A simple and rapid measurement method of encapsulation efficiency of doxorubicin loaded liposomes by direct injection of the liposomal suspension to liquid chromatography

A simple and rapid chromatographic measurement method for determining doxorubicin (DOX) encapsulation efficiency (EE) into PEGylated liposomes using nanoparticle exclusion chromatography (nPEC) was developed. In this work, Doxil (R) and two PEGylated liposomes spiked with DOX were employed as model liposomes, and unencapsulated DOX was measured by high performance liquid chromatography with diode-array detector using an N-vinylpyrrolidone modified nPEC column without any sample pretreatment. Only 5 mu L of an intact liposomal suspension and 3 min analysis time were required for the determination of the quantity of unencapsulated DOX. The method was validated in terms of linearity, accuracy, precision, and recovery in the range from 0.00-1.0 mg/mL (corresponding to 100-50% EE). Applicability of the method was confirmed using the measurement of time-dependent DOX loading% into the liposomes with the remote loading method using an ammonium sulfate gradient. Furthermore, it was found that the peak area of DOX-loaded liposomes in the chromatogram was proportional to DOX EE%. As this simple and rapid analytical method can measure the EE precisely, it is expected that this method will be applicable to the in-process control of liposome preparation manufacturing and the quality control of the liposome drug products.