Development Of Methodology For Quality Control Of Oxaliplatin Long Circulating Thermo-sensitive Liposomes

Anticancer agent oxaliplatin has great advantages in tumor treatment; but its use in clinic is limited by its severe side effects. In order to increase therapeutic effect and reduce toxicity, our laboratory has entrapped oxaliplatin into Long-circulating Thermosensitive Liposomes (LTL) to form Oxaliplatin Long-circulating Thermosensitive Liposomes (OLTL). There were few criterions or literature about the quality control of LTL at present. In this study, we developed methods for determining oxaliplatin content, Lyso-phosphatidyl Choline (LPC) content, impurities, entrapment efficiency and drug release to evaluate the quality of OLTL,First, High Performance Liquid Chromatography (HPLC) was used to quantitate the oxaliplatin content and LPC content of OLTL. The oxaliplatin content analysis was performed on Megress C18column (4.6mm×250mm,5μm), using a mobile phase consisting water-methanol (96:4v/v) with the flow rate of1.0mL-mL"’under UV detection at210nm. We found that the calibration curve of oxaliplatin was linear in the range of50.24-150.72μg-mL-1(r=0.9993) and mean Recovery was99.97%. Repeatability and intermediate precision were0.85%and0.92%respectively. The LPC content analysis was carried on Venusil XBP-C8(4.6mm X250mm,5μm), using a mobile phase consisting of water/methanol with Charged Aerosol Detection (CAD) at the flow rate of1mL-min-1. In the methodology validation, the calibration curve of LPC was linear in the range of15.06-45.18μg·mL-1(r=0.9993) and mean Recovery was100.18%, Repeatability was0.53%.Then, Impurity A, impurity B, impurity C and other impurities were also quantitive using HPLC. Impurity A was analysized on Hypersil ODS2C18column(4.6mm X250mm,5μm), using mobile phase consisting of the buffer (0.01mol·L-1potassium dihydrogen phosphate with0.001mol·L-1Tetrabutylammonium Hydroxide, pH6.0)-acetonitrile(85:15v/v) with UV detection at205nm at the flow rate of2.0mL·min-1; impurity B analysis was carried on ZORBAX Eclipse Plus C18(4.6mm×250mm,5μm), using a mobile phase consisting of the buffer (0.01mol·L-1potassium dihydrogen phosphate with0.005mol·L-1sodium heptanesulphonate, pH3.0)-acetonitrile (15:85, v/v) with UV detection at215nm at the flow rate of2.0mL·min-1; impurity C and other impurities analysis method was the same of oxaliplatin content determination method. Though the monitor of the preparation process of OLTL, we known buffer solution could accelerate the hydrolyze of oxaliplatin, lead to the amount of impurity A and impurity B increase; during the ultrasound stage, ultrasound would increase the amount of impurity C; in the isotropic stage, the amount of total impurities increase when through the100nm membrane.Thirdly, the mini-column centrifuge method was employed to evaluate the entrapment efficiency of OLTL. The height of mini-column was2.0cm; the leaching solution was phosphoric acid (pH6.5) and the rotate speed was1500rpm in the fractions (1-6tubes) and pH6.5phosphoric acid-methanol (95:5, v/v) and the rotate speed was2000rpm in the fractions (7-16tubes). In the methodology validation process, the calibration curve of oxaliplatin was linear in the range of1.01～50.06μg·mL-1(r=0.999) and mean recovery was99.97%. Repeatability and intermediate precision were1.55%,0.62%and1.42%,0.92%respectively.At last, The release rate of the drug from liposomes at37℃and41.5℃were studied in vitro. Only5%drug release from the liposomes in6h at37℃, however,58%drug release from the liposomes in30minutes at41.5℃in vitro.Through the methodology study above, we built the OLTL quality criteria protocol and these analysis methods appeared to be selective, precise and accurate which could be used for the quality control of OLTL.