Peak integration practice
Drop-line versus valley-to-valley, tangent skim, baseline placement, and the reporting threshold — where a purity figure is actually decided.
Chromatographic purity
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 is the area of the main peak expressed as a percentage of the total integrated area of all peaks in the chromatogram, excluding the injection disturbance and the solvent front. It is a relative quantity. It answers the question "of everything that eluted and absorbed at this wavelength, what fraction was the main peak" and it answers no other question. In particular it does not say how much peptide is in the vial, and it does not say what the main peak is.
Index reference method RG-2
| Parameter | Setting |
|---|---|
| Column | C18, 2.6 µm core–shell, 100 × 4.6 mm, 100 Å |
| Mobile phase A | Water + 0.1 % (v/v) trifluoroacetic acid |
| Mobile phase B | Acetonitrile + 0.1 % (v/v) trifluoroacetic acid |
| Gradient | 2 % B hold 2 min → 5 % B at 3 min → 65 % B at 30 min → 95 % B at 32 min, hold 3 min |
| Flow | 1.0 mL/min |
| Column temperature | 40 °C ± 1 °C |
| Detection | UV 214 nm, 4 nm bandwidth; secondary channel 280 nm where aromatic residues are present |
| Injection | 10 µL of a 0.5 mg/mL solution in mobile phase A |
| Run time | 35 min including re-equilibration |
The two-minute initial hold at 2 % B exists for one reason: compounds below about 500 Da elute within three minutes of injection, and without the hold their main peak partially merges with the solvent disturbance. Several reports submitted to the index have integrated solvent into a tripeptide main peak. The hold is not optional.
What the gradient length decides
Resolution between two peaks scales with the time the gradient spends passing through their elution window. Halving the gradient duration roughly halves the separation between neighbouring species — and the species that matter most on the acylated incretins are mass-identical diastereomers separated by 0.2 min on a 30-minute method. On a 15-minute method they are one peak.
The consequence is arithmetic rather than opinion: a laboratory running a 15-minute gradient will report a higher purity than a laboratory running a 30-minute gradient on the same vial, because it is integrating more material into the main peak. The index measures the offset at +0.24 pp overall and +0.51 pp on the incretin class. Both figures are on the inter-lab agreement page with their confidence intervals.
Acceptance criteria applied by the index
| Criterion | Requirement | If not met |
|---|---|---|
| Gradient duration | ≥ 25 min for the incretin class, ≥ 22 min for melanocortins, ≥ 15 min otherwise | Status set to METHOD LIMITED |
| Main-peak asymmetry (10 % height) | As ≤ 1.50 | Rule AX-R12; purity marked integration-limited |
| Resolution to nearest named impurity | R ≥ 1.20 | Rule AX-R12 |
| Plate count (main peak) | N ≥ 20,000 | Method note recorded |
| Retention within compound window | stated window after gradient normalisation | Rule AX-R11 |
| Baseline | no drift exceeding 2 % of main-peak height across the peak | Method note recorded |
Worked example
Integrated areas (µV·s), 214 nm: main peak 4 812 660 impurity A (−0.35) 22 140 impurity B (−0.68) 13 405 impurity C (+0.93) 7 890 impurity D (+1.62) 4 215 ────────────────────────────── total 4 860 310 purity = 4 812 660 / 4 860 310 × 100 = 99.019 % → 99.02 % total impurities = 0.98 % largest single impurity = 22 140 / 4 860 310 × 100 = 0.46 % check: 99.02 + 0.98 = 100.00 ✓ closes
Every record in this index closes that check to within 0.02 pp, and the build asserts it. A supplier certificate that does not close triggers rule AX-R02.
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.
- Ph. Eur. 2.2.29 — Liquid chromatography
- USP <621> — Chromatography
- Ph. Eur. 2.2.46 — Chromatographic separation techniques (asymmetry, resolution, plate count)
Related methods
Gradient design for peptides
Why 25 minutes is the index minimum for the acylated incretins, and how gradient slope trades against run time and against the impurities you can see.
Chromatographic purity
Diastereomers: the impurity no mass spectrum will find
Epimerisation during synthesis, why the resulting impurity is functionally significant and analytically invisible, and what it takes to see it.
Chromatographic purity
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