几种典型抗生素的磁分散固相萃取色谱质谱检测新技术研究

发布时间：2018-03-28 05:16

本文选题：磁性材料　切入点：四环素类抗生素　出处：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：近年来功能化磁性微球作为一种新型固相萃取材料获得越来越广泛的关注。通过对微球表面进行改性或修饰,可满足污染物在提取、分离和净化等多方面的需求。本研究利用不同功能化磁性材料对四环素类(四环素、土霉素、金霉素、强力霉素)、喹喏酮类(培氟沙星、诺氟沙星、氟罗沙星、左氧氟沙星、依诺沙星、洛美沙星、二氟沙星、司帕沙星、达氟沙星、环丙沙星、恩诺沙星、加替沙星)抗生素的吸附特性,建立了基于磁分散固相萃取的多种抗生素色谱质谱检测新技术,相关研究对于进一步探索和完善抗生素高灵敏度检测新技术,拓宽磁性材料的应用范围都具有重要的意义。主要的研究内容包括:1.基于氨基功能化磁性微球的四环素类抗生素HPLC检测新技术本研究以Fe_3O_4为磁性内核,制备了氨基修饰磁性功能材料,考察了不同材料对TCs的吸附性能。利用氨基功能化磁性微球(MNP-NH2)在有机相中对四环素类抗生素(TCs)高效的吸附特性,将分散固相萃取技术与磁固相萃取技术相结合,建立了4种TCs的磁分散固相萃取HPLC检测新技术,并应用于牛奶样品的检测。内容包括:(1)功能化磁性材料的制备:在实验室前期氨基修饰磁性微球基础上,将SBA-15大分子介孔材料引入磁性功能材料制备,并进一步优化了APTES用量,研究制备了不同比表面积及氨基含量的磁性材料Fe_3O_4@SBA-15@APTES和Fe_3O_4@SiO_2@APTES。傅里叶红外光谱,元素分析表征结果证实偶联成功,对硝基苯甲醛比色法对磁性材料表面氨基定量结果表明:通过控制合成过程中APTES用量可制备不同氨基含量的磁性功能材料。(2)功能化磁性材料对TCs吸附效果研究:Fe_3O_4@SiO_2@APTES在乙腈溶液中对TCs展示了良好的吸附特性。实验研究了萃取剂用量、磁性材料用量、pH值、吸附时间、洗脱液组分、洗脱时间和洗脱次数等因素对TCs吸附效果的影响,结果表明:以NaCl为盐析剂,MgSO4·7H2O为络合剂,乙腈为萃取剂,利用分散固相萃取可将TCs从水相萃取至乙腈溶液中;以Fe_3O_4@SiO_2@APTES作为吸附剂,1m L萃取液加入90μL浓度为1mol/L的NaOH,以20mg的Fe_3O_4@SiO_2@APTES吸附10min可实现TCs的高效吸附;水:乙腈:高氯酸比例为86:10:4(V:V:V)的混合溶液洗脱5min可获得较好的洗脱效果,4种TCs的吸附率在77.36%-99.04%之间。(3)牛奶样品中TCs测定:采用实验建立的方法对牛奶中四种TCs进行测定,方法线性范围为0.05-2.5mg/L,相对标准偏差在3.85-9.30%之间,检出限40μg/L,加标回收率在87.78-107.48%之间。相对于国标固相萃取方法,本研究建立的磁分散固相萃取方法具有操作简便、快速等优点,可满足牛奶样品TCs日常检测的需求。2.基于SBA-15新基质的喹诺酮类抗生素MALDI-TOF MS高通量检测新技术传统基质多为小分子有机酸,MALDI测定时在低分子量范围内(500Da)会产生很强的基质背景干扰。针对以上问题,实验在传统基质CHCA基础上,引入SBA-15大分子介孔材料,制得改性CHCA新基质SBA-15@APTES@CHCA并应用于喹诺酮类抗生素的检测,建立了基于MALDI-TOF MS新基质的12种喹诺酮类抗生素(QNs)检测新方法。利用传统CHCA基质和SBA-15反应所得的SBA-15@APTES@CHCA基质不仅保留了传统基质吸收和传递激光能量的性质,而且通过SBA-15大分子的引入可有效消除传统基质在低分子量测定时的基质背景干扰。主要研究内容包括:(1)SBA-15@APTES@CHCA新基质的合成:实验利用SBA-15表面的羟基基团,通过APTES水解反应制备了表面氨基化SBA-15@APTES前体;利用EDC和NHS酰胺反应,将CHCA的羧基进行活化后与SBA-15@APTES偶联,制备了SBA-15@APTES@CHCA新基质。傅里叶红外光谱,扫描电镜鉴定偶联成功。(2)SBA-15@APTES@CHCA使用条件优化:实验对基质溶剂种类、基质浓度、样品基质混合比、点样方法等条件做了优化,结果表明:选用乙腈:0.1%TFA(1+1)为基质溶剂,1mg/m L基质悬浮液,基质与样品体积比为1:6时,采用混合点样法可获得较好的MALDI-TOF-MS分析信号。(3)喹诺酮类抗生素MALDI-TOF MS检测方法建立:利用制备的新基质,在优化好的条件下对12种QNs用MALDI-TOF MS进行检测,可获得较好的分析信号,分别是:达氟沙星m/z 358(M+H)+,恩诺沙星m/z 360(M+H)+,环丙沙星m/z 332(M+H)+,二氟沙星m/z 400(M+H)+和m/z 422(M+Na)+,依诺沙星m/z321(M+H)+,加替沙星m/z 376(M+H)+,氟罗沙星m/z 370(M+H)+,洛美沙星m/z352(M+H)+,司帕沙星m/z 393(M+H)+,诺氟沙星m/z 320(M+H)+,左氧氟沙星m/z 362(M+H)+,培氟沙星m/z 334(M+H)+,12种QNs的方法检出限在0.5-1.0mg/L之间。3.石墨烯功能化磁性材料在喹诺酮类抗生素MALDI-TOF MS高通量检测中的应用本研究以氨基功能化磁性微球为基础,在其表面进一步修饰氧化石墨烯,制备了石墨烯功能化磁性材料(GO-NH-Fe_3O_4),并应用于喹诺酮类抗生素的分离富集。结合前期建立的MALDI-TOF MS检测技术,建立了基于石墨烯功能化磁性材料的喹诺酮类抗生素MALDI-TOF MS检测新技术,并应用于环境水样的检测。内容主要包括:(1)GO-NH-Fe_3O_4的制备:实验在利用Hummer’s改良法制备氧化石墨烯基础上,将氨基化磁性微球与氧化石墨烯偶联,制得石墨烯功能化磁性材料GO-NH-Fe_3O_4。FT-IR和SEM结果证明石墨烯氧化成功,氨基磁性微球成功偶联到了氧化石墨烯表面,且在水中具有较好的分散性。(2)GO-NH-Fe_3O_4对QNs吸附效果研究:实验对磁性材料种类、pH值、吸附反应时间、GO-NH-Fe_3O_4用量、洗脱液中氨水浓度、洗脱时间、洗脱次数进行了优化,结果表明:在弱酸性、中性或弱碱性条件下,3mL的GO-NH-Fe_3O_4与适量样品反应30min,可将样品中的喹诺酮类抗生素吸附到GO-NH-Fe_3O_4材料上,质谱图中12种目标物特征峰均能明显辨别,吸附后残液中检测不到目标物;用15%的氨水甲醇溶液对吸附剂洗脱15min,可获得较好的洗脱效果。(3)水样中QNs检测:在优化的条件下利用GO-NH-Fe_3O_4磁性功能材料,结合MALDI-TOF MS方法对自来水、海河水、井水等水样中的12种QNs进行检测,结果表明:结合GO-NH-Fe_3O_4的富集净化,利用MALDI-TOF-MS测定12种QNs方法的检出限可达10μg/L。实验建立的方法具有高通量、检测速度快等特性,可应用于环境水中多种QNs的快速高通量筛查需要。
