Solution Stability In Analytical Method Validation: How To Perform

Understanding Solution Stability in Analytical Method Validation: A Practical Case Study

Solution Stability In Analytical Method Validation is a critical parameter, ensuring the reliability and accuracy of analytical results over time. In this article, I will explore the importance of solution stability testing, explain the methodology used to assess it, and present a real-world case study to illustrate its practical application. By the end of this post, you will not only understand the purpose of solution stability testing but also gain the confidence to perform it independently and effectively in your own laboratory work.

Solution Stability In Analytical Method Validation

Solution stability is a crucial component of analytical method validation, ensuring that analytical results remain accurate and reliable over time. It involves evaluating how long standard and sample solutions can be stored under specific conditions – such as room temperature or refrigeration – without compromising their integrity. This is typically assessed by comparing the results of freshly prepared solutions with those stored over defined time intervals, allowing analysts to determine the acceptable duration for solution use in routine analysis..

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.

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Acceptance Criteria: Solution Stability In Analytical Method Validation

For assay test

The standard/sample solution is tested for up to 5 days by comparison of the response factor of an old solution (injected as a sample) to freshly prepared standard solutions.

The test for standard solution stability is also valid for sample solution stability because same diluent is used for sample-solution preparations

Acceptance criteria: The test passes the acceptance criteria if % RF (Response factor) difference between freshly prepared standard solution and stability solution is ≤ 2%.

For Related substances test

The sample solution is tested for up to 5 days for related substances stability testing. Result of old samples solution (stability solution) is compared with the freshly prepared sample solutions.

Acceptance criteria: No new peak higher or equal to QL (Quantitation limit) should appear in the old sample solutions

Solution Stability In Analytical Method Validation: How To Perform

1. Procedure of demonstrating solution stability of assay test
2. Procedure of demonstrating solution stability of Related substances test

1. 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/concentration) for each standard standard and sample solution by the following formula:

• Calculate the % difference of response factor (RF) by the following formulae:

Case studies: Solution Stability

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 perform 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

2. 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:

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• How to perform Specificity in Analytical Method Validation?
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• 3-Steps Recovery Calculation In Analytical Method Validation: Easy Tips
• How To Perform Accuracy In Analytical Method Validation: Calculation and Case Studies
• How to perform Detection Limit and Quantification Limit in AMV?
• How to Perform Linearity and Range in Method Validation?
• Relative Response Factor (RRF) in Pharmaceutical Analysis
• What is CSV (Computer System Validation): Learn in 12 Easy Steps
• Relative Response Factor (RRF) in Pharmaceutical Analysis: Learn In 5 Steps
• How To Calculate Solubility BY HPLC: Learn Easily in 11 Minutes
• ICPMS: A Key Tool For Elemental Impurities Analysis

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FAQs: Interview questions on Stability of solution

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.