Dr Pramod Kumar Pandey, PhD in Chemistry, is a Analytical expert with 31+ years of experience in pharmaceutical development and the founder of PharmaGuru.co, a global platform for pharmaceutical training and industry insights
Learn how to perform accuracy in analytical method validation with procedure, easy calculation and case Studies & FAQs
How To Perform Accuracy In Analytical Method Validation: Easy Calculation and Case Studies
Accuracy in Analytical Method Validation is one of the most critical parameters, directly impacting the reliability and credibility of test results. It ensures that the method measures exactly what it is intended to, without bias or error. In this article, we will explore how to perform accuracy testing in analytical method validation, supported by step-by-step calculations and real-world case studies. Whether you’re a beginner or an experienced analyst, this guide will help you enhance your understanding and confidently apply accuracy testing in both method development and validation processes.
How To Perform Accuracy In Analytical Method Validation?
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 :
How To Perform Accuracy In Analytical Method Validation: Easy Calculation and Case Studies 3
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 calculation formula
Case studies: Accuracy In Analytical Method Validation
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. The 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.
Concentration (mcg/ml)
Area response
5
154570
7.5
231850
10
309160
12.5
386423
15
463710
R2
1
Slope (m)
30914.12
Intercept (c)
1.4
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
Concentration level
True concentration
Calculated concertation
Accuracy
50%
5.2
5.0
96.2%
50%
5.2
5.0
96.2
50%
5.2
4.9
94.23
Average
–
–
95.54
Average deviation
–
–
102.04
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 a key parameter in analytical method validation, requiring both technical knowledge and practical experience to perform effectively. We hope this article has clarified your doubts and given you the confidence to independently carry out accuracy testing during method development and validation.
If you have any suggestions or feedback regarding this article, feel free to share them in the comments section. For further assistance, you may also reach out via the contact form.
You may also want to check out other articles on my blog, such as:
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.
