Direct, simultaneous measurement of liposome-encapsulated and released drugs in plasma by on-line SPE–SPE–HPLC

► Liposomal and released drug in plasma were separated by restricted-access media. ► During the extraction, drug release and/or leakage from liposomes was not observed. ► Liposomal and released drug were simultaneously measured by column-switching HPLC. A method for the simultaneous measurement of liposome-encapsulated and released drugs in mouse plasma by on-line solid phase extraction (SPE)–SPE–HPLC with direct plasma injection was developed using a doxorubicin (DXR)-containing liposome formulation as the model drug. During SPE, the released DXR was extracted on the 1st restricted-access media (RAM) SPE column, whereas the liposomes were eluted. The eluted liposomes were collapsed on-line, and the released DXR was delivered to the 2nd RAM SPE column for extraction. The retained DXR on the SPE columns was analyzed via HPLC–fluorescent detector by switching the valves. The method was validated and applied to the pharmacokinetic study of DXR in mice after intravenous injection of DXR-containing liposomes.