Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet-visible high-performance liquid chromatography

Abstract

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C(16) column (250 x 3 mm I.D., 5 microm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV-visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 microg/mL, with intra-day and inter-day coefficients of variation and percent error < or =10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.

Fig. 1

Chemical structures of DOX, DOXol, and DNR. (A) R1, doxorubicin; (B) R1, doxorubicinol; (C) R1, daunorubicin.

Fig. 2

Typical chromatogram following extraction of LDOX from human plasma. Conditions described in Section 2.2. (A) Retention times for LDOX and DNR are 4.8 and 10.1 min, respectively. (B) Blank plasma control injection.

Fig. 3

Typical chromatogram following extraction of DOX from human plasma. Conditions described in Section 2.2. (A) Retention times for DOX and DNR are 7.2 and 13.9 min, respectively. (B) Blank plasma standard extracted for control.

Fig. 4

Typical chromatogram following extraction of DOX and its major metabolite, DOXol, from human liver microsome metabolism studies. Conditions described in Section 2.2. (A) LDOX incubated with human liver microsomes in the presence of NADPH. Retention times for DOXol and LDOX are 8.8 and 12.9 min, respectively. (B) LDOX incubated under previous conditions without NADPH for control. LDOX retention time is 12.9 min.

Fig. 5

Concentration-time profiles for seven patients receiving single agent LDOX therapy. All patients received 60 mg/m² LDOX intravenously as a 2-h infusion. Concentrations of DOXol were below the LOQ and are not reported.