The below mentioned parameters are required to be complies during validation of HPLC method for Assay test.

1.0 Specificity :

Demonstrate the separation of the analyte from Placebo.

Conduct the following forced degradation studies to obtain degraded sample, preferably 10 – 50% degradation and demonstrate the separation of the analyte from degradants.

Heat, if necessary, aqueous, neutral, acidic and basic solutions or suspensions followed by neutralization.

If the compound is known to be susceptible to oxidation, expose to oxidative conditions such as reflux under oxygen or heating aqueous solution containing up to 10% hydrogen peroxide.

For light sensitive compound expose the drug substances to intense ultraviolet radiation up to minimum of 7 days.

Demonstrate the separation of the analyte(s) and the internal standard, if there is any, from the components of placebo mixture.

For a multi-component product, placebo formulation containing only one component should be forced to degrade by dry and moist heat (105°C), to demonstrate the separation from possible degradants of the other component.

Peak Purity :

Using an HPLC system, peak purity can be evaluated and the instrument will give the purity value. 

Peak purity shall be check for the above samples and criteria is “Purity angle shall be less than purity threshold”.   

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. As a part of method validation, a minimum of 6 injections of the standard preparation with an RSD of ≤ 2% is recommended.

Column Performance :

If an integrator is being used, collect at least one chromatogram of the Assay, System suitability, Standard Solution or a Resolution and calculate Column Efficiency (Theoretical Plates), Tailing Factor, Resolution and Capacity Factor.  Results should be within specifications.  Record the Retention Times for all components of interest.

3.0 Linearity :

Inject in duplicate at least 6 different standard concentrations covering range 80% to 120% of the target concentration into the HPLC system.  Plot average peak response versus the actual concentration.

The response may be considered linear, if the correlation coefficient is derived from the least squares fit of the data is not less than 0.999. In case of discrepancy still exists, 0.999 correlation, investigate and explain the reason.

4.0 Method Precision

Assay not less than 6 individual sample weights from a homogeneous blend preparations (as per manufacturing formula).  The relative standard deviation should not be more than 2.0%.

5.0 Recovery and Accuracy of Method

Recovery from the spiked Placebo: Prepare solutions in triplicate corresponding to approximately 80%, 100% and 120% of working concentration as per the test procedure by spiking placebo (amount of placebo should be equivalent to the weight of placebo present in the sample amount required for the sample preparation as per test method) with different amounts of the drug substance.

Assay the samples by following the test procedure and calculate the concentration found in % of working concentration. Calculate the mean recovery, the Relative standard Deviation for the 80%, 100% & 120% spiked placebo data.  The mean recovery should be between 97 – 103%

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

 6.0 Placebo Interference                                   

Inject placebo and ensure there is no interference of excipients.

7.0 Range

If the linearity and the accuracy of the method is acceptable and the Relative Standard Deviation of  the accuracy data does not exceed 2.0% then range of the method is the concentration range used for the accuracy of the method (75-125%).

8.0 Ruggedness

System to system variation :

System to system variability study should be conducted on two HPLC systems with two different columns (same or different manufacturer) using Six assay preparations prepared as per manufacturing formula at different times under similar conditions.

Results should be within test specifications. Make the system suitability comparison table and establish the limits. The relative standard deviation should not be more than 2.0% for both the systems.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP.  The relative standard deviation should not be more than 2.0% for both the columns.

Analyst to Analyst Variation :

Analyst to Analyst variability study should be conducted by two analysts on the same HPLC system at different times under similar conditions using Six different assay preparations prepared as per manufacturing formula.

Results should be within test specifications. Make the system suitability comparison table and establish the limits, if it is not in USP.  The relative standard deviation should not be more than 2.0% by both the analyst.

Laboratory to Laboratory Variation :

Laboratory-to-Laboratory variability study should be conducted in different labs on different HPLC systems at different times under similar conditions using Six different assay preparations prepared as per manufacturing formula.

Results should be within test specifications.  Make the system suitability comparison table and establish the limits, if it is not in USP. The relative standard deviation should not be more than 2.0% in both the laboratories.

9.0 Robustness :

Effect of variation in mobile phase composition:

Two mobile phases should be prepared having different concentrations of the method organic phase composition and inject into the HPLC system.

The system suitability values should be evaluated for peaks of interest using both the mobile phases and the results should be within the limits for mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation of pH of buffer in mobile phase :

Two mobile phases should be prepared having buffers with ± 0.5 of the method pH and inject into the HPLC system.

The system suitability values should be evaluated for peaks of interest using both the mobile phase.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

Effect of variation in flow rate :

Prepare the solution as per the test procedure and inject into the HPLC system at different flow rates (+ 10%).

Effect of variation in column temperature :

Prepare the solution as per the test procedure and inject into the HPLC system at ambient ± 5°C column temperatures.

The system suitability values should be evaluated for peaks of interest at both the column temperatures and the results should be within the limits.

If any of the system suitability value is not within the limits, narrow the range and establish the allowable range of variation.

10.0 Stability of standard and test preparations on Auto injector

The stability of test preparations on Auto injector should be established over the period time by injecting into the HPLC system at 4 hours intervals upto 24 hours. The relative standard deviation of peak area for standard and test preparations should be not more than 2.0%.

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