How to perform Robustness in Analytical Method Validation
Introduction and outcome
Robustness is one of the most important analytical parameters of analytical method validation. Apart from knowledge and skills, this test requires a lot of time. In this article, I will share skill-based knowledge with case study to perform this test in easy way.
7- Steps Strategies to Perform Robustness
The following are the 7- steps strategies to perform robustness:
- Step-1: Identify critical analytical variables of the analytical method ( e.g. pH, buffer concentration, solvent compositions, column-temperature, sample preparation procedures, etc. ) that are likely to influence the analysis result.
- Step-2: Define the variation range (lower limit and higher limit) for each analytical variables within which the experiment will be performed
- Step-3: Prepare the solutions and perform the robustness testing
- Step-4: Document the results with conclusion
- Step-5: Re-optimize the variation range (lower limit and higher limit) if robustness fails within the defined critical range
- Step-6: Perform the robustness test in the re-optimized range
- Step-7: Make the report and take approval of all concerned persons.
Acceptance criteria for Robustness test
Acceptance criteria of robustness test is decided on system suitability test of the method.
Inject system suitability test (SST) solution in each robustness (change) condition and note down system suitability test result. Robustness condition must meet the SST acceptance criteria.
Case studies:
Let us consider a drug substance D has the following specifications and we have to perform precision test:
- Impurity A NMT: 0.20%
- Impurity B NMT: 0.20%
- Any unknown impurity NMT: 0.10%
- Total impurity NMT 0.50%.
Sample concentration is 1.0 mg/ml. SST acceptance criteria is resolution (R) between main analyte peak D and impurity peak A should be ≥ 2.0 and following are the method chromatographic condition:
Column: C18, (150 x 4.6)mm, 5μm. Buffer 0.02M KH2PO4, pH 2.0 with 10 Phosphoric acid solution. Mobile phase is the mixture of buffer and acetonitrile in in ratio of 60:40. Flow rate of mobile phase is 1.0 ml/minute. Column temperature is 30oC, Injection volume is 20μl and detector wavelength is 254 nm.
Let us define the critical analytical variables and perform the robustness test.
For example pH may be changed 2.5 and 3.0 from 2.5 (±0,2). Flow rate may be changed 0.9 and 1.0 from 1.0 ml/minute and similarly other analytical variables can also be defined as in table-1
Table-1
| S.N | Robustness parameters | Nominal | Level (-1) | Level(+1) |
| 1 | pH | 2.7 | 2.5 | 3.0 |
| 2 | Flow rate (ml/minute) | 1 | 0.9 | 1.0 |
| 3 | Column temp | 30oC | 25oC | 35oC |
| 4 | KH2PO4 concentration | 0.02M | 0.01M | 0.03M |
| 5 | Mobile phase composition (Buffer : ACN) | 60:40 | 57:43 | 63:37 |
| 6 | Column | Make X | Make Y | Make Z |
Table-2
The following are the SST- Resolution between Main Analyte peak D and Impurity peak A in in different change conditions:
| S.N | Robustness parameters | Resolution (R) Nominal | Resolution (R) Level (-1) | Resolution (R) Level(+1) |
| 1 | pH | 3.1 | 3.5 | 5 |
| 2 | Flow rate (ml/minute) | 3.2 | 3.6 | 3.5 |
| 3 | Column temp | 3.4 | 3.6 | 5 |
| 4 | Buffer concentration | 3.6 | 4 | 4 |
| 5 | Mobile phase composition (Buffer : ACN | 2.8 | 2.5 | 2.9 |
| 5 | Column, make X, Y and Z | 4.2 | 3.7 | 4.1 |
Resolution R ≥ 2.0, at each lower level and a higher level of above analytical change variables and hence, analysis can be performed in that range.
Conclusion
Robustness testing is the critical step but very important in any analytical method validation. It assesses the analytical method’s capability to produce consistence and reliable result under extreme conditions. Now I hope this article has cleared all your doubts and now you can independently perform robustness testing during method development and method validation. For any opinion or suggestions related to this article, you can write in the comment section. For any further assistance you can contact me using contact form.
You may also want to check out other articles on my blog, such as:
- Analytical method mini-validation
- Analytical technology transfer
- Ultimate Guide in Analytical Method Validation
- How to perform Solution Stability
- How to calculate Recovery in AMF?
- How to perform precision
- How to perform Linearity
- How to perform DL and QL in AMV?
- How to perform Specificity?
- How to perform Accuracy?
- The best practices of Analytical Method transfer
- How to perform Forced Degradation Studies?
- How to perform Photostability?
References
- https://www.chromatographyonline.com/view/method-validation-and-robustness
- https://en.wikipedia.org/wiki/Robustness_validation
- https://www.chromatographyonline.com/view/robustness-tests
- https://www.fda.gov/regulatory-information/search-fda-guidance-documents/q2r2-validation-analytical-procedures
Abbreviations
- R: Resolution
- SST: system suitability test
Top interview questions on Robustness
How robustness is performed?
Identify critical analytical variables of the method ( e.g. pH, buffer concentration, solvent compositions, column-temperature, sample preparation procedures, etc. ) that are likely to influence the result. define the two extreme variable range. Inject the system suitability solution. Method must meet SST acceptance criteria in change condition. If fails then re-optimize the condition and perform the analysis in the modified condition.
Disclaimer: The numerical data used in the tables or calculations are not actual data. It is designed to explain the topic.