液相微萃取在药物分析中的应用
液相微萃取技术在药物分析中的应用
样品的前处理是分析化学的一个重要环节,甚至是制约复杂样品分析的关键环节。因此寻找一种高效、快速、简便、环境友好的前处理方法,成为复杂样品分析必需解决的问题,一直是分析化学的研究热点。液-液萃取作为经典的萃取方法,在样品分离上起着重要的作用,但其萃取耗时长,操作步骤多,消耗大量有机溶剂,易造成环境的二次污染[1,2]。
20世纪90年代中后期,He和Lee[3]、Jeannot和Cantwell[4],分别提出了较为成熟的液相微萃取(Liquid Phase Microextraction,LPME)方法。其基本原理是目标分析物在样品与微升级的萃取溶剂之间达到分配平衡,从而实现溶质的萃取和净化。即将样品前处理所涉及的多个步骤(萃取、浓缩、净化)以及直接进样进行GC分析加以组合,大大简化了样品前处理的操作,同时也实现了待测组分的富集。液相微萃取方法富集倍数可高达1000倍以上,操作简便、萃取速度快、操作成本低、不污环境,便于与GC、HPLC及CE等高效分离检测手段联用。通过对LPME技术的不断发展与改进,这种新型的萃取方法已成为现代仪器分析领域一种非常重要的样品前处理技术,在环境分析
析中应用广泛。 [5,6,7]、食品分析[8,9,10]和药物分
1. 液相微萃取的基本原理
1.1. 相平衡理论
液相微萃取的基本原理与经典的液-液萃取相似,可以通过相平衡理论解释。当目标分析物A在溶剂水(w)和萃取溶剂(o)之间进行萃取,达到萃取平衡时,目标分析物A在两相中的分配系数K为一常数,可表示为:
Kow=Co (1) Cw
式中Co,为萃取平衡时目标物A在有机相中的浓度,Cw,目标物A在水相中的浓度。根据质量平衡关系式,则:
Cw,initialVw=Co,eqVo+Cw,eqVw (2)
式中为Cw,initial为样本溶液中目标物A的最初浓度, Vw为样本溶液的体积,Vo为有机相体积。
由式(1)、式(2)可得:
Co,eq=KowCw,initial (3) 1+KowVo/Vw
1.2. 富集倍数(EF)
富集倍数(EF)是指当达到萃取平衡时,目标分析物A在萃取溶剂的浓度(Co,eq)和样品相中的最初浓度(Cw,initial)之比。即:
EF=Co,eq
Cw,initial (4)
将式(2)、式(3)代入得:
EF=1 (5) Vo/Vw+1/K
通过公式(5)可知,要获得较高的富集倍数,可通过降低有机相与水相的体积比Vo/Vw,或选择具有较高分配系数的两相组合来实现。因此,液相微萃取适合将非极性或中等极性的物质从水相中富集到有机萃取溶剂中。
1.3. 萃取回收率(ER)
萃取回收率(ER)为当达到萃取平衡时,目标分析物A被萃取到有机相得量(no)占样品相中分析物初始量(nw,initial)之比。即:
ER=no,eq
nw,initial⨯100%=Co,eqVo⨯100% (6) Cw,eqVw+Co,eqVo
将式(1)代入得:
ER=KowVo⨯100% (7) KowVo+Vw
在液相微萃取中,Vo较小,Vw较大,分析物的Kow适中,致使KowVo<<Vw,此时上式可表达为:ER=KowVo⨯100%。由此可见在降低有机相与水相的体积Vw
比Vo/VW,提高富集倍数的同时也会降低萃取效率。因此需要通过试验调整有机相与水相的体积比Vo/VW,达到合适的富集倍数和萃取回收率。
2. 液相微萃取的形式
经过近十几年研究,液相微萃取技术发展出多种不同的萃取方式。根据萃取过程的状态可以分为静态液相微萃取(Static liquid phase
