CN117030879A - 一种快速测定环境水中马兜铃酸的方法 - Google Patents
一种快速测定环境水中马兜铃酸的方法 Download PDFInfo
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Abstract
本发明公开了一种快速测定环境水中马兜铃酸的方法,该方法采用超声辅助分散液‑液微萃取(US‑DLLME)‑高效液相色谱(HPLC)联用技术测定环境水中马兜铃酸I(AA‑I)和马兜铃酸II(AA‑II)的方法。本发明方法简单、快速,前处理过程仅需12分钟。对浏阳河水样和湘江水样进行加标考察,相对标准偏差(RSD)均小于5.7%,加标回收率在91.7%‑97.8%之间。结果表明,该方法简单、环境友好、灵敏度高,可成功应用于环境水样中AA‑I和AA‑II的快速测定。
Description
技术领域
本发明涉及检测技术领域,特别涉及一种快速测定环境水中马兜铃酸的方法。
背景技术
马兜铃酸(AAs)是广泛存在于马兜铃科植物中的硝基菲类化合物,AA-I和AA-II是其中最常见的两种,结构式如下所示。早期因该类物质具有抗肿瘤活性、抗炎等作用,含AAs的中药在医药上长期广泛应用。近年来,马兜铃酸类物质被明确证实具有肾毒性、致癌和致基因突变作用,被认为是导致马兜铃酸肾病(AAN)、巴尔干地方性肾病(BEN)和慢性肾脏病(CKD)的主要原因之一。许多国家包括美国、英国、德国、澳大利亚等已明确禁止销售含有AAs的草药。
目前,AAs的检测方法主要包括高效液相色谱-紫外检测法(HPLC-UV)、高效液相色谱-荧光检测法(HPLC-FLD),液相色谱-质谱法(LC-MS)和毛细管电泳法(CE)等。其中,HPLC-UV法是马兜铃酸测定最常用的方法,也是中国药典收录的方法,但该法检测灵敏度较低,主要用于单味中药中较高浓度的AAs的检测。对于基质复杂、AAs含量低的制剂,借助选择性和灵敏度更高的LC-MS。遗憾的是,由于AAs的电离效率低,质谱分析受到限制。使用LC-MS进行AAs分析的检测灵敏度并不比使用HPLC-UV的检测灵敏度高。此外,由于CE在AAs分析中的重现性差,也很少使用。
Wan Chan等[W.Chan,N.M.W.Li,C.K.Chan,J.Liu,K.L.Deng,Y.N.Wang,B./>E.N./>Quantitation of aristolochic acids in corn,wheatgrain,and soil samples collected in Serbia:identifying a novel exposurepathway in the etiology of Balkan endemic nephropathy,J.Agric.Food Chem.64(2016)5928-5934.]提出了在酸溶液中将锌粉或铁粉将非荧光的马兜铃酸A还原成荧光的马兜铃内酰胺A然后再用HPLC-FLD或HPLC-MS进行测定的方法,该法选择性好、灵敏度高,且不需用到昂贵的质谱仪,但是该法需要经过提取、还原衍生、SPE净化等诸多步骤,繁琐费时。
通常,由于分析物的浓度低,且样品中存在大量干扰物,因此在测定分析物之前必须对样品进行预浓缩和净化。液-液萃取(LLE)是分离和提取马兜铃酸最常用的预处理方法。然而,LLE需要消耗大量有机溶剂,且难以实现分析物的富集和纯化。固相萃取(SPE)和磁性固相萃取(MSPE)与HPLC或LC-MS联用可成功地用于测定药用植物中的AAs。然而,这些方法通常需要使用固相萃取柱或合成特定的吸附材料。
发明内容
为此,本发明提供了一种快速测定环境水中马兜铃酸的方法,包含如下步骤:
