CN115453013A - 一种富集和检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮的方法 - Google Patents
一种富集和检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮的方法 Download PDFInfo
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Abstract
本发明公开了一种富集检测烟草中4‑甲基亚硝胺基‑1‑(3‑吡啶基)‑1‑丁酮(NNK)的方法,包括以下步骤:(1)制备烟草检测液;(2)富集NNK:采用磁性树枝状分子印迹识别吸附材料分离并富集烟草步骤(1)得到的甲醇溶液中的NNK;(3)检测:采用高效液相色谱法检测NNK。本发明的方法可以选择性富集和检测烟草中N‑亚硝胺类物质NNK,具有灵敏度高、选择性好、分析速度快、重现性好的优点。在烟草质量控制和成分分析中有广阔的应用前景。
Description
技术领域
本发明专利属于分析化学领域,具体涉及一种富集和检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的分析方法。
背景技术
烟草及烟草制品中的低丰度N-亚硝胺类物质与复杂基质成分共存,富集与检测存在样品处理方法繁杂和缺乏选择性的问题,难以达到针对性富集与简便快捷的检测要求。
现有技术中针对烟草NNK的分析检测研究,如专利CN108956802A和专利CN110646536A的检测方法,为该目标物的检测提供了易于推广应用的思路。但是,上述专利CN110646536A使用的分析方法,需要较长的时间和复杂的仪器设备;专利CN108956802A缺乏对某一具体目标物的选择识别性,在烟草这类复杂基质背景的样品分析中,易受到干扰,影响测定的准确性和灵敏度。因此需要建立一个选择性富集和简便快捷的检测NNK的分析方法。
分子印迹聚合物(molecularly imprinted polymers,MIPs)具有预定性、识别性以及广泛使用性,在食品药品和环境污染物或残留物的分离、富集和分析中的应用发展迅速。MIPs对特定目标化合物空间结构的“记忆”效应和对作用位点的“识别”作用,能够高选择性富集分离复杂体系中的目标分子。此外,与常规的“本体聚合”相比,“表面印迹”聚合技术使得残留模板大大减少,增加有效“印迹”容量,减少传质阻力,具有比表面积大、吸附量高、吸附和解吸动力学良好等特性。
树枝状聚合物(dendrimers)是一类新型高分子纳米材料,它的潜在应用价值已引起科学家的广泛关注。其结构规整、质量可控、通畅的分子骨架结构内外都能富含大量功能基团,并可针对MIPs纳米结构需求进行功能化设计。近年来基于磁性纳米材料表面树状纳米结构的MIPs研究报道较少。磁性材料使得富集过程极为简便,省去、离心、过滤等繁杂操作。将磁性树枝状分子印迹富集目标物和高效液相检测相结合,可更好满足产品质量控制的需求。为此提出本发明。
发明内容
本发明提供了一种富集和检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的方法。本发明的方法创新性地优化了目标物富集方法和液相检测方法,实现了烟草中NNK的高选择性、高灵敏度、低干扰、简便快捷的检测分析。
本发明的目的是通过以下技术方案来实现的。
一种富集检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的方法,包括以下步骤:
(1)制备烟草检测液:将烟草加入含有乙酸铵的水溶液中,超声处理一段时间,收集上清液,经微孔滤膜过滤得到滤液,氮气吹干,用甲醇复溶得到甲醇溶液;
(2)富集NNK:采用磁性树枝状分子印迹识别吸附材料分离并富集烟草步骤(1)得到的甲醇溶液中的NNK;