microextraction,S-LPME)[11]、动态液相微萃取(Dynamic liquid phase microextraction,D-LPME)[12]、连续流动液相微萃取(Continuous flow
microcxtraction,CFME)[13];根据萃取剂与样品的作用形式可以分为直接浸入式液相微萃取(Direct immersion liquid phase mieroextraction,DI-LPME)14和顶空液相微萃取(Headspace liquid phase microextraction,HS-LPME)15;根据萃取溶剂的状态不同可以分为单滴液相微萃取(Single drop liquid phase microcxtraction,SD-LPME)16和中空纤维膜液相微萃取(Hollow fiber based liquidphase microextraction,HF-LPME);根据作用相的多少可以分为两相液相微萃取和三相液相微萃取。
2.1. 单滴液相微萃取(Single-Drop liquid phase mieroextraction,SDLPME)
单滴液相微萃取是最早开发的液相微萃取方法。其主要方式见图1 ,是将1滴与水不相溶的有机溶剂液滴浸没在流动着的大水滴或浸入到被搅动着的样品溶液中对分析物进行萃取。通常使用微量注射器作为有机溶剂的载体,并在萃取完成后吸取萃取溶剂用于进样。在此基础之上发展得而来的动态单滴液相微萃取则是将有机溶剂抽进微量注射器中随后将含被测物的水溶液(样品溶液)也吸入装有有机溶剂的注射器中,反复推拉注射器活塞进行萃取。在反复推拉萃取的同时有机溶剂在微量注射器内壁形成一层有机溶剂薄膜,在每一次推拉萃取过程中,注射器腔内的萃取溶剂和腔壁有机液膜不断地进行更新和交换,实现了动态单滴液相微萃取。动态单滴液相微萃取不仅增加了被测物在两相中的扩散速度,并且提高了其富集倍数。Kamlesh Shrivas等利用单滴液相微萃取分析生物样本中的奎宁时,测得动态液相微萃取的富集倍数及静态的富集倍数分别为14 和3,最低检出限分别为0.15和0.8μM [18]。表明动态液相微萃取能提高监测灵敏度。 17
图 1单滴液相微萃取示意图
单滴液相微萃取适合用于萃取分子量相对较大、熔点和沸点较高、在水中有适当溶解度的化合物,这类化合物辛醇-水分配系数较大(KOW约在2000附近)。常用沸点相对较低的有机溶剂萃取(如:甲苯、二甲苯、正己烷),这类溶剂萃取后可直接进入GC,进行分析。而大多数极性分子(如酮类、醇类和胺类)极易与溶剂形成氢键或产生偶极作用,能被醇类溶剂有效萃取[19]。萃取得到的分析物也可采用HPLC或其他方法进行分析。当使用HPLC时,通常需要将有机萃取溶剂挥干,再用流动性或其他能与流动相互溶的溶剂溶解。
单滴液相微萃取具有溶剂用量少、仪器设备简单、操作快速等优点。但是该方法适合于萃取较为洁净的液体样品,对样品基质中含有固体颗粒、盐、不溶性的有机物或能乳化有机溶剂的可溶性蛋白等的复杂基质样品的萃取效果较差。而且悬在微量注射器针头的有机液滴在样品搅拌时易于脱落。因此,单滴液相微萃取在实际工作中的应用受到了限制。在用于体内药物分析时,最适合用于尿液的分析。
对于以离子形式存在的被测物,Ma和Cantwell[20]提出了液相微萃取-反萃取的模式。即通过调节样品相的pH使被测物质转变成进入有机相的中性分子而被有机相萃取,然后再调节接受相水溶液的pH,使被测物质转变成便于进入水相的离子型化合物而被接受相反萃取。这一萃取模式后来被定义为液-液-液微萃
取。该方法适合于酸性、碱性或离子型的极性化合物。由于接受相为水相,因此可直接进行HPLC、CE和ASS等分析。同时该方法也可服了单滴液相微萃取不适合复杂基质样品的分析,广泛应用于血液样品和中药的分析。
