Abstract: OBJECTIVE To determine encapsulation rate of irinotecan hydrochloride and 5-fluorouracil in compound liposome injection. METHODS The encapsulation rate was determined by sephadex column chromatography method with the pH 6.5 phosphate buffer solution as eluent. The sephadex column was 400 mm and the eluent flow rate was 1.0 mL·min^-1. Under the established HPLC-DAD chromatographic condition, it was used with Kromasil-C₁₈(250 mm×4.6 mm, 5 μm) by methanol-pH 6.6 phosphate buffer(5:95) as mobile phase A and methanol-pH 6.6 phosphate buffer(60:40) as mobile phase B. The column temperate was kept at 35℃. The flow rate was 1.0 mL·min^-1. The detector wavelength were set at 255, 268 nm, respectively. RESULTS The encapsulation drug and free drug could be separated effectively in compound liposome injection by this way. The recoveries of 3 concentrations of sephadex column for irinotecan hydrochloride were 98.9%, 100.5% and 100.4%, and for 5-fluorouracil were 98.2%, 100.5% and 100.2%. Under the applied chromatographic condition, irinotecan hydrochloride and 5-fluorouracil were separated from compound liposome injection. The calibration curves were linear in the range of 10.7-107.0 μg·mL^-1 for irinotecan hydrochloride(r=0.999 9, n=5) and 3.9-38.6 μg·mL^-1 for 5-fluorouracil(r=1.000 0, n=5). The average recoveries for irinotecan hydrochloride were 99.7%, 99.0% and 99.8%, and RSD were 0.20%, 0.48% and 1.38%. The average recoveries for 5-fluorouracil were 100.3%, 100.6% and 99.8%, and RSD were 0.72%, 0.09% and 0.67%. CONCLUSION The method is simple, accurate and reproducible. It can be used for the determination of encapsulation rate of irinotecan hydrochloride and 5-fluorouracil in compound liposome injection.