How to Perform Solutions Stability in Analytical Method validation: Case Study

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How to Perform Solutions Stability in AMV: Introduction and outcome

Solutions stability is one of the important parameters of analytical method validation. In this article, I will share with case study why solution solution stability is performed, and how solution stability is performed. After reading the post not only you know purpose of stability of solution but you will also able to perform this test independently.

Purpose of demonstrating stability of solution

During HPLC or GC analysis, several samples are prepared and analysed sequentially. Analysis may take several days to complete depending upon the run time and number of samples. All analysis result will be invalid if the standard or samples are not stable. To avoid such failure, stability of solution is demonstrated.

Procedure of demonstrating solution stability of assay test

Use the following steps when testing solutions stability test for an assay:

• Prepare the standard and sample solutions as per method given in the monograph
• Take six vials and Label these vials V0, V1, V2, V3, V4 and V5.
• Fill the solution in the above vials and use the these solution for stability study as given in the following table-1

• Prepare fresh standard solution at each time interval and inject in six replicates and then inject stability solution in duplicate
• Note down the area response
• Calculate response factor (area response/concertation) for each standard standard and sample solution
• Calculate the % difference of response factor by the following formulae

• % RF Difference = [(|standard RF – sample RF|)/Average RF ]x 100

Case studies:

A drug substance D having the following specifications for related substances and assay test:

Assay : Not less than 98.0%

Sample nominal concentration (assay analysis concentration) in the method is 1.0 mg/ml .

Let us demonstrate solution stability at different intervals:

Solution stability at 12 hours

• Prepare the standard 1mg/ml
• Transfer the solution in six different vials V0, V1, V2, V3, V4 and V5
• Use V1 solution to perfor solution stability at 12 hours
• Inject freshly prepared standard solution in six replicates and note down the area and calculate the RF (response factor)
• Inject V1 solution in duplicate and calculate the RF

Freshly prepared standard solution (V0)

V1 solution

% RF difference between freshly prepared standard solution and stability solution V1 (after 12 hours)
% RF Difference = [(|151970 – 151539|)/151755 ]x 100 = 431/151755 × 100 = 0.28%

Conclusion

The test passess the acceptance criteria at 12 hours since % RF difference between
V0 and V1 solution is 0.28% < 2%

Note:

1. Similarly perfor solution stability for V2, V3, V4, and V5 and conclude the result
2. The test for standard solution stability is also valid for sample solution stability because same diluent is used for solution preparations

Procedure of demonstrating solution stability of Related substances test

• Prepare the standard and sample solutions as per method given in the monograph
• Take six vials and Label these vials V0, V1, V2, V3, V4 and V5.
• Fill the solution in the above vials and use the these solution for stability study as given in the following table-1

• At each time interval inject stability solution (as per above table)
• Note down the result or calculate the result
• Calculate the % difference of result by the following formulae:

• % Result Difference = [(|Result at 0 hour – solution stability result|)/Average result ]x 100
• The difference in result must be within the acceptance criteria

Case studies:

A drug substance D having the following specifications for related substances and assay test:

Related substances test (by area normalisation method):

• Impurity A NMT: 0.20%
• Any unknown impurity NMT: 0.10%
• Total impurity NMT 0.50%

Sample nominal concentration (analysis concentration) in the method is 1.0 mg/ml. QL of the method is 0.04%.

Let us demonstrate solution stability at different intervals:

Solution stability at 12 hours

• Prepare the standard 1mg/ml
• Transfer the solution in five different vials V1, V2, V3, V4 and V5 and use V1 solution to perform the solution stability at 12 hours
• Inject freshly prepared standard solution (V0) in duplicate and note down the result
• Inject V1 solution in duplicate and note down the result

Freshly prepared standard solution

Conclusion

The test passess the acceptance criteria at 12 hours since the % difference of impurity A is 6.5% (< 10%) and not any new impurity (having value ≤ QL) forms in the chromatogram.

Note: Similarly perform solution stability for V2, V3, V4, V5 solution and conclude the result

Advantages

Solution stability data is very helpful in routine analyses especially when analysis is performed for longer time in sequence. It prevents failure of result solution stability analytical error.

Conclusion

I hope this article has helped you understand solution stability and its importance in pharmaceutical analysis. Now you can independently perform solution testing during method development and method validation. You may also want to check out other articles on my blog, such as procedure for performing accuracy, robustness and recovery.

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Interview questions on Stability of solution

Why stability of solution is performed?

During Chromatographic analysis, several samples are prepared and analysed sequentially. Analysis may take several days to complete depending upon the run time and number of samples. All analysis result will be invalid if the standard or samples are not stable. To avoid such failure, stability of solution is demonstrated

How stability of solution is performed in an analytical method validation?

Samples and solutions are prepared as per method given in the monograph. Solution are injected at different time interval such as 0 hour, 12 hour, 24 hours, 36 hours, 48 hours and 60 hours. The % differance of result is calculated at different time interval between freshly prepared solution and stability solution. The time period for which result remains within the acceptance criteria is called solutions stability.

What are the acceptance criteria for solution stability?

For assay, the % response factor difference between freshly prepared solution and stability solution ( after a particular given period) must be less than 2.0%.

References

• https://www.pharmacopeia.cn/v29240/usp29nf24s0_c1225.html
• https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r2-validation-analytical-procedures

Abbreviations

• RF: Response factor
• HPLC: High pressure liquid chromatography
• GC: Gas chromatography
• RF: Response factor
• ml: milliliter
• QL: Quantitation limit
• AMV: Analytical method validation
• NA: Not applicable

Disclaimer

The numerical data used in the tables or calculations are not actual data. It is designed to explain the topic.