(1)标准液配置:分别将AA-I和AA-II溶解在乙腈中,配置成AA-I标准储备液和AA-II标准储备液;AA-I标准储备液和AA-II标准储备液分别用乙腈稀释成AA-I标准液和AA-II标准液;
(2)分散液-液微萃取法:分别将AA-I标准液、AA-II标准液和待测的样品溶液作为料液相溶液置于离心管中,向离心管中加入乙腈和三氯甲烷的混合溶剂,加料完成后摇匀,混合物恒温至40~60℃超声处理,处理后离心,沉积相移出后用氮气吹干,在加入乙腈复溶获得色谱分析溶液;
(3)色谱分析:分别将所述步骤(2)采用AA-I标准液、AA-II标准液或待测的样品溶液制得的色谱分析溶液进行色谱分析,色谱柱:InertSustain C18柱(4.6mm×250mm,5μm);温度:30℃;流动相:乙腈-0.5%醋酸溶液(45:55,v/v);流速:1ml/min;进样量:4μL;检测波长:254nm。
进一步地,所述AA-I标准储备液中AA-I的浓度为400μg/mL;AA-II标准储备液中AA-II的浓度为500μg/mL;所述AA-I标准液中AA-I的浓度为0.01-2.00μg/mL,AA-II标准液中AA-II的浓度为0.01-2.00μg/mL。
进一步地,所述步骤(2)中,每5mL所述料液相溶液加入600μL所述混合溶剂,后续加入50μL乙腈复溶;所述混合溶剂中乙腈和三氯甲烷的体积比为乙腈:三氯甲烷=4~6:1。
进一步地,所述沉积相移出后用55~60℃的氮气吹干。
进一步地,所述混合物恒温至40~60℃超声处理3~4min,处理后4000rpm离心3~4min;超声波功率为100W,频率为40KHz。
进一步地,加入乙腈和三氯甲烷的混合溶剂前先将所述料液相溶液的pH调整至1~3。
本发明的有益效果在于:本发明所述方法可实现AA-I和AA-II的同时萃取,富集因子高,预处理时间短,提取溶剂消耗少。较高的富集因子确保该方法的灵敏度比现有技术中使用类似紫外检测器的方法高1-2个数量级。
附图说明
图1为萃取溶剂体积对富集因子的影响(萃取条件:料液相体积:5ml;萃取溶剂:氯仿;分散剂:乙腈;分散剂体积:500μL;超声时间:2分钟);
图2为分散剂溶剂体积对富集因子的影响(萃取条件:料液相体积:5ml;萃取溶剂:氯仿;萃取溶剂体积:100μL;分散剂:乙腈;超声时间:2分钟);
图3为超声波时间对富集因子的影响(萃取条件:料液相体积5ml;萃取溶剂:氯仿;萃取溶剂体积:100μL;分散剂溶剂:乙腈;分散剂体积:500μL);
图4为样品溶液pH值对富集因子的影响(萃取条件:料液相体积5ml;萃取溶剂:氯仿;萃取溶剂体积:100μL;分散剂:乙腈;分散剂体积:500μL;超声波处理时间:3分钟);
图5为US-DLLME后的进料溶液和重组溶液色谱图(1:AA-II;2:AA-I);
(A)含有5.0μg/mL AA-I和AA-II的标准溶液的色谱图(进样量:20μL);
(B)含有1.0μg/mL AA-I和AA-II的标准溶液的US-DLLME后的重组溶液色谱图(注射体积:4μL);
图6中,(A)20μL浏阳河水样,(B)4μL浏阳河水样US-DLLME后,(C)20μL 0.05μg/mL加标水样,(D)4μL 0.05μg/mL加标水样US-DLLME后的色谱图(1:AA-II;2:AA-I);
具体实施方式
下面结合实施例对本发明做进一步的说明。
1、用到的试剂和样品
马兜铃酸I(99.6%)购自上海安谱实验科技有限公司,马兜铃酸II(≥97%)购自西格玛化学有限公司。甲醇、乙腈为色谱纯,其它试剂均为分析纯,购于国药集团;实验室用水取自Milli-Q纯水系统。
