Range: the concentration range over which the method has been validated. Going outside the validated range is going outside the method's demonstrated performance.
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Analytics · Method validation · continued
Repeatability versus intermediate precision posts 31–60
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1 · go to the accepted answer.
Collapsed as off-topic by two members at trust level 3 or above
Worth separating two things that post #28 runs together.
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.
For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use.
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id.almeida, post #21: 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. Go to post
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.
On post #32 — agreed on the reasoning, with one qualification.
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.
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e.mbeki, post #19: For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use. Go to post
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).
bias_variance, post #28: For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use. Go to post
Two things before anyone answers the substance.
First, the context in the first post is clear and specific. Second, the question is framed so that an answer can actually address it. Both are the norm here and both matter more than they sound.
post #38 is right about the mechanism and I think understates the practical bit.
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.
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Practical note that does not fit anywhere else. Whatever you conclude from this topic, write down what you did and when. The single most useful thing in your own records is not any individual result; it is that they are dated and consecutive.
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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.
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d.oyelaran, post #24: 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. Go to post
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.
This follows post #41 rather than contradicting it.
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.
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Collapsed as off-topic by two members at trust level 3 or above
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.
l.sarkissian, post #1: Repeatability versus intermediate precision — setting out what I have, and where I think it stops being reliable. Posting the method first, because I know what the first three replies will otherwise be. Column: C8, 4.6 x 150 mm, 3.5 um Mobile phase: 0.1% TFA in water / 0.1% TFA in acetonitrile Gradient: 5% to 62% organic over 26 minutes… Go to post
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).
Coming back to post #45, because the follow-up matters more than the original answer.
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.
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Picking up post #45: 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.
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Worth separating two things that post #45 runs together.
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.
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post #49 is right about the mechanism and I think understates the practical bit.
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.
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z.cardoso, post #42: 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. Go to post
post #52 is right about the mechanism and I think understates the practical bit.
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.
Worth separating two things that post #50 runs together.
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.
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id.almeida, post #21: 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. Go to post
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.
On post #54 — agreed on the reasoning, with one qualification.
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.
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This follows post #56 rather than contradicting it.
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).
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I read post #58 twice before replying, because I had assumed the opposite.
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.
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