Determination of minor conformational changes of a doxorubicin–peptide conjugate under chromatographic conditions

Thermodynamic analysis of the reversed-phase retention behavior of a doxorubicin–peptide conjugate demonstrated that the degree of non-linearity observed in Van’t Hoff plots was impacted by mobile phase acetonitrile content over the 25–38% acetonitrile (v/v) range tested. Small decreases in the non-polar surface area of the doxorubicin–peptide conjugate as a function of temperature were estimated from these data using linear solvent strength relationships, suggesting that the retention behavior may be the result of minor analyte conformational changes during the chromatographic experiment. This hypothesis was supported via circular dichroism (CD), Raman and 1H NMR spectroscopic studies of the doxorubicin–peptide conjugate in selected chromatographic mobile phase compositions. The CD and Raman data indicated small changes to the apparent analyte microenvironment as a function of temperature and bulk solvent environment, while 1H NMR studies specifically demonstrated the environmental sensitivity of protons on three non-polar peptide residues and the proximal aromatic region of the analyte. Together, these data suggest that minor changes to the conformational order of the essentially random structure of the doxorubicin–peptide conjugate are sufficient to impact chromatographic performance.