In this study, a porous mixed-mode n-alkyl methacrylate-based monolith has been used in the separation of therapeutic peptides. While the sulfonic acid (SCX) moiety derived from 2-acrylamido-2-methyl-1-propanesulfonic acid supports the generation of a stable electroosmotic flow (EOF) at both acidic and basic pH values, the butyl ligands provide the nonpolar sites for chromatographic resolution. The performance of the monolith was evaluated regarding the influence of pH on chromatographic resolution of peptides. The suitability of the butylmethacrylate/SCX monolith for the analysis of therapeutic peptides containing basic centres, for example arginine, at moderately high pH 9.5 and the stability to repeat injections of a mixture of peptides was demonstrated. Separations with efficiencies as high as 5.0 x 10(5) plates/m were obtained and the migration behaviour of the peptides at both low (2.8) and high (9.5) pH values could be rationalised based on their charge, molecular mass/shape and relative hydrophobicities.

CE and hydrogen-deuterium (H/D) exchange MS are useful tools in the analysis and characterisation of peptides. This study reports the facile coupling of these tools in the H/D exchange CE-MS analysis of model and pharmaceutically important peptides, using a sheath flow interface. The peptides varied in mass from 556 (leucine enkephalin) to 1620 Da (bombesin), and in charge state from 0.33 (leucine enkephalin) to 3.0 (substance P). The application of a BGE composed of ammonium formate buffer (25 mM, pD 3.5 in D(2)O (>98% D atom)), a sheath liquid composed of formic acid (0.25% v/v in D(2)O) and ACN (30:70 v/v), and dissolving the samples in a mixture of ACN/D(2)O (50:50 v/v) facilitates complete H/D exchange. Because of complete H/D exchange the ESI mass spectra produced are easy to interpret and comparable to those obtained from LC-MS analysis. The CE-H/D-MS approach has the advantage of requiring lower volumes of deuterated solvents. The b- and y-series fragments produced by using in-source decomposition correspond to those predicted. With the peptides studied, the complete exchange H/D exchange observed with both the molecular and fragment ions helps to confirm both amino acid composition and sequence.