生物样品中硒形态分析前处理技术_张绿茵.pdf

2012 年6 月 June 2012 岩矿测试 ROCK AND MINERAL ANALYSIS Vol．31， No．3 407 ～412 收稿日期 2011 －11 －01; 接受日期 2012 －04 －20 基金项目 国家自然科学基金重点项目 2083600 ; 天津市科技支撑重点项目 11ZCKFGX2800 ; 天津市特聘教授人才引进启动基 金 20100405 ; 教育部博士学科点科研专项 20101208110003 作者简介 张绿茵， 硕士研究生， 海洋化学专业。E- mail lvyinhaohao163． com。 通讯作者 邓天龙， 教授， 博士生导师， 主要从事海卤水资源综合利用、 可再生资源利用技术和海洋化学研究。 E- mail tldengtust． edu．cn。 文章编号 0254- 5357 2012 03- 407- 06 Pretreatment Techniques for Selenium Speciation Analysis in Biosamples ZHANG L- yin1， YU Xiao- ping1， 2， GUO Ya- fei1， 3， DENG Tian- long1， 2， 3* 1． Tianjin Key Laboratory of Marine Resources and Chemistry，Tianjin University of Science and Technology，Tianjin300457，China; 2． Key Laboratory of Salt Lakes Resources and Chemistry，Qinghai Institute of Salt Lakes， Chinese Academy of Sciences，Xining810008，China; 3． College of Materials and Chemistry ＆ Chemical Engineering，Chengdu University of Technology， Chengdu610059，China Abstract Pretreatment processes play an important role in selenium speciation analysis． Selenium species extractive s including liquid extraction，microwave aided extraction，solid- phase microextraction and liquid- phase microextraction and the pretreatment s for biosamples are summarized in this paper． The new trends of pretreatment for trace and ultra- trace of selenium species in the future should focus on satisfying the automatic requirement with simple，rapid and eco- friendly characteristics，and be capable of integrating the different extraction techniques or new pretreatment s of direct and on- line coupling with specific selenium species detection． Key words selenium; speciation analysis; pretreatment techniques; biosamples 生物样品中硒形态分析前处理技术 张绿茵1，余晓平1， 2，郭亚飞1， 3，邓天龙1， 2， 3* 1． 天津科技大学， 天津市海洋资源与化学重点实验室，天津300457; 2． 中国科学院青海盐湖研究所盐湖资源与化学重点实验室，青海 西宁810008; 3． 成都理工大学材料与化学化工学院，四川 成都610059 摘要 样品前处理过程在硒形态分析中至关重要。文章对生物样品中硒形态分析的前处理方法， 包括液相 萃取、 微波辅助萃取、 固相微萃取、 液相微萃取等及其应用进行归纳总结， 指出了痕量、 超痕量硒形态分析前 处理技术的未来发展方向主要集中在满足简单、 快速和环境友好的自动化处理， 并着重于样品前处理的各种 萃取技术在线耦合以及与硒形态分析检测器联用技术。 关键词 硒; 形态分析; 前处理技术; 生物样品 中图分类号 O613． 52; O652． 4文献标识码 A 1Introduction Samplepretreatmentconstitutesabasicand crucial stepfortraceseleniumanalysis， andis generally analyte and matrix dependent，requiring a suitableoptimizationofitsdifferentinfluencing parameters．Somesequentialextractionprocesses which can distinguish the water- soluble and non- soluble selenium fractions areavailablesoasto 704 ChaoXing uate the availability of selenium inbiological materials． Due to the low- level presence of selenium in biosamples with complex matrices，its separation from other elements and the use of a pre- concentration step prior to determination are usually necessary．This paper mainly summarizes the development of sample pretreatment s for selenium speciation analysis in biosamples，including liquid extraction，microwave aidedextraction， solid- phasemicroextractionand liquid- phase microextraction． 2Selenium Species Extractive s in Biosamples 2． 1Liquid Extraction Extraction s adopted for selenium speciation analysis must be capable of quantitatively extracting selenium from biosamples without altering the individual selenium species under the extraction conditions． Liquid extraction is widely used as a mild pre- treatment techniqueforseparationandpre- concentrationof selenium species，and liquid extraction s at present mainly include aqueous extraction，enzymatic hydrolysis and pressurized liquid extraction． 2． 1． 