Investigations into the chromatographic behavior of a doxorubicin–peptide conjugate

HPLC impurity profile method development for a doxorubicin–heptapeptide conjugate included significant changes of the separation profile with diluent, eluent and pH. These separation variables were also temperature-dependent with a shift in retention from 35 to 45 °C. There was also a direct relationship of temperature with LC retention, and a pH minimum at 5.9. Atypical dependence of the impurity profile on diluent at a k′ of 18 led to further investigation. A large change in retention by several minutes was a function of both the organic eluent composition and temperature between 15 and 30 °C. Several Van’t Hoff temperature studies from 5 to 65 °C on several column types resulted in non-linear plots. Analysis of the molecular subunits suggested that the peptide portion of the analyte influenced the non-linear retention behavior. The stationary phase type was not a significant factor causing non-linearity. Circular dichroism–temperature studies indicated a notable transition in ellipticity for the amine regions (198–202 nm) that occurred between 39 and 44 °C. This transition temperature range coincided with the results of the Van’t Hoff analysis, between 35 and 44 °C, to indicate that these effects were not primarily stationary phase induced.