A new elliptic contour extraction method for reference hole detection in robotic drilling

Talanta46(1998)449–455Extraction procedures for the determination of heavy metals incontaminated soil and sedimentGemma RauretDept.Quı´mica Analı´tica,Uni6ersitat de Barcelona,Barcelona,SpainReceived25May1997;accepted14October1997AbstractExtraction tests are commonly used to study the mobility of metals in soils and sediments by mimicking different environmental conditions or dramatic changes on them.The results obtained by determining the extractable elements are dependent on the extraction procedure applied.The paper summarises state of the art extraction procedures used for heavy metal determination in contaminated soil and sediments.Two types of extraction are considered:single and sequential.Special attention is paid to the Standard,Measurement and Testing projects from the European Commission which focused on the harmonisation of the extraction procedures and on preparing soil and sediment certified reference materials for extractable heavy metal contents.©1998Elsevier Science B.V.All rights reserved. Keywords:Extraction procedures;Heavy metals;Contaminated soil;Sediment;Certified reference materials1.IntroductionTrace metals in soils and sediments may exist in different chemical forms or ways of binding.In unpolluted soils or sediments trace metals are mainly bound to silicates and primary minerals forming relatively immobile species,whereas in polluted ones trace metals are generally more mobile and bound to other soil or sediments phases.In environmental studies the determina-tion of the different ways of binding gives more information on trace metal mobility,as well as on their availability or toxicity,in comparison with the total element content.However,the determi-nation of the different ways of binding is difficult and often impossible.Different approaches are used for soil and sediment analysis,many of them focused on pollutant desorption from the solid phase;others are focused on the pollutant adsorp-tion from a solution by the solid phase.Among those approaches based on desorption,leaching procedures are the most widely accepted and used.Extraction procedures by means of a single extractant are widely used in soil science.These procedures are designed to dissolve a phase whose element content is correlated with the availability of the element to the plants.This approach is well established for major elements and nutrients and it is commonly applied in studies of fertility and quality of crops,for predicting the uptake of essential elements,for diagnosis of deficiency or excess of one element in a soil,in studies of the physical-chemical behaviour of elements in soils0039-9140/98/$19.00©1998Elsevier Science B.V.All rights reserved. PII S0039-9140(97)00406-2G.Rauret/Talanta46(1998)449–455 450and for survey purposes.To a lesser extent they are applied to elements considered as pollutants such as heavy metals.The application of extrac-tion procedures to polluted or naturally contami-nated soils is mainly focused to ascertain the potential availability and mobility of pollutants which is related to soil-plant transfer of pollutants and to study its migration in a soil profile which is usually connected with groundwater problems[1]. For sediment analysis,extraction is used to asses long term emission potential of pollutants and to study the distribution of pollutants among the geochemical phases.As far as heavy metals are concerned sediments are usually a sink but may also become a source under certain condi-tions,especially in heavily contaminated areas or in drastically changing environments.Chemical extraction of sediments has proven to be adequate for determining the metal associated with source constituents in sedimentary deposits[2],but the general aim of many studies involving chemical extraction is the determination of element distri-bution among different phases of a sediment. Single extractants are usually chosen to evaluate a particular release controlling mechanism such as desorption by increasing salinity or complexing by competing organic agents.Generally,fractions can be isolated more specifically by using sequen-tial extraction schemes.For sediments these pro-cedures are frequently used and are designed in relation to the problems arising from disposal of dredged materials.Extraction tests,either in soils and sediments, are always restricted to a reduced group of ele-ments and as far as soil is concerned they are applied to a particular type of soil;silicious,car-bonated or organic.In a regulatory context,two applications for leaching tests can be recognised: the assessment or prediction of the environmental effects of a pollutant concentration in the environ-ment and the promulgation of guidelines or objec-tives for soil quality as for example for land application of sewage sludge or dredge sediments. The data obtained when applying these tests are used for decision makers in topics such as land use of soil or in countermeasures monly used extraction procedures in soils During the last decades several extraction pro-cedures for extractable heavy metals in soils have been developed and modified.In this respect,two groups of tests must be considered:the single reagent extraction test,one extraction solution and one soil sample,and in the sequential extrac-tion procedures,several extraction solutions are used sequentially to the same sample although this last type of extraction is still in development for soils.Both types of extraction are applied using not only different extracting schemes but also different laboratory conditions.This leads to the use of a great deal of extraction procedures. In Table1a summary of the most common leaching test are given.Table1Most common single extraction testsType and solution strength Reference Group[3]HNO30.43–2mol l−1Acid extractionAqua regia[4]HCl0.1–1mol l−1[3]CH3COOH0.1mol l−1[5]Melich1:[6]HCl0.05mol l−1+H2SO40.0125mol l−1EDTA0.01–0.05mols l−1[3] Chelatingagents at different pH[7]DTPA0.005mol l−1+TEA0.1mol l−1CaCl20.01mol l−1Melich3:[8]CH3COOH0.02mol l−1NH4F0.015mol l−1HNO30.013mol l−1EDTA0.001mol l−1NH4–acetate,acetic acidBuffered salt[9]buffer pH=7;1mol l−1solution[3]NH4–acetate,acetic acidbuffer pH=4.8;1mol l−1Unbuffered salt CaCl20.1mol l−1[3]solutionCaCl20.05mol l−1[3][3]CaCl20.01mol l−1NaNO30.1mol l−1[10]NH4NO31mol l−1[3]AlCl30.3mol l−1[11]BaCl20.1mol l−1[12]G.Rauret/Talanta46(1998)449–455451 Table2Extraction methods proposed for standardisation or standardised in some European countriesMethod MethodCountry Reference[15]Mobile trace element determination1mol l−1NH4NO3Germany[16]Available Cu,Zn and Mn evaluation for fer-France0.01mol l−1Na2–EDTA+1mol l−1tilisation purposesCH3COONH4at pH=7DTPA0.005mol l−1+TEA0.1mol l−1+CaCl20.01mol l−1at pH=7.3Available Cu,Zn,Fe and Mn evaluation inItaly0.02mol l−1EDTA+0.5mol l−1[17]acidic soilsCH3COONH4at pH=4.6DTPA0.005mol l−1+TEA0.1mol l−1+CaCl20.01mol l−1at pH=7.3[18]Availability and mobility of heavy metals inCaCl20.1mol l−1Netherlandspolluted soils evaluationSoluble heavy metal(Cu,Zn,Cd,Pb and[19] Switzerland NaNO30.1mol l−1Ni)determination and ecotoxicity risk evalu-ationUnited Kingdom EDTA0.05mol l−1at pH=4[20]Cu availability evaluationFrom Table1it can be observed that a single extraction including a large spectra of extractants are used.It ranges from very strong acids,such as aqua regia,nitric acid or hydrochloric acid,to neutral unbuffered salt solutions,mainly CaCl2or NaNO3.Other extractants such as buffered salt solutions or complexing agents are frequently ap-plied,because of their ability to form very stable water soluble complexes with a wide range of cations.Hot water is also used for the extraction of boron.Basic extraction by using sodium hy-droxide is used to assess the influence of the dissolved organic carbon in the release of heavy metals from soils.A large number of extractants are reviewed by Pickering[13]and Lebourg[14]. The increasing performance of the analytical techniques used for element determination in an extract,together with the increasing evidence that exchangeable metals better correlate with plant uptake,has lead extraction methods to evolve towards the use of less and less aggressive solu-tions[10].These solutions are sometimes called soft extractants and are based on non buffered salt solutions although diluted acids and complex-ant agents are also included in the group.Neutral salts dissolve mainly the cation exchangeable frac-tion although in some cases the complexing ability of the anion can play a certain role.Diluted acids dissolve partially trace elements associated to dif-ferent fractions such as exchangeable,carbonates, iron and manganese oxides and organic matter. Complexing agents dissolve not only exchange-able element fraction but also the element fraction forming organic matter complexes and the ele-ment fractionfixed on the soil hydroxides.Nowa-days it is generally accepted that extractants are not selective and that minor variations in analyti-cal procedures have significant effects on the re-sults.Some leaching procedures for soils have been adopted officially or its adoption is under study in different countries with different objectives[14]. An account of these methods are given on Table 2.monly used extraction procedures in sedimentsAs for soils,exchangeable metal in sediments are selectively displaced by soft extractants.Other extractants used are less selective and they co-ex-tract the exchangeable fraction together with metals bound to different sediment phases moreG.Rauret/Talanta46(1998)449–455 452or less extensively.The phases considered relevant in heavy metals adsorption in sediments are ox-ides,sulphides and organic matter.Fractionation is usually performed by using sequential extrac-tion schemes.The fractions obtained,when apply-ing these schemes,are related to exchangeable metals,metals mainly bound to carbonates, metals released in reducible conditions such as those bound to hydrous oxides of Fe and Mn, metals bonded to oxidable components such as organic matter and sulphides and residual frac-tion.The extractants more commonly used in sequential extraction schemes are generally ap-plied according to the following order:unbuffered salts,weak acids,reducing agents,oxidising agents and strong acids.In Table3the extractants most commonly used to isolate each fraction are given[21].The water soluble fraction may be obtained by two ways,by sampling sediment pore solution using in situ filtration,dialysis tubes or bags,or by a leaching procedure in the laboratory.When this procedure is used the pH may be indeterminate because of the low buffering capacity of the extractant and problems with readsorption occurs.Exchangeable fraction uses an electrolyte such as salts of strong acids and bases or salts of weak acids and bases at pH7to prevent oxyhydroxy phases precipitation. The carbonate bound fraction generally uses an acid such as acetic or a buffer solution acetic acid-sodium acetate at pH5.These reagents are not able to attack all the carbonate content,as for example dolomitic carbonates,neither to attack carbonate selectively as they also remove partially organically bound trace metals.The fraction ob-tained when a reducing solution is used as extrac-tant is mainly related to metals bound to iron and manganese oxides.Hydroxylamine in acid solu-tion is the reducing agent most widely used to solubilise these oxides although iron oxide is not completely dissolved.Ammonium oxalate seems to be most effective when used in the dark,al-though some problems in heavy metals oxalate phase precipitation may occur even at low pH. The sodium dithionite/citrate/carbonate reagent dissolves the oxide and hydroxyoxides but can attack iron rich silicates.So reducing extractants are neither selective nor completely effective for iron and manganese oxides.Other group of ex-tractants used sequentially includes oxidising reagents which destroy organic matter and also oxidises sulphides to sulphates.The extractants most widely used in this group are H2O2and NaOCl.Hydrogen peroxide seems to be more efficient if used after the oxide extraction step. The most widely used extraction scheme is the one proposed by Tessier[22]which has been modified by several authors[23–25].Many of these modifications make more specific the isola-tion of the iron and manganese oxide and hydrox-ide phases.The Tessier procedure is schematised in Table4together with the modified procedures of Fo¨rstner[26]and of Meguelatti[24].4.Harmonisation and method validationOwing to the need of establishing common schemes in Europe for extractable trace metals in soils and sediments the EC Standards,Measure-ment and Testing Programme,formerly BCR(Bu-Table3Most common extractants used in sequential extraction schemesType and solution strenght GroupH2OWater soluble fractionExchangeable and weakly NaNO30.1mol l−1 adsorbed fractionKNO30.1mol l−1MgCl21mol l−1CaCl20.05mol l−1Ca(NO3)20.1mol l−1NH4OAc1mol l−1pH=7 Carbonate bound fraction HOAc0.5mol l−1HOAc/NaOAc1mol l−1pH=5Fractions bound to hydrous NH2OH.HCl0.04mol l−1 oxides of Fe and Mn in acetic or nitric acidNH4OxSodium ditionite,sodiumcitrate,sodium bicarbonate(DCB)Organically bound fraction H2O2NaOClG.Rauret/Talanta46(1998)449–455453 Table4Sequential extraction schemes2345Method1HF/HClO4NaOAc1mol l−1H2O28.8mol l−1Tessier et al.NH2OH.HCl0.04molMgCl2mol l−1l−1HNO3/NH4OAc residual pH7pH525%HOAcsilicate phaseorganic matter+sul-exchangeable carbonate Fe/Mn oxidesphideHNO3NH4Ox/HOx0.1mol H2O28.8mol l−1NH2OH.HCl0.1molFo¨rstner NaOAc1moll−1l−1l−1residualpH7NH4OAcpH5easily reducible pH3in darksilicate phasemoderately reducible organic matter+sul-exchan+carbphideNaOAc1mol l−1NH2OH.HCl0.1molMeguellati BaCl21mol l−1H2O28.8mol l−1+ashingl−1HNO3+HF/HClresidualpH525%HOAcorganic matter+sul-pH7phidesilicate phaseFe/Mn oxidesexchangeable carbonatereau Community of Reference),has sponsored from1987several projects focused on single ex-traction for soils and sequential extraction for soils and sediments.The project started with the intercomparison of existing procedures tested in an interlaboratory exercise[27].The next step was to adopt common procedures for single extraction of trace metals from mineral soils.The second step was to adopt a common procedure for se-quential extraction of sediment.As a conclusion of thefirst step,single extraction procedures using acetic acid,0.43mol l−1,and EDTA,0.005mol l−1for mineral soils and a mixture of DTPA, 0.005mol l−1diethylenetriamine pentaacetic acid, 0.01mol l−1CaCl2and0.1mol l−1tri-ethanolamine for calcareous soils were adopted for extractable Cd,Cr,Cu,Ni,Pb and Zn.In order to improve the quality of the determination of extractable metal content in different types of soil using the procedures previously adopted,the extraction procedures were validated by means of intercomparison exercises[28,29].Moreover the lack of suitable certified reference materials for this type of studies did not enable the quality of the measurements to be controlled.With the pur-pose to overcome this problem three certified reference materials:a terra rossa soil,a sewage amended soil and a calcareous soil have been prepared and their extractable trace metal con-tents were certified(CRM483,CRM484and CRM600)[30,31].The second step of the EC,Standards,Mea-surement and Testing was focused on a feasibility study on the adoption and validation of a sequen-tial extraction scheme for sediment samples.In a workshop held in1992in Sitges(Spain)a sequen-tial extraction scheme was proposed which in-cludes three steps:acetic acid,hydroxylamine hydrochloride or a reducing reagent and hydrogen peroxide or an oxidising reagent.This procedure is schematised in Table5.Moreover in this work-shop the main analytical limitations in sequential extraction of trace metals in sediments were thor-oughly discussed and practical recommendations were given[32,33].These recommendations deal with sampling and sample pre-treatment,practical experiences with reagents and matrices and ana-lytical problems after extraction.Once the scheme was designed,it was tested through two round robin exercises using two dif-ferent type of sediment,silicious and calcareous [34].In these exercises some critical parameters in the protocol were identified such as the type and the speed of the shaking and the need of an optimal separation of the liquid–solid phases af-ter the extraction.It was stated that the sedimentG.Rauret/Talanta46(1998)449–455 454should be continually in suspension during the extraction.In these intercomparison exercises an important decrease was noted on the acceptable set of values for concentration in the extract lower than10m g l−1,which illustrates the difficulties experienced by a number of laboratories in the determination of such concentration levels in these matrices.It was concluded that when elec-trothermal atomic absorption spectrometry is used for thefinal determination,the method of standard additions is strongly recommended for calibration.The results obtained in the round robin exercises encouraged to proceed with the organisation of a certification campaign in order to produce a sediment reference material follow-ing the sequential extraction scheme adopted.So the next step of the project was the preparation of a sediment certified reference material for the extractable contents of Cd,Cr,Cu,Ni.Pb and Zn,following the three-step sequential extraction procedure.A silicious type sediment with rather high trace metal content was chosen for this pur-pose.This material has been recently certified for five metals,Cd,Cr,Ni,Pb and Zn in thefirst step,Cd,Ni and Zn in the second step and Cd,Ni and Pb in the third step[35].Not all the elements were certified because the lack of reproducibility atributable to non adherence to the protocol,in the acceptance of too large tolerances in the con-ditions specified in it or in the existence of critical aspects in the procedure referred mainly to the second step.These aspects were mainly pH,redox conditions and possible losses of sediment in the transfer.The results obtained in the certification exercise recommended to continue the develop-ment of the extraction protocol in order to in-crease reproducibility.Consequently the causes of non reproducibility are now under study in a new SMT project.5.ConclusionsThe advantages of a differential analysis over investigations of total metal contents and about the usefulness of single and sequential chemical extraction for predicting long-term adverse effects of heavy metals from polluted solid material,soils and sediments,is beyond any doubt.The ad-vances in thisfield,especially to make available soil and sediment certified reference materials for extractable element contents by using harmonised procedures,is going to increase the quality of the results due to the possibility of verifying the ana-lytical quality control.Nevertheless some problems need to be solved with these procedures for example:(1)reactions are not selective and are influenced by the experi-mental conditions so it is necessary to identify the main variables which involves a lack of reproduci-bility when applying a procedure,to write very well defined protocols and to validate them;(2) labile fractions could be transformed during sam-ple preparation and during sequential extraction schemes application so problems encountered when preparing certified reference materials are not representing all the problems to be found when working with environmental samples such as wet sediments,some work in this area is needed;(3)analytical problems due to the low level of metals to be measured in the different fractions especially when using soft extractants; and(4)the procedures need to be optimised and validated for different type of soils,including organic soils and sediments.Table5EC Standard,Measurements and Testing procedureConditionsStep10.11mol l−1HOAc,V m−140ml g−1temp.20o C,shaking overnight20.1mol l−1NH2OH.HCl(pH=2with HNO3)V m−140ml.g−1temp.20o Cshaking overnight8.8mol l−1H2O2(pH=2–3with HNO3)3V m−1=10ml g−1room temperature1h.New addition10ml g−185o C for1h.reduce volume to few ml.1mol l−1NH4Oac(pH=2with HNO3)V m−1=50ml g−120o Cshaking overnightG.Rauret/Talanta46(1998)449–455455References[1]H.A.van der Sloot,L.Heasman,Ph.Quevauviller(Eds.),Harmonization of leaching/extraction test,Chap.3, 1997,41–56.[2]H.A.van der Sloot,L.Heasman,Ph.Quevauviller(Eds.),Harmonization of leaching/extraction test,Chap.5, 1997,pp.75–99.[3]I.Novozamski,Th.M.Lexmon,V.J.G.Houba,Int.J.Environ.Anal.Chem.51(1993)47–58.[4]E.Colinet,H.Gonska,B.Griepink,H.Muntau,EURReport8833EN,1983,p57.[5]A.M.Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)135–151.[6]C.L.Mulchi,C.A.Adamu,P.F.Bell,R.L.Chaney,Com-mon.Soil Sci.Plant Anal.23(1992)1053–1059.[7]W.L.Lindsay,W.A.Norvell,Soil Sci.Soc.Am.J.42(1978)421–428.[8]A.Melich,Common.Soil Sci.Plant Anal.15(1984)1409–1416.[9]A.M.Ure,R.Thomas, D.Litlejohn,Int.J.Environ.Anal.Chem.51(1993)65–84.[10]S.K.Gupta,C Aten,Int.J.Environ.Anal.Chem.51(1993)25–46.[11]J.C.Hughes,A.D.Noble,Common.Soil Sci.Plant Anal.22(1991)1753–1766.[12]C.Juste,P.Solda,Agronomie8(1988)897–904.[13]W.P.Pickering,Ore Geol.Rev.1(1986)83–146.[14]A.Lebourg,T.Sterckeman,H.Cielsielki,N.Proix,Agronomie16(1996)201–215.[15]DIN(Deutches Institut fu¨r Normung)(Ed.)Bo-denbeschaffenheit.Vornorm DIN V19730,in:Boden -Chemische Bodenuntersuchungsverfahren,DIN,Berlin, 1993,p.4.[16]AFNOR(Association Francaisede Normalization),AFNOR,Paris,1994,p.250.[17]UNICHIM(Ente Nazionale Italiano di Unificazione),UNICHIM,Milan1991.[18]V.J.G.Houba,I.Novozamski,T.X.Lexmon,J.J.van derLee,Common.Soil Sci.Plant Anal.21(1990)2281–2291.[19]VSBo(Veordnung u¨ber Schadstoffgehalt im Boden)Nr.814.12,Publ.eidg.Drucksachen und Materialzentrale, Bern,1986,pp.1–4.[20]MAFF(Ministry of Agriculture,Fisheries and Food),Reference Book427MAFF,London1981.[21]A.Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Re-port EUR14763EN.1993.[22]A.Tessier,P.G.X.Campbell,M.Bisson,Anal.Chem.51(1979)844.[23]W.Salomons,U.Fo¨rstner,Environ.Lett1(1980)506.[24]M.Meguellati,D.Robbe,P.Marchandise,M.Astruc,Proc.Int.Conf.on Heavy Metals in the Environment, Heidelberg,CEP Consultants,Edinburgh,1983,p.1090.[25]G.Rauret,R.Rubio,J.F.Lopez-Sanchez,Int.J.Envi-ron.Anal.Chem.36(1989)69–83.[26]U.Fo¨rstner,in:R.Lechsber,R.A.Davis,P.L’Hermitte(Eds.),Chemical Methods for Assessing Bioavailable Metals in Sludges,Elsevier,London,1985.[27]A Ure,Ph.Quevauviller,H.Muntau,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)135–151.[28]Ph.Quevauviller,chica,E.Barahona,G.Rauret,A.Ure,A Gomez,H.Muntau,Sci.Total Environ.178(1996)127–132.[29]Ph.Quevauviller,G.Rauret, A.Ure,R.Rubio,J-FLo´pez-Sa´nchez,H.Fiedler,H.Muntau,Mikrochim.Acta 120(1995)289–300.[30]Ph.Quevauviller,G.Rauret,A.Ure,J.Bacon,H.Mun-tau,Report EUR17127EN,1997.[31]Ph.Quevauviller,chica,E.Barahona,G.Rauret,A.Ure,A.Gomez,H.Muntau,Report EUR17555EN,1997.[32]Ph.Quevauviller,G.Rauret,B.Griepink,Int.J.Environ.Anal.Chem.51(1993)231–235.[33]B.Griepink,Int.J.Environ.Anal.Chem.51(1993)123–128.[34]Ph.Quevauviller,G.Rauret,H.Muntau,A.M.Ure,R.Rubio,J-F Lo´pez-Sanchez,H.D.Fiedler, B.Griepink, Fres.J.Anal.Chem.349(1994)808–814.[35]Ph.Quevauviller,G.Rauret,J-F.Lo´pez-Sanchez,R.Rubio, A.Ure,H.Muntau,Report EUR17554EN, 1997.. .。

