Analytical Method Validation

When to use

Any time the question is whether an analytical procedure is fit for its intended purpose: designing a validation study, evaluating validation data, verifying a compendial procedure, transferring a procedure to another laboratory or instrument, or defending any of these in a report.

The two rules

1. Establish which framework governs before designing anything. The same assay validates differently under ICH Q2(R2), USP <1225>, ICH M10, CLSI EP, and ISO/IEC 17025. They differ in which characteristics are required, how the studies are laid out, and whether numeric acceptance criteria are supplied at all. Blending them produces a protocol that satisfies none of them.

2. State acceptance criteria before collecting data. Criteria chosen after seeing results are not acceptance criteria, and deciding them post hoc is a standing audit finding. ICH Q2(R2) deliberately supplies almost no numeric criteria — they have to come from the specification, the analytical target profile (ICH Q14 section 3), or development data. ICH M10 is the exception: it supplies explicit numbers, and they differ between chromatographic assays and ligand binding assays.

Scope

This skill plans studies, computes the statistics correctly, and structures the documentation. It does not decide that a procedure is validated, release a batch, accept or reject a run, close an investigation, or substitute for the analyst, the technical reviewer, the quality unit, or the regulator. Every script reports; none of them concludes.

Copyright boundary

ICH guidelines are published openly and licensed for reuse with acknowledgement, so their requirements are encoded directly in this skill. USP general chapters, CLSI EP documents, and ISO standards are copyrighted and paywalled. For those, this skill supplies the designation, scope, and where to obtain an authorised copy — never the text, never invented thresholds. Do not ask an agent to retrieve, transcribe, or reconstruct their content. If a number matters and it lives in a paywalled document, read it from the authorised copy.

Frameworks

cd skills/analytical-method-validation/scripts
python3 plan_validation.py --list-frameworks

Key	Governs	Numeric criteria supplied
ich-q2r2	Release and stability testing of drug substances and products	Almost none — you derive them
ich-m10	Bioanalytical concentration measurement (PK, TK, BE)	Yes, and they differ by modality
usp-1220	Compendial procedure lifecycle, three stages	Paywalled
usp-1225 / usp-1226	Validation / verification of compendial procedures	Paywalled
clsi	Clinical laboratory measurement procedures (EP series)	Paywalled
iso-17025	Lab-developed and modified methods under accreditation	No — "to the extent necessary"

Q2(R2) replaced Q2(R1) in November 2023 and restructured the characteristics. Range is now the parent characteristic (section 3.2), containing response (linearity) and validation of lower range limits (DL/QL). Accuracy and precision are section 3.3 and may be evaluated in combination against a single criterion. Robustness is treated as a development activity and cross-refers to ICH Q14. Multivariate procedures are addressed explicitly (2.5 and 3.2.2.3), and Annex 2 adds worked examples for techniques Q2(R1) never covered — quantitative ¹H-NMR, NIR, quantitative LC/MS, qPCR, biological assays, and particle size. A Q2(R1)-shaped protocol — a flat list of linearity, range, accuracy, precision, specificity, LOD, LOQ, robustness — is out of date. Note also the error correction dated 30 November 2023 to Table 5 and Tables 6–11.

Scripts

cd skills/analytical-method-validation/scripts

Script	Question answered
plan_validation.py	Which framework, which characteristics, what study layout, what protocol?
check_response.py	Does the calibration model actually hold across the range?
check_accuracy_precision.py	What is the recovery, and how much of the variability is between days?
check_detection_limits.py	What are DL and QL by each allowed approach, and do they serve the reporting threshold?
check_bioanalytical_run.py	Does this run meet ICH M10 for its modality?
compare_methods.py	Are two procedures equivalent, at a pre-stated margin?

All take --format table|tsv|json. Provenance, guideline citations, and caveats go to stderr; data goes to stdout, so > out.tsv keeps them separate. Exit code is 0 for no findings, 1 when findings were raised, 2 for bad input — so any of them can gate a workflow.

Workflow

1. Fix the framework and the required characteristics

python3 plan_validation.py --framework ich-q2r2 --attribute assay --technique hplc --range-use assay

Q2(R2) Table 1 decides what is required from the measured attribute, not from the technique. For an assay: specificity, response, accuracy, repeatability, intermediate precision. For a limit test: specificity and DL only. For an identity test: specificity alone. Attributes accepted include assay, impurity (quantitative), impurity-limit, and identity.

