*Analytical, Bioanalytical, Stability-Indicating Methods: Key Part of Regulatory Submissions DOI: http://dx.doi.org/10.5772/intechopen.93566*

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 standard.


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

*Analytical Chemistry - Advancement, Perspectives and Applications*

matrix like bone marrow.

assessed before analysis.

case of potentially labile metabolites.

quantified is the intended analyte.

sion parameters in the validation.

range of expected concentrations.

be determined.

• The same biological matrix as the matrix in the intended samples should be used for validation purposes. It is necessary in case of limited availability of

• The stability at the time of matrix during collection and storage should be

• The stability of analyte in matrix from dosed subjects should be finalized in

• The parameters like accuracy, precision, reproducibility, response function, and selectivity of method for endogenous substances, metabolites, and known

• In case of selectivity the evidence should be produced that substance being

• The concentration range of analyte should be defined on standard samples including their statistical parameter which clears the standard curve.

• To define concentration and response relationship an enough sample should be analyzed. This relationship should be continuous and reproducible. For this purpose, the standard used should be from dynamic range and nature of the concentration-response relationship. Generally, six to eight concentrations excluding blank can be used to define standard curve. In case of nonlinear

• There should be proper demonstration to show the ability to dilute samples originally above the upper limit of the standard curve by accuracy and preci-

• In case of high throughput analyses like multiplexing, multicolumn and parallel systems, enough Quality control (QC) samples should be assessed to prove control of the assay. Based on the run size, the number of QC samples should

• There is a need to set a specific acceptance criterion for bioanalytical method to be considered as a valid method. That should be achieved for accuracy and

• There should be minimum six standard points for matrix based standard curve excluding blank, which may be single or replicates and should cover the entire

• Standard curve should explain the concentration-response relationship with

• The Lower limit of quantitation, (LLOQ ) should be measured with acceptable accuracy and precision which is the lowest concentration of the standard

degradation products should be set for biological matrix.

concentrations more standard may be recommended.

There should be proper placement of the QC samples in the run.

**6.2 Specific recommendations for bioanalytical method validation**

appropriate weighting and statistical tests for goodness of fit.

precision for validation of QC samples over the range of standards.

**160**

### *Analytical Chemistry - Advancement, Perspectives and Applications*

**Figure 3.** *Sequence showing development and validation of bioanalytical method.*