杨帆，霍志伟，朱雯，等. 超高压辅助胶束法提取落叶松中二氢槲皮素的工艺优化[J]. 食品工业科技，2023，44（23）：175−183. doi:10.13386/j.issn1002-0306.2023020026YANG Fan, HUO Zhiwei, ZHU Wen, et al. Optimization of Ultrahigh Pressure Assisted Micellar Extraction of Taxifolin from Larch[J]. Science and Technology of Food Industry, 2023, 44(23): 175−183. (in Chinese with English abstract). doi:10.13386/j.issn1002-0306.2023020026· 工艺技术 ·超高压辅助胶束法提取落叶松中二氢槲皮素的工艺优化杨　帆，霍志伟，朱　雯，赵修华*（东北林业大学化学化工与资源利用学院，黑龙江哈尔滨 150040）摘　要：为简化二氢槲皮素提取工艺，降低能耗与成本，提高提取效率，促进二氢槲皮素的综合应用，本研究采用黑龙江省的兴安落叶松为原料，运用超高压辅助胶束提取技术提取落叶松中二氢槲皮素，测定落叶松树根、树干等不同部位的二氢槲皮素总含量。

以此总含量为基础，对提取胶束进行筛选，采用响应面试验对提取工艺进行优化，考察了料液比、提取压力、提取次数及胶束浓度4种不同因素对二氢槲皮素提取率的影响，并与微波提取、超声提取、回流提取等不同提取工艺进行能耗与CO 2排放比较。

结果表明，最终确定提取胶束为茶皂素，最佳提取工艺条件为：茶皂素浓度8%，料液比1:11.5，提取压力157 MPa ，提取次数3次，保压时间5 min ，在此最佳条件下重复进行3次实验，二氢槲皮素实际提取率可达84.35%±1.20%，与预测值84.98%基本一致。

Journal of Chromatography A,1218 (2011) 171–177Contents lists available at ScienceDirectJournal of ChromatographyAj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o maRapid,highly efficient extraction and purification of membrane proteins using a microfluidic continuous-flow based aqueous two-phase systemRui Hu a ,Xiaojun Feng a ,Pu Chen a ,Meng Fu b ,Hong Chen c ,Lin Guo b ,Bi-Feng Liu a ,∗aBritton Chance Center for Biomedical Photonics at Wuhan National Laboratory for Optoelectronics –Hubei Bioinformatics &Molecular Imaging Key Laboratory,Department of Systems Biology,College of Life Science and Technology,Huazhong University of Science and Technology,Wuhan 430074,China bCollege of Life Science and Technology,Wuhan University,Wuhan 430072,China cKey Laboratory of Oil Crops Biology of the Ministry of Agriculture,Oil Crops Research Institute,Chinese Academy of Agricultural Sciences,Wuhan 430062,Chinaa r t i c l e i n f o Article history:Received 28July 2010Received in revised form 22October 2010Accepted 25October 2010Available online 30 October 2010Keywords:Microfluidic chipAqueous two-phase system Membrane proteins Purificationa b s t r a c tMembrane proteins play essential roles in regulating various fundamental cellular functions.To investigate membrane proteins,extraction and purification are usually prerequisite steps.Here,we demonstrated a microfluidic aqueous PEG/detergent two-phase system for the purification of mem-brane proteins from crude cell extract,which replaced the conventional discontinuous agitation method with continuous extraction in laminar flows,resulting in significantly increased extraction speed and efficiency.To evaluate this system,different separation and detection methods were used to identify the purified proteins,such as capillary electrophoresis,SDS-PAGE and nano-HPLC–MS/MS.Swiss-Prot database with Mascot search engine was used to search for membrane proteins from random selected bands of SDS-PAGE.Results indicated that efficient purification of membrane proteins can be achieved within 5–7s and approximately 90%of the purified proteins were membrane proteins (the highest extraction efficiency reported up to date),including membrane-associated proteins and integral mem-brane proteins with multiple transmembrane pared to conventional approaches,this new method had advantages of greater specific surface area,minimal emulsification,reduced sample con-sumption and analysis time.We expect the developed method to be potentially useful in membrane protein purifications,facilitating the investigation of membrane proteomics.© 2010 Elsevier B.V. All rights reserved.1.IntroductionMembrane proteins constitute approximately 30%of the pro-teome [1],playing essential roles in regulating various fundamental cellular functions,such as cell recognition,selective transportation of metabolites and receptor-mediated signal transduction [2,3].In addition,more than half of the known membrane proteins are pre-dicted to be pharmacological targets [4].However,researches in membrane proteins are relatively hampered since most membrane proteins are of natural low abundance.Thus,extraction and purifi-cation of membrane proteins is usually a prerequisite step in such investigations.Purification of membrane proteins has proven to be a chal-lenging task due to their hydrophobic nature as complexes of proteins and lipids [5].Consequently,solubilization of membrane proteins by detergents is necessary to separate them from crude cell extract.Currently,detergent/polymer aqueous two-phase system (ATPS)is a common approach for membrane protein enrichment∗Corresponding author.Tel.:+862787792203;fax:+862787792170.E-mail addresses:bfliu@ ,bifeng liu@ (B.-F.Liu).without denaturation [6–8].ATPS involves the use of two aque-ous phases to extract target molecules by vigorous agitation.Although it has been widely adopted in laboratories,the sepa-ration efficiency of ATPS still requires improvement.In addition,emulsification during agitation can also elongate the separation time [9].The emerging microfluidic technology has provided an oppor-tunity for the integration and miniaturization of existing biological tools to address issues like speed,throughput and sample cost [10–12].Previously,Kitamori and co-workers reported a microflu-idic liquid–liquid extraction system,which was applied to the isolation of metal ions based on multi-phase laminar flows.Extrac-tion of different metal ions was successfully realized,including Fe 2+,Co 2+,Ni 2+,K +,Na +and Al 3+[13–15].The extraction effect of laminar flows in microchannels was equivalent to that of vigorous agita-tion.However,the microfluidic-based method had advantages of enhanced extraction speed,simplified operation and potential for miniaturization.Recently,Meagher et al.developed a microfluidic aqueous two-phase system (␮ATPS)for isolating specific proteins from sub-microliter volumes of Escherichia coli cell lysate [16].In this method,PEG-salt two-phase system was realized in a Y-shaped microfluidic channel for continuous extraction of target proteins0021-9673/$–see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2010.10.090172R.Hu et al./J.Chromatogr.A1218 (2011) 171–177Fig.1.Microchip fabrication and system setup.(A)Schematic of the microfluidic chip design.(B)Size comparison of the fabricated PDMS microchip with a U.S.one cent coin.(C)Schematic of the system setup for ␮ATPS and image acquisition.into the PEG phase with reduced separation time,enhanced speed and throughput.Compared to the traditional batch techniques with agitation,␮ATPS has distinctive advantages of fast extraction rate,high separation efficiency and sample enrichment [17].In traditional methods,two liquid phases are highly scattered by vigorous agi-tation to maximize the specific interface area between the two phases and to improve the extraction rate and efficiency.With microfluidic based method,the laminar flow in microchannels usu-ally results in greater specific interface area,avoiding the use of agitation and preventing the occurrence of emulsification.In addi-tion,liquid–liquid extraction is typically a low-throughput batch technique in laboratory,but it is well-suited for continuous oper-ation on microfluidic chips [18,19].Therefore,we believe that the ␮ATPS method is potentially useful in the purification of membrane proteins [20,21].Here,we demonstrated the use of a PEG/detergent ␮ATPS sys-tem for the purification of membrane proteins from crude cell extract.Our ␮ATPS system combined the use of the zwitterionic detergent Zwittergent 3-10,sodium dodecyl sulfate (SDS)and the nonionic detergent Triton X-114,resulting in a complementary solubilization of proteins [22,23].The PEG/detergent two-phase system partitioning allowed successful removal of soluble pro-teins.Integral and peripheral membrane proteins remained in the detergent phase,while soluble proteins were found in the PEG-rich phase.Extraction of FITC-labeled IgG from detergent to PEG phase was first conducted to evaluate the developed ␮ATPS method.Capillary electrophoresis of the purified samples suggested effi-cient purification of IgG within 5–7s.We further applied the ␮ATPS method to the purification of membrane proteins from HeLa cell extracts.Results indicated that 90%of the extracted proteins are membrane proteins,including membrane-associated proteins and integral membrane proteins with multiple transmembrane domains,which represented one of the highest extraction effi-ciency among existing approaches.2.Experimental2.1.Chemicals and reagentsTris (hydroxymethyl)aminomethane (Tris),HCl,NaOH,KCl,NH 4HCO 3,ACN,NaCl,NaHCO 3,KH 2PO 4,Na 2HPO 4·12H 2O,formic acid,ethylene diamine tetra acetic acid (EDTA)were purchased from Tianjing Chemical Co.Ltd.(Tianjing,China).N,N -methylene Bisacrylamide,Coomassie Brilliant Blue G250,zwitterionic deter-gent Zwittergent 3-10were purchased from Fluka (MO,USA).Dithiothreitol (DTT),iodoacetamide (IAA),acrylamide,glycerol,bromophenol blue,␤-mercaptoethanol,polyacrylamide,glycine,polyethylene glycol #6000(PEG 6000),Trypsin (proteomics sequencing grade)were purchased from Amresco (OH,USA).N,N,N ,N -tetramethylethylenediamine (TEMED),ammonium per-sulfate (AP),sodium dodecyl sulfate (SDS),Triton X-114were purchased from Sigma–Aldrich (MO,USA).DMEM were pur-chased from GIBCO (Invitrogen corporation,USA).Membrane Protein Extraction Kit was purchased from XinHan (Shanghai,China).All reagents were of analytical grade unless specified otherwise.Water was purified by the Millipore-Q system (Mil-lipore,USA)before use for the preparation of all solutions.Samples and all buffer solutions were autoclaved (121◦C;0.12MPa)and filtered (0.45␮m microporous membrane filtration)before experiments.For chip experiments,the PEG-rich inlet stream was 35wt%PEG,and the detergent-rich inlet stream was pre-pared with a volume ratio of 9:5:1(20%(w/w)Zwittergent 3-10:100%(w/w)Triton X-114:100mM SDS),resulting in a pH of approximately 7.4.2.2.Chip design and fabricationWe designed the PDMS microchip with serpentine microchan-nels as shown in Fig.1A.The widths of the inlet channel a–o,b–o,c–o and the outlet channel p–e,p–f are 80␮m.The width of the outlet collection channel p–d is 40␮m.The separation channel o–p has a width of 180␮m and a total length of approximately 140mm.All channel depths are 50␮m (Fig.2C).We fabricated the microchip using previously reported protocols [24,25].Fabri-cated PDMS structures are then irreversibly bonded to a planar glass substrate (76mm ×26mm ×1mm)to form the final device.A comparison of the microchip with a US one-cent coin is given in Fig.1B.Micro-syringe pumps are used to control the fluid flow in the microchannels.Typical injection speed was 0.8–1.2␮L min −1of the PEG-rich inlet,and 3.5–5.0␮L min −1of the detergent-rich inlet.2.3.Image acquisitionExperiments were conducted on an inverted fluorescence microscope (IX 71,Olympus,Japan).A mercury lamp was used as the excitation source.For FITC and FQ,the light emitted from the mercury lamp was filtered by a 460–490nm band-pass filter,reflected by a 505nm dichroic mirror,and then focused on the microchannel by a 10×objective (NA 0.7)as illustrated in Fig.1C.During experiments,fluorescence images of the each local channel were collected through the same objective with a 510nm high-passR.Hu et al./J.Chromatogr.A 1218 (2011) 171–177173Fig.2.Extraction of membrane proteins by ␮ATPS.(A)Schematic of the ␮ATPS extraction mechanism.Side streams,PEG-rich phase;middle stream,crude membrane protein extract dissolved in detergent.Black arrows indicate the direction of flow.Hollow arrows indicate the direction of membrane protein migration.(B)Channel width dependence of specific interface area and diffusion time in microchip.S ,specific interface area;V ,volume;W ,diffusion distance;t ,diffusion time;D ,diffusion coefficient.Dotted line indicates the microchannel width of our device (180␮m).(C)Profile of extraction channel.L ,length;W ,width;H ,height.filter and monitored by a CCD camera (CoolSNAP cf2,Photometrics)with 200ms exposure.2.4.Cell cultureHeLa cells were grown in DMEM (Invitrogen Corporation,GIBCO,CA)supplemented with 10%NCS (Invitrogen Corporation,GIBCO,CA)and maintained in standard culture conditions (37◦C,95%humidified air,and 5%CO 2).Cells were allowed to grow toa density of 80%and then were harvested using sterile PBS/EDTA (pH 7.4)before experiments.As they spread out across the cell cul-ture dish,when two adjacent cells touch,this signals them to stop growing,loss of contact inhibition is a classic sign of oncogenic cells.2.5.Crude membrane protein preparationCrude membrane protein was prepared by using extraction kit.Briefly,HeLa cells ((5–10)×107)were collected bycentrifugationFig.3.Evaluation of the ␮ATPS method.(A)Fluorescence images of the extraction of FITC labeled IgG from the detergent phase to the PEG-rich phase.Microchannel outline is indicated by dotted lines.(B)Schematic of the microfluidic chip design.a–c,inlets;e–f,outlets.The red rectangles 1–4indicate the locations where the four fluorescence images in (A)were captured.(C)Capillary electrophoresis of standard FITC-IgG with concentrations of 0.01mg mL −1,0.05mg mL −1and 0.1mg mL −1.(D)Capillary electrophoresis of the FITC-IgG in the solutions before and after ␮ATPS purification.