Reportable range comes from the specification. Q2(R2) Table 2 gives worked examples — 80–120% of declared content for an assay, 70–130% for content uniformity, reporting threshold to 120% of the specification for an impurity.

2. Generate the protocol and fill in the criteria

python3 plan_validation.py --framework ich-q2r2 --attribute impurity --protocol > protocol.md

Every bracketed field is a decision to make and record before data collection. The protocol skeleton deliberately refuses to pre-fill acceptance criteria for Q2(R2) work, because there is no defensible default.

3. Evaluate the response

python3 check_response.py -i calibration.csv --max-back-calc-error 2

Input is level,response, one row per injection; repeated rows at the same level are replicates, and supplying them is what makes the linearity test possible.

Real output from a curve that a coefficient of determination would wave through:

statistic                           value
distinct levels                     5
slope                               166.6000
intercept                           2495.0000
intercept CI includes 0             no
coefficient of determination (r2)   0.9830
lack-of-fit F                       469.5294
lack-of-fit p                       1.5139e-06
runs test p                         0.0492

level     n  mean_response  mean_back_calculated  relative_error_pct
50.0000   2  10075.0000     45.4982               -9.0036
75.0000   2  15150.0000     75.9604               1.2805
100.0000  2  20050.0000     105.3721              5.3721
125.0000  2  24050.0000     129.3818              3.5054
150.0000  2  26450.0000     143.7875              -4.1417

r² = 0.983 and the model is unusable: −9.0% back-calculated error at the bottom of the range, lack-of-fit p = 1.5 × 10⁻⁶, non-random residual signs. r² is not evidence of linearity — it rises with range and is nearly insensitive to curvature. The lack-of-fit F test against pure error and the residual pattern are the evidence, which is why Q2(R2) 3.2.2.1 asks for an analysis of the deviation of points from the line rather than a correlation coefficient alone.

Add --weight 1/x2 for a wide-range curve. The script flags heteroscedasticity when the residual variance in the top third of the range exceeds the bottom third by more than 10×, because an unweighted fit then biases exactly the low end where a reporting threshold lives.

4. Evaluate accuracy and precision

python3 check_accuracy_precision.py -i ap.csv --accuracy-limit 2 --rsd-limit 1.0 --design-check assay

Input is level,measured,group, where group is the intermediate-precision factor — day, analyst, or instrument.

level  component                       sd      rsd_pct  df      ci90_low_sd  ci90_high_sd
100    repeatability (within group)    0.0707  0.0707   3       0.0438       0.2065
100    between-group                   1.6515  1.6515   2       n/a          n/a
100    intermediate precision (total)  1.6530  1.6530   2.0037  0.9554       7.2821

Repeatability of 0.07% RSD looks superb; intermediate precision is 1.65%, twenty-three times larger, because the variability lives entirely between days. Reporting the within-day figure as the procedure's precision would understate routine performance by more than an order of magnitude. This is why the script fits a one-way random-effects model rather than pooling.

Two traps the script handles for you:

• Precision is estimated within each level, never pooled across levels. Pooling 80/100/120% results into one standard deviation turns the range itself into apparent imprecision. The script reports per level, plus a level-independent view as percent of nominal.
• --require-ci-within-limit enforces that the whole confidence interval sits inside the limit, not just the mean. Q2(R2) 3.3.1.4 asks for the interval to be compatible with the criterion; a mean that scrapes inside on six replicates has not demonstrated much.

5. Establish DL and QL, and confirm them

python3 check_detection_limits.py --calibration lowcal.csv --blanks blanks.csv \
    --confirm-ql 0.05 --confirm-data ql_check.csv --reporting-threshold 0.05

approach                                          sigma   slope      DL      QL
sd-and-slope (sigma = residual SD of regression)  7.2816  5033.3490  0.0048  0.0145
sd-and-slope (sigma = SD of y-intercept)          4.3303  5033.3490  0.0028  0.0086
sd-and-slope (sigma = SD of 8 blanks)             3.7702  5033.3490  0.0025  0.0075

The same data give QL estimates spanning 1.9×, purely from the choice of σ. Q2(R2) 3.2.3.5 therefore requires the limit and the approach used to determine it to be reported, and an estimated limit to be confirmed with samples at or near it. For an impurity procedure the QL must be at or below the reporting threshold. Reaching for 3.3σ/slope reflexively, reporting one number with no named approach, and never confirming it are three separate findings.

