基于UFLC-QTrap-MS_MS结合多元统计分析的不同产地、不同部位乌药中核苷和氨基酸类成分分析.pdf

第45卷第2 期2024年3月质谱学报Journal of Chinese Mass Spectrometry SocietyVol.45No.2Mar.2024基于 UFLC-QTrap-MS/MS 结合多元统计分析的不同产地、不同部位乌药中核苷和氨基酸类成分分析罗益远，顾海燕，谢叶菲1，蒋新苗1，王蔡红蝶1，马舒伟1，陈宏降1（1.浙江药科职业大学中药学院，浙江宁波31510 0；2.启东市人民医院药剂科，江苏南通2 2 6 2 99）摘要：本研究建立了一种超快速液相色谱-三重四极杆/线性离子阱-串联质谱（UFLC-QTrap-MS/MS)同时测定乌药中11种核苷类和14种氨基酸类成分的分析方法，并结合多元统计分析比较不同产地、不同部位乌药的质量。采用WatersXBridgeAmide色谱柱（2.1mmX100mmX3.5m），以0.2%甲酸水溶液（A)-0.2%甲酸乙睛溶液（B)为流动相进行梯度洗脱；在电喷雾正离子模式下，以多反应监测（MRM）进行质谱检测；基于各成分的质量浓度，用方差分析（ANOVA）、主成分分析（PCA）、正交偏最小二乘法判别分析（OPLS-DA）、层次聚类分析（HCA）、逼近理想解排序法（TOPSIS）以及灰色关联度分析（G R A），对不同产地、不同部位乌药样品进行综合评价。结果表明，所测成分在一定浓度范围内具有良好的线性关系，相关系数均大于0.992 4；精密度、重复性、稳定性良好，平均加样回收率为94.8 6%106.47%，相对标准偏差（RSD)均小于4.55%。不同产地、不同部位乌药样品间存在一定的差异，HCA和PCA2种方法均将样品分为3类。OPLS-DA模型分析通过VIP值共筛选得到6 个差异性化合物，分别为尿苷、丙氨酸、胞嘧啶、黄嘌呤、腺嘌呤、鸟嘌呤。该方法简便、灵敏度高、重现性好，可用于乌药中核苷和氨基酸类成分的同时测定，为药材质量评价和综合利用提供方法参考。关键词：超快速液相色谱-三重四极杆/线性离子阱-串联质谱（UFLC-QTrap-MS/MS)；乌药；氨基酸；核苷；不同产地；不同部位中图分类号：0 6 57.6 3doi:10.7538/zpxb.2023.0049Analysis of Nucleosides and Amino Acids in Different Partsof Lindera aggregata(Sims)Kosterm.from Different Habitatsby UFLC-QTrap-MS/MS Combined with Multivariate Statistical AnalysisLUO Yi-yuan,GU Hai-yan,XIE Ye-fei,JIANG Xin-miao,WANG Juan,CAI Hong-die,MA Shu-wei,CHEN Hong-jiang(1.College of Chinese Medicine,Zhejiang Pharmaceutical College,Ningbo 315100,China;2.Pharmacy Department,Qidong Peoples Hospital,Nantong 226299,China)娟，文献标志码：A文章编号：10 0 4-2 997(2 0 2 4)0 2-0 2 6 9-12浙江省公益基础研究项目（LTGN23H280002）；浙江省药品监督管理局科技计划项目（2 0 2 2 0 0 9、2 0 2 30 14）；浙江省医药卫生科技计划项目（2 0 2 3KY298）；宁波市自然科学基金（2 0 2 3J306）；宁波市公益性科技计划项目（2 0 2 1S144）；浙江省教育厅一般项目（Y2 0 2 2 50 2 17）；浙江药科职业大学校级课题（2 0 2 2 12 8、2 0 2 2 134）本文通信作者顾海燕，陈宏降270Abstract:A method of ultra-fast liquid chromatography-triple quadrupole/linear iontrap tandem mass spectrometry(UFLC-QTrap-MS/MS)was established for simultane-ous determination of 1l nucleosides and 14 amino acids in Lindera aggregata(Sims)Kosterm.,which combined with multivariate statistical analysis to evaluate the quality ofdifferent medication parts of Lindera aggregata(Sims)Kosterm.from differenthabitats.The gradient separation was performed on a Waters XBridge Amide column(2.1 mmX100 mmX 3.5 m)with 0.2%formic acid in water(A)and 0.2%formicacid in acetonitrile(B)as the mobile phase,and the mass spectrometric detection wascarried out in electrospray positive ion mode under multiple reaction monitoring(MRM)mode.The samples of different habitats and medication parts were comprehensivelyevaluated by entropy weight ANOVA,PCA,OPLS-DA,HCA,TOPSIS and GRAaccording to the contents of 25 target constituents.All of the analytes had good linearityin the range of tested concentration with the correlation coefficient not less than0.992 4.The average recoveries were between 94.86%and 106.47%,with the relativestandard deviation(RSD)less than 4.55%.The analysis results showed that there weresignificant variations among the different medication parts of Lindera aggregata(Sims)Kosterm.samples.The contents of nucleosides and amino acids in tuber were signifi-cantly higher than those in taproot and leaf(p1为显著影响，共筛选得到6 个差异性化合物，分别质谱学报第45卷酸含量与直根和叶均有显著差异（p0.05);块根中的核苷含量最高，平均含量为7 4.7 8 6 g/g，其次是叶（55.40 1g/g)和直根（36.2 90 g/g)；乌药块根的氨基酸含量最高，平均含量为50.850g/g，其次是乌药的直根和叶，平均含量分别为43.2 13、35.6 7 6 g/g。