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Analytics · Method validation · continued

Spike-and-recover accuracy for a peptide assay posts 91–120

This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.

MA
m.almeidaTL210 Apr 2026#91

Two things can be true about spike-and-recover accuracy at once: the mechanism is plausible and the evidence for the size of the effect is thin. Most of the argument here is people defending the first against attacks on the second.

1 like 4mo
AK
a.kowalczykTL2Regular10 Apr 2026#92

I keep a log for spike-and-recover accuracy specifically because my memory of it turned out to be systematically wrong in one direction. Six weeks of notes cost nothing and settled it.

0 likes 4mo
CH
c.haddadTL210 Apr 2026#93
m.haddad, post #85: The most useful single question about a method: what would it fail to detect? Every method has an answer and few documents state it. I have said this before in a thread nobody could find, so it is worth repeating. Go to post

Answering the question post #89 raises rather than the one it answers.

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.

On reflection I would soften that slightly.

25 likes in reply to #85 4mo
PN
plateau_notesTL210 Apr 2026#94
AK
a.krastevTL210 Apr 2026#95

That is consistent with mine, for whatever one more account is worth.

0 likes 4mo
DB
d.bramleyTL3Regular10 Apr 2026#96

The confident answers on spike-and-recover accuracy and the well-sourced answers are not the same answers, which is the most useful thing I have learned reading this category.

0 likes 4mo
AN
a.nascimentoTL210 Apr 2026#97

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

The interesting part of this is the exception, and I do not understand the exception.

18 likes 4mo
KB
k.brandl_deTL3Translator · DE10 Apr 2026#98
d.ndiaye, post #58: Adding the measurement that post #57 says would settle it. 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. Go to post

Adding the measurement that post #97 says would settle it.

Small correction to my own earlier position on spike-and-recover accuracy. I had the units the wrong way round, which changes the conclusion by an order of magnitude and therefore changes it entirely.

7 likes in reply to #58 4mo
RP
r.petrovTL210 Apr 2026 · edited#99

Speaking only to spike-and-recover accuracy as I have actually seen it, rather than as it is usually described: the effect is real, it is smaller than the thread suggests, and the variance between people is larger than the effect.

7 likes 4mo
PE
ppm_errorTL3Analytical chemist10 Apr 2026#100

Confirming post #97 from a second method, which matters more than confirming it from a second person.

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.

1 like 4mo
LF
l.ferreiraTL210 Apr 2026#101

Adding the boring version of spike-and-recover accuracy, because the interesting version keeps getting posted and the boring one is usually right.

Check the ordinary explanations, in order, and stop when one of them accounts for what you are seeing. Most of the time the second one does.

0 likes 4mo
HK
h.koodziejTL2Member10 Apr 2026#102

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

Take the reasoning and check the arithmetic; I do not always get it right.

30 likes 4mo
AA
an.adeyemiTL210 Apr 2026#103

Worth separating two things that post #101 runs together.

The question underneath spike-and-recover accuracy is usually "how would I tell?" rather than "what is true?", and that one has a method attached to it.

Write down what you would expect to see under each hypothesis before you collect anything. If they predict the same observation, collecting it will not help.

15 likes 4mo
ES
e.silvaTL210 Apr 2026#104
a.iyer, post #36: Sensible. I would want the same detail before I acted on it either. Go to post

Following, with nothing to contribute beyond having asked the same thing elsewhere.

5 likes in reply to #36 4mo
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c.rasmussenTL210 Apr 2026 · edited#105

My experience of spike-and-recover accuracy contradicts the reply above. I am posting it as a data point rather than as a refutation, because one person's experience is exactly that.

0 likes 4mo
EL
e.lokkenTL210 Apr 2026#106

Narrowing post #105, because the general version has more than one answer.

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.

22 likes 4mo
JC
j.castellanosTL210 Apr 2026#107

Spike-and-recover accuracy is well covered in the tag pages, and the older discussions are better than the recent ones because they were argued out properly. Worth twenty minutes before adding to this one.

10 likes 4mo
JS
j.sorensenTL210 Apr 2026#108
c.inglethorpe, post #26: I read post #24 twice before replying, because I had assumed the opposite. Where a pharmacopoeial monograph exists, a method that follows it inherits a great deal of assurance. Almost nothing discussed here has one. The right answer here may simply be that it has not been measured. Go to post

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 have changed my mind on this once already, so take it as current rather than settled.

3 likes in reply to #26 4mo
SL
s.lundgrenTL210 Apr 2026#109

I had written a reply contradicting post #105 and deleted it. Here is what survived.

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).

2 likes 4mo
AS
a.stephanopoulosTL3Regular10 Apr 2026#110

The reason spike-and-recover accuracy 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.

0 likes 4mo
JL
j.lokkenTL210 Apr 2026 · edited#111

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

1 like 4mo
SS
s.stavrianosTL2Member10 Apr 2026#112
apostille_trace, post #78: Post #75 answers the question as asked. The question underneath it is different. Practical answer on spike-and-recover accuracy, since the theoretical one is upthread: do the simplest check first, write down the result, and only then decide whether the complicated explanation is needed. It usually is not. Go to post

Small methodological point on spike-and-recover accuracy: repeating a measurement is cheap and resolves most of what is being argued about here at no cost to anyone.

6 likes in reply to #78 4mo
LK
l.krastevTL210 Apr 2026#113

Picking up post #112: that is the part I would want checked first.

Where I would push back on the spike-and-recover accuracy consensus is the confidence, not the direction. The direction looks right. The confidence is borrowed.

16 likes 4mo
D
DKwiatkowskiTL3Regular10 Apr 2026#114

On post #110 — agreed on the reasoning, with one qualification.

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

31 likes 4mo
AV
a.vestergaardTL210 Apr 2026#115
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glossary_deskTL3Regular11 Apr 2026#116
bias_variance, post #31: Coming back to post #27, because the follow-up matters more than the original answer. 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. Happy to expand any of that if it is the useful part. Go to post

No notes. Posting so the count is not one.

3 likes in reply to #31 4mo
VS
v.stanescuTL211 Apr 2026#117

This follows post #114 rather than contradicting it.

Spike-and-recover accuracy is worth one more sentence than it usually gets, and the sentence is the one about how the number was arrived at.

11 likes 4mo
NM
n.marsdenTL1Member11 Apr 2026#118

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 the observation and leaving the explanation open deliberately.

23 likes 4mo
JS
j.solbergTL211 Apr 2026#119

Building on post #117 rather than restating it.

An update on my earlier spike-and-recover accuracy post: the pattern held for another six weeks and then stopped, which I did not predict and cannot explain.

6 likes 4mo
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LundqvistTL2Member11 Apr 2026#120

Post #118 put the caveat in the right place and I want to underline it.

Distinguishing three things in the spike-and-recover accuracy discussion that keep getting used interchangeably: the observation, the proposed mechanism, and the recommendation that gets attached to both.

15 likes 4mo