6. Bioanalytical runs under ICH M10

python3 check_bioanalytical_run.py --modality chromatographic --run run1.csv
python3 check_bioanalytical_run.py --modality lba --isr isr.csv
python3 check_bioanalytical_run.py --modality lba --criteria

--modality is mandatory and has no default, because the criteria genuinely differ:

	Chromatographic	Ligand binding assay
Calibration tolerance	±15%, ±20% at LLOQ	±20%, ±25% at LLOQ and ULOQ
Accuracy / precision	±15% / ≤15% CV (±20% / ≤20% at LLOQ)	±20% / ≤20% CV (±25% / ≤25% at LLOQ and ULOQ)
A&P design	4 QC levels, 5 replicates/run, ≥3 runs over ≥2 days	5 QC levels, 3 replicates/run, ≥6 runs over ≥2 days
Total error	no such criterion	≤30%, ≤40% at LLOQ and ULOQ
ISR agreement	±20% for ≥2/3 of repeats	±30% for ≥2/3 of repeats

Applying the ±15% chromatographic numbers to a ligand binding assay, or importing the LBA total-error criterion into a chromatographic method, are both common and both wrong.

The run check enforces the per-level rule that gets missed: at least 2/3 of all QCs and at least 50% at each level. A run can pass the overall fraction while a single level fails completely.

finding: QC level high: 0/2 within tolerance (0%); M10 requires at least 50% at each level

7. Transfer and method comparison

python3 compare_methods.py -i paired.csv --margin 2 --relative --slope-tolerance 0.05

mean difference (%)                       1.4646
TOST margin                               2.0000
TOST p-value                              1.0528e-13
90% CI (TOST)                             1.44127 to 1.48797
equivalent at stated margin               yes
--- for contrast only ---
paired t-test p (NOT equivalence)         0.0000
OLS slope (biased here)                   1.0396
Deming slope                              1.0398
Passing-Bablok slope                      1.0351

Two errors this replaces:

• "p > 0.05, no significant difference, therefore the methods are equivalent." Failing to detect a difference is not evidence of equivalence, and on a small transfer dataset that outcome is close to guaranteed. TOST tests the hypothesis that matters — that the true difference lies inside a pre-stated margin. Here the t test says the difference is highly significant and TOST says the methods are equivalent at ±2%; both are true, and only one answers the question.
• Ordinary least squares for method comparison. OLS assumes the reference values carry no error, which is false when comparing two procedures, and biases the slope toward zero. Deming (with a stated error-variance ratio) and Passing–Bablok (non-parametric, outlier-resistant) are the appropriate regressions and are reported side by side with OLS for contrast.

The script also flags proportional bias — when the difference trends with concentration, a single mean bias and its limits of agreement are misleading regardless of how tight they look.

What this skill exists to prevent

1. Validating against ICH Q2(R1)'s structure three years after Q2(R2) replaced it.
2. Acceptance criteria written after the data were seen.
3. r² presented as evidence of linearity.
4. Repeatability reported as the procedure's precision, with the between-day component invisible.
5. One DL/QL number with no named approach and no confirmation.
6. Chromatographic M10 criteria applied to a ligand binding assay, or the reverse.
7. A t test's non-significance presented as equivalence at a method transfer.

References

• references/framework-selection.md — which framework governs, and the questions that decide it
• references/ich-q2r2.md — structure, Table 1 and Table 2, per-characteristic recommended data
• references/ich-m10-bioanalytical.md — the full chromatographic and LBA criteria side by side
• references/compendial-and-clsi.md — USP, CLSI and ISO designations, scope, and how to cite them
• references/statistics.md — the statistical methods, why each one, and the common errors
• references/source-ledger.md — provenance and research dates for every claim in this skill

Assets

• assets/validation-protocol-template.md — protocol structure with criteria stated up front
• assets/validation-report-template.md — report structure with raw-data traceability