of concentration of analyte in biological matrix with which the accuracy and precision is determined. This can be performed by analyzing replicate sets of QC samples from same biological matrix. This QC sample should be representative of entire concentration range selected for standard curve. From which one concentration within LLOQ, one should be middle one that is middle QC (MQC) and last should be upper limit

• All outliers should be included in reported method validation data and accuracy and precision data. The values of outliers that are determined statistically

• The storage temperature stability in biological matrix should be determined for analyte. The freeze-thaw stability at minimum of three cycles of two concen-

• The ambient temperature stability of analyte should be determined over the time period equal to typical sample preparation, sample handling and analyti-

• In case of instrument failure, reinjection reproducibility should be evaluated to

• For determination of specificity of assay method, a minimum of six concentration of same matrix should be studied. In case of hyphenated techniques like mass-spectrometry based methods, it is not important to study six independent matrices. There should not any compromise to study matrix effect to ensure precision, selectivity and sensitivity in case of Liquid Chromatography-Mass spectrometry (LC-MS) and Liquid Chromatography-Mass spectrometry-Mass Spectrometry (LC-MS-MS) based procedures. The selectivity should be evaluated throughout method development, method validation and it should be continued up to the application of method to

The acceptance/rejection criteria for spiked, matrix-based calibration standards

and validation of QC samples should be based on theoretical concentration of analytes. For studying accuracy and precision, the specific criteria should be set in

can also be reported with the calculations of accuracy and precision.

*DOI: http://dx.doi.org/10.5772/intechopen.93566*

of standard curve that is High QC (HQC).

trations in triplicates should be studied.

determine an analytical run could be reanalyzed.

standard.

acceptable.

cal run times.

actual study samples.

the standard concentration range [13] (**Figure 3**).
