摘要
由于聚山梨酯80的组分复杂,分析耗时耗力,亟需寻找一种能够快速分析聚山梨酯80组分的方法。本研究采用ExcipientProfiler药用辅料智能分析系统,通过UHPLC-HRMS对收集到的国内外3家企业共10批样品进行分析,同时利用扩展数据库对分析结果进行补充解析。分析结果显示,ExcipientProfiler软件能够快速识别质谱图中加合了N
聚山梨酯80(polysorbate 80,PS80),商品名为吐温80,化学名为聚氧乙烯20山梨醇酐单油酸酯,结构见

Figure 1 Structure of polysorbate 80
在工业上,合成PS80主要有两种方法:一是先酯后醚,二是先醚后
但是有研究发现,PS80中含有的PIM组分具有强烈的致肥大细胞脱颗粒的作
现今,用于表征PS80组分的方法手段有很多,例如高效液相色谱结合蒸发光散射检测器(HPLC-ELSD
聚山梨酯80(南京威尔药业集团股份有限公司提供,A1批号20210304-1、A2批号20190405、A3批号20210401、A4批号20180606、A5批号20190527;上海卡乐康包衣技术有限公司提供,B1批号2020020001、B2批号2020040002、B3批号2020110003;赛诺菲制药有限公司提供,C1批号AA00144-55641、C2批号CA01016-63296,除A1和A2为注射级,其余均为普通级。其中南京威尔药业的样品合成过程为先醚后
甲酸(FA,美国ACS恩科化学)、乙腈(ACN,德国默克公司)均为质谱级;无水乙酸钠(色谱纯,上海阿拉丁试剂有限公司);实验用水为屈臣氏饮用水。
超高效液相色谱-高分辨质谱联用仪:Agilent 1290超高效液相色谱、Agilent 6546 Q-TOF质谱(美国安捷伦公司);IKA MS 3 Digital涡旋混匀器(德国艾卡公司)。ExcipientProfiler药用辅料智能分析系统(科迈恩(北京)科技有限公司,软件PS80库中结合脂肪酸的组分仅为聚氧乙烯山梨醇酐油酸酯类);Agilent MassHunter PCDL Manager PS80相关扩展数据库(安捷伦公司提供,该扩展数据库中结合脂肪酸的组分包含了聚氧乙烯山梨醇酐油酸酯类、聚氧乙烯山梨醇酐月桂酸酯类、聚氧乙烯山梨醇酐棕榈酸酯类和聚氧乙烯山梨醇酐硬脂酸酯类组分)。
参照文献以及ExcipientProfiler药用辅料智能分析系统的SOP拟定了以下实验条
色谱柱:Waters Acquity UPLC Protein BEH C4柱(2.1 mm × 100 mm, 1.7 μm);以0.1%甲酸水(含5 μmol乙酸钠)为流动相A,以乙腈为流动相B,进行梯度洗脱:0 ~ 15 min, 10% ~ 100% B;15 ~ 18 min,100% B;流速为0.4 mL/min;柱温:35 ℃;进样量:5 μL。
根据ExcipientProfiler软件中的SOP设置参数:以[PSM(n = 20) + Na