图 2 液-液-液微萃取示意图
表1 单滴液相微萃取在药物分析中的应用
2.2. 中空纤维液相微萃取(hollow-fiber liquid phase mieroextraction,
HF-LPME)
为了克服单滴液相微萃取的缺点(萃取液滴易于脱落,不适合复杂基质的分析),Pedersen 等人[25]提出了以多孔纤维膜为基础的新型液相微萃取,装置见图3。该方法将数微升的萃取溶剂(接受相)注入到浸没于水溶液(样品相)中的中空纤维管腔内进行萃取,达到萃取平衡后用微量注射器吸管腔内的接受相用于分析测定。这样较好地保护了萃取溶剂,同时由于中空纤维的多孔性,增加了溶剂与样品接触的表面积,从而提高了萃取效率,减小了溶剂损失,降低了复杂基质的影响,实现了在萃取的同时净化样品。中空纤维液相微萃取可分为两相中空纤维液相微萃取和三相中空纤维液相微萃取。
图 3 中空纤维液相微萃取示意图
(a) (b)
图 4两相(a)和三相(b)中空纤维液相微萃取原理图
2.2.1. 两相中空纤维液相微萃取
图4.a所示为两相中空纤维液相微萃取形式,中空纤维管腔内部的接受相和吸附在中空纤维壁孔中的液膜为同一种有机溶剂,水相中的被测物质由于萃取融入壁孔中及管腔内的有机溶剂中,达到富集、精华的作用。溶剂的体积通为10-20μl,萃取之后,吸取接受相直接用于GC分析;也有少数情况为吸取接受相之后挥干溶剂,在用合适的溶剂溶剂用于HPLC或CE分析。两相中空纤维液相微萃取适合于多数在有机相中溶解度大于其在水相中溶解度的弱极性物质。
两相中空纤维液相微萃取的萃取溶剂应满足三个条件[26]:对分析组分的溶解度需足够大,以获得较大的富集倍数;在水中的溶解度应小,以减少萃取过程中的损失;与中空纤维有较强的亲和力,能进入中空纤维壁的孔隙中,并能保持稳定,尽量避免其在萃取过程流失。常用的萃取溶剂有甲苯、正己烷等。
Qin Xiao等[27]建立了两相液相微萃取方法用于分析尿液中的四种酚噻嗪类药物。在他们的试验中pH=9.0 的3mL样品溶液被置于玻璃小瓶中,以甲苯为萃取溶剂,将在甲苯中浸泡过的15mm中空纤维浸没在样品溶液进行萃取。萃取温度为40℃,萃取时间为10分钟,磁力搅拌器速度为1000rpm。富集倍数为98~141。
2.2.2. 三相中空纤维液相微萃取
图4.b所示为三相中空纤维液相微萃取形式,纤维壁上的小孔中充满了有机相作为液膜,而纤维内部通常为水溶液接受相。在这种体系中分析物先从样品相被萃取到有机溶剂液膜相,再被置于纤维内部的接受相进行反萃取。
该方法适合用于酸性或者碱性可解离物质的前处理。通过调节供相和接受相的pH值,实现被测物质从供相到接受相的转移。对在于碱性物质,供相的pH值应调节至碱性,被测物处理游离状态,抑制其在水相中的溶解。而接受相的pH值相对较低,增加被测物质其中的溶解度。从而促使碱性物质从供相向接受相转移。Ugland等[28]通过对弱碱性药物的三相中空纤维液相微萃取的研究,表明接受相的pH应低于药物的pKa-3.3。相反,对于酸性物质供相的pH值应调节至酸性,而接受相的pH值相对较高。完成萃取之后,吸取水溶性的接受相用于HPLC和CE分析。
Mahnaz Ghambarian等[29]建立了以第二种有机溶剂代替水溶液作为接受相的三相中空纤维液相微萃取方法。由于有机接受相与正十二烷液膜之间的能斯特扩散层较水相与正十二烷液膜之间的扩散层薄,因此有助于被测物质从正十二烷中进入接受相。
表2 中空纤维液相微萃取在药物分析中的应用
注:NG-文献中为作考察
2.3. 分散相液相微萃取(dispersive liquid phase microextraction, DLPME)
