How to Perform Accuracy in Analytical Method Validation

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Introduction and outcome

Accuracy is one of the important parameters of the analytical method. In this article, I will discuss procedure to perform Accuracy test with case study.

Procedure to perform accuracy

• Accuracy is determined by deviation from the linearity
• It is calculated at 50%, 100% and 150% of the specification limit
• Prepare at least five concentration between QL or 50% to 150% of the specification limit of the analyte
• Inject 50%, 100% and 150% concentrations in triplicate and inject other concentration in singlets and generate the chromatograms
• Note down the area response of each concentration
• Plot the linearity graph between concentrations (at X-axis) and their corresponding area response (at Y axis)
• Calculate slope (m), intercept (c) and correlation coefficient (R²) using MS-Excel.
• Calculate concentration for each area response using the following formula :

y= mx + c and hence x = (y-c)/m

• where: m is slope, x is the concentration, c is the intercept and y is the area response
• Calculate the accuracy at each concentration using the following formulae
  • Accuracy = (Calculated concentration/True concentration) x 100

Case studies:

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

• Isomer I NMT: 1.0%

Sample concentration is 1.0mg/ml.

Standard preparation for Accuracy calculation

Since sample concentration is mg/ml or 1000 mcg/ml. There concentration of Isomer I at

• 50% will be 1000 x 0.5/100 = 5 mcg/ml
• 75% will be 1000 x 0.75/100 = 7.5 mcg/ml
• 100% will be 1000 x 1/100 = 10 mcg/ml
• 125% will be 1000 x 0.5/100 = 12.5 mcg/ml
• 150% will be 1000 x 1.5/100 = 15 mcg/ml

Use the above concentration to determine m (slope), c (intercept) and accuracy

Procedure for calculating m and c

• Inject 5 mcg/ml, 10 mcg/ml and 15 mcg/ml solutions in triplicate and remaining 7.5mcg/ml and 12.5mcg/ml solutions in singlets. Generate the chromatogram.
• Note down the area response
• Plot the linearity graph between concentrations x (at X-axis) and their corresponding area response y (at Y axis)
• Calculate slope (m), intercept (c) and correlation coefficient (R²) using MS-Excel.

Procedure for calculating x for each y using equation y= mx + c

• Calculate concentration for each y (area response) using the following formula :
  • y= mx + c or
  • x = (y-c)/m, where: m is slope, x is the concentration, c is the intercept and y is the area response

For y = 154570, x will be ( 231850 – 1.4)/30914.12 =5.0 mcg/ml

For y = 154600, x will be ( 154600 – 1.4)/30914.12 =5.0 mcg/ml

For y = 154200, x will be ( 154200 – 1.4)/30914.12 = 4.9 mcg/ml

and

For y = 309160, x will be ( 309160 – 1.4)/30914.12 =10 mcg/ml

For y = 463710, x will be ( 463710 – 1.4)/30914.12 =15.11 mcg/ml and similarly x can be calculated for other y (area response as well

Procedure for calculating accuracy

Calculate the accuracy at each concentration using the following formulae

Accuracy = (Calculated concentration/True concentration) x 100

Conclusion: Average deviation is less than 2.0% and hence method complies the accuracy test

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

Conclusion

Accuracy is one of the important parameter of the Analytical method validation. Therefore, both knowledge and experience are required to perform the same. Now I hope this article has cleared all your doubts and now you can independently preform accuracy 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.

References

• https://www.sciencedirect.com/science/article/abs/pii/S0099542805320090
• https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf

Abbreviations

• mcg: microgram
• ml: milli liter
• QL: Quantitation limit

FAQs

How do you perform accuracy in Analytical method validation?

Accuracy is performed at 50%, 100% and 150% of the specification level. Standard solution is injected in triplicate at each level. Concentration is calculated using equation y = mx +c. Accuracy is calculated using formula: Accuracy = (calculated concentration/true concentration) x 100

In which case accuracy is performed?

Accuracy is performed for Active pharmaceutical ingredients or its stages. It is not applicable for dosages forms.

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