[Abstract]:In recent years, magnetic microspheres function as a new solid phase extraction material to obtain more and more attention. The microspheres were modified or modified, which can meet the pollutants in the aspects of extraction, separation and purification and so on. This research use different functional magnetic materials of tetracyclines (tetracycline, oxytetracycline, chlortetracycline, doxycycline), quinolones (pefloxacin, norfloxacin, fleroxacin, levofloxacin, enoxacin, lomefloxacin, two levofloxacin, sparfloxacin, ciprofloxacin, enrofloxacin, danofloxacin, gatifloxacin) adsorption characteristics of antibiotics, the establishment of a new technology of antibiotics detection of magnetic chromatography mass spectrometry with dispersive solid phase extraction based on the related research for to further explore and improve the new technology of high sensitivity detection of antibiotics, are of great significance to broaden the application range of magnetic materials. The main research contents. Including: 1. based on the new technology of tetracycline antibiotics HPLC detection of amino functionalized magnetic microspheres on the basis of Fe_3O_4 magnetic core, amino modified magnetic functional materials were prepared. The adsorption properties of different materials on TCs were investigated. The amino functionalized magnetic microspheres (MNP-NH2) in the organic phase of tetracycline (TCs) adsorption the characteristics of high efficiency, the dispersive solid phase extraction technology and magnetic solid phase extraction technology, 4 kinds of TCs magnetic dispersion test technique of solid phase extraction HPLC was established and used to detect milk samples. Contents include: (1) functional magnetic materials prepared by modified in our laboratory based on amino magnetic microsphere SBA-15, molecular mesoporous material is introduced into the preparation of magnetic functional materials, and further optimize the dosage of APTES, different specific surface area and the amino content of magnetic materials Fe_3O_4@ SBA-15@APTES and Fe_3O_4 the preparation @SiO_2@APTES. Fourier transform infrared spectroscopy, elemental analysis results confirm the successful coupling of p-nitrobenzaldehyde colorimetric method of magnetic material surface amino quantitative results showed that through the APTES control in the process of synthesis of amount of magnetic functional materials were prepared with different amino content. (2) functional magnetic materials on adsorption effect of TCs: Fe_3O_4@SiO_2@APTES in acetonitrile solution the TCs shows good adsorption properties. Experimental study of the extraction agent, magnetic material dosage, pH value, adsorption time, eluent composition, influence factors, adsorption elution time and elution times on TCs results show that NaCl as the salting out agent, MgSO4 