60Bab40a200TuTa注：ac代表各组在p0.05水平上具有显著性差异图2 不同乌药样品中核苷（A)和氨基酸(B)类成分的方差分析Fig.2 ANOVA for nucleosides(A)and amino acids(B)in different Lindera aggregata(Sims)Kosterm.samplesaLTu为尿苷（2.8 498 5）、丙氨酸（1.990 17）、胞嘧啶（1.48 36 5）、黄嘌呤（1.3312 3）、腺嘌呤（1.2 5546）、鸟嘌呤（1.0 8 9 9 3），可用于区分不同部位乌药样品。2.4.3基于 TOPSIS和 GRA分析采用TOP-SIS和GRA分析法对不同产地、不同部位乌药样品进行质量分析，质量等级排序结果列于表5。可见，在TOPSIS模型中，相对接近程度值（C）越大，表示样本的质量越高；在GRA模型中，相对关联度（r）表示组分含量与样品之间的相对关联关系，r：值越高表示样本的质量越好。由表5可知，TOPSIS和GRA的分析结果基本一致，表示不同产地、不同部位乌药样品间的差异较大。乌药块根中，广东韶关样品（Tu-7)的C,值最大，而天台三洲（Tu-6）的ri值最大；乌药直根中，湖北咸宁（Ta-1)的Ci值和r值均为较高水平；乌药叶中，丽水遂昌(L-4)的C,值最大，而天台三洲（L-6）的ri值最大。另外，通过比较2 种分析方法的最大差异值，发现GRA的r最大差异值为12.0 8%，而TOPSIS的C，最大差异值高达53.2 3%，表明TOPSIS比GRA更适合区分乌药样品质量的优劣。TaL第2 期罗益远等：基于UFLC-QTrap-MS/MS结合多元统计分析的不同产地、不同部位乌药中核苷和氨基酸类成分分析a4202-4-6-10-8-6-4-20Cz(u100)z1(0)dleuwiglife0.5histidingoserineyaline-1Tu-9rosine1L240T-a-10a-2串Toglycine-0.5Ta-9alanine2773bTu-4Tu-6Tu-7Tu-5Tu-Tu-2Tu-1Tu-8Tu-9Ta-1Ta-3Ta-21aTa-Ta-7Ta-9isalenrineL-4L-20OL-61-T.-1OL-10L3OL-9OL-724 6 8threonineognanosineouradiloxathineLSlrwiaenieaindsmine-deoxyurL-7OL-810.50-0.5ouipun25d2015105工工FouisouenaEooruenaVar ID(Primary)Esuiongjos!Group!.Group2.Group3-1-0.5p(corr)1,(corr)1注：a.PCA分析；b.VIP得分图；c.Bioplot图；d.聚类分析树状图；图c中绿色圆点代表样品中2 5种核苷和氨基酸类成分，橙色六边形代表18 批不同产地、不同部位乌药样品图3不同乌药样品的多元统计分析Fig.3 Multivariate statistical analysis of different Lindera aggregata(Sims)Kosterm.samples表5不同乌药样品的质量等级排序Table 5Quality ranking of different Lindera aggregata(Sims)Kosterm.samples逼近理想解排序法TOPSIS灰色关联度分析GRA样品相对接近程度值SampleComparativecloseness(C,)Tu-10.2969Tu-20.3498Tu-30.4636Tu-40.4620Tu-50.4727Tu-60.4795Tu-70.5961Tu-80.3910Tu-90.3944Ta-10.4917Ta-20.4015Ta-30.4319Ta-40.4649Ta-50.3582Ta-60.2684Ta-70.333400.5排序Rank98453217614325861最大差异值Differenceof C./%50.2041.3122.2322.4920.7119.560.0034.4033.830.0018.3512.185.4627.1745.4132.20498相对关联度最大差异值排序Relative degreeRank(r)0.49570.46550.48990.50050.51790.52750.52440.48520.50850.53370.53160.52810.51640.50870.49750.5103Differenceof r:/%66.02911.7677.1255.1231.8310.0020.5888.0243.6010.0020.3831.0543.2464.6886.7754.37278样品SampleTa-8Ta-9L-1L-2L-3L-4L-5L-6L-7L-8L-9L-103结论本实验建立了超快速液相色谱-三重四极杆/线性离子阱质谱法测定不同产地、不同部位乌药中2 5种氨基酸和核苷类成分的含量,并结合方差分析、聚类热图分析对乌药进行综合评价。结果表明，不同产地、不同部位乌药中核苷和氨基酸类成分具有一定的差异。本方法快速灵敏、准确可靠，可为乌药不同部位的质量控制提供方法参考，同时为乌药资源的充分利用提供基础资料。参考文献：1国家药典委员会.中华人民共和国药典2 0 2 0 版（一部)M.北京：中国医药科技出版社，2 0 2 0：79.2李平。新“浙八味”将为相关中药材产业带来什么N台州日报，2 0 18-0 3-0 9.3杜伟锋，岳显可，吴瑶，葛卫红，陆兔林，王智民。基于HPLC与NIRS指纹图谱分析不同形态的乌药根J中国中药杂志，2 0 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