(3)检测:采用高效液相色谱法检测步骤(2)的甲醇溶液中的NNK。
优选地,步骤(1)烟草与含有乙酸铵的水溶液的质量比为1:(5-500);超声提取5-120分钟;微孔滤膜的孔径为0.1-0.5μm。
优选地,步骤(2)所述磁性树枝状分子印迹识别吸附材料制备步骤为:将 Fe3O4纳米颗粒外包覆SiO2后氨基硅烷偶联化修饰,得到氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2);然后加入醛功能试剂与1-5代聚酰胺-胺树枝的甲醇溶液;外磁场分离,洗涤干燥;然后加入虚拟模板分子N-(羟甲基)烟酰胺、交联剂、引发剂和功能单体进行分子印迹层的合成;磁性分离,洗脱虚拟模板分子,干燥;即得到所述磁性树枝状分子印迹识别吸附材料。
优选地,步骤(2)的富集步骤为:甲醇溶液中加入磁性树枝状分子印迹识别吸附材料,室温振摇一段时间,外磁场分离;向磁性树枝状分子印迹识别吸附材料加入甲醇-乙酸洗脱液(v:v=9:1)超声15分钟;外磁场分离,获取上清液,氮气吹干,使用0.1wt%乙酸铵甲醇溶液复溶。
优选地,步骤(3)的液相条件为,色谱柱:C18柱(5μm,150mm×4.6mm);流速:1.0mL/min;柱温:25-50℃;进样量:10μL;洗脱方式:梯度洗脱;检测器:二极管阵列检测器(DAD),全波长扫描(190-800nm),检测波长为230nm。
优选地,梯度洗脱条件为:0–4.5min,40wt%流动相A+60wt%流动相B; 4.5–10.0min,5wt%流动相A+95wt%流动相B。
优选地,流动相A为0.08wt%乙酸铵水溶液,流动相B为0.1wt%乙酸铵甲醇溶液。
本发明的有益效果:
1、本发明的方法采用磁性树枝状分子印迹识别吸附材料处理烟草检测液,可实现低丰度目标物NNK的高选择性识别和富集,排除了溶液中复杂基质的干扰,使得检测灵敏度显著提升。
2、本发明的磁性树枝状分子印迹识别吸附材料,表面接枝含有大量胺基功能团与空腔的PAMAM树枝状纳米材料,结构规整、质量可控、通畅;具有高度刚性和稳定性的分子骨架结构。与专利CN114853961A相比,树枝状结构提高了吸附容量与印记效率,缩短了吸附与解析时间。
3、本发明的富集方法简单,磁性树枝状分子印迹识别吸附材料在外磁场的定向控制下分离;与专利CN106674405A相比,省去了多次超声离心、静置过滤等繁杂操作,为分离富集过程带来了极大便利。回收率结果令人满意。
4、本发明的检测方法简单,实现了烟草及其制品中NNK的快速测定,检测结果准确。与专利CN110646536A采用的在线二位色谱串联质谱 (UPCC-HPLC-MS/MS)相比,仅使用高效液相色谱法,极大提高了分析方法的普适性,并在10分钟内即可完成检测,缩短检测时间。本方法具有准确性高,普适性好,重现性好的特点。可实现低丰度目标物NNK的高选择性、高灵敏度、低干扰的准确测定。
附图说明
图1为本发明的富集检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK) 的原理过程示意图;其中(a)为磁性树枝状分子印迹识别吸附材料的制备过程, (b)为富集检测实际烟草样品中NNK的分析过程。
图2为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料的透射电镜图、粒径分布图和高分辨透射电镜图;其中(a)、(b)和(c)分别为氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒 (Fe3O4@SiO2@PAMAM)和MIPs的透射电镜图下形貌;(d)、(e)和(f) 分别为Fe3O4@SiO2-NH2、Fe3O4@SiO2@PAMAM和MIPs的粒径分布图;(g)和(h)分别为MIPs的高分辨透射电镜图与局部放大图下的形貌。
