目的: 采用高效液相色谱(HPLC)、超高效液相色谱-四极杆串联静电场轨道阱高分辨质谱(UPLC-Q Orbitrap MS)技术对醋酸阿托西班注射液中杂质结构进行鉴定。方法: HPLC法采用Inertsil ODS-2 C18色谱柱(250 mm×4.6 mm,5 μm),流动相A为乙腈-甲醇-三氟乙酸溶液(pH 3.2)(15∶10∶75),流动相B为乙腈-甲醇(60∶40),梯度洗脱,流速1.2 mL·min-1,检测波长220 nm。UPLC-Q Orbitrap MS法采用BEH300 C18色谱柱(150 mm×2.1 mm,1.7 μm),流动相为0.1%甲酸水溶液(A)-0.1%甲酸乙腈溶液(B),梯度洗脱,流速0.2 mL·min-1;采用电喷雾离子源,选择正离子模式进行Full MS/dd-MS2扫描。结果: 对5家企业各1批醋酸阿托西班注射液进行了有关物质测定,以面积归一化计算,含量>0.1%的杂质峰个数分别为9、10、13、9和6,总杂含量分别为0.35%、0.63%、0.63%、0.32%和0.18%;对其中杂质个数较多的2家企业样品中的杂质峰进行了馏分收集和结构鉴定,其中1个企业样品中收集了9个杂质馏分,共鉴定到12个杂质,另外1个企业样品中收集了13个杂质馏分,共鉴定到15个杂质。鉴定到的杂质种类主要包括缺失肽、插入肽、错接肽、修饰肽等。结论: 采用液质联用技术可以使醋酸阿托西班注射液中的杂质结构得到有效鉴定,杂质结构的明确有助于各生产企业分析其产品中各种杂质产生的来源。
Objective: To identify the structure of impurities in atosiban acetate injection using high performance liquid chromatography(HPLC), ultra-high performance liquid chromatography coupled with quadrupole tandem orbitrap mass spectrometry(UPLC-Q Orbitrap MS). Methods: The HPLC analysis was performed on an Inertsil ODS-2 C18(250 mm×4.6 mm, 5 μm) column with the mobile phase A consisted of acetonitrile-methanol-trifluoroacetic acid solution(pH 3.2) (15∶10∶75) and mobile phase B consisted of acetonitrile-methanol (60∶40), with gradient elution at a flow rate of 1.2 mL·min-1, and the detection wavelength was 220 nm. The UPLC Q-Orbitrap MS analysis was performed on a BEH300 C18(150 mm×2.1 mm, 1.7 μm) column with 0.1% formic acid solution as mobile phase A and 0.1% formic acid-acetonitrile solution as mobile phase B, with gradient elution at a flow rate of 0.2 mL·min-1. Samples were analyzed by electrospray ionization source with positive ion mode and Full MS/dd-MS2 scan mode. Results: 5 batches of atosiban acetate injection produced by five different manufactures were analyzed of related substances by area normalization method. The number of impurity peaks with content >0.1% was 9, 10, 13, 9 and 6, respectively, and the total impurity content was as follows: 0.35%, 0.63%, 0.63%, 0.32% and 0.18%. The impurity peaks in the samples of two enterprises with a large number of impurities were collected and identified. Among them, 9 impurity distillate were collected in the sample of one enterprise, and the structures of 12 impurities were identified, 13 impurity distillate were collected and in the sample of another enterprise, and the structures of 15 impurities were identified.The identified impurities mainly included deletion, insertion, misconnection, modification of peptide and so on. Conclusion: The impurity structure in atosiban acetate injection can be effectively identified by using UPLC-MS, the determination of impurity structure is helpful for the manufacturers to analyze the origin of their product's various impurity occurrence.
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