将4.0mg AA-I或5.0mg AA-II分别溶解于10mL乙腈中,得到浓度分别为400μg/mLAA-I或500μg/mLAA-II的标准储备液。标准储备液用乙腈稀释得到标准系列溶液AA-I标准液和AA-II标准液,所述AA-I标准液中AA-I的浓度为1μg/mL,AA-II标准液中AA-II的浓度为1μg/mL。标准储备液和标准溶液均储存在4℃冰箱中。
样品溶液:2020年6月4日至8日从湖南农业大学附近的浏阳河和湖南师范大学附近的湘江采集的环境水样。
在萃取过程中,使用的标准溶液和样品溶液作为料液相溶液。
2、仪器和HPLC条件
LC-20AT高效液相色谱仪(日本岛津公司),配SPD-M20A紫外检测器。TG16-WS离心机(湖南湘仪离心机仪器有限公司),KQ-100E超声波清洗机(昆山超声波仪器有限公司),MD-200吹氮仪(杭州奥盛仪器有限公司)。
色谱柱:InertSustain C18柱(4.6mm×250mm,5μm);温度:30℃;流动相:乙腈-0.5%醋酸溶液(45:55,v/v);流速:1ml/min;进样量:4μL;检测波长:254nm。
2.3.分散液-液微萃取法
将5.00mL料液相溶液置于10mL尖底离心管中,快速注入600μL混合溶剂(含有500μL乙腈作为分散剂溶剂和100μL CHCl3作为萃取溶剂),摇匀。然后将混合物在50℃下超声处理3分钟(超声波功率为100W,频率为40KHz),并在4000rpm下离心3分钟。沉积相用100μL微量注射器移入5mL离心管中。考虑到有机溶剂对液相色谱柱的影响,实验中有机沉积相先55℃氮吹挥干、用50μL乙腈复溶后再进行色谱分析。
2.4.富集因子和相对回收率的计算
富集因子(EF)表示萃取过程中目标分析物的浓度增加的倍数,计算如下:
式中,cr,final和cf,initial分别表示复溶后溶液中分析物的最终浓度和萃取开始前分析物在料液相中的初始浓度。
相对回收率(R)计算如下:
式中,nr,final、nf,initial和nf,added分别为复溶溶液中分析物的最终物质的量、料液相中分析物的初始物质的量以及料液相中分析物加入的物质的量。
3、结果和讨论
3.1萃取条件的优化
3.1.1.萃取溶剂的选择
在US-DLLME中,萃取溶剂的选择是影响萃取效率和富集因子的重要因素。通常需满足两个条件:一是其密度大于水,这样才能通过离心的方法把水溶液与萃取剂分离,偶尔也有密度小于水的,需要采用合适的方法进行分离;二是萃取剂要不溶于水,且对待测物的溶解能力要大,以保证取得良好的萃取效率。实验考察了对AA-I和AA-II溶解度较好的氯仿、二氯甲烷、正辛醇和乙酸乙酯四种萃取溶剂对富集因子的影响,结果见表1。由表可知,因为乙酸乙酯与水溶液之间未实现分层,所以不适合用作萃取溶剂。以正辛醇、氯仿或二氯甲烷为萃取溶剂,均获得了56倍以上的富集因子。但正辛醇密度低,浮在上层,不容易操作。综合考虑到操作的便利性和更高的富集效果,选择氯仿作为最佳萃取溶剂。
表1萃取溶剂对富集因子的影响
3.1.2分散剂的选择
分散剂也是影响萃取效率的另一重要因素。合适的分散剂溶剂可以极大地增加萃取溶剂和分析物之间的接触面积,从而提高萃取效率。实验考察了乙腈、甲醇、乙醇和丙酮四种常用分散溶剂对US-DLLME富集因子的影响。结果表明,使用乙腈、甲醇、乙醇或丙酮作为分散溶剂时,AA-Ι的富集因子分别为63.18、56.35、54.23和57.17,AA-II的富集因子分别为62.13、57.85、55.99和58.83。由此可见,以乙腈为分散剂,可得到较高的富集因子,因此选择乙腈为分散剂。3.1.3萃取溶剂体积的影响