(For interpretation of the references to color in this figure legend,the reader is referred to the web version of the article.)174R.Hu et al./J.Chromatogr.A1218 (2011) 171–177at1000×g for5min at4◦C,then were resuspended in1mL of homogenate buffer with protease inhibitor cocktail in an ice-cold homogenizer and were homogenized on ice for30–50times after been washed once with1mL of ice cold PBS.The homogenate was centrifuged in700×g for10min at4◦C.The supernatant was trans-ferred to a new vial and centrifuged at10,000×g for30min at4◦C, cytosol fraction in the supernatant was collected.The pellet con-tained proteins from both plasma membrane and cellular organelle membrane.It was resuspended in200␮L lysis buffer with protease inhibitor cocktail for30min at4◦C,and centrifuged at10,000×g for 30min at4◦C,membrane protein in the supernatant was collected.2.6.Capillary electrophoresisCapillary electrophoresis–laser-inducedfluorescence detec-tion platform,including capillary,excitation and emission light path,high voltage power supply,optical signal conversion and collection,amplification,filtering devices,signal acquisition pro-grams and hardware,and other related components.The main components include Olympus IX71research-oriented inverted fluorescence microscope excitation,emission-axis optical system; high-voltage power supply(Institute of Nuclear Research,Shang-hai):the maximum voltage of30,000V,maximum current of 0.3mA;electrode diameter of0.6mm of P99platinum wire;argon ion laser(maximum power42mW)as afluorescence excitation light source;PMT photomultiplier tube and associated circuitry for fluorescence signal collection and amplification;hardware signal filter(Stanford Research System Model SR570);NI6035data acqui-sition card and LabView acquisition program.Uncoated fused-silica capillaries(i.d.75␮m,o.d.375␮m)were purchased from Yongnian Optical Fiber Factory(Hebei Province,China)and treated following the protocol introduced by Hjérten[26].2.7.SDS-PAGE and nano-HPLC–MS/MSA200-␮g sample of PM protein was separated by SDS-PAGE on a5%stacking gel and a12%separation gel run according to standard laboratory procedures.After electrophoresis,the gels were stained with Coomassie Brilliant Blue G250.In-gel digestion was performed as described previously with slight modification[27].In brief,random selected bands of SDS-PAGE gel was cut into many1mm3gel slices,each of which contained a stained protein(s).The resulting slices were washed with100mM NH4HCO3containing50%ACN(pH8.0)for3times,till the dye(Coomassie Brilliant Blue)was completely removed.After being dried in a SpeedVac concentrator,the gel-bound proteins were reduced in10mM DTT/50mM NH4HCO3(pH8.0)and then alkylated in55mM iodoacetamide/50mM NH4HCO3(pH8.0)for 30min in a darkroom.The gel pieces were then washed with10mM NH4HCO3,and again dehydrated with ACN and dried in a SpeedVac. The dry gel pieces were reswollen with35␮L of10mM NH4HCO3 containing0.5␮g of promega trypsin.Digestion was carried out at37◦C overnight.The peptides were extracted two times with 40␮L of60%ACN/5%formic acid by sonication/ultrasonic oscilla-tion/sonic oscillation for10min,then centrifuge for2min to collect the supernatant.The combined extracts were evaporated to about 2␮L in a SpeedVac and stored at−80◦C.The digested peptides were injected into a nanoLC system (Eksigent)andfirst desalted and preconcentrated on a CapTrap (0.5mm i.d.,2mm long;MICHROM)precolumn.The peptides were then eluted onto a C18column(100␮m i.d.,15cm long; MICHROM)coupled to a quadrupole time-of-flight(Q-TOF)hybrid mass spectrometer(QSTAR ELITE,Applied Biosystems)equipped with a MicroIonSpray ESI source.The gradient profile consisted of a linear gradient from5%to40%B(0.1%formic acid/1.9% H2O/98%acetonitrile,v/v)over40min into A(0.1%formic acid/1.9%acetonitrile/98%H2O,v/v),followed by10min of85%B and then 15min reconditioning with95%A.Theflow rate was300nL min−1. The peptides were detected in the positive ion MS mode or the data-dependent MS/MS mode.The data-dependent mode was used for survey scans(m/z400–1800)in order to choose up tofive most intense precursor ions.For collision-induced dissociation(CID) mass spectrometric(MS/MS)analysis,collision energies were cho-sen automatically as a function of m/z and charge.The collision gas was nitrogen.The temperature of a heated interface was150◦C and the electrospray voltage was2000V.3.Results and discussion3.1.Purification by ATPSWe demonstrate a continuousflow PEG/detergent␮ATPS.FITC labeled hydrophilic proteins migrated into the PEG phase,while hydrophobic plasma membrane protein from intact membrane protein complexes remained in the detergent phase.This opera-tion is illustrated schematically in Fig.2A.Sample stream of crude plasma membrane protein with unwanted(tagged)proteins are hydrodynamically focused between twoflowing streams contain-ing PEG.Theflow-rates of the liquid samples and extracting reagent were controlled by microsyringe pumps(model210,KD Scientific, Boston,MA)when precise control of the velocity of the sample stream in the mainflow channel was required.Each syringe nee-dle was connected to afilter through a fused-silica capillary tube (GL Sciences,0.25mm i.d.0.5mm o.d.)using epoxy-based glue.TheFig. 4.One-dimensional SDS-PAGE of the prepared crude membrane protein extract.CM,crude membrane proteins;PM,purified membrane proteins.Molecular mass markers are shown on the right.The gel was stained with colloidal Coomassie brilliant blue and12bands were randomly selected for further LC–MS/MS analysis.R.Hu et al./J.Chromatogr.A1218 (2011) 171–177175 outlets were also connected to a fused-silica capillary tube with EPcentrifuge tube for collection in the same way.For experiments thatdemonstrate the operations,the velocity of plugs in the mainflowchannel was∼5␮L min−1.Typically,we found the lower theflowrate,the better the extraction efficiency.However,the laminarflowbecomes unstable at too low aflow rate due to the limitation of thesyringe pumps.Thus,we chose5␮L min−1as the optimalflow ratefor the extraction of hydrophobic membrane proteins.3.2.Theory for purification by ATPSMicrofluidics has several characteristic features different frombulk scalefluidflow,such as short diffusion distance,high spe-cific interface area,and small heat capacity.These characteristicsof microfluidic systems are essential keys to control chemical unitoperations,such as mixing,reaction,extraction and separation.Especially,to control molecular transport in microfluidic channels,the molecular transportation time and the specific interface areamust be considered[28].The molecular transportation time is givenby:t=W2D(1)where t,W,and D are the molecular transportation time,diffusion distance and coefficient,respectively.The specific interface area, , can be expressed as:=SV∝1W(2)where S and V are the interface area and the volume,respectively.In our method,samples were injected into the separating chan-nel byflow focusing.For an ideal sandwich-type laminarflow with each stream occupying one third of the channel,the relationship between the microchannel width and the molecular transporta-tion time and the specific interface area is summarized in Fig.2B. Given the dimensions of our device(Fig.3C),the specific inter-face area is approximately167cm−1,which represents a dramatic increase compared to the conventional mechanical shaking method (1–10cm−1)and previously reported Y-shaped microfluidic sys-tems(80cm−1)[29,30].In consequence,a significant decrease in the transportation time can be expected.For molecules with a diffu-sion coefficient of10−9m2s−1,the transportation time is less than 5s.3.3.System evaluationExperiments were conducted withfluorescent tracer molecules to visualize the performance of the␮ATPS system.Fig.3A illus-trates thefluorescence images captured during experiments with locations indicated by the four rectangles shown in Fig.3B.As shown in Fig.3A,detergent phase containing water-soluble FITC-IgG was injected into the middle stream by hydrodynamic focusing. The FITC-IgG was continuously extracted from the detergent-rich stream into the two PEG-rich side plete extraction of FITC-IgG was observed at the end of the microchannel.Capillary electrophoresis was used to investigate the extraction efficiency of the developed␮ATPS system.Fig.3C illustrates a comparison of electropherogram of increasing concentrations of standard FITC-IgG solutions.To determine the recovery of extracted proteins, samples containing0.05mg mL−1FITC-IgG were quantitatively analyzed before and after␮ATPS extraction.As shown in Fig.3D, FITC-IgG only existed in the solutions collected from the two side streams(outlet e and f),consistent with the optical observations shown in Fig.3A.Quantitative analysis indicated a recovery of 90.8%.The loss of proteins could have resulted from the nonspecific adsorption of proteins on PDMS surfaces.Table1Categories of purified membrane proteins by␮ATPS.Identified membrane proteins Categories4F2cell-surface antigen heavy chain Plasma membrane Alkaline phosphatase,placental type Plasma membrane Alkaline phosphatase,tissue-nonspecific isozyme Plasma membrane Annexin A6Plasma membrane Antithrombin-III Plasma membrane Calcium-binding mitochondrial carrier proteinAralar2Plasma membrane Complement decay-accelerating factor Plasma membraneEzrin Plasma membrane Heterogeneous nuclear ribonucleoprotein M Plasma membrane Intestinal alkaline phosphatase Plasma membrane Junction plakoglobin Plasma membrane Lamin-A/C Plasma membrane Moesin Plasma membrane Olfactory receptor5AC2Plasma membrane Prostaglandin G/H synthase1Plasma membrane Scavenger receptor class B member1Plasma membrane Steryl-sulfatase Plasma membrane Transferrin receptor protein1Plasma membraneWD repeat and FYVE domain-containing protein3Plasma membrane78kDa glucose-regulated protein MembraneCarbamoyl-phosphate synthase[ammonia]MembraneCarnitine O-palmitoyltransferase2Membrane Cytoskeleton-associated protein4MembraneGlycerol-3-phosphate dehydrogenase MembraneGPI transamidase component PIG-S MembraneGPI transamidase component PIG-T MembraneLamin-B1MembraneNADH-ubiquinone oxidoreductase75kDa subunit MembraneNitric oxide synthase,brain MembraneSuccinate dehydrogenase[ubiquinone]flavoprotein subunitMembraneTrifunctional enzyme subunit alpha MembraneAFG3-like protein2Integral to membrane Calnexin Integral to membrane Dolichyl-diphosphooligosaccharide–proteinglycosyltransferase subunit1Integral to membrane Heterogeneous nuclear ribonucleoprotein R Integral to membrane Mitochondrial import receptor subunit TOM70Integral to membrane Protein disulfide-isomerase A4endoplasmic reticulum RNA-binding protein FUS NucleusATPase family AAA domain-containing protein3A CytoplasmElongation factor1-alpha1Cytoplasm3.4.Extraction of membrane proteins and SDS-PAGEFor membrane proteins,it usually involves a crude mem-brane protein extraction procedure before further purification and enrichment.Previously,Cao et al.reported the use of conventional aqueous two-phase agitation method for the purification of crude membrane protein extracts,yielding the highest extraction effi-ciency of67%[31].In this work,the developed␮ATPS system was used in combination with detergents for the purification of crude membrane proteins.After extraction,purified membrane proteins were separated by one-dimensional SDS-PAGE(Fig.4).Twelve bands were selected for further MS analysis to verify the devel-oped method,similar to approaches reported previously[22,23]. Proteins from the selected bands of SDS-PAGE were digested by trypsin;the tryptic peptides were then extracted from each gel band and further separated by reversed-phase nanoLC,and then detected and sequenced with waters Q-TOF micro mass spec-trometer.All MS/MS samples were analyzed using Mascot(Matrix Science,London,UK;version Mascot).Mascot was set up to search the SwissProt57.7database(selected for Homo sapiens)assuming the digestion enzyme trypsin.Mascot was searched with a frag-ment ion mass tolerance of0.40Da and a parent ion tolerance of 200ppm.176R.Hu et al./J.Chromatogr.A1218 (2011) 171–177Fig.5.Classification of the functional categories of the identified plasma membrane proteins in HeLa cells.(A)Subcellular localization of the identified proteins accord-ing to the GO annotation terms.(B)The functional categories of the characterized proteins.3.5.Identification of membrane proteinsAfter being processed with analytical software Analyst QS2.0, samples were utilized to search the Swiss-Prot database with Mas-cot search engine for protein identification.To assess the efficacy of the developed protocol for the enrichment of integral mem-brane proteins and to estimate contamination by other cellular organelles,including mitochondria and endoplasmic reticulum (ER),we classified the40identified proteins according to the gene ontology(GO)annotation and other currently available data (Table1).Of the annotated proteins,36(90%)were previously assigned as integral membrane or membrane-associated proteins. The90%purity of membrane proteins represented one of the highest extraction efficiency among existing approaches.Of the reminder proteins with a subcellular annotation,10%were anno-tated as cytoplasmics,nucleus and ER,this group may include proteins that exist at more than one site in the cell.These data indicate that the contamination by mitochondria and ER in the membrane fraction and soluble non-target protein in cytoplasmics was greatly reduced by use of the microfluidic aqueous two-phase extraction process.In Fig.5A,of the36PM proteins,19(47.5%)were plasma membrane,12(30%)were membrane proteins,5(12.5%) were integral membrane proteins.We also categorized the iden-tified proteins according to their functions,except for5.56%of the protein function is not clear,based on universal GO annota-tion terms Fig.5B:2.94%have signal activity,5.88%of proteins have electric carrier activity,17.65%have catalytic activity,41.18% are involved in cellular binding,and8.82%are structural pro-teins.In addition,17.65%of proteins are transport proteins which allow the passage of inorganic ions and other small,water-soluble molecules into the cells,5.88%proteins were not easily categorized and labeled“others”.Since the types of all membrane proteins are still unknown in HeLa cells,we did not compare the number of collected membrane proteins to the total number of membrane proteins.In addition,the loss of proteins during␮ATPS could not be determined due to unknown number of total proteins and possible loss of proteins during crude membrane protein extraction.4.ConclusionsIn this paper,we demonstrate a microfluidic aqueous PEG/detergent two-phase system for the purification of membrane proteins from crude cell extract.The method was applicable to hydrophobic proteins such as membrane proteins extracted from eukaryotic cells.Our␮ATPS combined the use of the zwitteri-onic detergent Zwittergent3-10,sodium dodecyl sulfate(SDS) and the nonionic detergent Triton X-114,resulting in a comple-mentary solubilization of proteins.The PEG/detergent two-phase system partitioning allowed successful removal of soluble pro-teins.Integral and peripheral membrane proteins remained in the detergent phase,while soluble proteins were found in the PEG-rich phase.Results indicated that approximately90%of the purified pro-teins were membrane proteins,including membrane-associated proteins and integral membrane proteins with multiple transmem-brane pared to conventional approaches,this new method had advantages of greater specific surface area,minimal emulsification,reduced sample consumption and analysis time.We expect the developed method to be potentially useful in membrane protein purifications,facilitating the investigation of membrane proteomics.AcknowledgementsThe authors gratefully acknowledgefinancial support from the National Basic Research Program of China(2007CB914203and 2007CB714507)and the National Natural Science Foundation of China(30970692,20875035and30800286).References[1]C.C.Wu,M.J.MacCoss,K.E.Howell,J.R.Yates,Nat.Biotechnol.21(2003)532.[2]A.Abbott,Nature426(2003)755.[3]M.C.King,C.P.Lusk,G.Blobel,Nature442(2006)1003.[4]C.C.Wu,J.R.Yates,Nat.Biotechnol.21(2003)262.[5]C.Smith,Nat.Methods2(2005)71.[6]M.W.Qoronfleh,B.Benton,R.Ignacio,B.Kaboord,J.Biomed.Biotechnol.2003(2003)249.[7]G.Munchow,F.Schonfeld,S.Hardt,K.Graf,Langmuir24(2008)8547.[8]H.Everberg,R.Peterson,S.Rak,F.Tjerneld,C.Emanuelsson,J.Proteome Res.5(2006)1168.[9]T.Chapman,Nature434(2005)795.[10]A.Hibara,M.Tokeshi,K.Uchiyama,H.Hisamoto,T.Kitamori,Anal.Sci.17(2001)89.[11]T.Minagawa,M.Tokeshi,T.Kitamori,Lab Chip1(2001)72.[12]K.Sato,M.Tokeshi,T.Sawada,T.Kitamori,Anal.Sci.16(2000)455.[13]H.Hisamoto,T.Horiuchi,K.Uchiyama,M.Tokeshi,A.Hibara,T.Kitamori,Anal.Chem.73(2001)5551.[14]M.Surmeian,A.Hibara,M.Slyadnev,K.Uchiyama,H.Hisamoto,T.Kitamori,Anal.Lett.34(2001)1421.[15]H.B.Kim,K.Ueno,M.Chiba,O.Kogi,N.Kitamura,Anal.Sci.16(2000)871.[16]R.J.Meagher,Y.K.Light,A.K.Singh,Lab Chip8(2008)527.[17]G.Munchow,S.Hardt,J.P.Kutter,K.S.Drese,Lab Chip7(2007)98.[18]J.Atencia,D.J.Beebe,Nature437(2005)648.[19]P.J.A.Kenis,R.F.Ismagilov,G.M.Whitesides,Science285(1999)83.[20]J.Blonder,M.B.Goshe,R.J.Moore,L.Pasa-Tolic,C.D.Masselon,M.S.Lipton,R.D.Smith,J.Proteome Res.1(2002)351.[21]K.K.Hixson,N.Rodriguez,D.G.Camp,E.F.Strittmatter,M.S.Lipton,R.D.Smith,Electrophoresis23(2002)3224.[22]H.Everberg,T.Leiding,A.Schioth,F.Tjerneld,N.Gustavsson,J.Chromatogr.A1122(2006)35.[23]H.Everberg,U.Sivars,C.Emanuelsson,C.Persson,A.K.Englund,L.Haneskog,P.Lipniunas,M.Jornten-Karlsson,F.Tjerneld,J.Chromatogr.A1029(2004)113.。

