3-Steps Recovery Calculation In Analytical Method Validation: Easy Tips

Recovery Calculation In Analytical Method Validation (AMV) plays a critical role, serving as a key parameter to assess the accuracy and reliability of an analytical method. It helps determine how effectively an analyte can be extracted and measured from a sample matrix. Recovery studies are an essential part of all types of analytical method validations – whether for assay, impurity, or residual solvent analysis. In this article, I will provide a detailed, step-by-step procedure for calculating recovery, supported by a real case study and a section addressing frequently asked questions (FAQs).

3-Steps Recovery Calculation In Analytical Method Validation

The following three steps are used for recovery calculation in AMV:

1. Solutions preparation
2. Solutions injections and their chromatograms generation
3. Recovery calculation

Step-1: Recovery solutions preparation

Prepare the following solutions:

• Prepare standard solution of main analyte (pharmaceutical) at nominal concentration
• Prepare standard solution at QL or 50%, 100% and 150% of the specification limit
• Prepare spiked solutions (main analyte at nominal concentration and impurities at 50%, 100% and 150%) of the specification level: prepare three pharmaceutical spiked solutions spiked with each impurities at 50%, 100% and 150% level

Note: In case of assay, recovered is performed between 80% to 120%

Step-2: Solutions injections and chromatogram generation

• Inject main analyte standard solution in duplicate
• Injection impurities standard solutions at each level (50%, 100% and 150%) in triplicate
• Injection spiked standard solution at each level (50%, 100% and 150%) in triplicate
• Calculate the recovery

Step-3: Recovery calculation formula

Use the following formula for recovery calculation:

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Case studies: Recovery Calculation In Analytical Method Validation

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

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

Sample nominal concentration in the method is 1.0 mg/ml to perform related substances test.

Recovery of Impurity A

Main analyte standard preparation 1.0 mg/ml (1.0 mg/ml sample concentration is equivalent 1000 mcg/ml)

Impurity A standard solution preparation at 50%

• Value of impurity A at 50% = 0.20 X (50 ÷100) = 0.10%
• Concentration of impurity D at 0.10% = 1000 x (0.10 ÷ 100) = 1 mcg/ml

Impurity A standard solution preparation at 100%

• Value of impurity A at 100% = 0.20%
• Concentration of impurity A at 0.20 X (100÷100) = 0.2 mcg/ml

Impurity A standard solution preparation at 150%

• Value of impurity A at 150% = 0.20 X (150 ÷100) = 0.30%l
• Concentration of impurity D at 0.30% = 1000 x (0.30 ÷ 100) = 3 mcg/ml

Injections

Inject the following solutions and generate the chromatogram and note down area response:

• Inject standard D solution in duplicate
• Injection impurity A standard solution at each level (50%, 100% and 150%) in triplicate
• Injection spiked standard D solution at each level (50%, 100% and 150%) in triplicate

Recovery calculation for impurity A at 50% level

Impurity A standard area (0.10% )

0.10% Impurity A area in spiked solution and corresponding recovery in triplicate injections;

Conclusion:

• Impurity A complies in recovery test at 50% level

Note: Similarly recovery can be calculated at 100 % level and 150% level

Conclusion

I hope this article has helped you gain a clear understanding of recovery calculation and its importance in analytical method validation. With this knowledge, you should now be able to independently perform and evaluate recovery testing during method development and validation. If you found this useful, you may also want to explore other related articles on my blog, including topics such as precision, linearity calculations, and determination of detection limit (DL) and quantitation limit (QL). If you have any suggestions or questions regarding this article, feel free to share them in the comments section below.

You may also want to check out other articles on my blog, such as:

1. How To Control Impurities In Pharmaceuticals: Get Mastery In …
2. What is the Analytical Method Validation?
3. The 7 Key Differences Between Validation and Verification
4. How to perform cleaning validation?
5. How to perform Specificity in Analytical Method Validation?
6. How to Perform Precision in Analytical Method Validation?
7. How To Perform Accuracy In Analytical Method Validation: Calculation and Case Studies
8. How to perform Detection Limit and Quantification Limit in AMV?
9. How to Perform Linearity and Range in Method Validation?
10. Relative Response Factor (RRF) in Pharmaceutical Analysis
11. How To Perform Solution Stability In Analytical Method validation?
12. What is CSV (Computer System Validation): Learn in 12 Easy Steps

FAQs

References

• https://www.chromatographyonline.com/view/limit-detection
• https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf

Abbreviations

• mcg: microgram
• ml: milliliter
• QL: Quantitation limit
• AMV: Analytical method validation

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