图3为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料的场发射扫描电镜与元素映射图;其中(a)、(b)和(c)分别为氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM) 和MIPs的场发射扫描电镜图下形貌;(d)、(e)、(f)、(g)和(h)为 MIPs的元素映射图。
图4为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料的表征图;其中(a)为X射线衍射分析(XRD),(b)为红外光谱图(FT-IR),(c) 为磁滞曲线图(VSM),(d)为N2吸附解析等温线,(e)为光电子能谱分析 (XPS)总谱,(f)为XPS碳元素精细谱。
图5为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料MIPs的吸附性能考察图;其中(a)为静态吸附曲线,(b)为动态吸附曲线,(c)为解析动力学曲线(NIPs是指不加入模板分子,其他步骤相同的同法制得的非分子印迹材料,用于对照比较分析)。
图6为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料MIPs与 NIPs对结构类似/非类似物化合物的选择性吸附考察。
图7为本发明实施例1制备的磁性树枝状分子印迹识别吸附材料MIPs对 NNK吸附与分析的重现性、重复性和稳定性考察;其中(a)为5个不同批次 MIPs吸附NNK标准溶液的测定结果;(b)为同一份MIPs重复吸附-解析循环使用7次,吸附NNK标准溶液的测定结果;(c)为1份MIPs置于4℃冰箱中保存30天,每隔6天对吸附NNK标准溶液的测定结果。
图8为烟草样品经本发明实施例1制备的磁性树枝状分子印迹识别吸附材料处理前后的高效液相色谱图对比;其中(a)为NNK标准对照溶液高效液相色谱图,(b)为样品经过分子印迹富集后的高效液相色谱图,(c)为样品未经过分子印迹富集的高效液相色谱图,(d)为样品经过分子印迹富集后的剩余残留溶液的高效液相色谱图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚,以下通过具体实施例,对本发明做进一步阐述,但本发明要求保护的范围并不局限于实施例表述的范围。实施例中所用试剂或仪器,未注明生产厂商者,均为可以通过购买获得的常规产品。
实施例1
对烤制型烟叶制品中的NNK含量进行富集检测,步骤如下:
步骤1、烟草检测液的制备:取市售烤制型烟草样品10g,加入25mL水(含 10mM乙酸铵),超声处理1小时,并在4℃冰箱中放置1小时。随后,收集上清液,0.45μm微孔滤膜过滤,氮气吹干,10mL甲醇复溶。
步骤2、采用磁性树枝状分子印迹识别吸附材料分离并富集烟草检测液中的 NNK:
(1)磁性树枝状分子印迹识别吸附材料的制备方法如下:(a)250mL三颈瓶中加入100mL除氧去离子水,800rmp/min磁力搅拌。氮气保护下加入9.5g 的FeCl3·6H2O和4.0g的FeCl2·4H2O,80℃逐滴加入浓氨水15mL,聚乙二醇 4mL,反应30分钟,磁性分离,去离子水洗至中性,干燥得黑色的四氧化三铁纳米粒(Fe3O4)。取0.3gFe3O4纳米粒,均匀分散于200mL乙醇和30mL去离子水中,保持200rmp/min搅拌速度,30℃水浴条件下加入4mL正硅酸四乙酯(TEOS)与40mL乙醇的混合溶液,再加入2mL浓氨水,反应30分钟,加入0.5mL 的3-氨丙基三乙氧基硅烷(KH550),继续反应6h,去离子水洗至中性,磁性分离,干燥得氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)。取100mg Fe3O4@SiO2-NH2,加入20wt%的戊二醛水溶液,振摇2小时,磁性分离,PBS 缓冲液洗涤后,加入1mL的10wt%的2代聚酰胺-胺树枝的甲醇溶液,25℃振摇6h。