Coming back to the opening post, because the follow-up matters more than the original answer. The most useful single question about a method: what would it fail to detect? Every method has an answer and few documents state it. The answer changed when I changed how I was measuring, which was informative.
Validating a method you did not develop — does this still hold? I have a specific reason for asking rather than idle curiosity, and the context is below.
Validating a method, and specifically the version of it that the documentation does not cover. The maintained page handles the general case well and stops exactly where my question starts.
Setting out the gap in case it is a gap in the page rather than a gap in what is known.
System suitability is the ongoing evidence that a validated method is still performing. Validation is done once; suitability is done every run, and it is the one that appears on a certificate.
The confident version of this sentence would be wrong, so here is the hedged one.
Worth separating two things that the opening post runs together.
Robustness: the method gives consistent results when minor parameters vary. Tested by deliberately varying pH, temperature, flow rate, and mobile phase composition within reasonable ranges and demonstrating that results stay within acceptance.
I would put the burden of proof on the interesting explanation, not the dull one.
Fair, and the limits you put on it are the part I will remember.
Coming back to the opening post, because the follow-up matters more than the original answer.
The most useful single question about a method: what would it fail to detect? Every method has an answer and few documents state it.
The answer changed when I changed how I was measuring, which was informative.
Specificity is the first question: does the method separate the analyte from everything reasonably expected to be present? A method that has not been challenged with its own degradation products has not answered it.
The mechanism is plausible, which is not the same as established.
Picking up post #5: that is the part I would want checked first.
Linearity across the working range is a routine demonstration and it constrains how far a result can be extrapolated. A method linear from 80 to 120 per cent of nominal says nothing about a sample at ten per cent.
Why two laboratories may disagree: after validating the same method, they may still report different purity on the same sample due to integration differences, column age differences, subtle differences in mobile phase pH or temperature. This is normal and not a sign that one is wrong.
Accuracy: the method measures what you intend to measure. For purity methods, this is tested by spike-and-recover experiments: add a known amount of impurity to a sample and measure whether you recover the added amount.
Anyone who has looked at this more carefully, please correct the record.
Post #8 put the caveat in the right place and I want to underline it.
Robustness testing deliberately varies the parameters most likely to drift — organic percentage, pH, temperature, flow — and shows the result does not. It is the part of validation that predicts whether a method will transfer.
I would be interested in a counterexample if anyone has one.
Forced degradation studies: deliberately stress the material with acid, base, oxidant, heat, light to generate degradation products and demonstrate that the method can separate them from the parent peak. Acceptance is that the method is stability-indicating.
Same experience here, different supplier, so it is at least not unique to one of them.
Post #12 is the version of this I will quote in future. One addition.
Limits of detection and quantitation: LOD is the lowest concentration that produces a signal above background. LOQ is the lowest concentration at which the method meets precision and accuracy acceptance criteria. Both are determined empirically.
That holds under the stated conditions and I have stated them.
Where I part company with post #12, and it is a narrow parting.
Validation is compound-specific and matrix-specific. A method validated for one peptide is a starting point for another and not a validated method for it.
Adding a data point of agreement rather than a data point.
A stability-indicating method is one demonstrated to resolve the analyte from its degradation products, usually through forced degradation. Calling a method stability-indicating without that work is a claim rather than a property.
Appreciated. The plain phrasing does more work here than a longer post would.
A method transferred between laboratories needs a demonstration that it performs equivalently, not just a document describing it. Transfer is where a great many between-laboratory disagreements originate.
The literature is thinner on this than the confidence in the thread implies.
Picking up post #21: that is the part I would want checked first.
Specificity: the method can distinguish the intended compound from related impurities and degradation products. Tested by comparing results on pure compounds, mixtures of compounds, and degraded samples.
If this contradicts something upthread, the upthread version may well be the better one.
I had written a reply contradicting post #21 and deleted it. Here is what survived.
The reason Validating a method keeps being re-asked is that the answer is conditional and people quote it without the condition. It is not that the answer is unknown.
Where a pharmacopoeial monograph exists, a method that follows it inherits a great deal of assurance. Almost nothing discussed here has one.
Stability-indicating method: one that can separate a compound from its degradation products. Critical for assay methods that claim to measure actual degradation (as opposed to purity, which is orthogonal).
A method that reports the same number for every lot is worth a second look. Real processes vary, and a total absence of variation is a statement about the measurement rather than the process.
The number is defensible. The precision I gave it is not.
Narrowing post #29, because the general version has more than one answer.
Transfer between laboratories: a method can be transferred from one lab to another, but the receiving lab needs to demonstrate that they can achieve the same performance. This requires comparative testing and sometimes small method refinements.
If that is already documented somewhere, ignore me and link it.