Journal of Pharmacy and Pharmacology 7 (2019) 110-118doi: 10.17265/2328-2150/2019.03.003Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunction in Adult Wistar RatsRunning Title: Newbouldia laevis Ameliorates Ovarian DisruptionAdeoye O. Oyewopo1, Kehinde S. Olaniyi2, Adesola A. Oniyide2, Olusola A. Sanya2, Oluwatobi A. Amusa2, Omosola F. Faniyan2and Olugbenga O. Eweoya31. Department of Anatomy, University of Ilorin, Ilorin, 240001, Nigeria2. Department of Physiology, Afe Babalola University, Ado-Ekiti, 360101, Nigeria3. Department of Anatomy, University of Abuja, Abuja, 900241, NigeriaAbstract: Background: The extract of Newbouldia laevis has been demonstrated to show antioxidant, antimicrobial, sedative, anticonvulsant, analgesic, anti-inflammatory, antinociceptive, hepatoprotective, anticancer, antiulcer, antihypertensive and antidiarrhea properties. However, its reproprotective effect is not well known. The present study was designed to investigate the ameliorative effect of Newbouldia laevis in cadmium- induced ovarian dysfunction. Method: Adult female Wistar rats were randomly allotted into groups; Vehicle (received distilled water), cadmium-treated (received 5 mg/kg b.w.), N. laevis-treated (received 200 mg/kg b.w.) and cadmium + N. laevis-treated groups. Cadmium sulphate was administered for 3 days (i.p.) followed by oral administration of N. laevis for 28 days. The body weight change was monitored using animal weighing balance (Olympia SCL66110 model, Kent Scientific Corporation, Torrington, CT06790, USA), biochemical assay and histology of ovaries were performed as previously described. Results: The results showed weight loss, severe disruption of ovarian follicles and significant reduction of gonadotropic hormones (FSH and LH) in cadmium-treated group compared with vehicle-treated group. These alterations were not associated with inflammatory response. However, concomitant administration of aqueous extract of N. laevis and cadmium sulphate significantly ameliorated ovarian disruption. Conclusion: The study demonstrates that administration of aqueous extract of N. laevis during treatment with cadmium sulphate preserves ovarian function, suggesting that possibly daily intake of aqueous extract of N. laevis prevents the onset of ovarian disorders.Key words: Cadmium, hypertrophy, Newbouldia laevis, ovarian dysfunction, reproprotective.1. IntroductionInfertility is a worldwide problem, development in the investigation, treatment and the dissemination of new knowledge has meant a new hope for infertile couples [1]. Infertility is one of the major gynaecological complications that affect women, and the majority of the focus has always been on expensive in-vitro fertilization rather than treating the cause of theCorresponding author:A. O. Oyewopo, Ph.D, associate professor of anatomy, head of reproduction and embryology unit, research field:anatomy. infertility [2]. Studies have shown that 25-30% of infertile women suffer from ovarian dysfunction[3], which does not occur only with disorders of the ovaries, but anything that affects the communication between the brain and ovary through hypothalamic-pituitary-gonadal axis [4, 5].Heavy metals such as cadmium are pollutants generated mostly through human activities, and they have high toxicological impact on humans and animals since they are likely ingested through food like seafood, meat offal, cereals, vegetables and fruits [6, 7]. Cigarette smoke is byfar the greatest source ofAqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats111cadmium exposure while diet remains a primary source of exposure in non-smokers [8]. Cadmium is a transitional metal that exists in different oxidational or transitional states. Acute exposure to cadmium in vivo causes dysuria, polyuria, chest pain, fatigue, headache, and hepatooxidative, where as chronic exposure through contaminated food or air results to organ dysfunction because of cell death through disruption of cellular mitochondrial function [9, 10]. Cadmium toxicity is associated with several clinical complications, renal dysfunction, bone diseases, hepatic dysfunction, testicular and ovarian dysfunction [11, 12].Newbouldia laevis is a medium sized angiosperm in the Bignoniaceae family. Phytochemical analysis of the root, root bark, stem and leaf of N. laevis revealed the presence of alkaloids, phenylpropaniod, glycosides, flavonoids, tanins, saponins, phenols, essential oils, terpenoids, triterpenoids, quinoids, ceramides among others[13].The roots and leaves are used in the treatment of dysentery, elephantiasis, migraines and seizures [14]. An extract of the leaves of N. laevis and mostly used as mouthwash has been shown to be bactericidal in dental caries[15]. Pharmacological studies of extracts of different parts of N. laevis have revealed the antioxidant [13], free radical scavenging[15], antimicrobial[16], sedative, anticonvulsant[17, 18], analgesic, antinociceptive[19], hepatoprotective[13], anticancer[20], uterine contraction[21], wound healing and antiulcer[22], antisickling[23], hypoglycemic, antihypertensive and entomocide activities[24, 25]. However, its reproprotective effect is not well known. The current study attempted to investigate the ameliorative effect of N. laevis on cadmium-induced ovarian dysfunction in adult female rats.2. Materials and Methods2.1 ChemicalsThe entire chemicals used in the study were of AR grade, which were obtained from Sigma Chemical, St. Louis, MO, USA.2.2 Preparation of the ExtractSamples of N. laevis were locally obtained. The leaf of the plant was botanically authenticated by Mr Bolu in the Department of Plant Biology, University of Ilorin, Ilorin. Authentication number was issued and the plant was deposited at the herbarium. The plant was air-dried and pounded into powder using pestle and mortar and kept in an air-tight container. 600 g of the sample was percolated in distilled water for 48 hours and stirred intermittently with magnetic stirrer. It was filtered and the filtrate was evaporated in steam bath until substantial water has been removed. It was later dried in the oven at 37 °C to make the extract concentrated.2.3 Animals, Grouping and ProtocolTwenty adult female Wistar rats weighing 110-250 g were obtained from the animal house, College of Medicine and Health Sciences, University of Ilorin, Ilorin, Nigeria. The rats were housed in wire mesh cages and maintained in a well ventilated room at 25 ±2 °C, on a 12-h light/12-h dark cycle. Rats had unrestricted access to standard rat chow and tap water. After acclimatized for two weeks, the rats were randomly allotted into groups (n= 5 each); Vehicle (received distilled water), cadmium-treated (received 5 mg/kg b.w.), N. laevis-treated (received 200 mg/kg b.w.) and cadmium + N. laevis-treated groups. Cadmium sulphate was administered for 3 days (i.p.) followed by oral administration of N. laevis for 28 days. The investigation was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and was approved by the Institutional Review Board of University of Ilorin, Ilorin, and every effort was made to minimize both the number of animals used and their suffering. Initial and final body weights were monitored using animal weighing balance (Olympia SCL66110 model, Kent Scientific Corporation, Torrington, CT06790, USA) and the body weight change was estimated.Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats1122.4 Sample Preparation and Biochemical AnalysisAt the end of treatment, the rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p). Blood was collected from the apex of the heart into EDTA and heparinized bottle and centrifuged at 3,000 rpm for 15 minutes using a bench centrifuge and the plasma was stored frozen until it was needed for biochemical assay. Biochemical analysis of plasma gonadotropic hormones (Follicle stimulating hormone; FSH, Luteinizing hormone; LH) were performed using ELISA kits obtained from Randox Laboratory Ltd. (Co. Antrim, UK). The blood collected with EDTA bottles was used for the analysis of hematological parameters (RBC; red blood cell, Hb; hemoglobin, HCT; hematocrit, PLT; platelet, WBC; white blood cell, PMN; polymorphonuclear, LYM; lymphocyte). Ratios of PLT/LYM and PMN/LYM were calculated as pro-inflammatory markers.2.5 HistologyThe ovaries were excised, blotted and weighed. After weighing, ovarian tissues were fixed in 10% buffered formol saline for histological examination using hematoxylin and eosin (H&E) staining techniques and examined microscopically.2.6 Statistical AnalysisAll data were expressed as means ± SEM. Statistical group analysis was performed with SPSS, version 22 of statistical software. One-way analysis of variance (ANOVA) was used to compare the mean values of variables among the groups. Bonferroni’s test was used to identify the significance of pair wise comparison of mean values among the groups. Statistically significant differences were accepted at p < 0.05.3. Results3.1 Effects of Newbouldia laevis on Body Weight in Cadmium-Treated Adult Female RatsTable 1 depicts the effect of administration N. laevis and cadmium on body weight. The results showed significant loss in body weight during treatment with cadmium sulphate alone when compared with vehicle-treated group. However, concomitant treatment with aqueous extract of N. laevis during treatment with cadmium sulphate significantly improved the body weight (p < 0.05).3.2 Effect of Newbouldia laevis on the Histology of Ovary in Cadmium-Treated Adult Female RatsHistopathological changes in the ovaries have been shown to influence the function of this organ. H&E stained section of ovaries of vehicle-treated rat, shows normal consistent proliferation of the follicles and normal arrangement of the ovarian epithelium (Fig. 1A), cadmium-treated rat, shows severe pathological lesions evident as hypertrophy of granulosa cells, haemolysis and severe deterioration of ovarian follicles (Fig. 1B), N. laevis-treated rat, shows moderate hypertrophy of ovarian follicles (Fig. 1C) and cadmium + N. laevis-treated rat, shows mild cellular hypertrophy (Fig. 1D).Table 1 Effects of Newbouldia laevis on body weight in cadmium-treated Wistar rats.vs. vehicle; #p < 0.05 vs. cadmium).Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats113Fig. 1 Photomicrograph of a section of ovary in cadmium-treated rat. Vehicle-treated rat, shows normal consistent proliferation of the follicles and normal arrangement of the ovarian epithelium (a); Cadmium-treated rat, shows severe pathological lesions evident as hypertrophy of granulosa cells, haemolysis and severe deterioration of ovarian follicles (b); N. laevis-treated rat, shows normal moderate hypertrophy of ovarian follicles (c); Cadmium + N. laevis-treated rat, shows mild cellular hypertrophy (d).(H & E paraffin stain; ×200, transverse section). PMF (primordial follicle); PF (primary follicle); GC (Granulosa cell); OC (OOcyte).3.3 Effects of Newbouldia laevis on Gonadotropic Hormones (FSH and LH) in Cadmium-Treated Adult Female RatsPlasma levels of gonadotropic hormones (FSH and LH) significantly decreased in cadmium-treated group when compared with vehicle-treated group. However, treatment with N. laevis significantly restored FSH and LH levels (Fig. 2).3.4 Effects of Newbouldia laevis on Hematological Parameters in Cadmium-Treated Adult Female RatsTreatment with cadmium sulphate significantly reduced red blood cells, hematocrit, platelet and white blood cells compared with vehicle-treated group. Whereas cadmium + N. laevis-treated group showed a significant increase in red blood cells, hematocrit, platelet and white blood cells compared with cadmium-treated group (Table 2). Hemoglobin, lymphocyte and polymorphonuclear remained unchanged in all the treated groups compared with vehicle-treated group.3.5 Effects of Newbouldia laevis on Pro-inflammatory Biomarkers (PMN/LYM and PLT/LYM) in Cadmium-Treated Adult Female RatsPMN/LYM and PLT/LYM are pro-inflammatory markers. Treatment with cadmium and cadmium + N. laevis did not significantly alter PMN/LYM and PLT/LYM when compared with control group (Fig. 3).Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunction114in Adult Wistar RatsFig. 2 Effect of N. laevis on circulating luteinizing hormone; LH (a) and follicle stimulating hormone; FSH (b) in cadmium-treated Wistar rats. Data are expressed as mean ± SEM. n = 5. Data were analysed by one-way ANOVA followed by Bonferroni post hoc test.*p < 0.05 vs. vehicle; # p < 0.05 vs. cadmium.Table 2 Effects of Newbouldia laevis on hematological parameters in cadmium-treated Wistar rats.vs. vehicle; #p < 0.05 vs. cadmium).RBC: red blood cell, Hb: hemoglobin, HCT: hematocrit, PLT: platelet, WBC: white blood cell, PMN: polymorphonuclear, LYM: lymphocyte.Fig. 3 Effect of N. laevis on pro-inflammatory biomarkers; PMN/LYM (a) and PLT/LYM (b) in cadmium-treated Wistar rats. Data are expressed as mean ± SEM. n = 5. Data were analysed by one-way ANOVA followed by Bonferroni post hoc test.*p < 0.05 vs. vehicle; # p < 0.05 vs. cadmium). PMN: polymorphonuclear; PLT: platelet; LYM: lymphocyte.Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats1154. DiscussionThe primary physiological function of the female reproductive system is to produce ovum necessary for progeny. Ovarian steroid hormones play a vital role in the production of ovum and other functions associated with reproductive behavior. The hormones secreted by the hypothalamus and pituitary also regulate ovarian functions. Cadmium has been documented to target ovary and suppress the synthesis and secretion of hormones and it is evident that cadmium disrupts reproductive endocrine functions by directly and/or indirectly affect the expression of StAR protein and P450scc during progesterone synthesis and thus interfere with progesterone synthesis[26]. The present study demonstrates significant loss in body weight, reduction of erythrocytes, hematocrit, platelet, white blood cells, severe pathological lesions evident as hypertrophy of granulosa cells, haemolysis, severe deterioration of ovarian follicles, and decrease in FSH or LH in cadmium-treated group compared with vehicle-treated group. However, administration of N. laevis to the group treated with cadmium significantly increased the body weight, erythrocytes, hematocrit, platelet, white blood cells and led to restoration of ovarian tissues/mild hypertrophy. These were associated with significant increase in FSH or LH but not inflammation when compared with cadmium-treated group.Our present result that administration of cadmium significantly reduced the body weight was consistent with earlier studies[5, 27]. This has been shown to be due to decrease availability and production of steroid hormone during cadmium administration[26]. However, treatment with aqueous extract of N. laevis significantly improved the body weight which implies that aqueous extract of N. laevis has the capacity to regulate body weight. Histopathological changes in the cytoarchitecture of ovarian tissue have been used as indicator of ovarian dysfunction. The present results revealed that treatment with cadmium causes severe pathological lesions evident as hypertrophy of granulosa cells, haemolysis and severe deterioration of ovarian follicles (Fig. 1B). This is also in line with previous observation that cadmium treatment induced disruption of ovarian histoarchitecture[28]. Gurel et al.[29] also reported the ovarian follicular cell damage in cadmium-treated female rats. Thus, the present study corroborates the ovo-toxic nature of cadmium. In addition, treatment with aqueous extract of N. laevis significantly restored the ovarian structure with mild hypertrophy when compared with cadmium/vehicle-treated rats respectively (Fig. 1D). This implies that aqueous extract of N. laevis significantly depletes cadmium-induced ovarian dysfunction. This is the first study to reveal the reproprotective effect of N. laevis in cadmium-induced ovarian disruptions.Furthermore, our current results revealed that the ovarian disruption in cadmium-treated animals was associated with significant decrease in circulating levels of FSH and LH. These observations were also in consonance with previous studies [5]. The decrease in FSH and LH levels may be due to impaired hypothalamic-pituitary-gonadotropin secretions reported in an earlier study[30]. Human reproductive toxicity of cadmium has been suggested by several epidemiologic studies[31-33]. The specific effect of exposure to cadmium on the female reproductive system has been previously associated with a reduction in LH binding and FSH as well as altered steroidogenesis in vitro in isolated granulose cells of rats[30]. Nevertheless, in the present study treatment with aqueous extract of N. laevis significantly increased circulating levels of FSH and LH in cadmium + N. laevis-treated group compared with cadmium-treated group (Fig. 2). This implies that aqueous extract of N. laevis significantly stimulates hypothalamic-pituitary-gonadotropin secretions. Hematological parameters indicate the sub-lethal effects of pollutants[34]. Anemia has been observed in rats, mice, rabbits, and monkeys exposed to cadmium.Aqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats116The present data on hematological indices indicate that the animals exposed to cadmium were in anemic condition due to significant reduction in erythrocytes and hematocrit. It is reported that oral cadmium treatment reduces gastrointestinal uptake of iron, which can result in anemia[35]. It was well known that anemia reduces the supply of oxygen to tissues by lowering the oxygen-carrying capacity of the blood. This finding is consistent with previous studies of anemia in animals exposed to cadmium[5]. The present study showed that treatment with cadmium leads to decreased platelet and white blood cells but no significant change in hemoglobin, lymphocyte and polymorphonuclear when compared with vehicle-treated rats. However, administration of aqueous extract of N. laevis significantly attenuated the anemic condition as observed in cadmium + N. laevis-treated group compared with cadmium-treated group through upregulation of erythrocytes and hematocrit.PMN/LYM and PLT/LYM ratios are pro-inflammatory markers. Our present observation that treatment with cadmium did not significantly alter PMN/LYM and PLT/LYM ratios when compared with vehicle-treated group implies that cadmium-induced ovarian disruption was not associated with inflammation and perhaps through oxidative stress as earlier reported [5, 36]. Administration of N. laevis did not also alter PMN/LYM and PLT/LYM ratios in cadmium + N. laevis-treated group compared with cadmium-treated group, which means that the ameliorative effect of aqueous extract of N. laevis in cadmium-induced ovarian dysfunction may probably be through its antioxidant properties as previously documented [5, 13].5. ConclusionsThe study demonstrates that administration of aqueous extract of N. laevis during treatment with cadmium sulphate depletes ovarian disruptions, suggesting that possibly daily intake of aqueous extract of N. laevis protects against the onset of ovarian disorders.AcknowledgementWe acknowledge Dr. Oyewopo I. C., Department of Anesthesia, University of Ilorin Teaching Hospital, Ilorin for her technical support.Conflict of InterestThe authors declare that there are no conflicts of interest.References[1]Amadi, C. N., Siminialayi, I. M., and Orisakwe, O. E.2011. “Male Infertility and Herbal Supplements: An Update.”Pharmacologia 2 (11): 323-48.[2]Kaplan, J. R., and Manuck, S. B. 2004. “Ovariandysfunction, Stress, and Disease: A Primate Continuum.”ILAR J 45 (2): 89-115.[3]Ryan, H. 2015. “About Ovarian Dysfunction.”/allwoman. 2015.[4]Henson, M. C., and Chendrese, P. J. 2004. “EndocrineDisruption by Cadmium, a Common Environmental Toxicant with Paradoxical Effects on Reproduction.” Exp Biol Med 229: 383-92.[5]Samuel, J. B., Stanley, J. A., Princess, R. A., Shanthi, P.,and Sebastian, M. S. 2011. “Gestational Cadmium Exposure-induced Ovotoxicity Delays Puberty through Oxidative Stress and Impaired Steroid Hormone Levels.”J Med Toxicol 7: 195-204.[6]Amir, W., Jahanzaib, A., Farhat, I., Ashif, S., Zahid, M.,and Ghulam, M. 2014. “Pollution Status of Pakistan: A Retrospective Review on Heavy Metal Contamination of Water, Soil, and Vegetables.”Bio Mededical Research International: 29.[7]Oumar, B., Ekengele, N. L., and Balla, D. 2014.“Évaluation du niveau de pollution par les métauxlourds des l acs Bini et Dang, Région de l’Adamaoua, Cameroun.”Afrique Science 10 (2): 184-98.[8]Zalups, R. K., and Ahmad, S. 2003. “Molecular Handlingof Cadmium in Transporting Epithelia.”Journal of Toxicology Applied Pharmacology 186: 163-88.[9]Suhartono, E., Triawanti, Y. A., Firdaus, R. T., andIskandar, T. F. 2013. “Chronic Cadmium Hepatooxidative in Rats: Treatment with Haruan Fish (Channastriata) Extract.”APCBEE Procedia 5: 441-5.[10]Bárány, E., Bergdahl, I. A., and Bratteby, L. E. 2005.“Iron Status Influences Trace Element Levels in HumanAqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats117Blood and Serum.”Environmental Research98: (2) 215-23.[11]Järup, L., Hellström, L., Alfvén, T., Carlsson, M. D.,Grubb, A., Persson, B., Pettersson, C., Spang, G., Schutz,A., and Elinder, C. 2000. “Low Level Exposure toCadmium and Early Kidney Damage: The OSCAR Study.”Journal of Environmental Medicine 57 (10): 668-72. [12]Koninger, A., Kauth, A., Schmidt, B., Schmidt, M.,Yerlikaya, G., Kasimir-Bauer, S. et al. 2013.“Anti-Mullerian-hormone levels during pregnancy and postpartum.”Reprod Biol Endocrinol 11 (1): 60.[13]Hassan, S. W., Salawu, K., Ladan, M. J., Hassan, L. G.,Umar, R. A., and Fatihu, M. Y. 2010. “Hepato-Protective, Antioxidant and Phytochemical Properties of Leaf Extracts of Newbouldia laevis.”International Journal of Pharmacology Technology Research 2: 573-84.[14]Odunbaku, O. A., and Amusa, N. A. 2012. “Antibacterialand Antifungal Effect of the Ethanol Leaf Extract of Newbouldia laevis.”Global Research Journal of Biological Sciences 3 (4): 370-2.[15]Arbonnier, M. 2004. Trees, Shrubs and Lianas of WestAfrican Dry Zones. CIRAD, MARGRAF Publishers GMBH, MNHN. CTA Wageningen Netherlands. [16]Akinmoladun, A. C., Obutor, E. M., and Farombi, E. O.2010. “Evaluation of Antioxidant and Free Radical Scavenging Capacities of Some Nigerian Indigenous Medicinal Plants.”Journal of Medicinal Food 10: 444-51.[17]Ejele, A. E., Duru, I. A., Ogukwe, C. E., and Iwu, I. C.2012. “Phytochemistry and Antimicrobial Potential of Basic Metabolities of Piper Umbellatum, Piper Guineense, Ocimum gratissimium and Newbouldia leavis Extracts.”Journal of Emerging Trends in Engineering and Applied Sciences 3 (2): 309-14.[18]Usman, H., and Osuji, J. C. 2007. “Phytochemical and invitro Antimicrobial Assay of the Leaf Extract of Newbouldia laevis.”African Journal of Traditional, Complimentary and Alternative Medicines 4 (4): 476-80.[19]Ainooson, G. K., Woode, E., Obiri, D. D., and Koffour, G.A. 2009. “Antinociceptive Effects of Newbouldialaevis(P.Beauv) Stem Bark Extract in a Rat Model.”Pharmacognosy Magazine 5: 49-54.[20]Kuete, V., Wabo, H. K., Eyon, K. O., Feussi, M. T.,Wiench, B., Krusche, B., and Effert, T. 2011. “Anticancer Activities of Six Selected Natural Compounds of Some Cameroonian Medicinal Plants.”PLoS ONE 6: e21762.[21]Bafor, E., Sanni, U., and Nworgu, Z. A. 2010. “In VitroDetermination of the Mechanism of the Uterine Stimulatory Effect of Newbouldia laevis.”Journal of Pharmaceutical Biology 48: 808-15.[22]Awemu, G. A., Okunrobo, L. O., and Awah, F. M. 2012.“Wound Healing and Antiulcer Activities of the Ethanol Extract of Newbouldia laevis Root Bark.”Journal ofPharmacy and Bioresources 9: 29-33.[23]Joppa, K. M., Vovor, A., Eklu-Gadegbeku, K., Agbonon,A., Aklikokou, K., and Gbeassor, M. 2008. “Effect ofMorindalucida Benth. (Rubiaceae) and Newbouldia leavis P. Beauv. (Bignoniaceae) on Sickling of Red Blood Cells.”Medecine Tropicale: Revue du Corps de Sante Colonial68: 251-6.[24]Enye, J. C., Onubueze, D. P. M., Chineke, H. N., andNweke, I. 2013. “The Antihypertensive Property of Methanolic Extract of Newbouldia laevis on Anaesthesized Cats.”Journal of Dental and Medical Sciences 8: 35-9.[25]Ogungbite, O. C., and Oyeniyi, E. A. 2014. “Newbouldialaevis (Stem) as an Enthomocide against Sitophilus oryzae and Sitophilus zeamais Infecting Maize Grain.”Jordan Journal of Biological Sciences 7: 49-55.[26]Nampoothiri, L. P., Agarwal, A., and Gupta, S. 2007.“Effect of Coexposure to Lead and Cadmium on Antioxidant Status in Rat Ovarian Granulose Cells.”Arch Toxicol 81: 145-50.[27]Diaby, V., Yapo, A. F., Adon, A. M., Dosso, M., andDjama, A. J. 2016. “Renal, Hepatic and Splenic Biotoxicity of Cadmium Sulphate in the Wistar Rats.”International Journal of Environmental Science and Toxicology Research 4 (6): 103-10.[28]Massanyi, P., Uhrin, V., Sirotkin, A. V., Toman, R., Pivko,J., Rafay, J., Forgacs, Z., and Somosy, Z. 2000. “Effects of Cadmium on Ultrastructure and Steroidogenesis in Cultured Porcine Ovarian Granulosa Cells.”Acta Vet Brno69: 101-6.[29]Gurel, E., Caner, M., Bayraktar, L., Yilmazer, N.,Dogruman, H., and Demirci, C. 2007. “Effects of Artichoke Extract Supplementation on Gonads of Cadmium-treated Rats.”Biol Trace Elem Res 119: 51-9.[30]Priya, P. N., Pillai, A., and Gupta, S. 2004. “Effect ofSimultaneous Exposure to Lead and Cadmium on Gonadotropin Binding and Steroidogenesis on Granulosa Cells: An in vitro Study.”Indian J Exp Biol 42: 143-8. [31]Wang, X., and Tian, J. 2004. “Health Risks Related toResidential Exposure to Cadmium in Zhenhe County, China.”Arch Environ Health 59 (6): 324-30.[32]Jackson, L. W., Zullo, M. D., and Goldberg, J. M. 2008.“The Association between Heavy Metals, Endometriosis and Uterine Myomas among Premenopausal Women: National Health and Nutrition Examination Survey 1999-2002.” Hum Reprod 23 (3): 679-87.[33]Al-Saleh, I., Coskun, S., Mashhour, A., Shinwari, N.,El-Doush, I., Billedo, G. et al. 2008. “Exposure to Heavy Metals (Lead, Cadmium and Mercury) and Its Effect on the Outcome of In-vitro Fertilization Treatment.”Int J Hyg Environ Health 211 (5-6): 560-79.[34]Larsson, A., Haux, C., and Sjobeck, M. L. 1985. “FishAqueous Extract of Newbouldia laevis Abrogates Cadmium-Induced Ovarian Dysfunctionin Adult Wistar Rats118Physiology and Metal Pollution: Results and Experiences from Laboratory and Field Studies.”Ecotoxicol Environ Saf 9: 250-81.[35]ATSDR. 1999. Toxicological Profile for Cadmium.Agency for Toxic Substances and Disease Registry,Atlanta.[36]Casalino, E., Sblano, C., and Landriscina, C. 1996. “APossible Mechanism for Initiation of Lipid Peroxidation by Ascorbate in Rat Liver Microsomes.”Int J Biochem Cell Biol 28: 137-49.。