1Aqueous Extraction The aqueous extraction process generally includes adding a highly selective reagent into the sample， waterbathshaking， centrifugingandfiltrating． Although hot water extraction is sometimes employed forseleniumspecies， lowrecoveriesareoften observed． Chen et al［1 ］used hot water to extraction selenium species［ selenomethionine SeMet ，selenite ［Se Ⅳ ］ ， selenate ［Se Ⅵ ］ ， selenocystein SeCys ，selenoethionineSeEt ，Se- methylselenocysteine MeSeCys ］ by reversed- phase high perance liquid chromatography RP- HPLC combinedwithinductivelycoupledplasmamass spectrometry ICP- MS ． The result showed that SeMet and SeCys were the main water soluble small molecule selenium species in Se- enriched yeast and Se- enriched clover， and only 20recoveries wereobtained． However，some acids such as hydrochloric acid，acetic acid and citric acid can improve the extract selenium species in some case． Wang et al［2 ］analyzed selenium species ［ SeMet，Se Ⅳ ，Se Ⅵ ，SeCys and SeEt］ in fresh mushroom and pork samples by HPLC- ICP- MS using citric acid as the extraction solution，and found that the recoveries were close to 100 for inorganic selenium and 85． 0- 95． 3 for SeMet，but were worse for SeCys and SeEt． Generally，acidic digestion was less appropriate for biosamples as some important organic selenium species may trans into other s． Besides， the alkaline extraction system sodium hydroxide showedahighperanceforthe extraction of high molecular weight fractions containing selenium．Somemixedreagents water- methanol， water- methanol- choro andbuffersolutions acidic ammonium acetate- EDTA mixture，Tris- HCl and KH2PO4 are also sometimes applied． Especially for Tris- HCl，it shows a great capability and selectivity for the extraction of water soluble proteins． Gergely et al［3 ］used NaOH and Tris- HCl respectively to extract selenium species ［ SeMet，Se Ⅳ ，Se Ⅵ ，SeCys， MeSeCys］from Agaricus bisporus and Lentinula edodes mushroom by reversed- phase ion- pairing HPLC- ICP- MS， andtheextractionefficienciesforAgaricus bisporus and Lentinula edodes mushrooms were 65． 20 －70． 30 and 69． 10 －78． 00，respectively． 2． 1． 2Enzymatic Hydrolysis Enzymatichydrolysisissuitabletoextract selenium protein bound compounds in biosamples． Enzymes such as protease ⅩⅣ，lipase Ⅶ，proteinase K，pronase E，pepsin，trypsin and protease Ⅷ are often used to extract various selenium species， of which protease ⅩⅣ is most often used． For example，Kuo et al［4 ］used Protease ⅩⅣ to extract selenium species ［ Se Ⅳ ，Se Ⅵ ，SeMet，Se- methylselenocysteine MeSeCys ］from milk powder and rice flour samples， and the extraction efficiency was no less than 90 for each samples． Chen et al［1 ］compared the extraction efficiencyofhotwaterextractionandenzymatic extraction for selenium species of SeMet，Se Ⅳ ， Se Ⅵ ，SeCys，SeEt，MeSeCys in Se- enriched yeast and Se- enriched clover samples by RP- HPLC，and it was found that the recoveries were higher for enzymatic extraction ranging from 94． 9to 116． 4when compared with 20 extraction efficiency with hot water extraction．However，the application of enzymes is limited by its time- consuming characteristics for a complete hydrolysis process，which needs about 5 －24 h． In view of this point，ultrasonic assisted enzymatic digestion USAED accelerated by ultrasound energy both ultrasonic water- baths and ultrasonic probes for extracting total selenium or selenium speciation during enzymatic hydrolysis biosamples is rapid ＜ 2 min ， easy to control and inexpensive． For example，Vale et al［5 ］hyphenated USAED with HPLC for total selenium and selenium speciation determination in supplements using low sample amounts 10 mgand extracting volume 1 mL ，and the results showed that total selenium contents in all samples were in agreement with the manufacture values with error no more than 30 and recoveries ranging from 85 to 110 for all selenium species． 