Formulation Research Laboratories, Pharmaceutical Science and Technology Core Function Unit, Medicine Development Center, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki, 300-2635, Japan.
GL Sciences Inc., 237-2 Sayamagahara, Iruma, Saitama, 358-0032, Japan.
Int J Pharm. 2018 Jan 30;536(1):21-28. doi: 10.1016/j.ijpharm.2017.11.035. Epub 2017 Nov 24.
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 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 μL 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.
建立了一种使用纳米粒子排除色谱法（nPEC）测定聚乙二醇化脂质体中阿霉素（DOX）包封效率（EE）的简单快速色谱测量方法。在这项工作中，使用 Doxil 和两种用 DOX 标记的 PEG 化脂质体作为模型脂质体，未包裹的 DOX 通过高效液相色谱-二极管阵列检测器进行测量，使用 N-乙烯基吡咯烷酮修饰的 nPEC 柱，无需任何样品预处理。只需 5μL 完整的脂质体悬浮液和 3 分钟的分析时间即可测定未包裹的 DOX 量。该方法在 0.00-1.0mg/mL 范围内（对应 100-50% EE）进行了线性、准确性、精密度和回收率验证。使用使用硫酸铵梯度的远程加载方法测定脂质体中 DOX 加载%的时间依赖性，验证了该方法的适用性。此外，还发现色谱图中负载 DOX 的脂质体的峰面积与 DOX EE%成正比。由于这种简单快速的分析方法可以精确测量 EE，因此预计该方法将适用于脂质体制备制造过程中的过程控制和脂质体药物产品的质量控制。
