Effect of dilution solvent and injection volume on the analysis of basic hydrophilic therapeutic polypeptide salts...

The chromatographic analysis of long-chain hydrophilic therapeutic peptides, with molecular weight mostly in the 3500-4500 Da range (31-34 amino acids), is explored with pressurized CO₂ in the mobile phase. The optimal method was obtained on a Torus 2-PIC column, with a gradient elution of 50-90% co-solvent in CO₂, which is relevant of enhanced-fluidity liquid chromatography (EFLC). Both UV (210 nm) and mass spectrometric detection modes were employed to assess the purity of the major peak and its resolution from impurities. Ten out of the eleven peptides in this set were basic, thus they were analyzed as acetate or trifluoroacetate salts. As significant peak distortion was observed in some cases, thorough examination of dilution solvent and injection volume was conducted to improve peak shape and resolution from impurities. Finally, the best injection volume was 1 µL, as any other volume (smaller or larger) yielded distorted peaks, and the best dilution solvent composition was the same as the mobile phase co-solvent (methanol comprising 5% water and 0.1 % methanesulfonic acid). However, not all peptide salts were fully soluble in this solvent so other alternatives (including more water in the dilution solvent), offering adequate dissolution but slightly inferior chromatographic performance should be chosen in such cases.