804 第3 期 岩矿测试 http ∥www． ykcs． ac． cn 2012 年 ChaoXing 2． 1． 3Pressurized Liquid Extraction PLE Whencomparedwithextrctviesof sonication， mechanical agitation， microwave， and Soxhlet extraction，PLE is a new approach to extract organic compounds at high pressure and temperature ore rapidly， using less solvent， and being automated． Gmez- Ariza et al［ 6 ］extracted selenium speciation ［ Se Ⅳ ，Se Ⅵ ， SeMet，SeCys，SeEt］ in yeast samples by PLE and analyzed it by liquid chromatography- hydride generation- atomic fluorescence spectrometry LC- HG- AFS ． They found that the highest recoveries were obtained using PLE when compared with mechanical agitation， sonication and Soxhlet extraction． The detection limits uated for PLE were 0．02， 0．03， 0．04， 0．01 and 0．02 μg/g for SeCys， SeMet， SeEt， Se Ⅳ and Se Ⅵ ， respectively． 2． 2Microwave Aided Extraction MAE MAE can be used for batch processing samples and is suitable for thermally unstable substances with the advantages of minimizing solvent consumption， generating less pollution，using less time and being safe． In order to enhance the dissolving capacity of the analyte，a certain amount of polar reagent is often added into the extraction reagents when non- polar reagents，such as benzene and normal hexane，are used． Some parameters including the kind of extraction solvent，power of the microwave，time，temperature and humidity can obviously influence the recoveries of MAE． Wang et al［7 ］proposed a MAE procedure with a microwave digester at 80℃ for 3 min to extract the selenium species in fish tissues， and the results showed that less than 35 of total selenium was extracted when 80 V/V methanolwasaddedintoa swordfish sample and only a small fraction of inorganic selenium ［ Se Ⅳ ，Se Ⅵ ］was extractable． Several attempts based on the use of microwave energy to speed up the enzymatic hydrolysis process found that better extraction efficiency of selenium species could be expectedbyusingmicrowaveassistedenzymatic extraction［8 ］． Hsieh et al［9 ］utilized a simple and rapid microwave assistedenzymatichydrolysis Protease ⅩⅣandLipase toextractselenium compounds of Se Ⅳ ，Se Ⅵ ，SeMet，SeCys， MeSeCys from fish liver and dietary supplement by using a CEM MARS microwave digester equipped with temperatureandpressuresensorssystemand programmed to maintain the water temperature at 37℃ for 30 min with a ramp time of 10 min． The recoveries for SeMet，MeSeCys，SeCys，Se Ⅳ ，Se Ⅵ were of 95－ 101and 95－ 101for dietary supplement and fish liver，respectively． 2． 3Solid- Phase Microextraction SPME Traditional sample preparation s are usually time- consuming， have a high risk and use large amounts of organic solvents，thus many solvent- free extraction s have been developed．SPME is one of the solvent- free extraction approaches in which the sorbent material is attached to the surface of a fiber rather than packaged into a cartridge tube or upon the surface of a flat disk，and it can be divided into two basic types direct immersion DI- SPMEand headspace HS- SPME ． DI- SPME is suitable for analyzing non- volatile organic compounds in gas and liquid samples，while HS-SPME is mainly applied to analyze volatile and half- volatile organic compounds in complex matrices． Many advantages including extremely small sample usage， short analytical time and low costs make SPME a simple and convenient technique． However， most water soluble seleniumspeciesareunsuitablefordirectSPME sampling．