7H2O as complexing agent, acetonitrile the extractant, using dispersive solid phase extraction can be TCs from aqueous extraction to acetonitrile; using Fe_3O_4@SiO_2@APTES as adsorbent, 90 L concentration added 1m L extraction solution is 1mol/L NaOH, 20mg F High e_3O_4@SiO_2@APTES adsorption 10min can achieve TCs; water: acetonitrile: perchloric acid ratio of 86:10:4 (V:V:V) of the mixed solution of 5min can obtain better elution elution, the adsorption rate of TCs 4 in 77.36%-99.04%. (3) the determination of TCs in milk samples: using the method of experiment based on four kinds of milk the determination of TCs, the linear range of the method was 0.05-2.5mg/L, the relative standard deviation between 3.85-9.30%, the detection limit of 40 g/L, the recovery rate was 87.78-107.48%. Compared with the national standard of solid phase extraction method, this study established the magnetic dispersion solid phase extraction method is simple, fast, and can meet the needs of daily milk samples TCs test.2. MALDI-TOF MS quinolones high-throughput SBA-15 new matrix detection technology based on the traditional matrix for small molecule organic acid, MALDI determination in low molecular weight range (500Da) will produce Matrix background of strong interference. To solve the above problems, the experiment in traditional matrix CHCA based on the introduction of SBA-15 molecular mesoporous materials, prepared modified CHCA matrix SBA-15@APTES@CHCA and applied to the detection of quinolone antibiotics, established 12 quinolones MALDI-TOF MS new matrix (QNs) based on a new method for the detection of SBA-15@APTES@CHCA matrix. By using the traditional CHCA matrix and SBA-15 reaction proceeds not only retains the properties of traditional matrix absorption and transmission of laser energy, and by introducing SBA-15 molecules can effectively eliminate the interference of matrix background of traditional matrix in low molecular weight determination. The main research contents include: (1) a synthetic SBA-15@APTES@CHCA matrix: experiments with hydroxyl groups the surface of SBA-15, the surface amino SBA-15@APTES precursor prepared by APTES hydrolysis; using EDC and NHS amide reaction, the carboxyl groups were CHCA After the activation of SBA-15@APTES@CHCA and SBA-15@APTES coupling, a new matrix was prepared. Fourier transform infrared spectroscopy, scanning electron microscopy identification successfully coupled. (2) SBA-15@APTES@CHCA using the optimal conditions: experiment on matrix solvent, substrate concentration, mixing ratio of the sample matrix, condition sampling method for optimization, the results show that the acetonitrile (1+1): 0.1%TFA as the matrix solvent, 1mg/m matrix and L matrix suspension, sample volume ratio was 1:6, the mixed sample method can obtain better MALDI-TOF-MS signal analysis. (3) MALDI-TOF MS quinolone antibiotics detection method established: using the new matrix preparation, the optimized conditions