Second pass at: LOD and LOQ: definitions people get wrong posts 31–52
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
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).
Linearity: the detector response is proportional to compound concentration across the working range. Demonstrated by running standards at multiple concentrations and showing R-squared values typically ≥0.99.
This follows post #31 rather than contradicting it.
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.
On post #31 — agreed on the reasoning, with one qualification.
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.
System suitability: injections run at the start of a batch to establish that the instrument and column are performing. Acceptance criteria typically include replicate precision (RSD ≤2%), peak tailing (0.8–1.5), theoretical plates (>2000), and resolution (>1.5).
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.
I disagree with the reply above, and I think the disagreement is substantive rather than terminological.
The distinction being drawn does not survive when you look at the published data for this specific question. I would be glad to be shown wrong on this, because the version I am arguing against is more convenient.
Range: the concentration range over which the method has been validated. Going outside the validated range is going outside the method's demonstrated performance.
post #39 is right about the mechanism and I think understates the practical bit.
Precision and repeatability: within-run and between-run variability of the method. Acceptance criterion is typically a relative standard deviation of ≤2% for area measurements.
Collapsed as off-topic by two members at trust level 3 or above
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.
This follows post #40 rather than contradicting it.
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 read post #42 twice before replying, because I had assumed the opposite.
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.
post #44 answers the question as asked. The question underneath it is different.
System suitability: injections run at the start of a batch to establish that the instrument and column are performing. Acceptance criteria typically include replicate precision (RSD ≤2%), peak tailing (0.8–1.5), theoretical plates (>2000), and resolution (>1.5).
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.
Coming back to post #46, because the follow-up matters more than the original answer.
Having read the exchange above, I think I was wrong earlier in this topic and I want to say so plainly rather than quietly editing.
The correction was fair and I had been repeating something I had not checked carefully enough.
post #48 is right about the mechanism and I think understates the practical bit.
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.
Worth separating two things that post #46 runs together.
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).
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.
This topic was referenced in
- When to suspect the method rather than the sampleAnalytics › Method validation · 115 replies
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