Limit of Detection and Limit of Quantitation

There is a difference between an impurity being absent and an impurity being invisible, and a purity figure does not distinguish them. Two thresholds govern which is which.

The two thresholds

Limit of detection is the smallest amount that can be reliably distinguished from baseline noise. Conventionally it sits around a signal-to-noise ratio of three: enough to say something is there, not enough to say how much.

Limit of quantitation is the smallest amount that can be measured with acceptable precision, conventionally around a signal-to-noise ratio of ten. Between the two thresholds an impurity is real but unmeasurable — visible on the trace, not reportable as a number.

The reporting threshold, which is a decision rather than a measurement

Separately from what the instrument can see, a method defines a reporting threshold: the level below which peaks are disregarded entirely. Anything under it is discarded before the area percentages are calculated.

This is where a purity figure quietly acquires its shape. If the threshold is 0.1%, then twenty impurities each present at 0.05% contribute a combined 1% of the material and none of them appear. The figure is not wrong — it is the correct output of a stated method — but it describes a sample from which a percent of real content has been excluded by rule.

Two laboratories using different thresholds will report different purities for identical material, which is one more mechanism behind disagreeing certificates. Why certificates of analysis disagree covers the others.

What sets the limits in practice

• Detector response. An impurity with a weaker chromophore than the target is harder to detect at the same concentration. At 280 nm, an impurity lacking aromatic residues may be effectively invisible while the target is plainly visible. At 214 nm the gap narrows because the peptide bond dominates.
• Baseline noise. A drifting or noisy baseline raises the detection limit directly. Gradient elution produces baseline rise as the organic content changes, which is why the end of a chromatogram is a less sensitive region than the middle.
• Injection load. Loading more sample lifts small peaks above the threshold, but overloading distorts the main peak and degrades resolution, which reintroduces co-elution. There is an optimum and it is compound-specific.

What “not detected” means on a report

It means not detected by that method, at that wavelength, at that load, above that threshold. It does not mean absent. A report stating “not detected” without stating the limit has made an unfalsifiable claim, and the limit is the part that carries the information.

This applies with particular force to species the method was never going to see. A diastereomer does not appear at any detection limit on a standard reversed-phase method, because it is not resolved rather than not present. Racemisation and chiral purity covers that blind spot.

What to ask for

The reporting threshold and the detection wavelength, both of which are one line on a method summary and neither of which usually appears on a customer-facing certificate. Together with the chromatogram they turn a percentage into something that can be checked. What a third-party peptide test actually measures covers the rest of the document.

All material is supplied for laboratory research use only. It is not a drug, not a supplement, and not for use in humans or animals.