10.1128/AEM.71.2.1051-1057.2005.2005, 71(2):1051. DOI:Appl. Environ. Microbiol. B. S. McSwain, R. L. Irvine, M. Hausner and P. A. WildererAerobic Flocs and Granular SludgeExtracellular Polymeric Substances in Composition and Distribution of/content/71/2/1051Updated information and services can be found at: These include:REFERENCES/content/71/2/1051#ref-list-1This article cites 20 articles, 1 of which can be accessed free at:CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on April 4, 2013 by guest/Downloaded fromA PPLIED AND E NVIRONMENTAL M ICROBIOLOGY,Feb.2005,p.1051–1057Vol.71,No.2 0099-2240/05/$08.00ϩ0doi:10.1128/AEM.71.2.1051–1057.2005Copyright©2005,American Society for Microbiology.All Rights Reserved.Composition and Distribution of Extracellular Polymeric Substances inAerobic Flocs and Granular SludgeB.S.McSwain,1,2*R.L.Irvine,1M.Hausner,2and P.A.Wilderer2Department of Civil Engineering and Geological Sciences,University of Notre Dame,Notre Dame,Indiana,1and Institute of Water Quality Control and Waste Management,Technical University of Munich,Garching,Germany2Received6July2004/Accepted27August2004Extracellular polymeric substances(EPS)were quantified inflocculent and aerobic granular sludge devel-oped in two sequencing batch reactors with the same shear force but different settling times.Several EPSextraction methods were compared to investigate how different methods affect EPS chemical characterization,andfluorescent stains were used to visualize EPS in intact samples and20-␮m cryosections.Reactor1(operated with a10-min settle)enriched predominantlyflocculent sludge with a sludge volume index(SVI)of120؎12ml g؊1,and reactor2(2-min settle time)formed compact aerobic granules with an SVI of50؎2mlg؊1.EPS extraction by using a cation-exchange resin showed that proteins were more dominant than poly-saccharides in all samples,and the protein content was50%more in granular EPS thanflocculent EPS.NaOHand heat extraction produced a higher protein and polysaccharide content from cell lysis.In situ EPS stainingof granules showed that cells and polysaccharides were localized to the outer edge of granules,whereas thecenter was comprised mostly of proteins.These observations confirm the chemical extraction data and indicatethat granule formation and stability are dependent on a noncellular,protein core.The comparison of EPSmethods explains how significant cell lysis and contamination by dead biomass leads to different and opposingconclusions.The efficiency of biological wastewater treatment depends,first,upon the selection and growth of metabolically capable microorganisms and,second,upon the efficient separation of those organisms from the treated effluent.Bacteria usually aggregate to form suspendedflocs,which can cause bulking and foaming problems iffilamentous bacteria are present.Ac-tivated sludgeflocs also settle relatively slowly,requiring large primary and secondary settling tanks before clear effluent can be released.Alternatively,aerobic granular sludge aggregates have been formed in sequencing batch reactors(SBRs)with shortfill periods and various substrates(1a,13,15).As op-posed toflocs,granules are dense and have high settling ve-locities.They can be described as a collection of self-immobi-lized cells into a somewhat spherical form and are considered to be a special case of biofilm growth(10).Microbial aggregates form biofilms by creating a network of cells and extracellular polymeric substances(EPS),which in-clude any substances of biological origin(9).The abbreviation “EPS”has often been expanded to extracellular polysaccha-rides or exopolysaccharides.However,EPS have been shown to be a rich matrix of polymers,including polysaccharides, proteins,glycoproteins,nucleic acids,phospholipids,and hu-mic acids.EPS are typically reported to aid in the formation of a gel-like network that keeps bacteria together in biofilms, cause the adherence of biofilms to surfaces,and protect bac-teria against noxious environmental conditions(24). Because EPS are a major component of cellflocs and bio-films,they are hypothesized to play a central role in all types of biofilm formation,includingflocculation and granulation.It is not fully understood what factors increase EPS formation, although several researchers hypothesize that hydraulic shear may contribute(21).Tay et al.(21)reported that increased aeration rates in a granular SBR resulted in an increased polysaccharide content and that granular sludge disintegrated when polysaccharides were lost from the system.Aerobic gran-ules have also failed to form in systems with reduced aeration rates(6,22).Researchers concluded that hydrodynamic shear force increases the production of cellular polysaccharides, which aid in the formation and stability of aerobic granules (11).However,several arguments exist against shear force being the necessary factor for granule formation.Most notably, granules were not stable in airlift reactors operated with longer settling times and high aeration rates(2).Therefore,the rela-tionship between shear force,EPS formation,and granule sta-bility is unclear.In the present experiment,two SBRs were operated with the same aeration rate and superficial upflow gas velocity but with two different settling times.Long and short settling times were utilized to formflocculent and granular sludge,respectively, under the same shear conditions.The EPS were extracted at steady-state to determine whether the polysaccharide and pro-tein contents varied betweenflocculent and granular sludge produced under the same aeration rate,and several extraction methods were compared.Intact,hydratedflocs and granules were alsofluorescently stained to visualize the distribution of cells,polysaccharides,and proteins in situ.MATERIALS AND METHODSReactor operation.Two5-liter column-type SBRs were operated for6months to developflocculant and granular sludge,respectively.The reactors were con-structed from Plexiglas shaped as a cylinder(height,100cm;diameter,9cm).*Corresponding author.Mailing address:Institute of Water Quality Control and Waste Management,Technical University of Munich, 85748Garching b.,Munich,Germany.Phone:49-89-289-13715.Fax:49-89-289-13718.E-mail:bmcswain@.1051 on April 4, 2013 by guest / Downloaded fromThey were aerated at a rate of 275liters h Ϫ1(superficial gas velocity of 1.2cm s Ϫ1)with a 50%volumetric exchange ratio.The reactors were inoculated with 5liters of activated sludge from a municipal wastewater treatment plant (initial mixed liquor suspended solid [MLSS]content of 2.5g liter Ϫ1).The walls of the reactors were cleaned every 2weeks,and the biofilm growth was discarded.Both reactors were fed from a common source of glucose and peptone with nutrients (similar to that used by (16))at a volumetric loading rate of 2.4kg of chemical oxygen demand (COD)m Ϫ3day Ϫ1(800mg of COD liter Ϫ1cycle Ϫ1).The total cycle time was 4h with six cycles per day (90-min static fill,120-min react,2-or 10-min settle,15-min draw,and 5-or 13-min idle).The only variation in oper-ating strategy was the settling and idle times (10-min settle for reactor 1and 2-min settle for reactor 2).Analytical procedures.MLSS and volatile suspended solid (VSS)content,effluent and volatile suspended solid (ESS and EVSS,respectively)content,and the sludge volume index (SVI)were measured one to three times per week,all according to APHA standard engineering methods (4).Substrate removal was measured weekly by the COD during the cycle and in the effluent by using nge COD kits (according to the colorimetric COD standard method)(nge GmbH,Du ¨sseldorf,Germany).The specific oxygen uptake rate (SOUR)during a cycle was measured weekly during startup,and the endogenous SOUR was measured twice a week (4,12).Endogenous oxygen uptake rate (OUR)samples were collected from the end of the react cycle and aerated for at least 2h before the OUR measurement,whereas beginning and end of the react OUR samples were measured immediately after sampling.The develop-ment of flocs and granules was observed by using a stereomicroscope (Leica Wild MPS 46/52;Leica,Vienna,Austria),and images were obtained with an attached Kodak digital camera.EPS extraction and chemical analysis.EPS was characterized from nonho-mogenized and homogenized samples by two extraction methods:the use of cation-exchange resin (Dowex)and alkaline treatment with heat (NaOH).Sample pretreatment.Approximately 0.5g volatile solids (VS)were taken from each reactor at the end of the SBR cycle.The reactor volume taken for the 0.5g of VS was estimated from previous VSS measurements.The actual mass of VS sampled for EPS extraction was determined from the MLSS and VSS con-tents measured at the time of sampling.The sample was centrifuged at 4°C and 10,000rpm for 15min.The supernatant was collected to determine chemical composition of reactor wash and loosely bound EPS.The samples were resus-pended in Milli-Q water and centrifuged again.For nonhomogenized samples,the remaining pellet was resuspended in phosphate buffer (7)to a total volume of 100ml.For homogenized samples,the pellet was resuspended in 40ml of phosphate buffer and divided into two aliquots.Each aliquot was homogenized for 10min in a homogenizer (RW 20DZM;Janke &Kunkel,Staufen,Germany)at a maximum rpm of 980,and the two homogenized parts were combined with phosphate buffer to a 100-ml total volume.Cation-exchange resin extraction.EPS extraction using a Dowex 50x8,Na ϩForm,cation-exchange resin (Fluka)was performed with a 0.5g of VS-to-35g of cation exchange resin ratio according to the method of Frølund et al.(7).The samples were stirred at 750rpm for 4h in the dark at 4°C.A blank sample with cation exchange resin and phosphate buffer (pH 7)was also tested.NaOH and heat extraction.Harvested samples (suspended in 100ml of buffer)were adjusted to pH 11by using 1M NaOH and placed in plastic bottles before heating to 80°C for 30min (modified from Tay et al.[21]).A blank with phosphate buffer adjusted to pH 11with NaOH was also measured.EPS harvesting and characterization.After the Dowex and NaOH extraction,respective samples were centrifuged at 10,000rpm for 1min.The supernatant was transferred to clean centrifuge tubes and again centrifuged 10min.After-ward,cell lysis was measured with a glucose-6-phosphate dehydrogenase kit (Fisher Scientific 345-A),and the remaining EPS supernatant was stored at Ϫ20°C in aliquots until chemical analyses were performed.Total organic carbon (TOC)was measured by using an Elementar High TOC II (Elementar,Hanau,Germany).Proteins and polysaccharides were measured according to the method of Frølund et al.(7)by using bovine albumin serum and glucose stan-dards,respectively.Cell lysis was measured directly after Dowex EPS extraction by using a glucose-6-phosphate dehydrogenase kit and was negligible.Cell lysis from NaOH EPS extraction could not be measured with the kit,since the kit measures the presence of the glucose-6-phosphate dehydrogenase enzyme.En-zymes are typically active only within a pH range of 5to 9,and controlled trials with the kit and NaOH extracts (pH 11)were not successful (23).Fluorescence staining and CLSM.Intact,living,and hydrated granules and flocs were stained with fluorescently labeled probes with different excitation and emission spectra in order to visualize the distribution of cells,polysaccharides,and proteins in samples.After being stained,whole samples were either visual-ized directly by confocal laser scanning microscopy (CLSM;LSM510META;Zeiss,Jeve,Germany)or embedded in cryosectioning compound (Microm,Wall-dorf,Germany)and cryosectioned into 20-␮m sections (CM 3050S Kryostat;Leica,Bensheim,Germany)for direct visualization.Staining.Samples were stained in 1.5-ml Eppendorf tubes,covered with alu-minum foil,and placed on a shaker table (100rpm)for 15min each.Fluorescein-isothiocyanate (FITC)(0.01%)is an amine reactive dye and stains all proteins and amino-sugars of cells and EPS (19).Concanavalin A (ConA)lectin conju-gated with Texas Red (100␮g ml Ϫ1)was used in the present study to bind to ␣-mannopyranosyl and ␣-glucopyranosyl sugar residues.Syto 63is a cell-perme-ative nucleic acid stain and was used to visualize all cells.All probes were purchased from Molecular Probes,and samples were washed with phosphate-buffered saline after each staining step.Granules were stained before and after cutting them in half in order to rule out any diffusion limitation of stains inside the granule structure.Based on these observations and reported diffusion coef-ficients of similar stains (5),there should have been no biases due to diffusiondifferences.FIG.1.Reactor inoculum and steady-state sludge (day 200)from reactors 1and 2(R1and R2;scale ϭ1mm).TABLE 1.Steady-state values for standard measurements (days 120to 220of operation)Property or performanceparameterSteady-state value ϮSD aReactor 1(10-min settle)Reactor 2(2-min settle)Sludge properties MLSS (g liter Ϫ1) 3.0Ϯ0.28.8Ϯ0.5VSS (g liter Ϫ1) 2.7Ϯ0.28.0Ϯ0.5SVI (ml g Ϫ1)120Ϯ1250Ϯ2ESS (mg liter Ϫ1)280Ϯ90170Ϯ50Reactor performance Endogenous SOUR b 12Ϯ18Ϯ2COD removal (%)96Ϯ196Ϯ1aValues are reported with 95%confidence (as determined by analysis of variance).bSOUR is reported as follows:mg of O 2/(g of VSS h).1052M C SWAIN ET AL.A PPL .E NVIRON .M ICROBIOL .on April 4, 2013 by guest/Downloaded fromCLSM.The probes were visualized on three channels with corresponding excitations and emissions:FITC (488nm,BP 505to 530nm),ConA (543nm,LP 560nm),and Syto 63(633nm,LP 650nm).Z-sectioning was performed on whole granules and rendered three-dimensionally by using the LSM 510Viewer software (Zeiss).RESULTSReactor operation and performance.A detailed report of granule development and performance in these reactors is published separately (14).Due to the difference in settling time,the washout of sludge during the first 2weeks of opera-tion was much greater for reactor 2(2min settling)(the MLSS dropped to 0.7g liter Ϫ1)than for reactor 1(10min settling)(the MLSS began to increase immediately after startup).After 1week of operation,granules were observed in both reactors.On day 56of operation,the SVIs of reactors 1and 2were 60and 63ml g Ϫ1,respectively,and the settling time seemed to have no effect on granule formation.However,after 80days of operation,the reactors began to diverge in terms of sludge characteristics,MLSS,and SVI.For reactor 1,the granules always coexisted with flocculent sludge,whereas the flocs in reactor 2were continuously washed out with the short settling time,leaving predominantly granular sludge.Images of reactor inoculum and steady-state sludge are presented in Fig.1.In parallel studies (not presented here),three separate reactorswere operated with the same strategy as reactor 2,and all formed granules with similar properties.The steady-state values for MLSS,VSS,effluent SS,SVI,OUR,and COD removal are summarized in Table 1.It is clear that both reactors performed well in terms of COD removal and OUR.The reactors differed in the properties of the sludge,MLSS content,and settling characteristics.Most significantly,the granular reactor 2developed an average MLSS of 8.8g liter Ϫ1and an SVI of 50ml g Ϫ1compared to an MLSS of 3.0g liter Ϫ1and an SVI of 120ml g Ϫ1for the flocculent reactor 1.EPS characterization.Initially,EPS extraction was per-formed by using only the Dowex cation-exchange method com-bined with homogenization (14),and the results were opposite those reported by using the NaOH heating method (21).To understand the influence of method on granular sludge char-acterization,the EPS from steady-state samples was chemically extracted and characterized by different methods.Nonhomog-enized and homogenized samples were extracted by using (i)alkaline treatment with NaOH at 80°C and (ii)stirring with cation-exchange resin.Table 2presents an overview of all results.The total TOC measurement indicates the amount of EPS extracted,and the values for each reactor are presented in Fig.2for both nonhomogenized and homogenized sludge samples.In general,the NaOH extraction yielded much more total TOC for each sample than the corresponding Dowex extraction.Triplicate NaOH extractions also had a greater variability than triplicate Dowex extractions,which is represented by the error bars in Fig.2.For the predominantly flocculent reactor 1,homogenization had little effect on the total TOC extracted by using either the NaOH or Dowex method.In contrast,homog-enization of granular reactor 2samples increased TOC yields by Ͼ200%for both extraction methods.The protein content was greater than the polysaccharide content of all EPS extracts (data presented in Table 2for homogenized samples).The alkaline treatment yielded Ͼ200%more proteins and carbohydrates than the correspond-ing Dowex treatment,which corresponded to the increase in TOC with alkaline extraction (Fig.2).Between reactors 1and 2,the EPS of granules (R2)had higher protein levels than EPS from flocs and granules (R1),whereas the carbohydrate con-tent increased onlyslightly.FIG.2.Total TOC of EPS extracted from reactor 1(flocculent)and 2(granular)by using each extraction methods.Gray bars represent homogenized samples.Error bars indicate the standard deviations of triplicate extractions.TABLE 2.EPS component analysis for each extraction methodSample and reactor PretreatmentMean value (mg g Ϫ1VSS Ϫ1)ϮSE aProteinCarbohydratesTOCPN/PS bNaOH extraction Reactor 1Nonhomogenized 199Ϯ2826Ϯ9227Ϯ447.8Reactor 1Homogenized 190Ϯ2830Ϯ9234Ϯ44 6.4Reactor 2Nonhomogenized 185Ϯ2818Ϯ986Ϯ4410.9Reactor 2Homogenized 210Ϯ2826Ϯ9203Ϯ447.9Dowex extraction Reactor 1Nonhomogenized 39Ϯ55Ϯ233Ϯ47.5Reactor 1Homogenized 50Ϯ58Ϯ244Ϯ4 6.6Reactor 2Nonhomogenized 23Ϯ53Ϯ215Ϯ47.6Reactor 2Homogenized73Ϯ511Ϯ269Ϯ46.7a Standard errors were calculated based on separate extractions of triplicate samples.bPN/PS,protein/polysaccharide ratio.V OL .71,2005EXTRACELLULAR POLYMERIC SUBSTANCES IN GRANULAR SLUDGE1053on April 4, 2013 by guest/Downloaded fromIn situ EPS staining and fluorescent microscopy.Fluores-cently labeled lectins such as ConA have been used to stain glycoconjugates in heterotrophic,multispecies biofilms,and FITC is an amine reactive dye that stains proteins and other amine-containing compounds (3,17,18,20).These stains may also bind with protein and glycoconjugate groups associated with cell walls,so a counterstain with Syto 63was used to distinguish EPS from cells.Figure 3shows the resultant stain-ing from a 20-␮m cryosection taken from the middle of a granule (ca.260␮m below the surface).A freshly sampled granule was stained before sectioning,and fluorescence was viewed on three separate channels for each stain.Figure 3shows that cells and polysaccharides were restricted to the outer edge of the granule structure,with the cells clustered within 100␮m of the surface.Figure 3C shows that FITC stained proteins throughout the entire granule,with no over-lapping of FITC with other stains in the center.At least 10cryosections were viewed from five different granules,all with similar observations.For several sections,the fluorescence intensity of each stain along one diameter of the granule was quantified by image analysis.The relative intensities,or percent total,are displayed in Fig.4for a section extracted 120␮m below the granule surface.The main distribution of cells and polysaccharides were at the edge of the section,whereas the protein stain was unevenly distributed across the entire diameter.This quantifi-cation confirms the microscopic observations shown in Fig.3.It also shows that the Syto and ConA stains were detected at the center of the granules albeit at low levels,indicating that the stains were able to diffuse into the entire sample.Whole flocs and granules were also observed under the microscope.One floc is displayed in Fig.5.EPS (both poly-saccharide and protein staining)was concentrated in a floccu-lent center,whereas some polysaccharides were present around the network of filamentous fungi.To view the EPS distribution on the outer edge of a granule,a stack of 10-␮m optical sections were taken into a depth of 130␮m within the granule.A projection of this stack of images is shown in Fig.5without the FITC channel,since it overlapped with both Syto 63and ConA staining at low magnification.The projection clearly shows that cells are embedded in a large network of polysaccharide material on the outer edge of the granule.This observation correlates with the cryosectioning datapresentedFIG.3.Fluorescent staining of 20-␮m cryosections from a reactor 2granule.Cells stained with Syto 63(A),polysaccharides stained with ConA (B),and proteins stained with FITC (C)are shown.Scale bar,100␮m.Images were obtained with a ϫ10objectivelens.FIG.4.Relative fluorescence of each stain through a 20-␮m cryo-section,cut 120␮m below the granule surface.The granule edge is marked by a dotted line,and an arrow marks the granule center.Cells (Syto 63)(A)and polysaccharides (ConA)(B)are clustered within the first 200mm of the granule edge,and proteins (FITC)are distributed unevenly throughout the granule (C).1054M C SWAIN ET AL.A PPL .E NVIRON .M ICROBIOL .on April 4, 2013 by guest/Downloaded fromin Fig.3and 4.Under a higher magnification,specific cell clusters were observed in association with polysaccharides and proteins.DISCUSSIONReactor operation and sludge properties.The effect of set-tling time on floc and granule formation followed previously reported research (1).Reactor 1,with a minimal settling ve-locity (␯min )of 2.4m h Ϫ1,formed predominantly flocculent sludge,and reactor 2(␯min of 12m h Ϫ1)formed predominantly granular sludge.However,granules were observed in both reactors,indicating that slow settling times do not prevent granule formation but rather prevent granular sludge from becoming dominant in the reactor.The short settling times cause continuous biomass washout,thus affecting species se-lection over time (14).EPS extraction and characterization.The results for EPS content differ strikingly with those reported by Tay et al.(21).For an SBR operated with a superficial gas velocity of 1.2cm s Ϫ1,the same as used in this experiment,Tay et al.reported a polysaccharide/protein ratio (PS/PN ratio)of 15.In that ex-periment,the polysaccharide content was hypothesized to con-tribute more to the granule structure and stability than the protein content (21).However,in the current experiment,the protein content was much higher than the polysaccharide ing a similar extraction method of NaOH and heat for nonhomogenized granules,the protein/polysaccharide ratio (PN/PS ratio)was calculated to be ca.11for reactor 2(see Table 2).This trend was confirmed by methods of homogeni-zation and Dowex cation-exchange extraction for both reactor 1and 2samples,and the data were correlated with a short EPS literature review shown in Table 3.For a variety of biofilm types and extraction methods,proteins have been reported more abundant than polysaccharides in EPS.The comparison of EPS extraction methods showed two trends with respect to homogenization and alkaline treatment.Homogenization had little effect on the amount of EPS ex-tracted from flocculent sludge,but it greatly aided the EPS extraction from granular sludge samples.Because granules can be very large (Ͼ1mm),they have a surface-to-volume limita-tion for both NaOH and cation-exchange resin.The resin ex-changes monovalent cations (in this case Na ϩ)with divalent cations (mainly Ca 2ϩand Mg 2ϩ)that are responsible for the cross-linking of charged compounds in the EPS matrix.By homogenizing the sample beforehand,the cation-exchange resin interacts with more total EPS,removing more total di-valent cations,and increasing the repulsion of EPS compo-nents and their water solubility.Alkaline treatment causes charged groups,such as carboxylic groups in proteins and poly-saccharides,to be ionized since their isoelectric points are typically below pH 4to 6.This also causes a repulsion between EPS components and increases their water solubility (18).Therefore,homogenization should be used when samples with vastly different shapes and sizes are compared.Homogeniza-tion eliminates a method bias for more efficient extraction from samples with a greater surface area/volume ratio,as was shown in Fig.2with the effect of homogenization for flocs versus granules.Only the homogenized Dowex extraction showed granules with more total EPS than flocs,although this observation was clearly made with the microscopic staining.When samples with a low surface area/volume ratio such as flocs are compared,homogenization is not essential.EPS extraction with alkaline and heat treatment produced much more TOC,proteins,and polysaccharides than Dowex extraction.This increase was most likely due to severe celllysisFIG.5.(A)Floc stained with Syto 63(red),ConA (blue),and FITC (green)and viewed with a ϫ10objective lens.Scale,100␮m.The majority of EPS is localized at a flocculent center,whereas some polysaccharides are present around filamentous fungi.(B)Three-dimensional projection of a 130-␮m stack of optical sections from the outer edge of a granule.Cells (red)and polysaccharides (blue)are evident.Scale,100␮m.Cells are embedded in a large polysaccharide matrix.V OL .71,2005EXTRACELLULAR POLYMERIC SUBSTANCES IN GRANULAR SLUDGE 1055on April 4, 2013 by guest/Downloaded fromcaused by the high pH and heat treatment.The extent of cell lysis during extraction is difficult to measure in undefined sam-ples,and the glucose-6-phosphate dehydrogenase kit was not applicable for alkaline samples,since high pH and heat are known to disrupt macromolecules such as enzymes and pro-teins.Previous studies reported that boiling and addition of NaOH cause severe cell lysis in activated sludge,whereas a few hours of mixing with Dowex does not cause strong lysis (18).The NaOH extraction also produced more total TOC from flocculent EPS (R1)than granular EPS (R2)as shown in Fig.2,but the Dowex extraction or microscopic staining of poly-saccharides and proteins did not confirm this observation.Therefore,extraction with NaOH and heat should be avoided.Fluorescence staining of EPS and microscopy.Flocs were comprised of a small center of EPS and cells surrounded by a network of filamentous bacteria and fungi.In contrast,the center of granules was labeled mostly with the protein stain,and cells and polysaccharides were isolated to the outer layer of granules,as shown with the cryosectioning data.This result correlates with another study by DeBeer et al.,in which an-aerobic flocs and granules were stained for EPS polysaccharide distribution.In loose flocs,the highest concentration of EPS was found in the center,whereas 50%of the EPS in granules was concentrated in a 40-␮m layer at the surface (5).This reflects the polysaccharide distribution stained by ConA in aerobic granules,with the outer layer being ca.100␮m thick.The center of the granules was mostly stained by FITC,which stains cells or free amino groups.A subsequent coun-terstaining with Syto 63resulted in few signals from the gran-ule center,suggesting that the majority of the granule volume was comprised of noncellular material.The origin of this ma-terial can be inferred by the microscopic staining of flocs and smaller granules,in which the flocculent center is comprised of cells and EPS together.The bacteria in these aggregates con-tinue to grow,creating an ever-larger granule.As the particle size increases,so does the mass transfer limitation of oxygen within the outer layer of active biomass (8).Mass transport limitations eventually create various layers of aerobic,anaer-obic,and dead biomass within granules.The aerobic layer of biomass has been reported to be 800␮m in diameter,which is much longer than observed in the present study (20).There-fore,the exact structure of aerobic granules is probably depen-dent on reactor operation,species selection,and biofilm growth morphology.The general observations suggest that granule centers are comprised of old aggregates of dead or dormant biomass and EPS,thus explaining the uneven distri-bution of FITC throughout the granule structure.The cells on the outer edge of granules are embedded in alarge network of polysaccharides stained by ConA (Fig.5),which were all counterstained by FITC and Syto 63.The mi-croscopic results suggest that the outer,active layer of cells excrete EPS with a large proportion of polysaccharides.The center of the granule was mostly stained by FITC but not by Syto 63.Therefore,the center could be composed of dead cells,which have leaked proteins and other amine-containing compounds into the granule center.The chemical extraction of EPS does not distinguish between proteins excreted by dead cells or live cells.Therefore,chemical extraction from granules is bound to contain some proteins and polysaccharides re-leased from dead cells at the center,which may constitute a large percentage of the total structure for a large granule.Given this consideration,the comparison of chemical EPS extraction data from different biofilm structures (flocs and granules)is difficult.Microscopic staining can be used in con-junction to understand the distribution of EPS in situ,provid-ing insight that the polysaccharides are significant components of EPS in the outer edges of granules,although they are but a small fraction of the total TOC extracted.Unfortunately,quantification of staining is difficult due to the specificity of lectins,which stain specific configurations of sugar residues,and the nonspecificity of FITC,which stain all amino groups.Overall.The method used for chemical extraction of EPS affects the total TOC,proteins,and polysaccharides extracted.Homogenization before extraction releases more total EPS from granule samples and has only a small effect on flocculent samples.Hot alkaline treatment with heat causes cell lysis that contaminates EPS with much higher levels of both proteins and polysaccharides than EPS extracted with cation-exchange resin.When comparing samples with different surface area/volume ratios are being compared,homogenization should be performed before chemical extraction in order to prevent method bias.Microscopic results showed that granular sludge has an outer layer (ca.200␮m thick)of biomass and EPS containing large amounts of polysaccharides.The center of the granule contained proteins as the major component and intact cells and polysaccharides to a lesser extent.ACKNOWLEDGMENTSThis study was supported by the German Research Foundation and a U.S.Department of Education GAANN Fellowship.We thank the MedTech Institute,Technical University of Munich,for the use of the Leica Kryostat.REFERENCES1.Batstone,D.J.,and J.Keller.2001.Variation of bulk properties of anaer-obic granules with wastewater type.Water Res.35:1723–1729.1a.Beun,J.J.,A.Hendriks,M.C.M.van Loosdrecht,E.Morgenroth,P.A.TABLE 3.EPS composition of different biofilm samples as determined by different methodsEPS extractionmethodSludge typeProtein (mg/g of VS)Carbohydrate (mg/g of SS)ReferenceHeating (70°C)UASB a8013Schmidt and Ahring (19a)Heating (80°C)Activated sludge 1218Frølund et al.(7)NaOH (pH 11)Activated sludge 9622Frølund et al.(7)Dowex and mixing Activated sludge 24348Frølund et al.(7)Dowex and mixing Sewer biofilm15412Jahn and Nielsen (10a)Dowex and mixingAnaerobic granules 14041Batstone and Keller (1)aUASB,upflow anaerobic sludge blanket.1056M C SWAIN ET AL.A PPL .E NVIRON .M ICROBIOL .on April 4, 2013 by guest/Downloaded from。