Figure 2 Total ion chromatogram of A1 (I-1), A3 (II-1), B1 (III-1) and C2 (IV-1); and component distribution map of A1 obtained by the ExcipientProfiler software (I-2), A3 (II-2), B1 (III-2) and C2 (IV-2)
*The fatty acids combined with PSM, PID, PM, PSD, PID, PD and PSTri in the table were oleic acid -: Not detected PEG: Polyethylene glycol; PS: Polyoxyethylene sorbitan; PI: Polyoxyethylene isosorbitan; PSM: Polyoxyethylene sorbitan monoleate; PIM: Polyoxyethylene isosorbitan monoleate; PM: Polyoxyethylene monoleate; PSD: Polyoxyethylene sorbitan dioleate; PID:Polyoxyethylene isosorbitan dioleate; PD: Polyoxyethylene dioleate; PSTri: Polyoxyethylene sorbitan trioleate
以样品A1、A3、B1、C2为例,PS80中PEG、PS、PI这3类组分的保留时间为2.50 ~ 4.50 min,PSM、PIM、PM这3类组分主要的保留时间为8.50 ~ 10.00 min,PSD、PID、PD这3类组分的保留时间则分布在11.75 ~ 13.50 min内,而PSTri组分的保留时间为13.50 ~ 15.50 min,详见
比较样品中各个组分不同聚合度化合物的数量发现,每批样品中PSM、PIM、PM这3类组分的组分个数均为最多,且这3类组分的组分数在注射级样品中占总个数约为70%,普通级样品中占总个数55% ~ 60%,即结合了一油酸酯的组分的组分数较多。从PS80的结构来看,这是因为已经结合了1分子油酸的聚氧乙烯侧链具有一定的空间位阻;同时由于结合一油酸酯的组分表面活性较强,争夺电荷的能力强,质谱响应高能被检出,所以结合二油酸酯和三油酸酯的组分组分数较低。但是注射级样品(A1、A2)较普通级样品组分总个数少,具体数据见
通过ExcipientProfiler软件对质谱结果的解析也能得到PS80样品各组分侧链中PEG的聚合度n(n = x + y + z + w)。经过统计,10批样品中PEG组分的聚合度n可能在13 ~ 34间,PS组分n = 9 ~ 39,PI组分n = 19 ~ 41,PSM组分n = 1 ~ 60,PIM组分n = 1 ~ 60,PM组分n = 1 ~ 60,PSD组分n = 4 ~ 25,PID组分n = 3 ~ 59,PD组分n = 4 ~ 60,PSTri组分n = 10 ~ 49。由此可见,PS80的单一组分中有不同聚合度的化合物,且各组分间的聚合度也不相同,因此对10批样品中各组分的聚合度进行整理归纳,聚合度以中位数表示,详见
*The fatty acids combined with PSM, PID, PM, PSD, PID, PD and PSTri in the table were oleic acid -: Not detected
本研究所用为安捷伦提供的PS80扩展数据库(Agilent database),该扩展数据库的构建原理与ExcipientProfiler软件中PS80库相同,均是依据文献中PS80各组分聚合度与保留时间之间存在的关系来建立数学模
因此利用数据库对PS80组分进一步分析发现,这10批样品中不单单含有聚氧乙烯山梨醇酐油酸酯类组分,还含有聚氧乙烯山梨醇酐其他脂肪酸酯类组分。注射级样品中含有结合了硬脂酸的PSM组分(PSM-stearate)、结合了棕榈酸的PIM和PM组分(PIM-palmitate、PM-palmitate)以及结合了月桂酸的PSTri组分(PSTri-laurate)。除了上述组分,普通级样品中还包含了结合月桂酸和硬脂酸的PIM组分(PIM-laurate、PIM-stearate)与PM组分(PM-laurate、PM-stearate),结合月桂酸和棕榈酸的PSD组分(PSD-laurate、PSD-palmitate),以及结合棕榈酸的PID组分(PID-palmitate)。说明油酸原料中不仅含有油酸,还有其他脂肪酸的存在,因此在PS80中具有一定量的其他脂肪酸酯类组分。此外,注射级样品的其他脂肪酸酯类组分数较普通级样品少(见
-: Not detected
ExcipientProfiler软件中SOP的质谱参数是依据Agilent 6550 iFunnel Q-TOF来设定的,而本研究采用的是Agilent 6546 Q-TOF,两者仪器硬件具有差异,故需要优化参数。通过改变干燥气流速、干燥气温度、鞘气流速、鞘气温度、雾化气压力以及碎裂电压的参数,来提高其离子响应。此外,由于本系统中[M+NH4
采用SPSS 26.0版软件,对ExcipientProfiler软件解析的结果进行聚类分析,以

Figure 3 Dendrogram of cluster analysis of results from different methodsA: Results obtained by the ExcipientProfiler software; B: Results obtained by the Agilent database
由于ExcipientProfiler软件的PS80库中仅包含聚氧乙烯山梨醇酐油酸酯类组分,没有收录聚氧乙烯山梨醇酐其他脂肪酸酯类组分,此外,现行版ExcipientProfiler软件仅能识别到加合了N
由于ExcipientProfiler软件和扩展数据库中均未收录PSTetra组分的相关信息,故不能确定PS80中是否含有四酯结构,因此,本研究在此基础上对得到的质谱图进一步分析,以A1样品为例,其PSTetra组分在14.83 ~ 15.28 min洗脱出峰,其EIC图见

Figure 4 EIC mass spectrum of PSTetra component peak of A1 sampleA: High m/z; B: Low m/z
将油酸相对分子质量以282.255 6计,PEG单体相对分子质量以44.026 2计,一失水山梨醇相对分子质量以164.068 5计,H2O的相对分子质量以18.010 6计,NH
本研究采用ExcipientProfiler软件及PS80相关扩展数据库对收集到的10批不同厂家PS80进行组分分析,并对组分数结果采用系统聚类分析,证明ExcipientProfiler软件能够快速便捷地分析PS80组分,具有能用来区分不同级别样品的优势。
但后续通过扩展数据库查找和质谱解析发现,PS80中还存在四酯组分和其他脂肪酸酯类组分,而ExcipientProfiler软件中缺少这些信息,故需要在扩展数据库的辅助下对PS80组分来进一步分析。因此,ExcipientProfiler软件后续需要经过进一步的实验验证来完善其包含的信息库,以便能更准确地应用于PS80的组分分析。
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