Determination of a peptide–doxorubicin, prostate-specific antigen activated prodrug, and its active metabolites in human plasma using high-performance liquid chromatography with fluorescence detection: Stabilization of the peptide prodrug with EDTA

A method for the determination of I, a peptide–doxorubicin conjugate that was evaluated for the treatment of prostate cancer, and two of its active metabolites, doxorubicin and leucine–doxorubicin is described. Blood samples were chilled immediately after being drawn in order to prevent ex vivo entry of the metabolites into red blood cells. EDTA (10 mg/ml final concentration) was used to prevent plasma-mediated degradation of the peptide portion of the prodrug. After the addition of internal standard, plasma was prepared for analysis using a C-8 solid-phase extraction column. In order to overcome secondary ionic interactions with the silica-based extraction column, the analytes were eluted with ammonium hydroxide in methanol. The extracts were evaporated to dryness, reconstituted, and assayed by step change, gradient, reverse phase HPLC with fluorescence detection. Two interfering metabolites found in post dose plasma were chromatographically separated by an adjustment of the mobile phase pH. The within-day reproducibility of the doxorubicin and leucine–doxorubicin chromatographic retention times was improved by a brief washing of the analytical column with 90% acetonitrile after each injection. The range of the standard curve was 12.5–1250 ng/ml for doxorubicin and 25–2500 ng/ml for I and leucine–doxorubicin.