2D-LC-QTOF_MS识别氨基葡萄糖液相含量测定方法中可疑色谱峰

目的：采用在线二维液相色谱-四极杆飞行时间质谱法（2D-LC-QTOF MS）对进口药品注册标准JX20030178中氨基葡萄糖液相含量测定法测定的3个可疑色谱峰进行结构分析。方法：第一维色谱采用Inertsil C₈-3色谱柱（250 mm×4.6 mm，5 μm），流动相为磷酸盐缓冲液（pH 2.5）-乙腈（80：20），流速0.5 mL·min^-1，检测波长195 nm，柱温30℃；第二维色谱采用Poroshell120 EC-C₁₈色谱柱（100 mm×3.0 mm，2.7 μm），0.1%（v/v）甲酸水溶液和0.1%（v/v）甲酸乙腈溶液梯度洗脱，流速0.3 mL·min^-1，柱温30℃。当第一维HPLC目标组分出峰时，通过阀切换将目标组分准确切割并捕获在40 μL定量环中，之后第二维HPLC泵将捕获的组分洗脱进行质谱检测。采用正负离子模式采集数据，对第一维谱图中3个色谱峰进行了结构分析。结果：第一维氨基葡萄糖液相含量测定法测定的3个色谱峰分别为氨基葡萄糖、硫酸根离子和氯离子，其中主峰为氯离子，而非活性组分氨基葡萄糖。结论：本实验建立的2D-LC-QTOF MS能用于氨基葡萄糖液相含量测定法测定的3个可疑色谱峰的结构分析。进口药品注册标准JX20030178中氨基葡萄糖液相含量测定法专属性差，该标准亟需改进和提高。

Objective:To develop a two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry(2D-LC-QTOF MS)method for detecting three peaks in HPLC of the content of glucosamine,which follows the drug registration standards JX20030178.Methods:An Inertsil C₈-3 column(250 mm×4.6 mm,5 mm)was used in the first dimensional chromatography,using phosphate buffer(pH 2.5)and acetonitrile(80:20)as the mobile phase with a flow rate at 0.5 mL·min^-1.The column temperature was maintained at 30℃ with a detection wavelength of 195 nm.A Poroshell 120 EC-C₁₈ column(100 mm×3.0 mm,2.7 μm)was used in the second dimensional chromatography.Gradient elution was applied using water containing 0.1%(v/v)formic acid and acetonitrile containing 0.1%(v/v)formic acid as the mobile phase,and the flow rate was 0.3 mL·min^-1.The column temperature was maintained at 30℃.The target component from the first dimensional LC was trapped in a 40 μL loop via switching valve,and then was eluted using an MS compatible mobile phase with a pump at the second dimensional HPLC for MS detection.The data was collected in positive and negative ion mode.The molecular formulas were determined by their exact masses and isotope distributions.And the structures in 3 peaks at the first dimensional chromatography were studied.Results:Three chromatographic peaks in the first dimensional HPLC of glucosamine were glucosamine,SO₄^2- and Cl_-,and the main peak was Cl_- instead of the active component(glucosamine).Conclusion:The established 2D-LC-QTOF MS method can be used for profiling three suspicious peaks in HPLC of glucosamine.The specificity of glucosamine HPLC method(the drug registration standards JX20030178)is poor,which needs to be improved.