磁性分离,去离子水洗,干燥后得到树枝状修饰的磁性纳米粒 (Fe3O4@SiO2@PAMAM);(b)取50mg树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和35mg虚拟模板分子N-(羟甲基)烟酰胺分散在甲醇:水(v:v=9:1)溶液中,加入70mg功能单体甲基丙烯酸MAA,氮气保护下预聚合1小时;再加入0.15g交联剂二甲基丙烯酸乙二醇酯(EGDMA)和25mg 引发剂偶氮二异丁腈(AIBN),氮气保护下120℃聚合5小时。反应结束后,磁性分离,用乙酸-甲醇混合溶液(v:v=1:9)超声洗脱30分钟,干燥,即得特异性识别4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的磁性树枝状分子印迹识别吸附材料(MIPs)。为了便于对照比较分析,同法制备非分子印迹材料(NIPs),除不加虚拟模板分子外,其余制备过程与MIPs一致。
4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的磁性树枝状分子印迹识别吸附材料(MIPs)的表征与吸附性能如下:
图2的a-c表明氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和分子印迹识别吸附材料(MIPs) 均为包覆有硅胶层的具有均匀粒径的规则球形;图2d-f粒径分布图表明它们的平均粒径随着进一步的修饰和印迹层的覆盖有一定程度的增加;图2g-h为MIPs 的高分辨透射电镜图与局部放大图下的形貌,可观察其磁性球外包覆的聚合物层的厚度约为14nm,同时也展现了其具有属于Fe3O4的0.242nm的晶格间距。
图3的a-c分别为氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和分子印迹识别吸附材料 (MIPs)场发射扫描电镜图;图3a表明硅胶磁性球具有光滑的表面;图3b表明树枝状聚合物修饰后,表面变得粗糙和不均匀,边界不清晰;图3c表明在树枝状大分子表面合成印迹聚合物后,表面变得光滑,出现大孔印迹空腔。此外,图3d-h为MIPs的元素映射图,图3d显示了元素的整体分布,图3e-h表明了主要由铁、硅、氮和氧元素组成。
图4a为Fe3O4和MIPs的X射线衍射图,可看出包覆硅胶后在17°至24°的 2θ角处表现出属于非晶态二氧化硅的宽谱带,同时表明,在二氧化硅涂层、树枝状簇修饰和印迹合成过程中,Fe3O4纳米颗粒的晶体结构没有被破坏;图4b 为Fe3O4纳米粒、氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和分子印迹识别吸附材料(MIPs) 的傅里叶红外图谱,各材料均出现了对应于自身化学结构的官能团特征峰,证实了硅胶包覆、树枝状纳米团簇修饰和表面分子印迹的成功合成。图4c为树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和分子印迹识别吸附材料(MIPs) 的N2吸附解析等温线。结果表明,两种材料均表现出介孔材料(IV型)的典型特征。该H1磁滞吸附等温线具有饱和吸附平台,这反映了孔径分布相对均匀。此外,在对Fe3O4纳米颗粒进行多步功能化后,磁滞回线形状没有明显变化,这表明孔穴形状相似,原始孔穴没有受到明显损坏。树枝状结构修饰的纳米颗粒提供了大量孔径大于30nm的介孔结构。图4d为氨基修饰的Fe3O4@SiO2纳米粒(Fe3O4@SiO2-NH2)、树枝状修饰的磁性纳米粒(Fe3O4@SiO2@PAMAM)和分子印迹识别吸附材料(MIPs)的磁滞曲线图(VSM),表明上述材料的饱和磁化强度分别为17.69、12.84和10.70emu/g,同时展现了其实际磁性分离效果,在外磁场作用下,15秒即可实现固液分离,并可快速实现再分散。图4e为X射线光电子能谱分析图(XPS),表明MIPs由铁、硅、碳、氮和氧组成,结论与图3d-h的元素映射图结果一致。图4f为MIPs的XPS碳元素精细谱,存在属于 C=O、C-O、C-N和C-C结合能的特征峰,证明了树枝状修饰纳米材料和表面分子印迹的成功合成。