于德涵，黎莉，苏适. 天然低共熔溶剂提取黄酮类化合物的研究进展[J]. 食品工业科技，2023，44（24）：367−375. doi:10.13386/j.issn1002-0306.2023020204YU Dehan, LI Li, SU Shi. Research Progress on Extraction of Flavonoids Using Natural Deep Eutectic Solvents[J]. Science and Technology of Food Industry, 2023, 44(24): 367−375. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023020204· 专题综述 ·天然低共熔溶剂提取黄酮类化合物的研究进展于德涵*，黎　莉，苏　适（绥化学院食品与制药工程学院，黑龙江绥化 152061）摘　要：天然低共熔溶剂是一种新型绿色溶剂，有望替代传统有机溶剂实现对黄酮等天然产物的高效提取。

为了阐明天然低共熔溶剂在黄酮化合物萃取方面的应用，本文对近5年发表的相关研究论文进行了整理和分析，综述了天然低共熔溶剂提取黄酮的研究现状，并详细讨论了影响提取率的各种因素。

天然低共熔溶剂在黄酮、黄酮醇、二氢黄酮、花色素、异黄酮等多类天然黄酮产物的提取方面表现良好，其萃取率普遍优于甲醇、乙醇等传统溶剂，且萃取产物活性更高；低共熔溶剂的组成、摩尔比、含水量和温度等条件会显著影响其对黄酮化合物的萃取。

文章还对天然低共熔溶剂在未来的发展趋势作出展望，希望能为黄酮化合物的高效、绿色提取提供有益参考。

关键词：低共熔溶剂，黄酮类化合物，绿色溶剂，提取本文网刊: 中图分类号：TQ28、TS201 文献标识码：A 文章编号：1002−0306（2023）24−0367−09DOI: 10.13386/j.issn1002-0306.2023020204Research Progress on Extraction of Flavonoids Using Natural DeepEutectic SolventsYU Dehan *，LI Li ，SU Shi（Food and Pharmaceutical Engineering Department, Suihua University, Suihua 152061, China ）Abstract ：The natural deep eutectic solvent is a new type of green solvent that is expected to replace traditional organic solv-ents for efficient extraction of natural products such as flavonoids. In order to clarify the application of natural deep eutectic solvents in the extraction of flavonoids, the author summarizes and analyzes relevant research papers published in the past 5years. This article provides a review of the current research status of natural deep eutectic solvents for extracting flavonoids,and discuss in detail the various factors that affect the extraction rate. The natural deep eutectic solvents perform well in the extraction of various natural flavonoid products such as flavonoids, flavonols, flavonones, anthocyans, and isoflavones.Their extraction rates are generally better than traditional solvents such as methanol and ethanol, and the extracted products have higher activity. The composition, molar ratio, water content, and temperature of deep eutectic solvents signi-ficantly affect their extraction of flavonoids. Finally, the development trend of natural eutectic solvents in the future is prospected. This paper aims to provide reference for the efficient and green extraction of flavonoids.Key words ：deep eutectic solvents ；flavonoids ；green solvent ；extraction黄酮是植物细胞中一种重要的次级代谢产物，能够消除人体内自由基，有较强抗氧化、抗衰老的功能[1]，在抗菌、抗病毒、抗炎、降血糖、降血脂等方面也颇有功效[2]。

Extraction of Cottonseed Oil Using SubcriticalWater TechnologyWael Abdelmoez,Rehab Abdelfatah,and Aghareed TayebDept.of Chemical Engineering,Elminia University,61519Elminia,EgyptHiroyuki YoshidaDept.of Chemical Engineering,Center of Excellence Project,Osaka Prefecture University,Osaka 599-8531,JapanDOI 10.1002/aic.12454Published online December 15,2010in Wiley Online Library ().This work represents the extraction of cottonseed oil using subcritical water.The extraction efficiencies of different range temperatures (180–280 C),having mean parti-cle size range from 3mm to less than 0.5mm,water:seed ratios of 0.5:1,1:1,and 2:1,and extraction times in the range of 5–60min were all investigated.The composi-tion of the extracted oil,using the subcritical water,was analyzed by gas-liquid chro-matography and compared with that extracted using traditional hexane extraction.The results showed that the optimum temperature,mean particle size,water:seed ratio,and extraction time were 270 C,\0.5mm,2:1,and 30min,respectively.In addition the extracted oil was identical to that extracted using the traditional hexane method.VC 2010American Institute of Chemical Engineers AIChE J,57:2353–2359,2011Keywords:extraction,cottonseed oil,subcritical water,oil extraction,water as a solventIntroductionCotton plants are widely distributed and mainly cultivated for both their flowers (the cotton itself)and seeds in differ-ent countries,especially in Egypt where the soil and weather are most suitable for its cultivation.In the past,the cottonseed oil extraction was done by apply-ing manual powered mechanical pressure to squeeze the oil from the seed.This was very labor intensive and at best one half of the oil contained in the seed could be extracted.Now-adays,cottonseed oil is mostly extracted using the solvent extraction technique,mainly hexane is used as the solvent.The extraction process could be simply described as a leaching process in which solid or liquid components are removed and recovered from a solid mass by solvent extraction.Cottonseed oil extraction in modern factories involves a number of steps.These steps are tedious and incorporate many complicated pro-cedures.Those include seed preparation,primer pressing,andsteam cooking to facilitate the solvent extraction process.In addition,the extraction step itself takes a very long time approaching almost 8h as an extraction residence time.Lately,subcritical water technology has attracted many researchers for its versatile applications in the field of envi-ronment as a green alternative process than solvent extrac-tion.On heating within the critical point of water (tempera-ture \374 C,pressure 22.1\MPa)under enough pressure to maintain the liquid state.Water (subcritical water in this state)was reported to have distinctive properties,such as a low dielectric constant and a high ion product.1,2Many ex-tractable components from different biomass could be easily extracted under these conditions.The development of such technique has passed over many periods.In 1950,Fred Zimmerman and his staff developed a completely new method of obtaining vanillin (a pleasant smelling aromatic compound)directly from pulping liquor.3Then in 1966,John Connolly of Standard Oil Corporation published remarkable data on hydrocarbon solubility in water at high temperatures and pressures.4In 1970GerhardCorrespondence concerning this article should be addressed to W.Abdelmoez at drengwael2003@.V C 2010American Institute of Chemical Engineers AIChE Journal 2353September 2011Vol.57,No.9Schneider suggested the extension of wet air oxidation to higher temperatures for disposal of organic materials.5In the early1970s,the properties of super-criticalfluids(e.g., changes in solubility and diffusivity with temperature and pressure)were discovered at Max Planck Institute.6Since 1982many patents by General Atomics Company(GA) including reactor designs,solids handling,effluent quench-ing,reaction rate enhancers,heat transfer,and corrosion resistant materials have been developed.7Authors of this article have carried out many extraction processes using the subcritical water technology as well as synthesis of new materials.8–11Extraction of compounds from natural sources is the most important application of subcritical water.In1999,Hiroyuki Yoshida,showed that a relatively large amount of oil,organic acids,and amino acids could be extracted fromfish,squid en-trails and meat wastes by subcritical water treatment.12,13 The essential oil from plants has been extracted by using subcritical ually the oil in these plants is tradition-ally extracted either using steam distillation5,6or solvent extraction.12–15These techniques present some shortcomings, namely losses of volatile compounds,low extraction effi-ciency,long extraction time,degradation of unsaturated com-pounds,and toxic solvent residue.That encourages the use of alternative techniques for the extraction of essential oils.16 Continuous subcritical water extraction presented a powerful alternative for solid sample extraction.17–19Its use in thefield of essential oils is recent and seems to be very promising.20,21 The aim of this work was to investigate the use of subcriti-cal water to extract oil from the cottonseed.With a typical weight of10g,the results were compared with those obtained by traditional techniques to study the advantages of using sub-critical water method for the extraction of cottonseed oil. Materials and MethodsMaterialsAll chemicals and materials used in this work were locally manufactured in Egypt,where the extraction processes pass through many steps that use different materials.The cotton-seeds used in this study were obtained from Henderson fac-tory(Elminia,Egypt),which were the same as those used in the traditional solvent extraction process employed in the fac-tory.Hexane used for measuring the extracted oil in water is produced by El-Nasr Chemical(Cairo,Egypt).Distilled water,prepared at Elminia University in Egypt,was used as an extracting solvent in subcritical water extraction.A mix-ture of edible oil(sunflower oil)and paraffin wax was used as the heating medium for subcritical water extraction.This mix-ture could reach up to290 C without boiling.Both com-pounds were obtained from local markets in Elminia,Egypt. Sample Preparation.Fresh seeds were stored in polyeth-ylene bags at room temperature and samples of10g of seed were used for the extraction process.Before the seeds were used they underwent many procedures such as dehulling and cleaning to remove impurities.MethodsHexane Extraction.Before starting the experiments,the amount of oil contained in the seeds was measured.Dried cottonseeds used in this study were dehulled and the oil was extracted by hexane.Ten grams of dehulled seeds were con-tained in a50-ml plastic container and40ml of hexane were added to the seeds.The plastic container was then tightly closed and mixed using a rotating mixing machine, which was put inside an oven adjusted at45 C.Subcritical Water Extraction.The subcritical water extraction was carried out in laboratory–built apparatus con-sists of a heating bath installed with agitator.After the prep-aration step,the subcritical water extraction was carried out in stainless steel pipes SUS316,i.d.0.0168Â0.15m2(with a reactor volume of34Â10À6m3)with Swadgelok caps. The seeds and estimated amount of water were then charged into the reactor tube.The reactor was then sealed and immersed in a preheated oil-paraffin bath(Thomas Kagaku). It is very important to note here as a safety comment that the sealed tube should not be overfilled to prevent hydrolytic pressure of the expanding liquid from the fracturing the tube vessel The extraction was carried out in the range of180–280 C,and the pressure inside the reactor was estimated from the steam table for the subcritical conditions(saturated steam).After the desired reaction time,the reactor was im-mediately cooled down by immersing it into a cold-water bath.The extraction product was separated into three phases, the oil phase,the aqueous phase(including oil and water), and the solid phase,using a centrifuge andfilter to separate the oil phase from the aqueous phase and the solid phase. For the separated aqueous phase,recovery of all oil pro-duced from the water layer was done by adding hexane to extract any oil that could be emulsified into the water.Then, the hexane was evaporated by heating in a furnace at80 C. The extracted oil was then weighed and the data was recorded.The experiments were done in some cases in dou-ble or triple to avoid any uncertainty.Analyzing the extracted OilThe composition of the extracted oil using,using the sub-critical water,was analyzed by gas liquid chromatograph model HP6890,equipped with a BD-23column(60mÂ0.32mmÂ0.23l m)using carrier gases of H2,N2,and air withflow rates of40,3,450ml/min,respectively.The de-tector was operating at250 C,with injector at230 C. Results and DiscussionThefirst step was to measure the amount of oil contained in the cottonseeds used in the study.Following the proce-dures described in the materials and methods,it was found that the oil content represents15%by weight based on the dry weight of the dehulled seeds.This percent was taken as the maximum oil content of the used cottonseed.The yield of oil extraction using subcritical water was based on this oil content and is calculated as:Yield%¼The weight of extracted oilThe weight of dehulled seed used in the experimentÂ0:15Â1002354DOI10.1002/aic Published on behalf of the AIChE September2011Vol.57,No.9AIChE JournalNow to discuss the extraction conditions,the extraction process was carried out under different operating conditions to find the optimum conditions as shown in Figure 1,so in each time only one parameter changed while the other extraction parameters were kept constant.The extraction parameters were classified as temperature of extraction,extraction time,solvent (water)to seed ratio,particle size,and seeds pretreatment.First,the effect of temperature on the extraction process by changing the extraction temperature and keeping other parameters constant.The temperatures tested were:180–280 C.Figures 2a,b show the effect of extraction tempera-ture on the overall yield of extracted oil and the total solid residual for 10min of extraction time using 10g of dehulled and uncrushed seed (with 3mm diameter).The separation of oil was done through a filtration procedure to measure the extracted oil content accurately.The calculations were done based on a maximum extraction yield of 15%oil content.The results showed that the maximum yield of extraction could be obtained at 270 C.The maximum value was 0.33g of extracted oil (which represents 21.7%of the maximum oil content of 1.5g).However,the measurements of solid resid-ual during the extraction course showed a linear reduction in the solid residual content when increasing the temperature.Second was to determine the optimum extraction time by changing the extraction time.The extraction was carried out at temperature 190,200,210,230,250,and 270 C,only the figures represent the results of 190,250,and 270 C are shown to minimized the number of presented data,and water to seed ratio of 2:1(optimum extraction temperature and water to seed ratio,respectively).Extraction times were tested in the range of 5–60min while keeping other parame-ters constant.Based on these conditions,the experiments were accomplished and the optimum extraction time and solid residue were determined.The results in Figures 3a,4a,and 5a show that when increasing the extraction temperature,the yield of oil extracted increased while the residual solid decreased.Also,the peak (maximum yield at applied tem-perature)is reached earlier as the temperature increases.However,it was observed that the yield of oil extracted increased up to a maximum value then it started todecreaseFigure 2.Effect of extraction temperature on the oilextraction (a)and the solid residual (b)at 10min,water:seed ratio 2:1,using 10g of seed within 3mm diameter.Figure 1.(a)Photographs of the extraction products ofthe cottonseed extraction at different temper-atures in the range of 170–210 C,(b)the solid residual,and (c)the final extracted oil in the range of 190–280 C.[Color figure can be viewed in the online issue,which is available at .]AIChE JournalSeptember 2011Vol.57,No.9Published on behalf of the AIChEDOI 10.1002/aic2355by increasing the extraction time.Such reduction in oil yields by increasing the extraction time is due to the decom-position of some extracted oil.Also,it was observed that increasing the yield is done through increasing the extraction temperature.Maximum extraction yield was in the range of 20–40min and recorded0.6,19,and56%,for extraction temperatures of190,250,and270 C,respectively.It is im-portant to note here that the extraction time was longer at extraction temperature of270 C than other temperatures.It is important to note here that for extraction temperature of 270 C,the optimum extraction time was different from that of other temperatures,which it was longer.This could be explained in the bases of transformation of the fat into the oil phase as a free fatty acid due to the higher temperature and extraction time in this case.Moreover,it was not possi-ble to measure the solid residual after reaction time of20 min because the solid became veryfine and very law in its content.However,Figures3b,4b,and5b showed that the solid residual values were decreasing with the increase in the extraction time until it almost reached a plateau.Also, the rate of reduction in the solid residual was increasing by the increase in extraction temperatures.The optimum extraction temperature and time have been determined.Now,the effect of solvent-feed ratio on the extraction yield was studied,where the solvent is water and the feed is the cottonseed.Accordingly,the extraction pro-cess was carried out under different water:seed ratios and the extraction yields were compared.Figure6shows the effect of the water:seed ratio on the yield of oil in the range of 0.5:1to2:1.Experiments were carried out at270 C for10 min extraction time using dehulled-uncrushed seeds.Itwas Figure3.Effect of extraction time on the oil extraction(a)and solid residual(b)at190 C,water:seedratio2:1,using10g of seed within3mmdiameter.Figure4.Effect of extraction time on the oil extraction(a)and solid residual(b)at250 C,water:seedratio2:1,using10g of seed within3mmdiameter.2356DOI10.1002/aic Published on behalf of the AIChE September2011Vol.57,No.9AIChE Journalclearly found that by increasing the ratio of water (the sol-vent)to seed the yield of extraction increased too.The maxi-mum extraction yield was 22%at water:seed ratio of 2:1.It was found that there was no significant difference between the results of 1:1and 2:1ratios.In addition,it is very impor-tant to note that increasing the amount of water above 2:1could not happen,because of the dramatic increase of the pressure inside the reactor which hindered the easy opening of the reactor and led to explosion-like performance during the opening of the reactor to recover the extraction product.Regarding the previous optimum data,now the effect of the particle size of the seeds on extraction yield will be dis-cussed.It is considered as an important parameter in oil extraction due to its effect in minimizing the diffusion limi-tations during the extraction process.The extraction process was done at 270 C using water to seed ratio of 2:1for 10min for three different particle sizes.The results in Figure 7show that the extraction yield increases with the decrease in the particle size,where a maximum yield of 51%was obtained from seeds with particle size of 0.5–0.75mm.Finally,it is important to obtain the maximum oil yield,where the extraction process was followed under the opti-mum conditions.Figure 8shows the effect of theextractingFigure 5.Effect of extraction time on the oil extraction (a)and solid residual (b)at 270 C,water:seed ratio 2:1,using 10g of seed within 3mmdiameter.Figure 6.Effect of the water:seed ratio on the yield ofthe oil extraction at 270 C for 10min extrac-tion time and using 10g of seed within 3mmdiameter.Figure 7.The effect of the particle size on the yield ofthe oil extraction at 270 C for 10min extrac-tion time and water:seed ratio2:1.Figure 8.The effect of extraction time on the yield ofthe oil extraction under the obtained opti-mum conditions.AIChE JournalSeptember 2011Vol.57,No.9Published on behalf of the AIChEDOI 10.1002/aic2357time on the yield of extraction at 270 C using powdered seeds (\0.5mm)and water:seed ratio of 1:1.The results showed that the maximum yield was 64%from the oil con-tained originally in the seeds.Accordingly,the optimum conditions of subcritical water extraction of cottonseed oil would be as follows:•Extraction temperature:270 C •Time:30min.•Water:seed ratio:1:1•Particle size:\0.5mmAnalysis of the extracted oilThe last step in this research was to make sure that the obtained oil was not decomposed during the extraction pro-cess as well as it being safe to be used as edible oil.The result of the gas-liquid chromatograph is shown in Figure 9.Table 1shows a comparison between the fatty acid composi-tions for cottonseed oil extracted with hexane and subcritical water.The obtained results indicated practically no difference in the fatty acid compositions of the cottonseed oil extracted with the subcritical water method with that extracted by hexane.ConclusionIn this work subcritical water has been used as a new pathway for cottonseed oil extraction.The main conclusionsfrom this work are that the optimum oil extraction tempera-ture is 270 C,the optimum extraction time is 30min,the optimum solvent:feed ratio is 1:1,and the optimum particle size is \0.5mm.Comparing this technique with the traditional hexane extraction method,it is found that the new technique has a shorter time of extraction providing an environmentally friendly proceeding,since it uses water as the solvent and no organic solvents are involved.Literature Cited1.Deshpande GV,Holder GD,Bishop AA,Gopal J,Wender I.Extrac-tion of coal using supercritical water.Fuel .1984;63:956–960.2.Daimon H,Kang K,Sato N,Fuje K.Development of marine waste recycling technologies using sub-and supercritical water.J Chem Eng Jpn .2001;34:1091–1096.3.Abdelmoez W,Yoshida H.An overview of the applications of the subcritical water hydrolysis technology in waste reuse,recycle,and treatment.Proceedings of El-Minia International Conference,Towards a Safe and Clean EnVironment,TSCE’05.Elminia,Egypt,15-17April,2005;E3–4.4.Connelly J.Solubility of hydrocarbons in water near the critical so-lution temperature.Chem Eng Data .1966;11:13–16.5.Schneider G.Phase equilibria in fluid mixtures at high pressures.in I.Prigogine and S.A.Rice,eds.,Advances in Chemical Physics .1970,Vol 17:39.6.Zosel K.The process for the decaffeination of green coffee beans ,Ger.Pat.DBP 2,005,293,1970.7.Kiran E,Debenedetti P,Peter C,editors.Supercritical Fluids-Funda-mentals and Applications.Dordrecht:Kluwer Academic Publishers,2000,425–37.8.Abdelmoez W,Yoshida H.Kinetics and mechanism of the synthesis of a novel protein-based plastic using subcritical water.Biotechnol prog 2008;24:466–475.9.Abdelmoez W,Yoshida H.Synthesis of a novel protein-based bio-degradable plastic from the BSA using the sub-critical water tech-nology.AIChE J .2006;52:2607–2616.10.Abdelmoez W,Yoshida H.Mechanical and Thermal Properties of aNovel Protein-Based Plastic Synthesized Using Subcritical Water Technology.Macromol J .2007;40(26):9371–9377.11.Abdelmoez W,Yoshida H.Modeling and simulation of fast reac-tions in batch reactors under sub-critical water condition.AIChE J .2006;52:2607–2617.Figure 9.The gas-liquid chromatographic of the extracted cottonseed oil using subcritical water.Table parision Between the Compositions of the OilExtracted by Hexane and Subcritical WaterFatty acid Linoleic Palmatic Oleic Stearic Myristic Heaxane extraction 47–5025–2822–22.70–0.40.8–0.9Subcritical water Extraction4725210.30.92358DOI 10.1002/aic Published on behalf of the AIChE September 2011Vol.57,No.9AIChE Journal12.Yoshida H,Tavakoli Q.Effective recovery of harmful metal ionsfrom squid wastes using subcritical and supercritical water treat-ment.Environ Sci Technol J.2005;39:2357–2363.13.Tester J,Holgate H,Armellini F,Webley P,Killilea W,Hong G,Barner,H.Supercritical water oxidation technology.In:Tedder DW,Pohland FG,editors.Emerging Technologies in Hazardous Waste Management III,Washington DC:American Chemical Soci-ety,1993.14.Tester J,Pohland F.Supercritical Water Oxidation Technology:AReview of Process Development and Fundamental Research.In: Tedder DW,Pohland FG,editors.Emerging Technologies in Haz-ardous Waste Management III,ACS Symposium Series#518,Chap-ter3,American Chemical Society Symposium Series No.518,1993.15.Modell M.SCWO Historical Perspective.Supercritical Water Oxida-tion–Achievements and Challenges in Commercial Applications Strategic Analysis,Inc.200116.Hutchenson K,Foster N.Innovations in Supercritical Fluids.ACS Sympo-sium,Series608,Washington,1995.17.Akgerman A,Madras G.Supercritical Fluids-fundamentals forApplication.NATO ASI Ser.E273,Dordrecht:Kluwer,1994,669–695.18.Yoshida H,Terashima M,Takahashi Y.Production of organic acidsand amino acids fromfish meat bysubcritical water hydrolysis.Bio-technol Prog.1999;15:1090–1094.19.Yoshida H,Katayama Y.Production of useful substances fromwood wastes by subcritical water hydrolysis.Proceedings of the 10th APCChE Congress.2004,3P-03–025,A272.20.McHugh M,Krukonis V.Supercritical Fluid Extraction:Principlesand Practice,1st ed..Stoneham,MA:Butterworths.April1986;2nd ed,512January1994.21.Cansell F,Botella P,Garrabos Y,Six J,Gnanou Y,Tufeu R.Frac-tionation of poly(ethylene oxide)star samples by supercriticalfluids.Polymers J.1997;29:910–916.Manuscript received Apr.21,2010,and revision received Sept.12,2010.AIChE Journal September2011Vol.57,No.9Published on behalf of the AIChE DOI10.1002/aic2359。

