Uncertainty budget for a purity figure
Nobody publishes one. Here is what it would contain, and what the combined standard uncertainty on a peptide purity result actually looks like.
Quality system
Method validation to ICH Q2(R2)
The eight validation characteristics, what each one demonstrates, and which of them the reports in this index actually document.
A method that has not been validated has produced a number of unknown meaning. Validation is the documented demonstration that a method is suitable for its purpose, and ICH Q2(R2) sets out which characteristics must be demonstrated for which kind of test.
Characteristics
| Characteristic | Demonstrates | Typical acceptance | Required for |
|---|---|---|---|
| Specificity | The response is due to the analyte and nothing else | Peak purity by PDA or MS; resolution ≥ 1.5 from nearest peak | Identity, assay, impurities |
| Linearity | Response is proportional to concentration over the range | r² ≥ 0.999, y-intercept within ±2 % of the 100 % response | Assay, quantitative impurities |
| Range | The interval over which linearity, accuracy and precision hold | 80–120 % of nominal for assay; LOQ–120 % for impurities | Assay, impurities |
| Accuracy | Closeness to the true value | Recovery 98–102 % for assay; 90–110 % at impurity levels | Assay, quantitative impurities |
| Repeatability | Precision under the same conditions, same session | RSD ≤ 1.0 % for assay (n=6) | Assay, impurities |
| Intermediate precision | Precision across days, analysts, instruments | RSD ≤ 2.0 % | Assay, impurities |
| Detection limit | Lowest detectable amount | S/N ≥ 3 | Limit tests |
| Quantitation limit | Lowest reliably quantifiable amount | S/N ≥ 10, RSD ≤ 10 % | Quantitative impurities |
| Robustness | Tolerance to deliberate small method changes | System suitability maintained at ±2 °C, ±0.02 pH, ±2 % organic | All |
What the index actually holds
Of the six laboratories contributing to this index, two publish a validation summary for their peptide purity method, one publishes system-suitability limits without a full validation package, and three publish neither. None of the twenty suppliers publishes a validation summary for the methods behind their certificates.
That is worth stating plainly rather than treating as a footnote. Most of the numbers in this index — and effectively all of the numbers on supplier certificates — come from methods whose accuracy, linearity and intermediate precision have not been demonstrated to anyone outside the laboratory that runs them. The index's inter-lab agreement analysis is a partial substitute: it measures whether two undocumented methods agree, which is weaker than either being validated but is not nothing.
Normative references
Cited because they are the documents the acceptance criteria above are taken from. The index applies them as written and states every deviation.
- ICH Q2(R2) — Validation of analytical procedures (2023)
- ICH Q14 — Analytical procedure development (2023)
- USP <1225> — Validation of compendial procedures
- USP <1226> — Verification of compendial procedures
Related methods
System suitability
The injections that establish a chromatographic system was fit to produce the result, and the parameters the index checks before accepting a purity figure.
Quality system
Reference standards, and what "area percent" means without one
The difference between an assay and an area percent, why most research peptides have no available standard, and what that costs the numbers on this site.
Quality system