CEI-MS-19 — Purity determination for peptides not retained by conventional reverse-phase…

§1Scope

§1.1Scope

Applies to peptides below approximately 800 daltons, to peptides with a net negative charge and minimal hydrophobic surface, and to any peptide whose main peak elutes with a retention factor below 1.0 under CEI-MS-01. This class includes several widely supplied compounds for which the purity figures in general circulation cannot be correct.

§2Principle

A retention factor below 1.0 means the analyte spends less time in the stationary phase than in the mobile phase, and impurities differing only slightly in polarity are not separated from it or from the injection disturbance. Adequate separation requires either a stationary phase that retains polar analytes, or a separation mechanism other than hydrophobic partition. Three approaches are available: aqueous-compatible or polar-embedded reverse phase; hydrophilic-interaction chromatography, in which retention increases with polarity; and ion-pairing or ion-exchange separation.^[1,2]

§3Apparatus and reagents

§3.1Apparatus

• HPLC with a low-dispersion flow path and an injector capable of small injection volumes
• Aqueous-compatible C18, polar-embedded, or hydrophilic-interaction stationary phase
• Ultraviolet detector at 214 nm, and a charged-aerosol or refractive-index detector where the analyte lacks a chromophore

§3.2Reagents and reference materials

• Water and acetonitrile of chromatographic grade
• Ammonium formate or ammonium acetate buffer for hydrophilic-interaction separation
• Ion-pair reagent where ion pairing is selected

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. European Directorate for the Quality of Medicines & HealthCare. General Chapter 2.2.29 Liquid Chromatography. European Pharmacopoeia 2023;11th edition. identifier not held by the Institute
2. Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, Tripet BP, Hodges RS. HPLC analysis and purification of peptides. Methods in Molecular Biology 2007;386:3–55. doi:10.1007/978-1-59745-430-8_1 · PMID 18604941

Identifiers are reproduced only where the Institute holds them. Where a digital object identifier or PubMed identifier is not shown, the Institute has recorded the journal and year and has not constructed an identifier.