Question 1

What is the difference between LOD and LOQ?

Accepted Answer

LOD (Limit of Detection) is the lowest analyte concentration that can be reliably distinguished from zero — it is detected but not necessarily quantified. LOQ (Limit of Quantitation) is the lowest concentration that can be measured with acceptable precision and accuracy. ICH defines LOD = 3.3σ/S and LOQ = 10σ/S, where $\sigma$ is the residual standard deviation and S is the calibration slope.

Question 2

How is σ (sigma) calculated?

Accepted Answer

This tool uses the residual standard deviation method: $\sigma$ = √(Σresiduals² / (n-2)). The residuals come from the OLS regression of the calibration curve. An alternative is the standard deviation of blank responses (y-intercept method), which this tool will support in a future update.

Question 3

What R² value does ICH Q2 require for linearity?

Accepted Answer

ICH Q2(R2) does not specify an exact $	ext{R}^{2}$ cutoff. $	ext{R}^{2}$ $\geq$ 0.999 is a common industry rule of thumb for chromatographic methods, but it is not a regulatory requirement. For biological assays, $	ext{R}^{2}$ $\geq$ 0.99 may be acceptable. Adjust the threshold in the settings panel.

Question 4

How many calibration levels and replicates do I need?

Accepted Answer

ICH recommends a minimum of 5 concentration levels across the expected range, with replicates (typically triplicates) at each level. More levels and replicates improve the reliability of LOD/LOQ estimates.

Question 5

What precision criteria should I use?

Accepted Answer

For standard analytical methods: %RSD $\leq$ 2% at each level (repeatability). At the LOQ level, %RSD $\leq$ 10% is typically acceptable. For biological assays, wider limits (e.g., %RSD $\leq$ 15%) may be appropriate per FDA bioanalytical guidance.

Question 6

What is % recovery and how is it assessed?

Accepted Answer

% Recovery = (measured/expected) $	imes$ 100. For standard methods, 98-102% recovery is typical. At LOQ, 80-120% is often acceptable. Spike-and-recovery experiments at multiple levels (low, mid, high) demonstrate accuracy across the range.

ICH Q2(R2) LOD/LOQ Calculator.

Calculator

Calibration Data

Accuracy / Recovery Data (Optional)

LOD/LOQ from regression can underestimate true limits

R² alone does not prove linearity

Include zero concentration in calibration

%RSD is unreliable near zero

Method

Validated

How to cite

How to Cite

ICH Q2(R2) Method Validation Framework

LOD and LOQ Calculation Methods

Frequently asked

Next steps

Spectrophotometers

Analytical Balances

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