of 12 kinds of QNs MALDI-TOF MS for testing, can obtain better signal analysis, respectively is: m/z 358 + danofloxacin (M+H), enrofloxacin (M+H) m/z 360 +, m/z 332 + ciprofloxacin (M+H), two m/z (M+H) loxacin 400 + and m/z 422 + (M+Na), enoxacine M /z321 (M+H) + m/z (M+H), gatifloxacin 376 +, m/z 370 + (M+H) of fleroxacin, lomefloxacin (m/z352 M+H) + m/z (M+H), sparfloxacin 393 +, m/z 320 + norfloxacin (M+H), levofloxacin (M+H) m/z 362 +, pefloxacin (M+H) + m/z 334 12 kinds of methods, the detection limit of QNs between 0.5-1.0mg/L application of.3. functionalized graphene magnetic materials in MALDI-TOF MS high-throughput detection of quinolone antibiotics in this study on amino functionalized magnetic microspheres based on the surface modification of graphene oxide to graphene, functional magnetic materials were prepared (GO-NH-Fe_3O_4), and separation applied to quinolones enrichment. Combined with MALDI-TOF MS detection technology pre established, established a new technique for the detection of quinolone antibiotics MALDI-TOF MS functionalized graphene based magnetic materials, and apply in the detection of environmental samples. The main contents include: (1) GO-NH-Fe_3O_4 Preparation: the experiment in the use of Hummer s prepared by a modified method of graphene oxide based on the coupling of magnetic microspheres and amino functionalized graphene oxide, prepared graphene functional magnetic materials GO-NH-Fe_3O_4.FT-IR and SEM results show that graphene oxide, amino magnetic microspheres successfully coupled to the graphene oxide surface, and has good dispersion in the water. (2) study on the effect of adsorption of GO-NH-Fe_3O_4 on QNs: the experiment of magnetic materials, pH value, adsorption time, dosage of GO-NH-Fe_3O_4, the elution time of elution solution, ammonia concentration, were optimized. The results showed that: in the times of elution of weakly acidic, neutral or alkaline conditions, the amount of GO-NH-Fe_3O_4 and 3mL sample reaction 30min, can be in a sample of quinolones adsorbed to the GO-NH-Fe_3O_4 material, the mass spectrum of 12 target peaks can significantly distinguish, after adsorption residue was not detected in. Standard; 15min elution adsorbent with ammonia of 15% methanol solution, elution can obtain better effect. (3) QNs in water detection: using GO-NH-Fe_3O_4 magnetic functional materials under the optimized conditions, combined with the MALDI-TOF MS method of tap water, sea water, 12 QNs wells in water samples were detected, results show that according to GO-NH-Fe_3O_4 enrichment and purification, detection limit of up to 10 g/L. to establish experimental determination of 12 QNs by MALDI-TOF-MS method with high throughput, fast detection speed and other characteristics, rapid high-throughput screening can be applied to a variety of environmental water QNs.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ465;O657.63

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