CEI-MS-01 — Determination of chromatographic purity of synthetic peptides by reverse-phase…

§1Scope

§1.1Scope

Applies to synthetic peptides of 3 to 60 amino-acid residues supplied as lyophilised powders or solutions, including acylated analogues. Does not apply to recombinant glycoproteins, antibody conjugates, undefined extracts, or non-peptide small molecules; separate standards cover those.

§2Principle

Peptides are separated on a hydrophobic stationary phase under an acidified aqueous–organic gradient. Retention is governed principally by the hydrophobic surface area presented by the folded or extended chain and, for acylated analogues, by the fatty-acid conjugate. Detection is by ultraviolet absorbance at 214 nm, where the amide backbone absorbs; area percentage of the main peak relative to total integrated area is reported as chromatographic purity. The measurement is a relative area, not a mass fraction, and the distinction is the single most frequently misunderstood point in this field.^[1,2]

§3Apparatus and reagents

§3.1Apparatus

• Gradient HPLC or UHPLC system with quaternary or binary proportioning, degasser and thermostatted column compartment
• Diode-array or variable-wavelength ultraviolet detector capable of operation at 214 nm with a path length appropriate to the expected concentration
• Column: C18 or C8 silica, 1.7–5 µm particle, 100 Å pore for peptides below 3 kDa and 300 Å pore above 3 kDa
• Analytical balance readable to 0.01 mg
• Calibrated micropipettes and Class A volumetric glassware
• Sample vials of low-adsorption polypropylene or silanised glass

§3.2Reagents and reference materials

• Water, ≤0.055 µS/cm at 25 °C
• Acetonitrile, gradient grade
• Trifluoroacetic acid, ≥99.5 %, or formic acid ≥98 % where the sample will subsequently be analysed by mass spectrometry
• Reference standard of the peptide under test, where available, with a stated assigned content

References cited on this page

References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.

1. United States Pharmacopeial Convention. General Chapter ⟨1225⟩ Validation of Compendial Procedures. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute
2. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q2(R2) Validation of Analytical Procedures. ICH Harmonised Guideline 2023;Step 4 version, 1 November 2023. identifier not held by the Institute

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