In a series of posts, we are going to talk about method validation.

1. Part 1: Introduction-Is it valid, invalid or non-validated?
2. Part 2: What is method validation?
3. Part 3: Can we use someone else’s validated method?
4. Part 4: What triggers verification, re-validation or out right new validation of a method?
5. Part 5: What are the essential terms in method validation?
6. Part 6: What is quality assurance and quality control?

According to Dr. Ludwig Huber in his book Validation and Qualification in Analytical Laboratories

Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results; it is an integral part of any good analytical practice.

The United States Pharmacopeia (USP) is a non–governmental, official public standards–setting authority for prescription and over–the–counter medicines and other healthcare products manufactured or sold in the United States. The USP also sets widely recognized standards for food ingredients and dietary supplements. The USP sets standards for the quality, purity, strength, and consistency of these products–critical to the public health. The USP’s standards are recognized and used in more than 130 countries around the globe. These standards have helped to ensure public health throughout the world for close to 200 years.

The USP has published specific guidelines for method validation for compound evaluation. USP defines eight steps for validation:

1. Accuracy (Bias)
2. Precision
3. Specificity
4. Limit of detection
5. Limit of quantitation
6. Linearity and range
7. Ruggedness
8. Robustness

Put basically and simply, a validated method means that there has been some sort of rigorous method of testing of the instructions of the assay and its calibration procedure to produce data that shows and proves that the method is suitable for its intended purpose.

It might be useful to use a concrete example so we can translates the words and the concepts into the demonstrable concept. In Headspace analysis by Gas Chromatography with Flame Ionization Detector (HS-GC-FID) for EtOH, the intended purpose is to:

1. correctly and uniquely identify the target analyte—ethanol—to the exclusion of everything else (that may likely be in the matrix—the blood—or in the environment of the collection, storage, transportation, sample preparation, or analysis—contamination) and therefore be specific in its qualitative measure.
2. correctly and uniquely identify the quantity of the target analyte—ethanol—to the exclusion of all other possible interfering compounds, and therefore have a specific measure in terms of the quantitative measurement.

You may be thinking to yourself, ” Great. But as practicing attorneys, why should we care?“

Without a valid method, we can never achieve a validated result. If there is deviation, this will result in a non-validated result.

A good analogy is that of a foundation.

Of course, how you lay the foundation is going to affect the quality and the durability of the building it rests upon. If the foundation is poor, it doesn’t matter how perfect the framing, how perfect the roof, how perfect the blue board or drywall, and how perfect the interior supports are in the subsequent building the house, the building is in jeopardy and will eventually collapse.

Just as in laying the correct concrete foundation, there are certain correct ways of doing it such as:

1. Determine how thick the slab will be. This will be determined by the use and size.
2. Excavate or dig down twice the thickness of the slab. If the concrete foundation is going to be 3 inches, then you need to go down 6 inches.
3. Remove any large stones or garbage from the area you excavated, then rent a compactor and compact the ground.
4. Put down sand or gravel or something for drainage so water will not come up through the slab.
5. Lay down a vapor barrier, especially if the concrete foundation is going to be used for interior use such as a house.
6. Build a wood frame to lay a concrete foundation. Make screed rails from 2-by-4 inch boards and put them on opposite sides around the outside of the foundation. Span the screed rails with a straightedge. This slides across the rails, removing excess concrete and helping to make the concrete level.
7. Pour the concrete for the foundation and allow the straightedge to slide, leveling the concrete as it goes. Once the concrete is firm enough, remove the straightedge and rails.
8. Finish the concrete foundation you are laying with hand or power trowels. Remove any imperfections the straightedge has left, then let the concrete foundation sit and cure.

so there are the correct ways of performing method validation. However, there is no “one way” to design and perform validation experiments. As Dr. Huber wrote in his book, based on his experience, for example in trying to develop a liquid chromatographic method, the following sequence has proven to be useful in terms what experiments to run and in what order:

1. Selectivity of standards (optimizing separation and detection of standard mixtures if selectivity is insufficient)
2. Linearity, limit of quantitation, limit of detection, and range
3. Repeatability (short-term precision) of retention times and peak areas
4. Intermediate precision
5. Selectivity with real samples
6. Trueness/accuracy at different concentrations
7. Ruggedness (interlaboratory studies)

According to Dr. Huber:

Once the method has been developed and validated, a validation report should be prepared that includes the following:

• Objective and scope of the method (applicability, type).
• Summary of methodology.
• Type of compounds and matrix.
• All chemicals, reagents, reference standards, QC samples with purity, grade, their source or detailed instructions on their preparation.
• Procedures for quality checks of standards and chemicals used.
• Safety precautions.
• A plan and procedure for method implementation from the method development lab to routine analysis.
• Method parameters.
• Critical parameters taken from robustness testing.
• Listing of equipment and its functional and performance requirements, e.g., cell dimensions, baseline noise and column temperature range. For complex equipment, a picture or schematic diagram may be useful.
• Detailed conditions on how the experiments were conducted, including sample preparation. The report must be detailed enough to ensure that it can be reproduced by a competent technician with comparable equipment.
• Statistical procedures and representative calculations.
• Procedures for QC in routine analyses, e.g., system suitability tests.
• Representative plots, e.g., chromatograms, spectra and calibration curves.
• Method acceptance limit performance data.
• The expected uncertainty of measurement results.
• Criteria for revalidation.
• The person(s) who developed and validated the method.
• References (if any).
• Summary and conclusions.
• Approval with names, titles, date and signature of those responsible for the review and approval of the analytical test procedure.