Journal of Environmental Sciences 19(2007)874–878Cloud point extraction coupled with HPLC-UV for the determination ofphthalate esters in environmental water samplesWANG Ling 1,2,JIANG Gui-bin 2,∗,CAI Ya-qi 2,HE Bin 2,WANG Ya-wei 2,SHEN Da-zhong 11.School of Chemistry and Chemical Engineering,Shan Dong University,Jinan 250100,China.E-mail:qd wling@2.State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Beijing 100085,ChinaReceived 11August 2006;revised 31December 2006;accepted 8January 2007AbstractA method based on cloud point extraction was developed to determine phthalate esters including di-ethyl-phthalate (DEP),di-(2-ethylhexyl)-phthalate (DEHP)and di-cyclohexyl-phthalate (DCP)in environmental water samples using high-performance liquid chromatography separation and ultraviolet detection (HPLC-UV).The non-ionic surfactant Triton X-114was chosen as extraction solvent.The parameters a ffecting extraction e fficiency,such as concentrations of Triton X-114and Na 2SO 4,equilibration temperature,equilibration time and centrifugation time were evaluated and optimized.Under the optimum conditions,the method can achieve preconcentration factors of 35,88,111and detection of limits of 2.0,3.8,1.0ng /ml for DEP,DEHP and DCP in 10-ml water sample,respectively.The proposed method was successfully applied to the determination of trace amount of phathalate esters in e ffluent water of the wastewater treatment plant and the lixivium of plastic fragments.Key words :phthalate esters;cloud point extraction;Triton X-114;non-ionic surfactant,HPLC-UVIntroductionPhthalate esters (PAEs)are a class of chemical com-pounds primarily used as plasticizers for polyvinyl chloride resins,cellulose film coating,and adhesives (Prokupkov´a et al .,2002).It is estimated that the produc-tion of these compounds in the world is several million tons per year (Polo et al .,2005).During the process of their production,manufacture,usage and disposal,significant migration of them into the environmental compartments is possible.Certain phthalates and /or their metabolites are suspected to be human endocrine disruptors,and carcino-gens (Wezel et al .,2000).Several of them have been included in the priority pollutants of endocrine disrupter compounds,due to their potential risks for human health and environment (Li et al .,2004).Environmental research-es are mainly focused on the aquatic environments adjacent to or downstream from industrial sites.It was reported that PAE concentrations are in the range of 0.1–300µg /L for surface marine water and freshwater sites,and 0.1ng /g–100µg /g for river sediments (Yuan et al .,2002).The most widely used methods for analyzing phtha-late esters are chromatographic techniques such as GC or HPLC,but their sensitivity and selectivity limit theirProjected supported by the National Basic Research Program (973)of China (No.2003CB415001)and the Pilot Program of Knowledge Innovation Program of Chinese Academy of Sciences (No.KZCX3-SW-431).*Corresponding author.E-mail:gbjiang@.direct use for determination of these contaminants at a very low levels of concentration in environmental samples with complex matrix.Therefore,a sample pretreatment prior to chromatographic analysis,such as liquid-liquid extraction and solid-phase extraction,is usually necessary.Unfortunately,all of these methods are time-consuming and need a large sample volume.In particular,the tradi-tional liquid-liquid extraction method is also dangerous to analysts because of the large volume of volatile or-ganic solvent used.As a result,the green liquid-liquid extraction method–cloud point extraction (CPE)has been employed in analytical chemistry to preconcentrate organic compounds (Bai et al .,2001;Casero et al .,1999;Carabias-Martinez et al .,1999)and metal ions (Chen and Teo,2001;Manzoori and Karim-Nezhad,2003;Yuan et al .,2005)in the last pared with the traditional organic liquid-liquid extraction,cloud point extraction requires a very small amount of relatively nonflammable and non-volatile surfactants that are friendly to the environment.Another important merit is that no solvent concentration procedures,which may cause analytes loss,are needed under appropriate conditions such as temperature,con-centration of surfactant and Na 2SO 4,equilibration time,the solution containing the surfactant becomes turbid and separates into two phases:a surfactant-rich phase (at a very small volume)and a larger volume aqueous solution phase (bulk amount)with a diluted surfactant concentration,which approximates to its critical micelle concentration邻苯二甲酸酯No.7Cloud point extraction coupled with HPLC-UV for the determination of phthalate esters in environmental water samples875(CMC).The hydrophobic analytes of the solution are extracted into the small volume of surfactant-rich phase with a high enrichment factor.As a promising alterna-tive to traditional solvent extraction,CPE,especially the extraction of environmental pollutants is still at its initial stage.Only a few reports can be found on the extraction of polycyclic aromatic hydrocarbons(PAHs)(Rodenbrock et al.,2000;Collen et al.,2002;Sirimanne et al.,1996), polychlorinated biphenyls(PCBs)(Manzoori and Karim-Nezhad,1998),dibenzofurans(PCDFs),polychlorinated dibenzop-dioxins(PCDDs),pesticides(Fern´a ndez et al., 1999),vitamins(Pinto et al.,1992)and other organic compounds such as chlorinated phenols(Saitoh and Hime, 1991).These reported results indicate that cloud point extraction has a great analytical potential as an effective enrichment method.However,no researches focused on the extraction of phthalate esters from the water by CPE, have been published.In the present study,a method was developed for the determination of trace levels of PAEs in water by CPE using Triton X-114as the extraction solvent.1Materials and methods1.1MaterialsAll reagents used were HPLC grade,and purified water from a Milli Q system was used throughout the experi-ments.Standard stock solutions containing phthalate esters compounds were prepared by dissolving an appropriate amount of these compounds in methanol.Working solu-tions were prepared daily by an appropriate dilution of the stock solutions.The non-ionic surfactant Triton X-114was from Acros Organics,New Jersey,USA.Na2SO4(Beijing Chemical Factory,China)was prepared immediately be-fore each experiment.Analysis of phthalate esters in environmental samples may pose a serious problem because high blanks are often encountered due to PAEs presence in many laboratory products,including chemicals and glassware.Therefore, the vessels used for trace analysis were washed with methanol and purified water before use.1.2InstrumentationThe HPLC system includes an Agilent1100series binary pump,an Agilent1100series VWD detector and a Rheodyne7225i injector.The separations were performed on an ODS-C18column(250mm×4.6mm,particle size 5µm).A mixture of acetonitrile and water was used as mobile phase with the gradient:0–10min,75:25(v:v; acetonitrile:water);10–11min,the organic phase increased from75%to100%;11–22min,100%acetonitrile.Aflow rate of1ml/min was selected.The226nm wavelength of the UV detector was selected for all of the three kinds of phthalate esters.A personal computer equipped with an Agilent Chemstation program for LC systems was used to acquire and process chromatographic data.Peak area was used as the analytical measurement.A thermostatic bath (TB-85Therma Bath,Shimadsu,Japan),maintained at the desired temperature,was used to obtain cloud point pre-concentration.Centrifugation with calibrated centrifuge tubes(Beijing Medicinal Instrument Company,China) were used to accelerate the phase separation process. Easypure deionized water was used in this study(Model D7382-33,Barnstead Thermolyne Corporation,Dubuque, IA,USA).The injector volume was20µl.1.3Cloud point procedureFor the preconcentration of phthalate esters,aliquots of10ml of the sample solution containing the analytes, 0.25%Triton X-114and0.4mol/L Na2SO4,were kept for 60min in the thermostatic bath at45°C.Then the phase separation was accelerated by centrifugation for5min at3500r/min.After phase separation,the bulk aqueous phase was removed,and20µl of the remaining surfactant-rich phase was injected directly in the HPLC loop for subsequent analysis.1.4Sample preparationSample1:since the phthalate esters are not chemically but only physically bound to the polymer chains,they may be leached into food and beverages from the plastic containers(Li et al.,2004).It was assumed there are free phthalate esters in the lixivium of plastic fragments. Broken down30g plastic package into fragments and mixed with50ml purified water.The sample was subjected to the ultrasonic bath for agitation for5h.Sample2:the effluent water from Gaobeidian Wastewa-ter Treatment Plant(WWTP),Beijing,China.1.5Extraction of phthalate esters in real samplesThe real water samples werefiltered through a0.45-µm pore-size membranefilter to remove the suspended particular matter.A10-ml real water sample was submitted to the cloud point extraction procedure using0.25%Triton X-114and0.4mol/L Na2SO4.After phase separation, 20µl of surfactant-rich phase was injected directly to the loop of HPLC coupled UV detector for the analysis. Standard solutions containing40,60,50ng/ml of di-ethyl-phthalate(DEP),di-(2-ethylhexyl)-phthalate(DEHP)and di-cyclohexyl-phthalate(DCP)were added to a10-ml real water sample for the recovery test,respectively.2Results and discussionTriton X-114with a cloud-point temperature of 23°C(Toerne et al.,2000;Delgado et al.,2004)is one of the most common nonionic surfactants used in the bibliography in the cloud point extraction(Delgado et al., 2004).There are several different parameters that may influence the extraction efficiency.They were investigated in this experiment excluding the initial concentrations which were proved to no contribution to preconcentration factor(Li and Chen,2003).2.1Effect of the concentration of surfactantThe theoretical preconcentration factors depend on the volume of the surfactant-rich phase,which varies with the876WANG Ling et al.V ol.19surfactant concentration in solution at the same time (Del-gado et al .,2004).The nonionic Triton X-114surfactant was chosen for its low cloud point temperature and high density,phase separation was facilitated by centrifugation (Shemirani et al .,2005).The e ffect of the surfactant concentration ranging from 0.125%–0.75%was examined and the results are shown in Fig.1.It can be found that the highest extraction e fficiency is obtained at 0.25%.When the concentration of surfactant is below 0.25%,the volume of surfactant-rich phase is too small to be separated from the bulk solution,Moreover,such small volume is not enough for triple injections.Based on these experimental results,0.25%Triton X-114was adopted as the optimum amount to achieve the best analytical signals and highest extraction e fficiency.2.2E ffect of equilibration time,temperature and con-centration of Na 2SO 4Surfactant can exhibit di fferent behaviors when the equilibration time varies (Manzoori and Karim-Nezhad,1998).Triton X-114surfactant exhibits a similar behavior for all the phthalate esters under the given concentration range.Fig.2a shows an increase in the enrichment e ffect from 27to 70min,and then a decrease as the time goes.As a consequence,60min was adopted as the optimum equilibration time.The addition of salt to the solution may influence the extraction process.In the case of most non-ionic surfactant,the presence of salts may facilitate phase separation since it increases the density of the aqueous phase (Carabias-Martınez et al .,2000).Availableelec-Fig.1Extraction e ffect of the phthalate esters as a function of Triton X-114concentration.trolytes can also change the cloud-point temperatures of nonionic surfactant.The relevant electrolytes are usually in high concentrations (exceeding 0.1mol /L)(Purkait et al .,2004).The salting-in and salting-out e ffects can be used to interpret the electrolyte e ffects on the cloud-points of nonionic surfactant (Pino et al .,2002).In this work,di fferent concentrations of Na 2SO 4,ranging from 0.2to 0.6mol /L were added to the solution.The results are in agreement with other studies.The final surfactant-rich phase volume was not noticeably influenced by the increased ionic strength (Pino et al .,2002).When the concentration is higher than 0.4mol /L,the surfactant-rich phase will be on the surface of the solution,which may make it more di fficult to separate the extraction system into two phases and the accuracy and reproducibility were probably not satisfactory.As shown in Fig.2b,the highest extraction e fficiency can be obtained at a concentration of 0.4mol /L.When the cloud point extraction procedure was pro-cessed at equilibration temperature of the surfactant,the best extraction e fficiency was achieved (Raymond et al .,1994).If the temperature is lower than the cloud point,the phase separation is di fficult to be formed.In order to obtain the maximum phase separation,the lowest equilibration temperature need to be examined.Theoretically,the op-timal equilibration temperature of the extraction occurs when the equilibration temperature is 15–20°C higher than the cloud point temperature of surfactant (Raymond et al .,1994).Fig.2c shows the e ffects of equilibration temper-ature on the extraction e fficiency.The maximum signals were presented between 40–50°C.Therefore,45°C was selected as the working equilibration temperature.2.3E ffect of centrifugation timeThe e ffect of centrifugation time on phase separation was studied in the range of 2–20min at 3500r /min.The results showed 5min is available for a complete phase separation.2.4Characteristics of analytical methodTable 1shows some characteristics of the proposed method.The linearities of the three kinds of phthalate esters were in the range of 8–200ng /ml for DEP,10–200ng /ml for DEHP,5–200ng /ml for DCP,respectively.The detection limits based on 3levels of the ratio of signal to noise were 2.5ng /ml for DEP,3.8ng /ml for DEHP,1.0ng /ml for DCP respectively.There issignificantFig.2E ffect of the equilibration time (a),ionic strength (b)and temperature (c)on the extraction e fficiency.No.7Cloud point extraction coupled with HPLC-UV for the determination of phthalate esters in environmental water samples 877Table 1Analytical characteristics of the methodCompoundsEnrichment Detection RSD R 2Linear factor limit (%)range (ng /ml)a (ng /ml)DEP 35 2.5 3.890.99828–200DEHP 88 3.8 3.710.996410–200DCP1111.01.870.99985–200a .Determined as 3levels of the ratio of signal tonoise.Fig.3Chromatogram of the standard solution of the three kinds of phtha-late esters.(a)standard solution in the bulk aqueous before enrichment (y-axis on the right).(b)standard solution in the enrichment-phase(y-axis on the left).(1)DEP;(2)DEHP;(3)DCP.di fference on the preconcentration factor between DEP and DEHP or DCP.This can be explained by their hydrophobic properties K o /w (Saitoh et al .,2004).Fig.3has compared the enrichment e fficiency of phthalate esters before and after enrichment as the concentration in aqueous solution is 2.5µg /ml for DEP,3.74µg /ml for DEHP,3.2µg /ml for DCP,respectively.2.5Analysis of real samplesIn order to validate the accuracy and precision of the proposed method under the selected conditions,WWTP e ffluent water sample,the lixivium of plastic fragments and spiked samples had been tested by HPLC-UV .The results are shown in Table 2.No PAEs were detected out in the selected real WTTPTable 2Determination and recoveries of three compounds in spikedwater samples Compounds Added conc.Detected conc.a Recovery b (ng /ml)(ng /ml)(%)Sample 1DEP –nd –40.039.598.7±1.9DEHP –nd –60.051.986.5±5.0DCP–nd –50.049.498.8±5.9Sample 2DEP–nd –12.512.9103.2±10.340.040.9102.7±0.9125.0107.385.8±4.5DEHP–nd –18.718.9101.1±9.860.058.498.0±6.7150.0151.9101.3±5.3DCP–nd –16.016.2101.3±11.050.051.8101.5±9.0128.0127.499.5±4.9aMean for three determinations;b mean and standard deviation for three determinations;nd:not detected.samples.It may be that the PAE concentrations in the real samples were below method detection limits.In all cases,the spiked recoveries were satisfied,showing no obvious matrix interferences.3ConclusionsThe cloud point technique was applied as an e ffec-tive method for the extraction of three kinds of PAEs (DEP,DEHP,DCP)in aqueous samples.Experimental results showed that high recoveries and precision can be obtained at the optimized parameters:concentration of Triton X-114,0.25%;for Na 2SO 4,0.4mol /L;equilibration temperature,45°C,equilibration time,60min and cen-trifugation time,5min.Furthermore,the proposed method is a simple,rapid,and e ffective for the simultaneous determination of three kinds of phthalate esters with low concentration levels in environmental water.ReferencesBai D S,Li J L,Chen S B et al .,2001.A novel cloud-pointextraction process for preconcentrating selected polycyclic aromatic hydrocarbons in aqueous solution[J].Environ Sci Technol,35:3936–3940.Carabias-Martınez R,Rodrıguez-Gonza´ıo E,Dominguez-Alvarez J et al .,1999.Cloud point extraction as apreconcentration step prior to capillary electrophoresis[J].Anal Chem,71:2468–2474.Carabias-Mart´ınez R,Rodr´ıguez-Gonza´ıo E,Moreno-Cordero Bet al .,2000.Surfactant cloud point extraction and precon-centration of organic compounds prior to chromatography and capillary electrophoresis[J].J Chromatogra A,902:251–265.Casero I,Sicilia D,Rubio S et al .,1999.An acid-induced phasecloud point separation approach using anionic surfactants for the extraction and preconcentration of organic com-pounds[J].Anal Chem,71:4519–4526.Chen J R,Teo K C,2001.Determination of cobalt and nickelin water samples by flame atomic absorption spectrometry after cloud point extraction[J].Anal Chim Acta,434:325–330.Collen A,Persson J,Linder M et al .,2002.A novel two-step extraction method with detergent /polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I[J].Biochim Biophys Acta,1569:139–150.Delgado B,Pino V ,Ayala J H,2004.Nonionic surfactantmixtures:a new cloud-point extraction approach for the determination of PAHs in seawater using HPLC with flu-orimetric detection[J].Analy Chim Acta,518:165–172.Fern´a ndez A E,Ferrera Z S,Rodrłguez J J S,1999.Application ofcloud-point methodology to the determination of polychlo-rinated dibenzofurans in sea water by high-performance liquid chromatography[J].Analyst,124:487–491.Frankewlcht R P,Hinze W L,1994.Evaluation and optimizationof the factors a ffecting nonionic surfactant-mediated phase separations[J].Anal Chem,66:944–954.Li J L,Chen B H,2003.Equilibrium partition of polycyclic aro-matic hydrocarbons in a cloud-point extraction process[J].J Colloid Interf Sci,263:625–632.Li X J,Zeng Z R,Chen Y et al .,2004.Determination of phthalateacid esters plasticizers in plastic by ultrasonic solvent878WANG Ling et al.V ol.19extraction combined with solid-phase microextraction using calix[4]arenefiber[J].Talanta,63:1013–1019.Manzoori J L,Karim-Nezhad G,1998.Determination of poly-chlorinated biphenyls by liquid chromatography following cloud-point extraction[J].Analy Chim Acta,358:145–155. Manzoori J L,Karim-Nezhad G,2003.Selective cloud point extraction and preconcentration of trace amounts of silver as a dithizone complex prior toflame atomic absorption spectrometric determination[J].Anal Chim Acta,484:155–161.Pino V,Ayala J H,Afonso A M et al.,2002.Determina-tion of polycyclic aromatic hydrocarbons in seawater by high-performance liquid chromatography withfluorescence detection following micelle-mediated preconcentration[J].J Chromatogra A,949:291–299.Pinto C G,Luis J,Pavon P et al.,1992.Cloud point preconcentra-tion and high-performance liquid chromatographic analysis with electrochemical detection[J].Anal Chem,64:2334–2338.Polo M,Llompart M,Garcia-Jares C et al.,2005.Multivariate optimization of a solid-phase microextraction method for the analysis of phthalate esters in environmental waters[J].J Chromatogra A,1072:63–72.Prokupkov´a G,Holadov´a K,Poustka J et al.,2002.Development of a solid-phase microextraction method for the determina-tion of phthalic acid esters in water[J].Anal Chim Acta, 457:211–223.Purkait M K,DasGupta S S,De S,2004.Separation of congo red by surfactant mediated cloud point extraction[J].Dyes and Pigments,63:151–159.Rodenbrock A,Selber K,Egmond M R et al.,2000.Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems[J].Bioseparation,9:269–276.Saitoh T,Hime W L,1991.Concentration of hydrophobic organic compounds and extraction of protein using alkylammonio-sulfate zwitterionic surfactant mediated phase separations (Cloud Point Extractions)[J].Anal Chem,63:2520–2525. Saitoh T,Matsushima S,Hiraide M,2004.Aerosol-OT-γ-alumina admicelles for the concentration of hydrophobic organic compounds in water[J].J Chromatogra A,1040:185–191. Shemirani F,Baghdadi M,Ramezani M et al.,2005.Determi-nation of ultra trace amounts of bismuth in biological and water samples by electrothermal atomic absorption spec-trometry(ET-AAS)after cloud point extraction[J].Analy Chim Acta,534:163–169.Sirimanne S R,Barr J R,Patterson D G,1996.Quantification of polycyclic aromatic hydrocarbons and polychlorinated dibenzo-p-dioxins in human serum by combined micelle-mediated extraction(cloud-point extraction)and HPLC[J].Anal Chem,68:1556–1560.Toerne K,Rogers R,Wandruszka R,2000.Fluctuating clouding behavior in mixed micellar solutions[J].Langmuir,16: 2141–2144.Wezel A P,Vlaardingen P,Posthumus R et al.,2000.Environ-mental risk limits for two phthalates,with special emphasis on endocrine disruptive properties[J].Ecotoxicol Environ Saf,46:305–321.Yuan S Y,Liu C,Liao C S et al.,2002.Occurrence and microbial degradation of phthalate esters in Taiwan river sediments[J].Chemosphere,49:1295–1299.Yuan C G,Jiang G B,He B et al.,2005.Preconcentration and determination of tin in water samples by using cloud point extraction and graphite furnace atomic absorption spectrometry[J].Microchim Acta,150:329–334.。

