Analytical method validation

Analytical method validation is the documented demonstration that an analytical procedure is suitable for its intended purpose. It is not a demonstration that a method is correct in the abstract: a method validated for assay of a drug substance is not thereby validated for detection of a trace impurity, and the purpose is part of the claim.^[1]

The characteristics assessed are specificity, linearity, range, accuracy, precision (repeatability and intermediate precision), the detection and quantitation limits where relevant, and robustness. Which of these apply depends on what the method is for, and the compendial treatment of the same subject is framed the same way.^[1][2]

For peptides the critical characteristic is specificity — the ability to determine the analyte in the presence of everything else that may be there. Peptide impurities are structurally close to the peptide, so a method that separates the analyte from unrelated substances may still fail to separate it from a deletion sequence differing by one residue.^[3]

Robustness is the characteristic most relevant to reading a third-party result. A method sensitive to a two-degree change in column temperature or a small change in mobile-phase pH will give different answers in different laboratories, and a difference between two results may reflect that rather than a difference in material.^[1]

Specificity is demonstrated by showing that the analyte peak is resolved from known related substances, from process impurities, and from degradation products generated by forced degradation.^[3]

Forced degradation is the part most often omitted outside licensed manufacture. Deliberately stressing the material with heat, light, acid, base and oxidant generates the degradation products the method must be able to see, and a method that cannot resolve them is not stability-indicating. A certificate describing its method as stability-indicating is making a claim that forced degradation supports; one that does not make the claim leaves the question open.^[4]

The peptide-specific difficulty is that the closest impurities are the most likely. A deletion sequence missing one residue, a peptide with one residue oxidised, a deamidated variant — all are structurally similar to the parent and may elute within a fraction of a minute of it. Resolving them requires a gradient with adequate duration and shallowness, which is precisely what a short run sacrifices.

Validation is about the method, not about the material or the sample. A fully validated method applied to a non-representative sample produces a precise, accurate and irrelevant number. See Blind sampling.^[1]

Nor does it transfer between laboratories automatically. A method validated in one laboratory and run in another requires verification that it performs equivalently there — the same characteristics, demonstrated locally. Two laboratories running "the same" method may differ in column lot, instrument dwell volume and mobile-phase preparation.

For certificates in research-chemical supply, validation status is usually simply unstated. That is a gap in the document rather than a defect in the material, and the correct inference is that the figure is uncharacterised rather than that it is wrong.^[3]

• Limit of detection
• Reference standard
• High-performance liquid chromatography
• Area percent purity
• Deamidation
• Blind sampling