How to Calculate Recovery in Analytical Method Validation

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Recovery; Introduction and outcome

Recovery is the important parameters of any analytical method and it is performed in all type of validations. In this article, I will discuss procedure to calculate recovery with case study.

3-Steps recovery calculation procedure

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

Recovery calculation

% Recovery = (Area response of impurity in spiked solution ÷average standard impurity area response) x 100

Case studies:

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

https://pharmaknowledgeforum.com/how-to-perform-linearity-and-range-in-amv-case-study/ I hope this article has helped you understand Recovery calculation and its importunacy. Now you can independently perform and calculate the recover testing during method development and method validation. You may also want to check out other articles on my blog, such as precision, linearity calculation, DL and QL calculation. For any opinion or suggestions related to this article, you can write in the comment section

FAQS

How to calculate recovery in validation?

Recovery is canulated by the following formulae:

% Recovery = (Area response of impurity in spiked solution ÷average standard impurity area response) x 100

At what range recovery is performed?

In related substances test, recovery is performed between 50 to 150% of the impurity limit. In assay , it is performed between 80n to 120% of the assay limit.

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.