Coming back to: When to suspect the method rather than the sample posts 121–136

Sample stability during analysis is part of validation and is routinely ignored. A preparation that degrades in the autosampler over eight hours produces a sequence-dependent result.

Happy to be the one who is wrong here if it settles the question.

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.

That is what I would do. It may not be what is correct.

Adding the measurement that post #122 says would settle it.

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 limit of quantitation determines what the impurity table can honestly contain. Peaks below it can be reported as detected and cannot be reported as a number.

Reporting a result to more decimal places than the method's precision supports is a small dishonesty that appears everywhere. A method with a two per cent relative standard deviation does not support a figure quoted to a hundredth.

It is the sort of thing that seems obvious in retrospect and was not at the time.

I had written a reply contradicting post #124 and deleted it. Here is what survived.

Range and working range are different things and a certificate rarely distinguishes them. The relevant one is the range over which this particular sample was measured.

That has been true for the cases I have seen and I have not seen many.

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.

Where a pharmacopoeial monograph exists, a method that follows it inherits a great deal of assurance. Almost nothing discussed here has one.

The evidence for this is thinner than the way I have phrased it suggests.

Taking post #126 at face value and following it one step further.

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.

A modest claim, modestly supported.

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.

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.

Noting that I have skin in this question and have tried to discount for it.

That is a fair summary of where the discussion has got to.

On post #131 — agreed on the reasoning, with one qualification.

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.

Picking up post #131: that is the part I would want checked first.

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.