Detection Limit (DL) and Quantification Limit (QL) In Method Validation

• Facebook
• LinkedIn
• WhatsApp
• Mail

Introduction and outcome

Detection Limit and Quantification Limit are the important parameters of any analytical method. In this article, I will discuss procedure to calculate Detection Limit (DL) and Quantification Limit (QL) with case study

Procedure to Calculate Detection Limit and Quantification Limit Calculation

Detection Limit

Three methods are available to calculate Detection Limit. In which Signal to noise ratio (S/N) method is widely used in the industries. Prepare several analyte solutions at lower concentrations. Inject each solution one by one and check the S/N ration. The lowest concentration which gives S/N ratio about 2:1 to 3:1 will be DL concentration.

Acceptance criteria

• Detectability of the peak in all three replicate injections
• The Signal to Noise ratio (S/N) should be between 2:1 to 3:1

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

Prepare Impurity A at (lower concentration) 0.01% that is 1000 x 0.01/100 = 0.1mcg/ml. Inject this solution in triplicate and note down S/N ration in each injection.

Conclusion

• Peak is detected in all three injections
• S/N ratio is between 3 to 4.
• Therefore 0.01% will be Detection Limit or DL concentration for impurity A

Note: Similarly DL can also be calculated for unknown impurities by preparing main analyte concentration at lower level

Quantitation Limit

Three methods are available to calculate Quantification Limit. In which Signal to noise ratio (S/N) method is widely used in the industries. Prepare several analyte solutions at lower concentrations. Inject each solution one by one and check the S/N ration. The lowest concentration which gives S/N ratio about 10 is the QL concentration.

Acceptance criteria

• The Signal to Noise ratio (S/N) should be about 10
• The RSD of area response of six injections should be
  • NMT 10% if QL is between 0.05% (500ppm) to 0.50%
  • NMT 15% if QL is between 100 ppm to less than 500 ppm
  • NMT 30% if QL is less than 100 ppm

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

QL of Impurity A

1.0 mg/ml sample concentration is equivalent 1000 mcg/ml

Prepare Impurity A at (lower concentration) 0.03% that is 1000 x 0.03/100 = 0.3mcg/ml. Inject this solution in six times. Calculate the RSD of six injections and note down S/N ratio of each injection.

Conclusion:

• Peak is detected in all three injections
• S/N ratio is more than 10
• Therefore 0.03% will be Quantification Limit or Ql concentration for impurity A

Conclusion

DL and QL are the important parameters of the Analytical method validation. Both knowledge and experience are required to perform the same. Now I hope this article, cleared all your doubts and now you can independently perform DL and QL 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.chromatographyonline.com/view/limit-detection
• https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf

Abbreviations

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

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