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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (3): 467-476. doi: 10.19723/j.issn.1671-167X.2019.03.014

• 论著 • 上一篇    下一篇

拉洛他赛的波谱解析及高效液相色谱法对其脂质体含量的测定

李雪琦1,李建伟1,2,3,4,李秋红1,4,阎妍1,段嘉伦1,崔一诺1,苏展博1,罗倩1,许佳瑞1,杜亚菲1,王桂玲1,谢英1,吕万良1△()   

  • 收稿日期:2019-03-14 出版日期:2019-06-18 发布日期:2019-06-26
  • 作者简介:吕万良,北京大学药学院教授、博士生导师,药学院党委副书记,分子药剂学与新释药系统北京市重点实验室副主任,中国药学会药剂专业委员会副主任委员,中国颗粒学会常务理事、生物颗粒专业委员会副主任委员,《北京大学学报(医学版)》、《中国药学杂志》、《中国新药杂志》、《中国药学(英文版)》、Asian Journal of Pharmaceutical Sciences、Precision Nanomedicine、BioMed Research International等国内外学术期刊编委。
    吕万良教授针对以乳腺癌、脑肿瘤、肺癌等为代表的肿瘤和肿瘤干细胞进行了持续性研究,在肿瘤细胞转运体、肿瘤干细胞、肿瘤线粒体及内源性耐药基因、乳腺癌拟态血管作用机制及其功能靶向性给药系统的研究方面取得了重要进展。作为课题负责人,先后主持了国家高技术研究发展计划(863计划)、国家重点基础研究发展计划(973计划)、国家自然科学基金、北京市自然科学基金重点项目等纵向课题20余项,获新药证书1项和临床批件4项。研究成果在Advanced Drug Delivery Reviews、Biomaterials、Journal of Controlled Release、Clinical Pharmacology & Therapeutics等学科顶尖学术期刊上发表论文160余篇,在国内外学术界产生了重要影响,有关论文在美国《科学引文索引》数据库(Science Citation Index,SCI)中被列为高被引论文。连续入选2014—2018年度 Elsevier 发布的在国际具有重要学术影响力的高被引中国学者榜单,研究成果分别获得了2009年度教育部自然科学奖一等奖(第1完成人)、2012年度教育部自然科学奖一等奖(第2完成人)、2018年度北京市科学技术奖二等奖(第2完成人)等。
  • 基金资助:
    国家“重大新药创制”科技重大专项基金2018ZX09301-018-004

Spectrometric analyses of larotaxel and larotaxel liposomes quantification by high performance liquid chromatography

Xue-qi LI1,Jian-wei LI1,2,3,4,Qiu-hong LI1,4,Yan YAN1,Jia-lun DUAN1,Yi-nuo CUI1,Zhan-bo SU1,Qian LUO1,Jia-rui XU1,Ya-fei DU1,Gui-ling WANG1,Ying XIE1,Wan-liang LU1△()   

  • Received:2019-03-14 Online:2019-06-18 Published:2019-06-26
  • Supported by:
    Supported by the National Key Grant for New Drug Innovation 2018ZX09301-018-004

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摘要： 目的 拉洛他赛是国内外均未上市的新结构药物,未见质量研究相关报道。对拉洛他赛进行波谱解析以验证其分子式、相对分子质量和化学结构式,同时建立一种定量方法用于拉洛他赛脂质体制剂的含量测定。方法 利用质谱、红外吸收光谱、核磁共振波谱测定方法,对拉洛他赛进行药物结构和光谱学解析;利用紫外-可见分光光度法对拉洛他赛进行全波长扫描,确定其吸收波长;利用高效液相色谱法,建立拉洛他赛定量方法并用于脂质体拉洛他赛包封率的测定。结果 揭示了拉洛他赛的四大光谱学特征并制订相应的标准图谱。确认了拉洛他赛的结构为三环二萜,分子式为C45H53NO14,相对分子质量为831.900 1。建立了拉洛他赛的高效液相色谱定量方法,其色谱柱为C18硅胶反相色谱柱 (5 μm, 250 mm×4.6 mm),流动相为乙腈-水(体积比75 ∶25),检测波长为230 nm,该方法可用于测定脂质体制剂中拉洛他赛的包封率,稳定性、回收率和精密度高。此外,新制备了拉洛他赛脂质体,该脂质体粒径大约105 nm,均一性良好,药物包封率大于80%。结论 制订了拉洛他赛的质谱、红外吸收光谱、核磁共振波谱和紫外-可见光谱图谱,验证了拉洛他赛的分子式、相对分子质量和结构式,建立了拉洛他赛的高效液相色谱定量方法,该方法可用于拉洛他赛脂质体的质量控制。

关键词: 拉洛他赛, 波谱解析, 高效液相色谱法

Abstract: Objective: Larotaxel is a new chemical structure drug, which has not been marketed worldwide. Accordingly, the standard identification and quantification methods for larotaxel remain unclear. The spectrometric analyses were performed for verifying weight molecular formula, molecular weight and chemical structure of larotaxel. Besides, a quantification method was developed for measuring larotaxel in the liposomes.Methods: The molecular formula, molecular weight and chemical structure of larotaxel were studied by using mass spectrometry (MS), infra-red (IR), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis) spectrometric techniques. The absorption wavelength of larotaxel was investigated by UV-vis spectrophotometry full-wavelength scanning. Besides, a quantification method was developed by high performance liquid chromatography (HPLC), and then validated by measuring the encapsulation efficacy of larotaxel liposomes. Results: The four spectral characteristics of larotaxel were revealed and the corresponding standard spectra were defined. It was confirmed that larotaxel had the structure of tricyclic diterpenoids, with the molecular formula of C45H53NO14, the molecular weight of 831.900 1, and the maximum absorption wavelength of 230 nm. The quantitative method of larotaxel was established by using HPLC with a reversed phase C18 column (5 μm, 250 mm×4.6 mm), a mobile phase of acetonitrile-water (75 ∶25, volume/volume), and a detection wavelength of 230 nm. The validation study exhibited that the established HPLC method was stable, and had a high recovery and precision in the quantitative measurement of larotaxel in liposomes. In addition, a new kind of larotaxel liposomes was also successfully prepared. The particle size of the liposomes was about 105 nm, with an even size distribution. And the encapsulation efficiency of larotaxel in the liposomes was above 80%.Conclusion: The present study offers reference standard spectra of larotaxel, including MS, IR, NMR, and UV-vis, and confirms the molecular formula, molecular weight and chemical structure of larotaxel. Besides, the study develops a rapid HPLC method for quality control of larotaxel liposomes.