Somevolatilealkylselenidessuchas dimethylselenide DMSe anddimethyldiselenide DMDSe are ideal targets for HS- SPME sampling． For example， Campillo et al［ 10 ］used HS- SPME coupled with GC- AES to analyze the species of DMSe and DMDSe in different beverage samples，and it was found that the bonded carboxen/polydimethylsiloxane fibers were the most suitable for pre- concentrating the analyte from the headspace of the sample solution with more than 88 recoveries of DMSe and DMDSe． Meanwhile，Campillo et al［ 11 ］also studied the influence of some SPME parameters including sample volume or mass，ionic strength，temperature，adsorption and desorption time on extraction effect during the analysis of DMSe and DMDSe in milk and its by- products by SPME- GC． Average recovery of DMSe and DMDSe for the four samples were all within 89． 5 15． 5 under the optimum conditions of adsorption for 5 min at 40℃ in 30 w/Vof ionic strength of NaCl solution and desorption of 1 min at 1300 r/min in 2 mL of extraction solution． Of course，SPME also has some disadvantages such as limited lifetime，weak fibers and the possibility of sample carry- over during runs． 2． 4Liquid- Phase Microextraction LPME LPME has been developed based on liquid- liquid extraction technologyandincreasinglyappliedfor selenium speciation analysis in both inorganic and organic samples．It normally takes place between several microliters of water- immiscible solvent known as extractant or acceptor and an aqueous phase also known in some cases as donor phase containing the analyte［12 ］．Threeprocessesincludingextraction， concentrationandsampleintroductioncan simultaneously be finished at a step using LPME． LPME can be divided into three modes single drop microextraction SDME ， dispersiveliquid- liquid 904 第3 期张绿茵， 等 生物样品中硒形态分析前处理技术第31 卷 ChaoXing microextraction DLLME and hollow fiber- LPME HF- LPME ．The HF- LPME technique has been developed based on the application of a supported liquid membrane． The organic solvent in HF- LPME is injected into the lumen of a porous hollow fiber acting as an interface between the sample solution and the extraction phase．Saleha et al［13 ］used HF- LPME coupled with HPLC for the determination of ultra- trace amounts of Se Ⅳafter derivatization in biological under natural water samples． The results showed that the maximum extraction efficiency of piazselenol the reactant of Se Ⅳwith ophenylenediamine was obtained at these conditions 20 μL of 1- octanol at pH 2 extraction solvent ， 30 min extraction time， 500 r/min of stirring rate and 30 w/VNaCl the addition of salt can decrease the solubility of the analyte and enhancepartitioningintotheorganic phase ． The RSD of this was no more than 3． 7 for different concentrations of Se Ⅳ ． Sarkouhi et al［14 ］presented a novel to determine selenium based on the ation of piazselenol by derivatization andsubsequentLPMEandgaschromatography separation． A dynamic linear range of 20 －1000 μg/L was obtained under optimized conditions for Se Ⅳ ． The pre- concentration factor and detection limit in this were 91 and 0． 9 μg/L，respectively． 3Pretreatment of Biosamples The analytical results of biosamples are always unsatisfactory compared with the recommended values of standard reference material for complex matrices of different samples． Also，some components in samples retained in chromatographic column will finally lead to low column efficiency． Therefore，choosing a suitable pretreatment for actual samples prior to analysis is of great importance． Generally，the actual samples for selenium speciation analysis can be roughly divided into fluid and solid samples． 3． 1Fluid Biosamples Most papersreportedforseleniumspeciation analysis in fluid samples involve urine，milk，serum and waters of many kinds． Usually，fluid samples need certain pretreatment s before injecting into a chromatographiccolumnincludingdilutionor concentrationandsubsequentlyfiltratio