Validation of HPLC Method For Cleaning Validation


1.0  Specificity

Demonstrate the separation of the analyte from available Diluent, and Blank (swab) with standard solution using the HPLC system.

Note:  No other peak should be observed in diluent and Blank (swab) at the retention time of analyte.

2.0 System Precision

The precision as measured by multiple injections of a homogenous standard solution indicates the performance of the HPLC instrument under the chromatographic conditions and day tested.  As a part of method validation, a minimum of 6 injections of the standard preparation with an RSD of ≤ 5.0 % is recommended.

3.0 Linearity

Inject in duplicate at least 5 different standard concentrations covering range LOQ to 150% of the target concentration into the HPLC system.  Plot average peak response versus the actual concentration. The Correlation coefficient should be greater than 0.99.

4.0 Limit of detection

The limit of detection shall be arrived by diluting the standard solution and injecting the diluted solutions. Estimate the concentration, which gives the ratio between 2 and 3 for signal over noise from the same run.  Run the concentration approximately above, and at below level that actually produces S/N ratio of 2 to 3.  The LOD is considered the concentration which produces peak with a S/N = 2 to 3.  Measure a series of diluted solution with a very low concentration and inject six replicate into the HPLC system. Estimate the concentration, which gives the response % RSD between 10.0% and 33.0%.

5.0 Limit of Quantification

Inject six replicate injections of standard solution to give about 3.0 times the LOD concentration. Relative standard deviation of response should be between 3.0 % and 10.0 %.

6.0 Stability of Standard solution

The stability of Standard solution should be established over the period time by injecting into the HPLC system at 0, 24, 36,48, 60 and 72 hours. The difference in assay from initial should be not more than 3.0%

 7.0  Percentage Recovery

Standard Solutions: Prepare standard solutions corresponding to approximately 50%, 100% and 150% of Acceptance Limit concentration as per the test procedure and inject each standard solution in triplicate.

Spiked Swab solution: Prepare spiked swab solutions corresponding to approximately 50%, 100% and 150% of Acceptance Limit as per  the test procedure and inject each spiked swab solution in triplicate.

Calculate the %Recovery of spiked swab using the following  formula. 

 % Recovery =  (Spiked swab Area / Average Standard Area) x 100

Average % Recovery should be not less than 80.0%. 

In case of discrepancy repeat the experiment. If these criteria are still not met, investigate the reason.

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