Key words: Larotaxel, Spectrometric analysis, High performance liquid chromatography

中图分类号: 

  • R927

图1

拉洛他赛的高分辨飞行时间质谱图"

图2

拉洛他赛的红外吸收光谱"

图3

拉洛他赛的核磁共振碳谱及氢谱"

图4

拉洛他赛的二维核磁共振谱"

图5

拉洛他赛的结构式和相对分子质量"

表1

拉洛他赛的13C NMR和1H NMR波谱解析数据"

Positon Larotaxel
13C 1H
1 79.40
2 80.06 5.68 (1H, d, J=7.7 Hz)
3 38.56 4.07 (1H, d, J=7.7 Hz)
4 79.60
5 84.86 4.75 (1H, d, J=3.3 Hz)
6 26.06 2.46 (1H, dt, J=16.1, 4.4 Hz)
7 32.04 1.38 (1H, m)
8 35.12
9 201.79
10 75.69 6.35 (1H, s)
11 133.63
12 140.40
13 72.17 6.29 (1H, t, J=8.4 Hz)
14 35.86 2.40 (1H, m) 2.23 (1H, m)
15 42.92
16 26.06 1.28 (3H, s)
17 21.48 1.30 (3H, s)
18 14.59 1.87 (3H, s)
19 15.63 2.25 (1H, m)
1.66 (1H, t, J=5.8 Hz)
20 75.43 4.32 (1H, d, J=8.8 Hz)
4.03 (1H, d, J=8.8 Hz)
1' 172.80
2' 73.74 4.63 (1H,br.s)
3' 56.00 5.32 (1H, d, J=8.8 Hz)
4' 138.50
5', 9' 126.61 7.39 (2H, d, J=7.0 Hz)
6', 8' 128.88 7.35 (2H, t, J=7.0 Hz)
7' 128.04 7.33 (1H, t, J=7.0 Hz)
10' 155.20
11' 80.10
11'-(CH3)3 28.12 1.28 (9H, s)
1'' 167.49
2'' 129.26
3'',7'' 130.30 8.16 (2H, d, J=7.7 Hz)
4'',6'' 128.73 7.51 (2H, t, J=7.7 Hz)
5'' 133.63 7.61 (1H, t, J=7.7 Hz)
4-OAc 22.26, 169.70 2.45 (3H, s)
10-OAc 20.87, 169.64 2.27 (3H, s)
1-OH 2.07 (1H, s)
2'-OH 3.31 (1H,br.s)
3'-NH 5.39 (1H, br.d,
J=9.5 Hz)

图6

拉洛他赛甲醇溶液(250 mg/L)在200~800 nm 波长范围内的紫外吸收扫描光谱"

图7

高效液相-紫外色谱法测定拉洛他赛的浓度与色谱峰面积相关曲线及典型色谱图"

表2

拉洛他赛甲醇溶液HPLC-UV测定时的稳定性"

Concentration/(mg/L) Peak area/(mAU·min)
0 h 2 h 4 h 6 h 8 h RSD/%
5.0 59.1±0.0 59.1±0.1 59.1±0.0 59.1±0.1 59.1±0.1 1.2
10.0 118.2±0.1 118.2±0.1 118.2±0.0 118.2±0.1 118.2±0.2 1.9
25.0 295.5±0.2 295.5±0.1 295.5±0.1 295.5±0.2 295.5±0.1 1.7

表3

拉洛他赛对照品及脂质体中用高效液相-紫外色谱法进行拉洛他赛含量测定的回收率"

Substances Concentration/(mg/L) Recovery/% RSD/%
Larotaxel1
 5.0 99.62±0.39 0.51
10.0 99.17±0.10 0.33
25.0 101.34±0.07 0.16
Larotaxel2
 5.0 98.07±0.93 1.21
10.0 99.61±0.06 0.03
25.0 102.68±0.15 0.14

表4

拉洛他赛对照品及脂质体中用高效液相-紫外色谱法进行拉洛他赛含量测定的精密度"

Concentration added/(mg/L) Intra-day (n=5) Inter-day (n=5)
Precision/% RSD/% Precision/% RSD/%
2.01 99.23±0.49 1.26 98.00±1.05 1.37
10.01 99.69±0.22 0.31 98.02±2.76 1.20
50.01 98.89±0.43 0.25 98.32±2.03 0.75
2.02 98.41±1.49 1.43 98.03±2.03 1.73
10.02 99.70±0.83 0.09 98.11±1.76 1.02
50.02 99.64±0.33 0.12 98.23±1.53 1.39

图8

拉洛他赛脂质体的透射电镜图像"

表5

拉洛他赛脂质体的理化表征"

Formulations Particle size/nm PDI Zeta potential/mV Encapsulation efficiency/%
Blank liposomes 82.13±1.94 0.131±0.032 0.25±0.01
Larotaxel liposomes 105.73±2.30 0.190±0.072 0.24±0.02 87.73±2.70
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