Analytical Guide

LOD and LOQ: What "Not Detected" Actually Means

Detection limit and quantitation limit describe a method, not a sample. What each one means, how they are established, and why not detected never means absent.

[Research Use Only] This guide is for controlled laboratory research workflows only. It is not for human or veterinary use and does not provide applied-use guidance.

A result about the method, not the material

"Not detected" reads like a finding about a sample. It is closer to a finding about an instrument. Every analytical procedure has a floor below which it cannot distinguish a real signal from background, and that floor moves with the method, the matrix, the instrument and the day. Two laboratories can analyse the same material, both report nothing detected, and mean materially different things — because they were asking with different sensitivity. Understanding where those floors sit is what turns a negative result from a reassurance into a piece of information.

The two limits, defined

ICH Q2(R2) defines both, and the definitions are narrower than common usage. The detection limit is the lowest amount of an analyte in a sample which can be detected but not necessarily quantitated as an exact value. The quantitation limit is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy — a parameter used particularly for the determination of impurities and degradation products.

Detection limit (DL, or LOD): the lowest amount that can be detected
Quantitation limit (QL, or LOQ): the lowest amount that can be measured with suitable precision and accuracy
Detection is a weaker claim than quantitation, and comes first
Both are properties of the analytical procedure as applied, not of the substance

Four states, not two

A concentration is not simply detected or not. There are four distinguishable states, and reports that collapse them into a binary lose most of the information.

Below the detection limitNothing is distinguishable from background. A statement about the sensitivity of the method, not a demonstration of absence.
At the detection limitPresence can be detected, but not quantitated as an exact value.
Between DL and QLThe signal is real and present, but reporting a number for it would claim more precision than the method supports.
At or above the quantitation limitThe amount can be determined with suitable precision and accuracy.

Three of these four states are routinely summarised as "not detected" or "none found". The middle two are where most misreading happens: something was seen, and the honest report is that it cannot be reliably quantified.

How the limits are established

ICH Q2(R2) sets out several approaches for estimating the lower range limits of a procedure. They are alternatives rather than a ranking, and which one was used changes what the number means — which is why the guideline asks that both the limit and the approach be presented.

Approach	How the limit is established	What it depends on
Visual evaluation	Analyse samples of known concentration and establish the minimum level at which the analyte can be reliably resolved and detected or quantitated	The judgement applied, and the quality of the samples used
Signal-to-noise	Compare measured signals from samples of known low concentration against blank samples, or against an appropriate baseline region	The procedure exhibiting measurable baseline noise — not all do
Standard deviation of the response and the slope	Calculate from the variability of the response and the slope of the calibration curve	How the standard deviation is estimated — from blanks, or from the regression line
Accuracy and precision at the lower range limit	Validate the quantitation limit directly by measuring accuracy and precision at that level	Direct measurement rather than estimation

A reported limit with no stated approach is difficult to compare against another laboratory’s limit, because the two may not be measuring the same idea.

The conventional numbers

Two sets of figures appear so often that they are worth stating explicitly, along with what they are and are not. Where the signal-to-noise approach applies, ICH Q2(R2) describes a signal-to-noise ratio of 3:1 as generally acceptable for estimating the detection limit, and a ratio of at least 10:1 for the quantitation limit. Where the limits are derived from response variability instead, the guideline expresses them as formulae.

What a negative result licenses

A result reported as not detected supports a bounded statement, and only a bounded one: that if the analyte was present, it was present below the detection limit of the procedure applied. That is genuinely useful — but only if the limit is stated. Without it, the claim has no floor and is not checkable.

Supported: not detected at or above the stated detection limit of this procedure
Not supported: the substance is absent
Not supported: the substance is present at zero
Not supported: a more sensitive method would also find nothing
Unverifiable: not detected, with no limit stated anywhere in the report

Why limits are context-dependent

The same procedure does not have the same limits everywhere. Sensitivity depends on the detector and its response to the particular analyte, on how much the sample matrix interferes, on the condition of the instrument and column, and on where in the run the measurement sits. ICH Q2(R2) is specific even about where noise should be assessed — within a defined region and, if possible, situated equally around the place where the peak of interest would be found. A limit quoted without its method is a number without a unit.

Detector response varies by analyte; a detector blind to a substance has no limit for it
Matrix interference can raise the floor considerably
Instrument and column condition move the baseline
Noise must be assessed in a defined region, near where the peak would appear

The relationship to validation

Detection and quantitation limits are not standalone figures; they are part of establishing that a procedure is fit for its purpose. Q2(R2) treats them as validation of the lower range limits — relevant where the attribute being measured requires the procedure to work close to the bottom of its range, which is exactly the situation for impurities and degradation products. Where a limit has been estimated by calculation or extrapolation, the guideline notes that the estimate can subsequently be validated by independent analysis of a suitable number of samples known to be near, or prepared at, the limit.

Reading a report that quotes limits

The useful questions are about whether the numbers are anchored to anything. A limit is only comparable if you know how it was obtained and what it applies to.

Is the limit stated numerically, with units?
Is the approach used to determine it stated?
Does the limit apply to the analyte in question, or to the procedure generally?
Are results between the detection and quantitation limits reported as such, or rounded to zero?
Is "not detected" accompanied by the limit it is not detected against?

Research Checklist

Confirm batch identity and records.
Document all preparation inputs.
Keep use within controlled laboratory workflows.
Do not infer applied-use suitability from guide content.

Frequently Asked Questions

Does "not detected" mean the substance is absent?

No. It means the procedure applied could not distinguish it from background. The detection limit is a property of the method, so a negative result is bounded by that limit: the substance was not present above it. Whether it was present below it is a question the method did not answer.

Why is there a gap between the detection limit and the quantitation limit?

Because detecting something and measuring it are different achievements. ICH Q2(R2) defines the detection limit as the lowest amount that can be detected but not necessarily quantitated as an exact value, and the quantitation limit as the lowest amount that can be determined with suitable precision and accuracy. Between the two, a signal is real but a number attached to it would overstate what the method supports.

Are 3:1 and 10:1 fixed rules?

They are conventions described in ICH Q2(R2) as generally acceptable for the signal-to-noise approach, and that approach only applies to procedures which exhibit baseline noise. Other approaches — visual evaluation, calculation from the standard deviation of the response and the slope, or direct validation by accuracy and precision — produce limits without reference to those ratios at all.

Can two laboratories report different limits for the same substance?

Routinely, and legitimately. Limits depend on the procedure, the detector, the matrix, the instrument and the approach used to establish them. Two different limits are not evidence that one laboratory is wrong; they are evidence that the two asked the question with different sensitivity.

Sources

The technical statements in this guide are drawn from the following. Where a definition is contested or a figure depends on method, the guide says so rather than picking one.

ICH Q2(R2): Validation of Analytical ProceduresInternational Council for Harmonisation, via the European Medicines AgencyUsed for the definitions of detection limit and quantitation limit, the four approaches to determining them, the 3:1 and 10:1 signal-to-noise conventions, the DL and QL formulae, guidance on where noise is assessed, and the recommendation that the approach used be presented alongside the limit.
Q2(R2) Validation of Analytical Procedures: Guidance for IndustryU.S. Food and Drug AdministrationThe same guideline as adopted by the FDA.