1.0 Sink Conditions for non-compendial Dissolution Method

Determine the solubility of active drug substance in the dissolution medium at room temperature.  In case of very soluble compounds demonstrate that two times the amount of active drug substance in one tablet or capsule will completely dissolve in the designated volume of dissolution medium at room temperature.  The room temperature solubility should be more than two times the final concentration of the solution of one tablet or capsule in the designated volume of dissolution medium. Otherwise determine the solubility at 37°C.  The 37°C solubility generally should be at least three (3) times that of the final solution of one tablet or capsule in the designated volume of medium.

2.0 System Precision

The precision is 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.

 3.0 Linearity

Inject in duplicate at least 6 different standard concentrations covering range 25% to 150% 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

Prepare six test preparations as per the test procedure.  Dilute as specified in the dissolution and inject into the HPLC system.  The Q value should meet the specifications.  The relative standard deviation should not be more than 2.0%.

5.0 Recovery and Accuracy of the 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    Ruggedness

 System to system variation

System to system variability study should be conducted on two HPLC and Dissolution systems (same or different manufacturer) using five 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, if it is not in USP.  The Q value should meet the specifications.  The relative standard deviation should not be more than 3.0% for both the systems.

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 five 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 Q value should meet the specifications.  The relative standard deviation should not be more than 3.0% by both the analysts.

8.0 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 phases having buffers with ± 0.5 of the method pH 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 in flow rate

Prepare the solution as per the test procedure and inject into the HPLC system at different flow rates.

The system suitability values should be evaluated for peaks of interest with both the flow rates 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.

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.

9.0 Stability of standard and test preparations on Auto injector

The stability of standard and test preparations on Auto injector should be established over the period time by injecting into the HPLC system at 0, 24, 48 hours. The difference in assay from initial should be 3.0%

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