萃取剂体积直接影响分散液相微萃取的萃取率和回收率,和富集倍数密切关系。一般来说,随着萃取剂体积增加,目标化合物的富集倍数逐渐减小,而如果有机沉积相的体积太小,则不易移取。实验考察了50、75、100、150、200和300μL等不同萃取溶剂体积对富集因子的影响,结果如图1所示。由图可知,随着萃取溶剂体积从50μL增加到100μL富集因子出现明显增加。随后,随着萃取溶剂体积的进一步增加,富集因子增长缓慢。一般来说,需要足够大体积的萃取溶剂,以保证萃取效率高,有机相收集方便。然而,过量使用有机溶剂会造成环境污染,增加氮吹的时间和成本。因此,使用100μL萃取溶剂进行进一步研究。
3.1.4分散剂体积的影响
分散剂的体积直接影响溶液均匀分散的程度。分散剂过少时,萃取溶剂和料液相之间的混合不够,萃取剂分散不均匀,萃取率低;分散剂过多时,分析物在样品溶液中的溶解度增大而不易被萃取,萃取率就会降低。实验进一步考察了100、200、500、800和1000μL等不同体积分散剂对富集因子的影响,结果如图2所示。由图可知,当分散剂体积小于500μL时,富集因子随着分散剂体积的增加而逐渐增加。当分散剂体积大于500μL时,富集因子随分散剂体积的增大而逐渐减小。所以,选择500μL分散剂作为最佳分散剂体积。
3.1.5超声时间的影响
超声辅助萃取可以通过增加进料溶液与萃取溶剂的接触面积来提高萃取效率。如果超声萃取时间过短,萃取不完全,也会大大影响萃取效率。超声时间对富集因子的影响结果见图3所示。从图中可以看出,分析物的富集因子先随着超声时间的增加而逐渐增加,当超声时间达到3分钟时,混合溶液通常变得浑浊。进一步延长超声时间,溶液开始轻微乳化,富集因子不再显著增加,且由于超声会产生热量,长时间的超声可能造成试剂的挥发,从而影响萃取效果,因此,选择3分钟作为最佳超声时间。
3.1.6料液相pH值的影响
在US-DLLME中,料液相pH值在萃取的传质过程中起着非常重要的作用。为了将分析物从水溶液中提取到有机相中,分析物应处于非电离状态。根据AA-I(pKa=3.3±0.1)和AA-II(pKa=3.2±0.1)的pKa值,萃取前,需要调节样品溶液的pH值至3左右使目标物去离子化以降低其在水相中的溶解度。实验考察了料液相pH在1.0-5.0范围内对富集因子的影响,结果见图4。图4表明,随着料液相pH值从5.0降低至3.0,分析物非电离形式的比率增加,富集因子缓慢增加。当pH值从3.0进一步降低到1.0时,富集因子不再有明显增加。因此,在后面的实验中,需要通过加入盐酸溶液将料液相pH值调为3.0。
3.1.7料液相中盐浓度的影响
在其他条件不变的情况下,通过向料液相中添加不同量的NaCl(0-6%,w/v),考察料液相中盐浓度对富集因子的影响。结果表明,盐的加入对AA-I和AA-II的富集因子没有明显影响。因此,在随后的实验中选择不加盐。
综上所述,AA-I和AA-II的最佳US-DLLME条件为:100μL氯仿作为萃取溶剂,500μL乙腈作为分散剂,超声波时间为3分钟,料液相pH值为3.0。在最佳萃取条件下,AA-I和AA-II的富集倍数分别高达95.9和93.4。标准溶液直接进样和标准溶液进行US-DLLME后的色谱图如图5所示。与图5A相比,图5B中AA-I和AA-II的峰强度均增加约4倍。结果表明,采用US-DLLME后,分析物得到了有效富集。
3.2分析特性和样品分析