图5a为分子印迹识别吸附材料(MIPs)和非分子印迹识别吸附材料(NIPs) 的静态吸附特性。结果表明,MIPs和NIPs的最大吸附量分别为1243.4μmol/g 和228.7μmol/g。说明印记过程中,MIPs的印记空穴及结合位点对目标物具有较高的亲和能力。同时,NIPs的吸附是因为大量功能基团的存在,增加了非特异性吸附,但MIPs的印迹因子是NIPs的5.43倍,仍说明MIPs对目标物NNK 具有较好的识别能力。图5b-c为MIPs的动态吸附及解析动力学曲线。得知MIPs 在20分钟可达到吸附平衡,15分钟可达到解析平衡,其具有较好的吸附动力学表现是由于表面印迹的传质阻力较低,这一特性归因于树枝状材料的高比表面积、规则的树枝状纳米结构。
图6为本实施例1制得的MIPs的选择性吸附试验;选择了结构类似物3- 甲基吡啶和烟酰胺,以及结构非类似物氯霉素和β-雌二醇,进行选择性吸附,考察其特异性识别能力。由图可见,MIPs对所有类似物的吸附能力均高于NIPs。虽然3-甲基吡啶和烟酰胺结构与NNK类似,但本发明的MIPs仍仅对目标化合物NNK具有较高的选择性和较强的吸附能力,原因是分子印迹材料特定的三维空穴和识别位点,只会特异性的识别吸附目标物,表明合成的MIPs高选择性的能力。
图7为本实施例1制得的MIPs的重现性、重复性和稳定性试验:制备5个不同批次MIPs,采用本发明实施例的分析方法,对10mM浓度NNK标准溶液进行吸附量考察(图7a-c)。测定结果为5个不同批次MIPs初次使用其对10mM 浓度NNK标准溶液吸附容量的RSD为3.16%;同一份MIPs重复吸附-解析循环使用7次后吸附容量仍能维持在初次使用时吸附容量的93.6%,表明MIPs可以重复利用,测定重现性和重复性良好。另制备1份MIPs置于4℃冰箱中保存 30天,采用本发明实施例1的分析方法,每隔6天对NNK标准溶液进行吸附容量考察。MIPs在4℃储存30天后吸附容量仍能维持在初始吸附容量的97.1%,表明制得的MIPs稳定性较好。
(2)取5mL步骤1中烟草检测液,加入20mg磁性树枝状分子印迹识别吸附材料(MIPs),室温振摇20分钟,外磁场分离,弃去上清液。加入甲醇-乙酸洗脱液(v:v=9:1)超声15分钟,外磁场分离,获取上清液,氮气吹干,500μL 的0.1wt%乙酸铵甲醇溶液复溶。
步骤3、采用高效液相色谱法检测NNK的含量:
高效液相色谱条件为,色谱柱:C18柱(5μm,150mm×4.6mm);流速:1.0mL/min;柱温:25℃;进样量:10μL;洗脱方式:梯度洗脱;检测器:二极管阵列检测器(DAD),扫描范围:190-800nm;检测波长为230nm。流动相A为 0.08wt%乙酸铵水溶液,流动相B为0.1wt%乙酸铵甲醇溶液。梯度洗脱条件为: 0–4.5min,40wt%流动相A+60wt%流动相B;4.5–10.0min,5wt%流动相A+95wt%流动相B。
标准溶液的配制:取NNK标准液适量,精密称定,配置250μg/mL的储备液。依次配置0.01-250μg/mL不同浓度的的系列标准溶液,液相测定峰面积。结果为:NNK的标准线性方程为Y=31639X-68384,相关系数R2为0.9993,检测限为2.5ng/mL,定量限为6.2ng/mL。
采用该富集方法,结果为:测得市售烤制型烟草样品1和烟草样品2中NNK 的含量分别是8.32和7.16ng/g。
