skills/analytical-method-validation/references/ich-m10-bioanalytical.md
Research basis: 2026-07-27, read from the ICH Harmonised Guideline Bioanalytical Method Validation and Study Sample Analysis M10, Step 4 dated 24 May 2022. ICH licenses its documents for reuse with acknowledgement. Confirm the current text and your region's implementation at https://database.ich.org/sites/default/files/M10_Guideline_Step4_2022_0524.pdf.
M10 harmonised what had been separate FDA and EMA bioanalytical guidance for studies in its scope: methods quantifying drug and metabolite concentrations in biological matrices supporting nonclinical and clinical studies, plus the analysis of study samples.
Chromatographic assays (section 3) and ligand binding assays (section 4) have different numeric criteria throughout. They are not stylistic variants of one set. Applying chromatographic tolerances to an LBA is the most common error in this area, and importing the LBA total-error criterion into a chromatographic method is its mirror image.
| Chromatographic | Ligand binding assay | |
|---|---|---|
| Calibration levels (minimum) | 6, including LLOQ | 6, including LLOQ |
| Calibration standard tolerance | ±15% | ±20% |
| … at LLOQ | ±20% | ±25% |
| … at ULOQ | ±15% | ±25% |
| Calibration standards that must pass | ≥75% | ≥75%, excluding anchor points |
| Accuracy | ±15% | ±20% |
| … at limits | ±20% at LLOQ | ±25% at LLOQ and ULOQ |
| Precision (%CV) | ≤15% | ≤20% |
| … at limits | ≤20% at LLOQ | ≤25% at LLOQ and ULOQ |
| A&P QC levels | minimum 4 | 5 (LLOQ, low, medium, high, ULOQ) |
| A&P replicates per level per run | ≥5 (within-run) | ≥3 |
| A&P runs | ≥3 runs over ≥2 days | ≥6 runs over ≥2 days |
| Total error | no such criterion | ≤30%; ≤40% at LLOQ and ULOQ |
| Routine run QC tolerance | ±15% | ±20% |
| Routine run QC pass rule | ≥2/3 of all QCs and ≥50% at each level | same rule, ±20% |
| Dilution integrity | mean within ±15% | mean within ±20% |
| Stability | mean at each QC level within ±15% | mean within ±20% |
| ISR agreement | within ±20% for ≥2/3 of repeats | within ±30% for ≥2/3 of repeats |
| Selectivity sources/lots | ≥6 individual sources | ≥6 individual sources |
| Carry-over in blank | ≤20% of LLOQ analyte response and ≤5% of IS response | per guideline |
Verify any figure against the guideline before using it in a protocol; regional implementation and subsequent revisions can change the picture.
Accuracy and precision validation QCs at a minimum of four concentration levels:
For runs that are not accuracy-and-precision runs, low, medium and high QCs may be analysed in duplicate; these plus the calibration standards form the basis for accepting or rejecting the run.
Calibration standards and QCs should be prepared from separate stock solutions, to avoid a bias that is not a property of the analytical performance. If a single stock must serve both, verify the accuracy and stability of that stock. A single source of blank matrix may be used if it is free of interference and matrix effects.
Calibration curves for accuracy and precision assessment should use freshly spiked standards in at least one run; if other runs use frozen standards, demonstrate their stability.
Report everything. Validation data and the determination of accuracy and precision must include all results obtained, including individual QCs outside the acceptance criteria — except cases where errors are obvious and documented. Silently dropping an out-of-criteria QC is a data integrity problem, not a rounding decision.
Within-run accuracy and precision are reported per run. If the within-run criteria are not met in every run, calculate an overall estimate of within-run accuracy and precision for each QC level. Between-run (intermediate) accuracy and precision combine data from all runs.
Trend within a run. It is recommended to demonstrate accuracy and precision over at least one run sized like a prospective study-sample run, so time-dependent drift is visible.
ISR repeats the analysis of a subset of study samples in separate runs, to verify that measured concentrations in real samples are reproducible. It is not a substitute for QCs — QCs are spiked, incurred samples are not, and only incurred samples can reveal metabolite back-conversion, protein binding effects, or matrix instability.
(repeat value - initial value) / mean value x 100 -- assessed
against the mean of the two, not against the initial value.For nonclinical studies in scope, ISR should in general be performed; the guideline notes incurred samples need only be included if available, so inclusion was not felt to be mandatory in every case. Confirm the situations requiring ISR against the guideline text for your study type.
ISR is a method-reliability check. Reanalysis of study samples for a reportable-value decision is different, and the reasons for reanalysis, the number of replicates, and the criteria for selecting the value to report must be predefined in the protocol, study plan, or SOP before study sample analysis begins. Deciding after the fact which of two values to report is the classic finding.
M10 addresses partial validation (a change to a validated method — matrix, anticoagulant, species,
instrument, or a range change) and cross validation (comparing data from two methods or two
laboratories contributing to the same study). Both are scoped by the change and the risk; consult
the guideline for what each requires. For a cross validation between sites or methods, the
statistics in compare_methods.py — equivalence testing against a pre-stated margin, and a
regression that allows error in both measurements — are the appropriate treatment.
M10's scope centres on drug and metabolite concentration measurement. Biomarker assays, immunogenicity assays, and diagnostic measurements are addressed differently or fall outside scope; do not assume the concentration-assay criteria transfer. Where a biomarker assay supports a regulatory decision, the fit-for-purpose framework and the applicable regional guidance govern the extent of validation.