Analytical methods (HPLC, MS)

The sample is separated on a C18 column using a water/acetonitrile gradient containing 0.1% TFA, with UV detection at 214–220 nm (peptide bond absorbance).

Purity is reported as the main-peak area as a percentage of total integrated peak area. Related impurities — deletion sequences, truncations, oxidized or deamidated species — appear as separate peaks.

Electrospray ionization MS (ESI-MS) or MALDI-TOF determines the molecular mass. Multiply charged ions are deconvoluted to the parent mass and compared with the theoretical value calculated from the sequence.

LC-MS combines both techniques, assigning masses to individual chromatographic peaks.

Small-molecule products are characterized by HPLC purity, LC-MS identity and, where applicable, ¹H-NMR consistent with structure.

On request: amino acid analysis or nitrogen content (net peptide content), Karl Fischer water content, and ion chromatography for counter-ion content.