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.
Ph. Eur. 2.2.29USP <621>
Method references
28 pages covering every measurement the index publishes: the procedure, the acceptance criteria, a worked calculation, and — the part most sources omit — what the method cannot establish. A result is only interpretable alongside the method that produced it, so every record on this site links here.
7 methods
Chromatographic purity
The reference method, integration practice, gradient design and the impurities a chromatogram can and cannot resolve.
Related-substances profiling
Naming impurities instead of totalling them: what it enables, how assignment works, and why an impurity fingerprint identifies a bulk.
ICH Q3A(R2)Ph. Eur. 5.10
Retention-time normalisation across gradients
How the index compares a retention time measured on a 15-minute gradient with one measured on a 35-minute gradient, and where the approximation breaks.
Snyder & DolanUSP <621>
Peak integration practice
Drop-line versus valley-to-valley, tangent skim, baseline placement, and the reporting threshold — where a purity figure is actually decided.
ICH Q3A(R2)Ph. Eur. 2.2.46
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.
USP <621>Ph. Eur. 2.2.46
Detection wavelength and response
214 nm, 220 nm, 280 nm — what each one sees, and how the choice changes a purity figure.
Ph. Eur. 2.2.25USP <857>
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.
Ph. Eur. 2.2.46ICH Q6A
4 methods
Identity
Mass spectrometry: what a mass measurement establishes, what resolution buys, and where the arithmetic goes wrong.
Identity by LC-MS
What a mass measurement establishes, what resolution buys you, and the specific compound pairs where unit resolution is not enough.
Ph. Eur. 2.2.43USP <736>
Mass accuracy and calibration
What ppm means at 340 Da and at 4,813 Da, how calibration drifts, and the checks that separate an instrument problem from a material problem.
Ph. Eur. 2.2.43USP <736>
Charge-state deconvolution
Recovering a neutral mass from an electrospray envelope, the arithmetic, and the failure modes that produce confident wrong answers.
Ph. Eur. 2.2.43ICH Q6B
Monoisotopic versus average mass
A 3 Da distinction that produces a 620 ppm error when confused. The most common single mistake in submitted identity data.
Ph. Eur. 2.2.43IUPAC
5 methods
Content and mass
The measurements that determine how much peptide is actually in the vial. The most consequential and least reported group.
Peptide content versus purity — the distinction most buyers get wrong
Purity is a ratio of areas. Content is a fraction of mass. A 99 % pure vial can contain 78 % peptide, and the difference is the whole ball game.
Ph. Eur. 2.2.56USP <1057>
Water content by Karl Fischer titration
Coulometric KF on lyophilised peptides: sample handling, the two things that ruin the measurement, and what a high result actually tells you.
Ph. Eur. 2.5.32USP <921>
Counter-ion quantification
Acetate, trifluoroacetate, mesylate and the rest — by ion chromatography, with the stoichiometry check that tells you whether the number is plausible.
Ph. Eur. 2.4.2USP <1065>
Amino-acid analysis for peptide content
Acid hydrolysis followed by chromatographic quantification of the liberated residues — the reference method for content where no standard of the intact peptide exists.
Ph. Eur. 2.2.56USP <1052>
Residual solvents by GC headspace
The mass in the cake nobody measures: DMF, DMSO, acetonitrile, DCM and TFA, to ICH Q3C(R8) classes and limits.
ICH Q3C(R8)USP <467>
2 methods
Microbiological
Endotoxin and sterility — the two properties no chromatogram bears on.
Bacterial endotoxin testing
Kinetic chromogenic, turbidimetric and gel-clot LAL; why the index specification is a screening threshold and not a limit; and the dose arithmetic that gives a real limit.
USP <85>Ph. Eur. 2.6.14
Sterility testing
Membrane filtration and direct inoculation to USP <71>: the media, the fourteen days, and why a passed sterility test on one vial says very little.
USP <71>Ph. Eur. 2.6.1
4 methods
Alternative techniques
Where reverse-phase chromatography is the wrong tool: short peptides, metal complexes, conjugates and folded proteins.
Ion-pair and HILIC chromatography for short and charged peptides
Why a dipeptide at 3.9 minutes on C18 is not a separation, and what to run instead.
USP <621>Ph. Eur. 2.2.29
Size-exclusion chromatography for aggregates and conjugates
The only routine method that sees an aggregate, and the only sensible way to characterise a polydisperse conjugate.
USP <621>Ph. Eur. 2.2.30
Disulfide mapping and the scrambling problem
A correct mass proves nothing about how the cysteines are paired. What does, and how rarely it is done.
ICH Q6BPh. Eur. 2.2.55
ICP-MS for metal-complexed peptides and elemental impurities
GHK-Cu and thymulin are half-measured without a metal number, and the mobile phase is the reason.
ICH Q3D(R2)USP <233>
2 methods
Physical tests
Appearance, clarity, pH and forced degradation. Cheap, informative, routinely reduced to one word.
Appearance, reconstitution clarity and pH
The three cheapest measurements on a vial, what each one actually diagnoses, and why the index prints them verbatim.
Ph. Eur. 2.2.3USP <791>
Forced degradation and stability-indicating capability
Deliberately damaging a sample to prove the method can see damage — and what the resulting degradant profile says about storage.
ICH Q1A(R2)ICH Q1B
4 methods
Quality system
Validation, uncertainty, system suitability and reference standards — the documentation that gives a number its meaning.
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.
ICH Q2(R2)ICH Q14
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.
ISO/IEC Guide 98-3 (GUM)Eurachem/CITAC CG 4
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.
USP <621>Ph. Eur. 2.2.46
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.
USP <11>Ph. Eur. 5.12