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Analytics · Method validation · continued

Coming back to: When to suspect the method rather than the sample posts 91–120

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.

H
HHidalgoTL2Member18 Dec 2024#91

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 it because I spent an afternoon working it out and nobody should have to twice.

31 likes 19mo
SD
st.dialloTL218 Dec 2024#92

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

Someone should write this up properly, and it should probably not be me.

0 likes 19mo
GV
g.valckenaereTL3Regular18 Dec 2024 · edited#93

Post #90 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.

6 likes 19mo
AL
a.lindqvistTL218 Dec 2024#94
e.kimani, post #34: 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. Caveat: everything above assumes the paperwork is… Go to post

Coming back to post #92, because the follow-up matters more than the original answer.

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.

16 likes in reply to #34 19mo
JV
j.vandermolenTL3Regular18 Dec 2024#95

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.

I would call that likely rather than established.

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IA
i.amankwahTL219 Dec 2024#96

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.

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TN
t.nardoneTL3Regular19 Dec 2024#97

Post #94 answers the question as asked. The question underneath it is different.

Documented validation is what separates a number from an opinion expressed numerically. That is the whole reason to ask for the procedure identifier rather than the method name.

If anyone can point at the primary source I would be grateful.

3 likes 19mo
CS
c.serranoTL219 Dec 2024#98
j.hartmann, post #45: 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. Scoping that to what I have actually seen rather than what I have read. Go to post

An independent laboratory's method being different from the supplier's is not a discrepancy. It becomes one only when the results differ by more than both methods' demonstrated precision.

None of the above is medical advice and I am not qualified to give any.

11 likes in reply to #45 19mo
B
BBramleyTL3Regular19 Dec 2024#99

Building on post #98 rather than restating it.

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.

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TT
t.tullochTL219 Dec 2024#100

Post #96 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.

3 likes 19mo
FD
f.danquahTL220 Dec 2024#101

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.

Noting that the question and the thing people usually mean by it are different.

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CC
crossref_checkTL3Wiki editor20 Dec 2024#102

This is the answer, and the reason it is the answer is the more useful part.

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PK
p.krastevTL220 Dec 2024#103
V
VPoulsenTL3Regular20 Dec 2024 · edited#104

Everything in post #101 holds. The case it does not cover is the one I have.

Range: the concentration range over which the method has been validated. Going outside the validated range is going outside the method's demonstrated performance.

A guess, clearly labelled as one.

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VB
v.bergstromTL220 Dec 2024#105

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.

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RJ
r.jhannsdttirTL3Regular20 Dec 2024#106

Post #104 and I disagree about the size of the effect, not about the direction.

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.

0 likes 19mo
AV
a.vermeulenTL221 Dec 2024#107
m.stephanopoulos, post #85: 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. I am describing what is, rather than arguing for what should be. Go to post

Post #104 answers the question as asked. The question underneath it is different.

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.

The claim is narrower than it sounds, and deliberately so.

3 likes in reply to #85 19mo
TK
t.kulkarniTL3Regular21 Dec 2024#108
figure_review, post #3: Noted, and thank you for writing it out rather than summarising it. Go to post

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.

That holds for the case as described. Change the assumptions and it may not.

10 likes in reply to #3 19mo
VK
v.kirchnerTL221 Dec 2024#109

Right, and stated more narrowly than I would have dared to state it.

1 like 19mo
AF
a.finnegan_rdTL2Dietitian21 Dec 2024#110

Worth separating two things that post #106 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).

I am not the right person to answer the follow-up to this.

5 likes 19mo
NT
n.torrenceTL3Regular21 Dec 2024#111
a.lindqvist, post #94: Coming back to post #92, because the follow-up matters more than the original answer. 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. Go to post

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.

29 likes in reply to #94 19mo
EK
e.krastevTL222 Dec 2024#112
vial_slope, post #70: Documented validation is what separates a number from an opinion expressed numerically. That is the whole reason to ask for the procedure identifier rather than the method name. Go to post

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.

Two sources, same conclusion, and I could not rule out that one copied the other.

14 likes in reply to #70 19mo
SP
s.poulsenTL3Regular22 Dec 2024 · edited#113

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.

The confident version of this sentence would be wrong, so here is the hedged one.

5 likes 19mo
AP
a.petrovTL222 Dec 2024#114

This follows post #113 rather than contradicting it.

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.

I keep a log of this specifically because memory is unreliable about it.

0 likes 19mo
K
KnowltonTL3Regular22 Dec 2024#115
m.stephanopoulos, post #85: 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. I am describing what is, rather than arguing for what should be. Go to post

I read post #113 twice before replying, because I had assumed the opposite.

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.

22 likes in reply to #85 19mo
SA
s.achebeTL222 Dec 2024#116

The distinction between a qualified instrument and a validated method is worth keeping. Both are needed and they fail in different ways.

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V
VThorvaldsenTL3Regular22 Dec 2024 · edited#117

The most useful single question about a method: what would it fail to detect? Every method has an answer and few documents state it.

Worth checking against a second source before it gets quoted onward.

2 likes 19mo
IR
i.rasmussenTL223 Dec 2024#118

Marking my place. If it changes for me I will come back and say so.

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KF
k.farrugiaTL3Regular23 Dec 2024#119
j.vandermolen, post #76: Worth separating two things that post #74 runs together. 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 the practical version. The rigorous version is longer and says the same thing. Go to post

Right — I had this wrong and I am glad to have read it before it mattered.

0 likes in reply to #76 19mo
CB
c.balogunTL223 Dec 2024#120

Narrowing post #117, because the general version has more than one answer.

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.

28 likes 19mo