分散液相微萃取(DLPME)是自2006年后迅速发展起来的液相微萃取技术。分散相液相为萃取技术以传统的液-液萃取和浊点萃取技术为基础。该技术是将数微升的有机萃取溶剂通过一定的方法(如加入分散剂,或/和超声、旋涡震荡等)形成极其细微的小液滴,并分散到样品水溶液中形成白色云雾状的微乳液,对目标物质进行萃取。待达到萃取平衡后,对乳状液体进行离心或采用其他方法实现两相分离,最后用微量注射器吸取有机相进行分析。分散相液相微萃取最初被用于分析有机化合物如多环芳烃、有机磷农药和氯苯化合物等[37]。
分散相液相微萃取技术的基础是:a.萃取溶剂能够以微液滴的形式分散到样品溶液中,使萃取相和水相有极大的接触面积;b.萃取之后两相能通过离心等手段进行相分离。
与其他液相微萃取方法相比,分散相液相微萃取具有巨大的优势。由于萃取溶剂与水相的接触表面积大,使得萃取平衡能迅速达到。因此,具有富集倍数高、操作时间短(一般能在5-10分钟内完成)、重复性好等优点。
2.3.1. 传统的分散相液相微萃取(Conventional dispersive liquid phase
microextraction, Conventional-DLPME)
传统的分散相液相微萃取使用密度大于水的萃取溶剂,如二氯甲烷、三氯甲烷、四氯化碳、氯苯等。通过使用将萃取溶剂和分散剂快速注入到样品的水溶液中,形成微乳状液体使萃取溶剂与水相充分作用,实现被测物质的萃取。加入分散剂的目的是促进萃取溶剂在水相中分散形成微小液滴。合适的分散剂即能与萃取溶剂又能与水互溶,能够促进两者之间的相互作用。但是分散剂的存在也影响了萃取之后两相的分离。
由于需要使用密度大于水的液体作为萃取溶剂,可供选择的有机溶剂有限,而且毒性较强。同时这些溶剂密度太大、黏度较高,用微量注射器吸取时较为困难。因此出现了很多新的分散相液相微萃取方法,其中应用较为广泛的是低密度溶剂分散相液相微萃取和漂浮固体液相微萃取。
2.3.2. 低密度溶剂分散相液相微萃取(Low density solvent dispersive liquid
phase microextraction, LDS-DLPME)
顾名思义,低密度溶剂分散相液相微萃取使用的萃取溶剂的密度小于水。该方法扩大了萃取溶剂的范围,可使用许多毒性较小的溶剂,如辛醇、甲苯、正己烷等。但是萃取之后,萃取溶剂处于水相的上层,聚集不完整,难以分离收集。因此不同文献中先后出现多种萃取设备,如注射器、细口塑料瓶等。 其主要作用都是将分散萃取溶剂集中便于收集。
Li-Sha Zhang等[38]将以上两种分散相液相微萃取方法进行结合,提出了三相的分散相液相微萃取。采用两种密度不同的溶剂-庚醇和离子流体作为萃取溶剂。萃取完毕后,庚醇位于水层的上方含有黄酮类化合物,离子流体位于水层下方含有蒽醌类化合物。从而实现了通过一次操作,萃取得到两类性质不同的化合物。
2.3.3. 漂浮有机液滴凝固液相微萃取(Dispersive liquid–liquid
microextraction based on the solidification of floating organic droplets, DLLPME-SFOD)
DLLPME-SPOD与上述两种DLLPME基本原理一致。但选用的有机萃取溶剂具有其特殊性:密度比水小且凝固点接近室温。便于有机溶剂对分析物进行萃取并漂浮于样品溶液表面,当温度降低时,含有分析物质的有机溶剂能够凝固,实现相分离。因此该方法适合于高度或中等亲脂性物质的分离。常用的有机溶剂有:十一醇、1-十二醇、2-十二醇、1-溴代十六烷、正十六烷等。
