How to perform Specificity in Analytical Method Validation

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Introductions and Outcome

Specificity is one of the important parameters of Analytical method validation. In this article, I will discuss the procedure of performing the specificity such as sample and standard preparation, injection procedure, acceptance criteria, case study and frequently asked questions. After reading this post you will easily replicate the same during method validation or during method development.

Specificity in Analytical method validation

Specificity tells about the the efficiency of the analytical method or in other words capability of the method to separate different components like main analyte peak and impurity peaks.

Standard and sample preparation

1. Sample and standard solution: Prepare sample and standard at nominal concentration as per standard test procedure (STP).
2. Known specified impurity solution: Prepare each known impurity at the specification level
3. Known unspecified impurity solution: Prepare unknown specified impurity at 0.10% level
4. Spiked solution preparation: Prepare a solution containing the main analyte at the nominal concentration and each known specified impurity at specification limit and known unspecified impurity at 0.10%.

Injection procedure

Inject the following solutions in the HPLC system containing a PDA or DAD detector and generate the chromatogram as per chromatographic condition given in the method::

• Inject blank or diluent solution
• Inject each known specified impurity
• Inject each known unspecified impurity
• Inject main analyte sample standard solution
• Inject spiked solution

Specificity acceptance criteria

• There should not be any interference of any known specified impurity with the main analyte
• There should not be any interference of any known unspecified impurity with the main analyte
• There should not be any interference of blank peak with the main analyte
• There should not be any interference between known specified impurities and each peak should be separated
• There should not be any interference between known unspecified impurities and each peak should be separated
• There should not be any interference between known specified impurities and known unspecified impurity each peak should be separated
• Each peak should be homogeneous and pure. For example, peak purity should be less than peak threshold

Case study

Let us consider the following are the related substances specification of an API and we have to perform specificity test:

• Impurity A: NMT 0.50%
• Impurity B: NMT 0,20%
• Any known unspecified impurity NMT: 0.10%
• Any unknown unspecified impurity NMT: 0.10%
• Total impurity NMT: 1.0%

The sample concentration in the method is 1000 mcg/ml

Standard and sample preparation

1. Prepare Impurity A at concentration, 1000 x 0.5/100=5 mcg/ml inject
2. Prepare Impurity B at concentration, 1000 x 0.2/100=2 mcg/ml inject
3. Prepare each known unspecified impurity at concentration, 1000 x 0.1/100=1 mcg/ml inject
4. Spiked solution preparation: Prepare a solution containing the main analyte at the 1000 mcg/ml concentration and Impurity A at a concentration 5 mcg/ml, Impurity B at concentration 2 mcg/ml and each known unspecified impurity at concentration 1 mcg/ml and inject

Acceptance criteria

• Impurity A must be separated with main peak amd Impurity B and any known or unknown unspecified
• Impurity B must be separated with main peak amd Impurity A and any known or unknown unspecified
• There should not be any interference blank peak with main analyte and with impurity A and Impurity B
• Peak must be pure and homogeneous. Peak purity should be less than peak threshold

Specificity test in stability indicating method (SIM) validation

In Stability indicating method validation sample is exposed to the following stress conditions to perform the specificity test:

• Heat
• Sun light or Sun cabinet
• Basic treatment
• Acidic treatment
• Oxidation treatment

Difference between Specificity and Selectivity

Conclusion

Specificity is one of the vital parameters of any analytical; method validation and therefore each impurity (known, unknown and degradant) must be considered while performing the validation.

FAQS

What is the specificity of method validation?

Specificity tells about the efficiency of the method to separate peak of interest from the adducent component peak. Secondly, there should not be any interference of diluent peak with the paek of interest

What are the differences between selectivity and specificity?

Specificity deals with the separation of the peak of interest from the adjacent component peak or impurity peak. Selectivity deals with the separation between each component in the chromatogram.

For example assay method is specific whereas the related substance method is selective

What is the specificity of an assay?

There should not be any interference of the main peak or assay peak with any impurity peak or diluent peak.

What is specificity used for?

The peak of interest must be separated from other component peaks or diluent peak

What is specificity and sensitivity?

Specificity tells about the efficiency of the method to separate peak of interest from the adjacent component. There should not be any interference of diluent peak with the paek of interest. Sensitivity telles about the capacity of detector to quantify lowest amount of analytes. For sensitivity, the ratio of signal to noise (S/N) should be more than or equal to 10

Is specificity same as accuracy?

No. Specificity is related to the separation of the main component from the other component peaks whereas accuracy is related exactness of the method to quantify the analyte

References

• ICH, Q2 (R1)
• www.sciencedirect.com

Abbreviations

• PDA: Photodiode array detector
• DAD: Diode array detector
• API: Active pharmaceutical ingredient
• SIM: Stability indicating method