Key points
- Number of ISR samples: 10% of the first 1,000 study samples plus 5% of the samples above 1,000.
- % difference = (repeat - original) / mean of the two × 100.
- Chromatography: at least two-thirds of the repeats within ±20% of their mean (ligand-binding assays: ±30%).
What ISR shows
Calibration standards and QCs are prepared in clean matrix. Study samples are not: they may contain metabolites, concomitant medication, or be affected by protein binding, sample inhomogeneity or instability. Incurred sample reanalysis - repeating a subset of real study samples in a separate run on a different day - shows whether the method gives reproducible results for the samples that matter.
When ISR is required
ICH M10 expects ISR at least in:
- all pivotal bioequivalence studies;
- the first clinical trial in humans and the first trial in patients;
- the first trial in patients with impaired hepatic and/or renal function;
- non-clinical: pivotal toxicology studies, once per species (generally).
How many samples
ISR covers 10% of the first 1,000 study samples and 5% of the number of samples exceeding 1,000. Samples are selected around Cmax and in the elimination phase, and across as many subjects as possible.
| Study samples | Calculation | ISR samples |
|---|---|---|
| 600 | 10% × 600 | 60 |
| 1,000 | 10% × 1,000 | 100 |
| 1,500 | 10% × 1,000 + 5% × 500 | 125 |
| 4,000 | 10% × 1,000 + 5% × 3,000 | 250 |
How to calculate
For each pair of original and repeat results:
% difference = (repeat − original) / ((repeat + original) / 2) × 100
For chromatographic methods, the pair passes when the % difference is within ±20%. ISR as a whole is acceptable when at least two-thirds (66.7%) of the repeated samples pass. For ligand-binding assays the limit is ±30%.
Example: original 120 ng/mL, repeat 101 ng/mL. Mean 110.5; difference −19; % difference −17.2% - within ±20%, pass. With 125 ISR samples, at least 84 must pass (125 × 2/3 = 83.3, rounded up).
When ISR fails
A failed ISR does not by itself invalidate the study, but it must be investigated and the impact assessed and documented before the study is reported. Typical areas to examine:
- Back-conversion of metabolites (for example acyl glucuronides or N-oxides converting to the parent drug).
- Stability of the analyte in study samples under the actual handling and storage conditions.
- Sample inhomogeneity, mixing before aliquoting, or haemolysed / lipaemic samples.
- Matrix effects and IS response in the original and the repeat runs.
- Pattern: are failures concentrated in one subject, one time point, one run or one concentration range?
The investigation should follow a pre-defined SOP; trends, not just the overall pass rate, should be reviewed.
Good practice
- Plan ISR in the study plan, including the selection rules, before the first study sample is analysed.
- Use the same validated method; the repeat runs follow normal run acceptance.
- Keep original and repeat results linked - ISR tables are among the first things auditors ask for.
Educational summary for chromatographic (LC-MS/MS) bioanalysis. Always apply the current ICH M10 text, the expectations of the regulator concerned and your laboratory's SOPs. Ligand-binding assays have wider limits in ICH M10.
ARISENTO Insights is published by Autofai Technologies Private Limited, the makers of ARISENTO bioanalytical software.