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FDA Guidelines for Assays


US FDA Guidelines for Bioanalytical Validation

Bioanalytical Validation Method

US FDA Guidelines

Selectivity (specificity)

Analyses of blank samples of the appropriate biological matric (plasma, urine or other matrix) should be obtained from at least six sources. Each blank should be tested for interference and selectivity should be ensured at LLOQ.

 

Accuracy

Should be measured using a minimum of six determinations per concentration. Minimum of three concentrations in the range of expected concentrations is recommended for determination of accuracy. The mean should be ±15% of the actual value except at LLOQ, where it should not deviate by ±20%. This deviation of mean from the true values serves as the measure of accuracy.

 

Precision

Precision should be measured using a minimum of five determinations per concentration. Minimum of three concentrations in the range of expected concentrations is recommended. The precision determined at each concentration level should not exceed 15% of the CV except for the LLOQ, where it should not exceed 20% of the CV.

 

Recovery

Recovery experiments should be performed at three concentrations (low, medium and high) with unextracted standards that represent 100% recovery.

 

Calibration curve

Should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard) and six to eight non-zero samples covering the expected range, including LLOQ.

 

LLOQ

Analyte response should be five times the response compared to blank response. Analyte peak should be identifiable, discrete and reproducible with a precision of 15-20% and an accuracy of 80-120%.

 

Freeze-thaw stability

Analyte stability should be determined after three freeze-thaw cycles. At least three aliquots at each of the low and high concentrations should be stored at intended storage temperature for 24 hours and thawed at room temperature. When completely thawed, refreeze again for 12-24 hours under same conditions. This cycle should be repeated two more times, then analyze on third cycle. Standard deviation of error should be <15%. If analyte is unstable, freeze at -70°C for three freeze-thaw cycles.

 

Short-term stability

Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this temperature for 4-24 hours and analyzed. Percent deviation should be <15%.

 

Long-term stability

At least three aliquots of each of low and high concentrations at same conditions as study samples. Analyze on three separate occasions. Storage time should exceed the time between the date of first sample collection and the date of last sample analysis.

 

Stock-solution stability

Stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours. Percent deviation should be <15%.

 

QC samples

QC samples in duplicates at three concentration levels (one near the 3 x LLOQ, one in mid-range, one close to high end) should be incorporated at each assay run. At least four out of every six should be within 15% of the respective nominal value. Two of the six may be outside of 15% but not both at the same concentration. Minimum number QCs should be at least 5% of total number of unknown samples or six total QCs, whichever is greater.