Analytical methods
Assay validation: proving a method is fit for its intended purpose
Assay validation is an intended-use argument supported by experiments. The required characteristics depend on whether the method identifies, quantifies, compares, or monitors a measurand and on the matrix and decision it supports.
Educational content only. Not medical advice.
Validation begins with intended purpose
A method for identity, impurity detection, quantitative assay, potency, or exploratory screening faces different requirements. The target measurand, matrix, reportable range, acceptance criteria, and decision must be defined before testing performance. A method can be valid for one intended use and inadequate for another without contradiction.
Accuracy and precision address different errors
Accuracy concerns agreement with an accepted reference value, while precision concerns variability among repeated measurements. Repeatability examines closely matched conditions; intermediate precision introduces changes such as day, analyst, instrument, or run. A precise method can be consistently biased, and an accurate average can hide poor repeatability.
Specificity, sensitivity, and range need operational definitions
Specificity or selectivity tests whether the method distinguishes the intended signal from impurities, related species, matrix, and interference. Detection and quantitation limits address low-level performance under stated error criteria. Range identifies where accuracy, precision, and response are acceptable; it should not be inferred from a fitted line that lacks validation samples near its boundaries.
Robustness and system suitability protect routine use
Robustness evaluates deliberate small method changes, while system-suitability checks confirm that the instrument and procedure are functioning before or during a run. Blanks, negative and positive controls, reference materials, matrix spikes, replicates, and predefined acceptance criteria create an auditable quality system. Validation is not permanent if the method, matrix, instrument, or purpose materially changes.
Evidence limits
- Validation characteristics and acceptance criteria depend on the intended analytical purpose.
- A validated method can still fail through poor sample handling, calibration, or execution.
- Performance in one matrix, instrument, or laboratory may not transfer without verification.
Sources and further reading
These sources ground the definitions and evidence boundaries on this page. A citation is a route for verification, not an endorsement of a product or personal use.
U.S. Food and Drug Administration / ICH
Q2(R2) Validation of Analytical Procedures
Official framework for demonstrating that an analytical procedure is fit for its intended purpose.
Open sourceAssay Guidance Manual, NCBI Bookshelf
Glossary of Quantitative Biology Terms
Institutional definitions for EC50, IC50, assay selectivity, accuracy, controls, and quantitative biology terms.
Open sourceNational Institute of Standards and Technology
Reference Materials
Official distinctions among certified Standard Reference Materials, reference materials, and research-grade test materials.
Open sourceCommon questions
Is sensitivity the same as specificity?
No. Sensitivity concerns detecting change or low levels; specificity concerns distinguishing the intended signal from alternatives and interference.
What is the difference between detection and quantitation limits?
Detection indicates reliable evidence of presence, while quantitation requires acceptable numerical performance under stated criteria.
Does a high R-squared validate an assay?
No. Linearity alone does not establish accuracy, precision, specificity, range, robustness, or suitability.
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