图4 分散相液相微萃取示意图 (A)传统分散相液相微萃取、(B)漂浮有机液滴凝固液相微萃取、(C)低密度溶剂分散相液相微萃取
表3 分散相液相微萃取在药物分析中的应用
3. 液相微萃取技术的新发展
3.1. 离子流体在液相微萃取中的应用
离子液体(ILs),又称室温离子液体,一般由结构不对称的有机阳离子和无机阴离子组成,是在室温或接近室温下呈液态的有机盐[55]。离子液体具有蒸汽压低、液程宽、热稳定性和溶解性能好、可降解、可设汁性和多样性等特点[56],因此非常适合作为液相微萃取的萃取溶剂。
由于离子流体具有较大的黏度和密度,恰恰能解决单滴液相微萃取中萃取溶剂易于从微量注射器针尖脱落的问题。离子流体的密度通常大于水,由于其环境友好、毒性低。因此在传统分散相液相微萃取中,离子流体逐渐取代高毒性的有机溶剂作为高效的萃取剂[46、47]。三相中空纤维液相微萃取的中介溶剂必须与两侧的溶剂均不互溶,且粘度高,因此只有少数几种有机溶剂满足条件。而与水不互溶的具有非挥发性、粘度大等特性的疏水性离子液体,恰恰成为最理想的中介萃取剂。
但是离子流体的非挥发性限制其用于GC分析。同时其用于HPLC分析时,若流动相中水相比例较大时,也会造成色谱峰变形。Changqin Xiao等[49]通过将离子流体用醋酸溶液进行后萃取再进行HPLC分析,解决了峰变形的问题,同时达到了较高的灵敏度。
3.2. 加电中空纤维膜萃取
传统的中空纤维膜液相微萃取机制是依靠被目标物质分子被动的扩散作用从供相进入接受相。而且为了实现萃取,在两相微萃取时,目标分析物需要有较大的油水分配系数。而对于三相液相微萃取,同时还需要满足目标分析物在接受相的溶解度远远大于其在有机液膜中的溶解度。虽然通过调节供相和接受相的的pH值,能实现液相微萃取,但是分析过程需要十几分钟甚至半个小时才能达到萃取平衡。
为了克服中空纤维膜液相微萃取耗时长的缺点,Pedersen-Bjergaard[57]等于2006年提出了加电中空纤维膜萃取用于分析哌替啶等碱性药物。两段直径为
0.2mm的金属线作为电极分别被插入到样品溶液(pH≈2)和中空纤维膜中心的接受相(10mM盐酸溶液)中,中空纤维膜的空隙中充满邻硝基苯辛醚。在电极之间加入一个300V的电源,正极位于样品溶液,负极位于接受相。碱性药物在酸性溶液中与H集合而带正电,在电压作用下向与负极相连接的接受相迁移,萃取时间缩短至5分钟,大大节省了分析时间。该方法尤其适合于酸性或碱性物质的液相微萃取。
3.3. 清洁能源辅助的液相微萃取
超声、微波和紫外辐射可作提高传质能力的清洁能源。将微波辅助萃
(Microwave extraction, MAE) 技术与HF-LPME 技术相结合 ,利用微波对微观粒子瞬时极化所带来的大量热能和快速传质,达到缩短HF-LPME 平衡时间、提高萃取效率的目的。林珊珊[58]+等利用微波辅助中空纤维液相微萃取测定牛奶中的抗生素残留。与传统的中空纤维液相微萃取比较,该方法将萃取时间从60分钟缩短到小于13分钟;并将富集倍数提高数十倍。
超声辅助[43、59、60]及漩涡振荡辅助[61、62]技术被广泛应用于分散相液相微萃取。超声及漩涡振荡有利于萃取剂在水相中的分散,形成许多小的有机萃取单元,一定程度上能代替分散剂的作用。但如果仅使用超声辅助而不使用分散剂如甲醇、丙酮等,萃取平衡时间会大大延长。因此在超声辅助的同时在水相中加入表面活性剂63,可以促进微乳状液的形成,完全取代有机分散剂的作用,能够大大提高萃取效率。 1 张红,陈玲,陈皓,彭中良.液相微萃取技术及其在环境水样预处理中的应用[J].环境监测管理与技术,2004,16(6):8-11.
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