Assay Method Validation

[Liza]

Hello all:


Could you help a beginner with assay method validation -


(1) What are the general validation parameters? (I know of System Precision, Method Precision, Standard Stability, Sample Stability, Linearity, LOQ, Filter Test. Any else I'm missing? Any guidance document/article I can refer to?)

(2) How does one go about validating each parameter? (For System Precision, simply make multiple injections & check the RSD. For Standard & Sample Stability, simply inject after one or two days & calculate average % recovery. For Linearity, perform serial dilutions and plot concentration vs. peak area graph. For LOQ, make multiple injections of proposed lowest concentration solution prepared from the linearity experiment & check the %RSD. For Filter Test, compare average % recovery of filtered and unfiltered std solutions. How do I go about validating Method Precision? What about Accuracy? Can someone explain spike std in detail?)


Thanks a million.


Liz

[Sunjay]

Dear Liz,

There are number of articles available on the method validation. U can read ICH guideline for method validation Q2A and Q2B. In addition there is CDER guideline for Chromatograohic Method Validation.

Method Precision is determination u will get form ICH guideline. Six preparations and checking the RSD. For accuracy for drug product, u have to take placebo and API and analyse it at three levels and three differnet preparation at each level ie total nine preparation at 80%, 100% and 120%.

Hope this will work.

[Prometheus]

Dear Liz,

Here are the list of documents and articles that could help you.

- FDA (2001): Guidance for Industry: Bioanalytical Method Validation. Center for Drug Evaluation and Research, U.S. Food and Drug Administration
- FDA (1994): Reviewer Guidance: Validation of Chromatographic Methods. Center for Drug Evaluation and Research, U.S. Food and Drug Administration
- ICH Q2A (1995): Text on Validation of Analytical Procedures. International Conference on Harmonization of Technical Requirements for the Registration of Drugs for Human Use
-ICH Q2B (1997): Validation of Analytical Procedure Methodology. Harmonization of Technical Requirements for the Registration of Drugs for Human Use
-Shah, V.P., K.K. Midha, et al. (1992): Analytical Method Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies. Journal of Pharmaceutical Sciences, Vol. 81, No. 3
-Hartmann, C., D.L. Massart, and R.D. McDowall (1994): An analysis of the Washington Conference Report on bioanalytical method validation. Journal of Pharmaceutical & Biomedical Analysis, Vol. 12, No. 11
-Shah, V.P., K.K. Midha, et al. (2000): Bioanalytical Method Validation – A Revisit with a Decade of Progress. Pharmaceutical Research, Vol. 17, No.
-Swartz, M. and I. Krull (2003): Validation of Bioanalytical Methods – Highlights of FDA’s Guidance. LCGC North America, Vol. 21, No.2
-Dadgar, D., P. E. Burnett, et al. (1995): Application issues in bioanalytical method validation, sample analysis and data reporting. Journal of Pharmaceutical & Biomedical Analysis, Vol. 13, No. 2
-Hartmann C., J.S. Verbeke, et al. (1998): Validation of bioanalytical chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis, Vol. 17.
-Dadgar, D. and P.E. Burnett (1995): Issues in evaluation of bioanalytical method selectivity and drug stability. Journal of Pharmaceutical and Biomedical Analysis, Vol. 14.
-Peters, F.T. and H.H. Maurer (2001): Review: Bioanalytical method validation – How, how much and why? Toxichem + Krimtech, Vol. 68, No. 3

[Albany-12303]

Wow that's a tall order.

Generally, as Sunjay posted, it's a good idea to start with the ICH guidelines. By 'Assay' do you mean analysing the active, or Impurities (its easier to validate active methods).

Do you come under the auspices of the FDA? If so, you will likely need an internal SOP for validation.

All the details of validation are too long and complicated to be summed up on a discussion board (and my boss would not be too happy if I were to post our sop )

If you are analysing a drug product then you are missing accuracy (spiking placebo with a known amount of impurities and/or active and measuring the amount with the method). Also there is specificity, which includes stressing your active/product and making sure that all of the degs separate.

Additionally, there is 'robustness' which is done by changing the HPLC parameters slightly (for example, 2% more organic) to measure whether the method still works OK with these small variations.

Also, if the method is to be used elsewhere, you will need to validate for 'method transfer'.

Good luck!

[Mary Carson]

Some of the details of method validation can vary depending on your field of analysis. The references listed above are really good if you are in human drugs--either dosage form quality control or clinical PK assays. However, there are other protocols for validation out there. FDA/EPA pesticide methods are validated slightly differently, and many other food-type methods are also validated differently. U.S. and EU aren't quite harmonized in those areas yet, though it is getting better. Talk to some people in your specific field and find out what they are doing.

AOAC International is running a course on single-laboratory validation of methods this September if you happen to be anywhere close to Minneapolis. It says it is for Dietary Supplements, but the application is much broader than that.

Other organizations also routinely offer training in method validation.

[Liza]

Thanks very much.