图8为烟草样品经本实施例1制备的磁性树枝状分子印迹识别吸附材料处理前后的高效液相色谱图对比,其中(a)为NNK标准对照溶液高效液相色谱图, (b)为样品经过分子印迹富集后的高效液相色谱图,(c)为样品未经过分子印迹富集的高效液相色谱图,(d)为样品经过分子印迹富集后的剩余残留溶液的高效液相色谱图。结果表明,由于烟草样品中基质成分复杂,如未经MIPs处理而直接进样,则基质干扰严重,目标物色谱峰接近基线噪音,无法准确测定(图 8c)。而经过MIPs富集后,富含印记空穴和识别位点的MIPs对目标化合物具有明显的特异性识别作用,因此大部分干扰物被去除,目标物被选择性富集,可采用常规HPLC定量测定(图8b),极大提高了分析方法的灵敏度和普适性。同时,与样品经过分子印迹富集后的剩余残留溶液的高效液相色谱图进行比较 (图8d),并结合表1的回收率结果,表明MIPs可较完全的吸附、富集目标物,为实现准确定量分析提供保证。此外,MIPs在外磁场作用下,成功地迅速分离富集复杂基质中的目标物,与常规的固相萃取相比,省去了繁琐的离心、过滤等步骤。HPLC分析显示了MIPs优越的去基质干扰能力,并具有良好的回收率和重复性。为复杂样品中微痕量物质监测提供了一种有效的分析手段。
加样回收率试验:烟草样品中分别加入低、中、高3个浓度NNK的标准溶液,按照步骤1和步骤2处理样品,经高效液相色谱法检测,并计算目标物NNK 的回收率如表1所示。由表1可以看出NNK的平均回收率在87.8-97.3%之间, RSD≤2.71%,表明本方法具有较好的准确度和精密度,可用于实际样品中NNK 的富集检测。
表1本发明实施例1分析检测烟草样品中NNK的含量与加样回收率
【注:烟草样品均从市场上购买得到。含量是指市售烟草样品中被检测出NNK的量;加入量指的在烟草样品中按照含量约80%,100%,120%加入标准物质NNK的量;测得量是加入标准物质NNK后,在烟草样品中最终实际测得的总NNK含量;回收率系指向已知被测物含量的样品中精密加入一定量的被测物对照品,依法测定。用测得的总量与供试品的本底量之差,除以加入对照品量计算。加标回收率=(加标样品测得量-原样品含量)÷加入量×100%】
将本实施例分析方法与文献报道的关于NNK其它分析方法进行比较,见表 2。由表2可知,本实施例分析方法采用分子印迹(MIPs)进行前处理,磁性分离省去了离心、过滤等繁杂步骤,操作更加简便快速;分析方法选择性好,成本低廉,无需复杂昂贵的仪器。与其它方法比较,本方法分析时间短,并有较好的检测限和较宽的线性范围。
表2本发明分析方法与文献报道的NNK其它分析方法比较
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实施例2
对晾晒烟叶粉末中的NNK含量进行富集检测,步骤如下:
步骤1、烟叶粉末检测液的制备:取干燥的烟烟叶粉末0.5g,加入5mL水 (含有10mM乙酸铵),超声处理,并在4℃冰箱中放置30分钟。随后,收集上清液,0.22μm微孔滤膜过滤,氮气吹干,5mL甲醇复溶。
步骤2、采用磁性树枝状分子印迹识别吸附材料分离并富集烟草检测液中的 NNK;操作过程同实施例1。
步骤3、采用高效液相色谱法检测NNK的含量:
高效液相色谱条件为,色谱柱:C18柱(5μm,150mm×4.6mm);流速: 0.8mL/min;柱温:35℃;进样量:1μL;洗脱方式:梯度洗脱;检测器:二极管阵列检测器(DAD),检测波长为235nm。流动相A为水(含10mM乙酸铵),流动相B为甲醇(含10mM乙酸铵)。梯度洗脱条件为:0–5.0min,50wt%流动相A+50wt%流动相B;5.0–10.0min,10wt%流动相A+90wt%流动相B。
标准溶液的配制同实施例1。根据NNK标准溶液的浓度和峰面积,获得本次实验的标准线性方程为Y=31090X-4506.8,相关系数R2为0.9993,检测限为 4.1ng/mL,定量限为8.1ng/mL。测得晾晒烟叶粉末1和烟叶粉末2中NNK 的含量分别是6.74和5.98ng/g。
实施例3
对新鲜烟叶中的NNK含量进行富集检测,步骤如下:
步骤1、新鲜烟叶检测液的制备:准确称取冻干新鲜烟叶5g,加入50mL 水(含有10mM乙酸铵),超声处理,并在4℃冰箱中放置过夜。收集上清液, 0.45μm微孔滤膜过滤,氮气吹干,10mL甲醇复溶。
步骤2、采用磁性树枝状分子印迹识别吸附材料分离并富集烟草检测液中的 NNK。操作过程同实施例1。
步骤3、采用高效液相色谱法检测NNK的含量:
高效液相色谱条件为,色谱柱:C18柱(5μm,150mm×4.6mm);流速:1.0mL/min;柱温:50℃;进样量:20μL;洗脱方式:梯度洗脱;检测器:二极管阵列检测器(DAD),检测波长为230nm。流动相A为水(含10mM乙酸铵),流动相B为甲醇(含10mM乙酸铵)。梯度洗脱条件为:0–4.5min,35wt%流动相A+65wt%流动相B;4.5–10.0min,8wt%流动相A+92wt%流动相B。
标准溶液的配制同实施例1。根据NNK标准溶液的浓度和峰面积,获得本次实验的标准线性方程为Y=32308X-8165,相关系数R2为0.9997,检测限为3.8 ng/mL,定量限为7.2ng/mL。测得新鲜烟叶样品1和新鲜烟叶样品2中NNK 的含量分别是5.42和3.94ng/g。
以上显示和描述了本发明的主要特征和本发明的优点。本领域的技术人员应该了解,本发明不受上述实施例的限制,以上所述仅为本发明的较佳实施例,并不用以限制本发明。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (7)
1.一种富集检测烟草中4-甲基亚硝胺基-1-(3-吡啶基)-1-丁酮(NNK)的方法,其特征在于,包括以下步骤:
(1)制备烟草检测液:将烟草加入含有乙酸铵的水溶液中,超声处理一段时间,收集上清液,经微孔滤膜过滤得到滤液,氮气吹干,用甲醇复溶得到甲醇溶液;
(2)富集NNK:采用磁性树枝状分子印迹识别吸附材料分离并富集步骤(1)得到的甲醇溶液中的NNK;
(3)检测:采用高效液相色谱法检测NNK。
2.根据权利要求1所述的方法,其特征在于,步骤(1)烟草与含有乙酸铵的水溶液的质量比为1:(5-500);超声提取5-120分钟;微孔滤膜的孔径为0.1-0.5μm。
3.根据权利要求1所述的方法,其特征在于,步骤(2)所述磁性树枝状分子印迹识别吸附材料制备步骤为:将Fe3O4纳米颗粒外包覆SiO2后氨基硅烷偶联化修饰,得到氨基修饰的Fe3O4@SiO2纳米粒;然后加入醛功能试剂与1-5代聚酰胺-胺树枝的甲醇溶液;外磁场分离,洗涤干燥;然后加入虚拟模板分子N-(羟甲基)烟酰胺、交联剂、引发剂和功能单体进行分子印迹层的合成;磁性分离,洗脱虚拟模板分子,干燥;即得到所述磁性树枝状分子印迹识别吸附材料。
4.根据权利要求1所述的方法,其特征在于,步骤(2)的富集步骤为:甲醇溶液中加入磁性树枝状分子印迹识别吸附材料,室温振摇一段时间,外磁场分离;向磁性树枝状分子印迹识别吸附材料加入甲醇-乙酸洗脱液(v:v=9:1)超声15分钟;外磁场分离,获取上清液,氮气吹干;然后使用0.1wt%乙酸铵甲醇溶液复溶。
5.根据权利要求1所述的方法,其特征在于,步骤(3)的液相条件为,色谱柱:C18柱(5μm,150mm×4.6mm);流速:1.0mL/min;柱温:25-50℃;进样量:10μL;洗脱方式:梯度洗脱;检测器:二极管阵列检测器(DAD),全波长扫描(190-800nm),检测波长为230nm。
6.根据权利要求5所述的方法,其特征在于,梯度洗脱条件为:0–4.5min,40wt%流动相A+60wt%流动相B;4.5–10.0min,5wt%流动相A+95wt%流动相B。
7.根据权利要求6所述的方法,其特征在于,流动相A为0.08wt%乙酸铵水溶液,流动相B为0.1wt%乙酸铵甲醇溶液。
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