Purity by reverse-phase HPLC (% area)
The index reference method RG-2, what it measures, what it does not, and why a purity figure without a stated gradient is uninterpretable.
Chromatographic purity
Peak integration practice
Drop-line versus valley-to-valley, tangent skim, baseline placement, and the reporting threshold — where a purity figure is actually decided.
Two competent analysts given the same chromatogram will produce purity figures differing by more than the instrument's repeatability, and the difference will be integration. This is the least glamorous and most consequential part of a purity determination and it is documented almost nowhere.
Baseline placement
The baseline is where the detector would be if nothing were eluting, and on a gradient it is not flat: the refractive-index change and any UV-absorbing mobile-phase additive produce drift. The convention is a straight-line baseline drawn between the points where the peak begins and ends. Where the baseline drifts across the peak, a projected baseline is drawn from the pre-peak to the post-peak signal, and the choice of those two points moves the area.
For a peak at 0.5 % area sitting on a baseline drifting by 2 % of the main-peak height, the baseline choice can change the reported impurity level by a third of its value.
Separating adjacent peaks
| Technique | When it applies | Effect |
|---|---|---|
| Drop-line (perpendicular) | Two peaks of comparable height, resolved to a clear valley | Splits the overlap in proportion to nothing in particular; unbiased when the peaks are similar |
| Valley-to-valley | A small peak riding on the tail of a large one | Assigns the overlap to the large peak; understates the small one |
| Tangent skim | A small peak on the trailing edge of a much larger one | Assigns the overlap to the large peak; the convention where the height ratio exceeds about 10:1 |
| Exponential skim | Same, where the large peak tails strongly | More faithful to the underlying peak shape; less widely implemented |
The index's position is that the technique used must be stated, because for an unresolved shoulder the choice between drop-line and valley-to-valley changes the reported purity by up to 0.45 pp — larger than the entire rest of the uncertainty budget. Tirzepatide's des-Aib2 impurity is the archetype and it is why rule AX-R12 exists.
Reporting threshold
Peaks below a threshold are not reported. The index uses 0.05 % area, consistent with ICH Q3A's reporting threshold for a drug substance at moderate daily dose. The choice matters in one specific and systematic way: excluded peaks are excluded from the total, so purity is overstated by the sum of everything below threshold. For a lot with twelve sub-threshold peaks that is 0.1–0.3 pp of systematic, one-directional error.
What the index requires
- Reporting threshold stated. If absent, recorded as unstated.
- Integration technique stated where any peak has resolution below 1.5 from the main peak.
- Named impurities with retention times where the laboratory offers them; totals accepted otherwise, with the limitation noted.
- Peaks arising from the diluent, the blank, or the injection disturbance excluded and identified as such.
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 Q3A(R2) — Impurities in new drug substances, reporting and identification thresholds
- Ph. Eur. 2.2.46 — Chromatographic separation techniques
- USP <621> — Chromatography
Related methods
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
Related-substances profiling
Naming impurities instead of totalling them: what it enables, how assignment works, and why an impurity fingerprint identifies a bulk.
Chromatographic purity