为了评价方法的可行性,在最佳萃取条件下,对方法的线性范围、准确度、精密度、检测限(LOD,S/N=3)和定量限(LOQ,S/N=10)等进行了方法学考察。以标准溶液的浓度为横坐标,以标准溶液US-DLLME后分析物的峰面积为纵坐标,绘制校准曲线。结果表明,AA-I和AA-II在0.01-2.00μg/mL浓度范围内均具有良好的线性,其相关系数(r2)分别为0.9994和0.9997,LOD分别为2.5和3.0ng/mL,LOQ分别为8.0和9.5ng/mL。以0.1和1.0μg/ml两个不同浓度的混合标准溶液考察方法的精密度,得到AA-I的日内精密度(n=3)分别为5.1%和3.2%、日间精密度(n=3)分别为5.4%和3.6%,AA-II的日内精密度分别为5.4%和3.0%、日间精密度(n=3)分别为5.6%和3.4%。
将所开发的方法应用于浏阳河实际水样中AA-I和AA-II的含量测定,相关色谱图如图6所示。图6A和6B表明,无论是样品溶液直接进样分析,还是经过US-DLLME进样分析,均未在水样中发现分析物。图6C和6D分别是浏阳河水加标和浏阳河水加标US-DLLME后的色谱图。与图6C相比,图6D表明,使用US-DLLME作为预处理方法实现了对分析物的有效富集。连续5天的监测结果表明,水样中未发现AA-I和AA-II。将方法用于湘江水样的测定,获得了与浏阳河水相似的测定结果,说明浏阳河水未被AA-I和AA-II污染,相关测定结果见表2。
表2实际样品的测定结果及实际样品加标方法学考察结果
ND=未检出
为了评价方法的基质效应和准确性,考察了浏阳河水样和湘江水样在三个不同浓度(0.02μg/mL、0.20μg/mL和1.50μg/mL)加标下的相对回收率和RSD(见表2)。表2表明,浏阳河水样的相对回收率在91.7%至96.9%之间,RSD小于5.7%;湘江水样的相对回收率在91.9%至97.8%之间,RSD小于5.1%。结果表明,该方法精密度高、准确性好,可用于环境水样中AA-I和AA-II的分析。
进一步将本方法与文献报道的测定AA-I和AA-II的方法进行了比较,结果见表3。如表3所示,与文献中报道的方法相比,本方法具有预处理时间短、萃取溶剂消耗量少、且LOD较低等优点。结果表明,所提出的US-DLLME-HPLC方法灵敏度高、准确度高,可成功用于环境水样中AA-I和AA-II的测定。
表3与文献报道方法的比较
表中文献如下:
[1]J.Yuan,L.Nie,D.Zeng,X.Luo,F.Tang,L.Ding,Q.Liu,M.Guo,S.Yao,Simultaneous determination of nine aristolochic acid analogues in medicinalplants and preparations by high-performance liquid chromatography,Talanta 73(2007)644-650.
[2]C.H.Kuo,C.W.Lee,S.C.Lin,I.L.Tsai,S.S.Lee,Y.J.Tseng,J.J.Kang,F.C.Peng,L.W.Chu,Rapid determination of aristolochic acids I and II in herbalproducts and biological samples by ultra-high-pressure liquid chromatography-tandem mass spectrometry,Talanta 80(2010)1672-1680.
[3]Y.Wang,W.Chan,Determination of aristolochic acids by high-performance liquid chromatography with fluorescence detection,J.Agric.FoodChem.62(2014)5859-5864.
[4]F.Ji,R.Jin,C.Luo,C.Deng,Y.Hu,L.Wang,R.Wang,J.Zhang,G.Song,Fastdetermination of aristolochic acid I(AAI)in traditional Chinese medicine soupwith magnetic solid-phase extraction by high performance liquidchromatography.J.Chromatogr.A 1609(2020)460455.
[5]H.Shu,Y.Ge,X.Y.Xu,P.Q.Guo,Z.M.Luo,W.Du,C.Chang,R.L.Liu,Q.Fu,Hybrid-type carbon microcoil-chitosan composite for selective extraction ofaristolochic acid I from Aristolochiaceae medicinal plants,J.Chromatogr.A1561(2018)13-19.
4.结论
本研究表明,US-DLLME作为HPLC-UV分析之前的样品预处理步骤,可实现AA-I和AA-II的同时萃取,富集因子高,预处理时间短,提取溶剂消耗少。较高的富集因子确保该方法的灵敏度比文献中使用类似紫外检测器的方法高1-2个数量级。结果表明,所提出的US-DLLME-HPLC-UV方法是一种可行、方便和实用的环境水样中AA-I和AA-II分析技术。
以上对本发明所提供的技术方案进行了详细介绍,对于本领域的一般技术人员,依据本发明实施例的思想,在具体实施方式及应用范围上均会有改变之处,综上所述,本说明书内容不应理解为对本发明的限制。
Claims (6)
1.一种快速测定环境水中马兜铃酸的方法,其特征在于,包含如下步骤:
(1)标准液配置:分别将AA-I和AA-II溶解在乙腈中,配置成AA-I标准储备液和AA-II标准储备液;AA-I标准储备液和AA-II标准储备液分别用乙腈稀释成AA-I标准液和AA-II标准液;
(2)分散液-液微萃取法:分别将AA-I标准液、AA-II标准液和待测的样品溶液作为料液相溶液置于离心管中,向离心管中加入乙腈和三氯甲烷的混合溶剂,加料完成后摇匀,混合物恒温至40~60℃超声处理,处理后离心,沉积相移出后用氮气吹干,在加入乙腈复溶获得色谱分析溶液;
(3)色谱分析:分别将所述步骤(2)采用AA-I标准液、AA-II标准液或待测的样品溶液制得的色谱分析溶液进行色谱分析,色谱柱:InertSustain C18柱(4.6mm×250mm,5μm);温度:30℃;流动相:乙腈-0.5%醋酸溶液(45:55,v/v);流速:1ml/min;进样量:4μL;检测波长:254nm。
2.根据权利要求1所述的一种快速测定环境水中马兜铃酸的方法,其特征在于,所述AA-I标准储备液中AA-I的浓度为400μg/mL;AA-II标准储备液中AA-II的浓度为500μg/mL;所述AA-I标准液中AA-I的浓度为0.01-2.00μg/mL,AA-II标准液中AA-II的浓度为0.01-2.00μg/mL。
3.根据权利要求1所述的一种快速测定环境水中马兜铃酸的方法,其特征在于,所述步骤(2)中,每5mL所述料液相溶液加入600μL所述混合溶剂,后续加入50μL乙腈复溶;所述混合溶剂中乙腈和三氯甲烷的体积比为乙腈:三氯甲烷=4~6:1。
4.根据权利要求1所述的一种快速测定环境水中马兜铃酸的方法,其特征在于,所述沉积相移出后用55~60℃的氮气吹干。
5.根据权利要求1所述的一种快速测定环境水中马兜铃酸的方法,其特征在于,所述混合物恒温至40~60℃超声处理3~4min,处理后4000rpm离心3~4min;超声波功率为100W,频率为40KHz。
6.根据权利要求1所述的一种快速测定环境水中马兜铃酸的方法,其特征在于,加入乙腈和三氯甲烷的混合溶剂前先将所述料液相溶液的pH调整至1~3。
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