Journal of Chromatography B,877 (2009) 3572–3580Contents lists available at ScienceDirectJournal of ChromatographyBj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o mbJoint GC–MS and LC–MS platforms for comprehensive plant metabolomics:Repeatability and sample pre-treatmentRuben t’Kindt a ,Kris Morreel b ,Dieter Deforce c ,Wout Boerjan b ,Jan Van Bocxlaer a ,∗aLaboratory of Medical Biochemistry and Clinical Analysis,Ghent University,Harelbekestraat 72,B-9000Ghent,BelgiumbDepartment of Plant Systems Biology,Flanders Interuniversity Institute for Biotechnology,Ghent University,B-9052Ghent,Belgium cLaboratory of Pharmaceutical Biotechnology,Ghent University,Harelbekestraat 72,B-9000Ghent,Belgiuma r t i c l e i n f o Article history:Received 7May 2009Accepted 26August 2009Available online 1 September 2009Keywords:RepeatabilityMetabolite extraction GC–MS LC–MSPlant metabolomics Enzymatic activitya b s t r a c tMetabolomics nowadays mostly comprises the application of both LC–MS and GC–MS based approaches.Here we investigate different extraction set-ups for these two established analytical platforms in the field of plant metabolomics.Six extraction approaches for Arabidopsis thaliana leaves,varying in extraction sol-vent composition,extraction temperature and order of solvent addition within the extraction sequence,were analyzed on the two platforms.Our aim was to establish the most suitable analysis protocol,prac-ticable for both LC–MS and GC–MS analysis,in order to obtain as extensive as possible metabolome coverage.One single sample preparation procedure would save time and valuable sample while still offering the complementary datasets generated by GC–MS and LC–MS.All extraction approaches were evaluated based on the following criteria:number of detected m /z -retention time pairs,heat maps of the detected peaks,and residual enzymatic activity of invertase and phosphatase in the plant leaf extracts.Unsupervised principal component analysis (PCA)was used to evaluate grouping trends between the different extraction approaches.Quality controls,a blend of aliquots of the different extracts,were used to establish a paired evaluation of the repeatability performance of the GC–MS and LC–MS analysis.We conclude that the use of chloroform in the extraction solvent is counterproductive in an untargeted LC–MS metabolomics approach as is heating.Below room temperature (instead of heated)extraction does not significantly degrade GC–MS performance but one should be more cautious with respect to residual enzymatic activity in the plant extract.© 2009 Elsevier B.V. All rights reserved.1.IntroductionMetabolomics plant analyses aim at the simultaneous detec-tion of all metabolites in plant tissues.While GC–MS is mainly suited for compound classes appearing mainly in the primary metabolism,i.e.amino acids,fatty acids,carbohydrates and organic acids [1],LC–MS is more practicable towards the overall biochem-ical richness of plants.The latter technique analytically covers the large (semi-polar)group of plant secondary metabolites such as alkaloids,saponins,phenolic acids,phenylpropanoids,flavonoids,glucosinolates,polyamines and derivatives thereof;next to var-ious primary metabolites depending on the type of stationary phase used [2].Nevertheless,as no single analytical technique is entirely competent in covering the broad metabolic picture,com-bining multiparallel technologies in metabolomics applications∗Corresponding author at:Laboratory of Medical Biochemistry &Clinical Anal-ysis,Faculty of Pharmaceutical Sciences,Ghent University,Harelbekestraat 72,B-9000Ghent,Belgium.Tel.:+3292648131;fax:+3292648197.E-mail address:Jan.VanBocxlaer@UGent.be (J.Van Bocxlaer).has become indispensable [3],thus aiming at a comprehensive metabolome coverage.Apart from the analytical platform used,sample preparation too has a vital contribution in defining the array of metabolite classes covered.Any sample preparation protocol in a metabolomics perspective is a compromise between complete recovery of (ulti-mately)all compound classes,avoiding chemical or physical breakdown of labile metabolites as well as enzyme mediated metabolite conversions,and producing a sample compatible to the separation technique to be used [4].Sample preparation most of the time starts with immediately quenching metabolism by flash-freezing fresh plant tissues in liquid nitrogen [2,5,6].Other techniques are freeze clamping or acidic treatment of plant mate-rial,although the latter can result in a severe reduction in the number of detectable metabolites [7].Freeze-drying of samples can also be performed.However,extraction of frozen tissues that still contain the original amount of water may prove more advanta-geous when compared to extracting freeze-dried samples.Indeed,freeze-drying may potentially lead to the irreversible adsorption of metabolites on cell walls and membranes [8].Conversely,others promote the use of freeze-drying due to the variable water con-1570-0232/$–see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jchromb.2009.08.041R.t’Kindt et al./J.Chromatogr.B877 (2009) 3572–35803573tent in fresh plants,causing lower extraction reproducibility and enhanced degradation of metabolites[9].No matter how,thaw-ing of plant material must be avoided as long as proteins are not fully precipitated.At ambient temperatures,some enzymes such as hydrolases or phosphatases tend to remain or become active even in methanol extraction solutions[10].An interest-ing criterion for evaluating residual enzymatic activity in plant biomass is consequently monitoring the degradation of sucrose to glucose and fructose by invertase[1]and the degradation of glucose-6-phosphate and fructose-6-phosphate to phosphate by phosphatase[11].If enzymatic activity is not immediately stopped during sample preparation,these substrates will disappear.Clearly, a successful metabolomics analysis protocol consists of much more than mere LC–MS,GC–MS or NMR measurements and subsequent data treatment.In a recent publication,we already focussed on the optimiza-tion of pre-LC–MS procedures[12].The current study extends the search for a comprehensive homogenization/extraction set-up as described in this previous work,hereby focussing on sample stability and expanding the pre-LC–MS procedure to provide com-patibility with parallel GC–MS analysis.Having one single sample preparation procedure for both GC–MS and LC–MS saves valuable time and sample,especially taking into consideration that present day metabolomics studies have evolved from a couple of samples to numerous samples in each treatment group.In that respect sam-ple stability is also a vital aspect.Several alternative approaches in restraining the enzymatic activity in harvested Arabidopsis thaliana plant material during sample treatment are compared in this study, including the use of chloroform instead of methanol for precipitat-ing proteins and the influence of temperature during extraction on this process(4◦C versus70◦C).As the order in which the differ-ent extraction solvents are added also proves to be crucial[1],the initial addition of a two-phase system to the frozen plant material (e.g.,methanol and water together)is compared with successively adding each solvent during the extraction procedure.All investi-gated procedures were aimed at obtaining a comprehensive plant leaf extract for subsequent GC–MS or LC–MS analysis.Performance evaluation of the different sample treatment approaches was based mainly on number of extracted mass-retention time(m/z-tR)pairs, heat maps of the detected peaks,and the residual activity of inver-tase and phosphatase.Unsupervised principal component analysis (PCA)was used to evaluate grouping trends between the different extraction approaches.Finally,the analytical set-up was designed to provide an indi-cation of repeatability performance of two conventional tools in metabolomics research,i.e.electron-impact gas chromatography mass spectrometry(EI GC–MS)and micro-LC electrospray ionisa-tion quadrupole time-of-flight mass spectrometry(LC–ESI QTOF MS).Quality controls,as described by Sangster et al.[13],were analyzed regularly throughout both analytical runs in an effort to provide proof of the validity of a contiguous set of metabolomics experimental analyses.For target compound analysis,the FDA recommends a coefficient of variation(CV)of15%regarding the analytical variability(except for concentrations close to the detec-tion limit(LOQ)where a CV of20%is acceptable).Although metabolomics is of a whole different fundamental analytical nature, the FDA guidance is used as a benchmark towards the repeatability evaluation of both metabolomics approaches.2.Experimental2.1.ChemicalsRibitol,leucine-enkephalin,methoxyamine hydrochloride, pyridine,N-methyl-N-(trimethylsilyl)-trifluoroacetamide,and C12,C15,C19,C22,C28,C32,C36n-alkanes were purchased from Sigma–Aldrich(Bornem,Belgium).Chloroform HPLC grade,ace-tonitrile and methanol LC–MS grade were supplied by Biosolve B.V.(Valkenswaard,The Netherlands).Formic acid was obtained from Acros Organics(New Jersey,USA).A Synergy185system (Millipore Corporation,Bedford,MA,USA)was used to generate high purity water for the preparation of all aqueous solutions.2.2.Plant growth and extraction of the biological matrixPlants were grown in the Flanders Institute for Biotechnology (VIB,Department of Plant Systems Biology,Technologiepark927, Ghent,Belgium).Seeds of A.thaliana Columbia-O were sown on a 0.5×MS growth medium.After sowing,media were conserved two days at4◦C,after which they were placed in a temperature control room with the following conditions:a light intensity of±350lx from6a.m.to10p.m.,a relative humidity of50%and a temper-ature of21◦C.Fresh plant leaves were harvested with a pair of micro-scissors and immediately frozen in liquid N2.Homogeniza-tion was performed with mortar and pestle in liquid N2,after which 150±5mg of pooled homogenized plant material was weighed in an Eppendorf tube.Each extraction procedure was performed in triplicate on the same pool of plant leaves.The extraction solvent combinations,comprising300␮l of liquid for each extraction,were spiked in advance with ribitol(internal standard,GC–MS)obtain-ing a concentration of180␮g/ml.Extraction procedures A–C are based on liquid extraction with MeOH/H2O:(A)Plant material was extracted with300␮l cold MeOH/H2O80/20(v/v)in a Thermomixer(Eppendorf AG,Hamburg,Germany) during15min(1250rpm,4◦C)[12].(B)240␮l of cold MeOH was added to the plant material contain-ing Eppendorf tube,after which the samples were extracted in a Thermomixer(15 ,1250rpm,4◦C).60␮l of H2O was added sub-sequently and samples were mixed again(5 ,1250rpm,4◦C).(C)Identical as procedure B,except that the Thermomixer tem-perature was set at70◦C during extraction with MeOH.After 1min of incubation,the eppendorf tubes are opened for a short moment to relieve built-up gas pressure.The vials remain closed for the rest of the incubation.Immediately after the incu-bation,all samples are cooled down to4◦C.As such,all gaseous solvent is liquefied again through condensation.Extraction procedures D–F are based on liquid extraction with a one-phase CHCl3/MeOH/H2O mixture:(D)Plant material was extracted with300␮l coldCHCl3/MeOH/H2O20/60/20(v/v)in a Thermomixer(15 , 1250rpm,4◦C).(E)60␮l of cold CHCl3was added to the plant material containingEppendorf tube,after which the plant samples were extracted ina Thermomixer(5 ,1250rpm,4◦C).240␮l of MeOH/H2O60/20(v/v)was added subsequently and samples were placed in the Thermomixer again(15 ,1250rpm,4◦C)[1].(F)180␮l of cold MeOH was added to the plant material,after which the samples were placed in a Thermomixer for 15min(1250rpm,70◦C,with relief of build-up pressure,see above).60␮l of CHCl3was then added and samples were mixed again(5 ,1250rpm,4◦C).Finally,60␮l of H2O was added to the Eppendorf tubes.This method was adopted from the Golm Metabolome Database(http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/analytic/gmd prot.html).All extracts werefinally sonicated for5min(Bransonic Ultra-sonic Cleaner1210,Danbury,CT,USA)and centrifuged(Sigma 3-18K,Sartorius AG,Göttingen,Germany)for15min(4◦C, 15,000rpm).The supernatant(300␮l)was isolated and used for3574R.t’Kindt et al./J.Chromatogr.B877 (2009) 3572–3580subsequent LC–MS analysis.For GC–MS analysis,a50␮l aliquot of the metabolite sample supernatant was further derivatized by methoxyamination,using a20mg/ml solution of methoxyamine hydrochloride in pyridine,and subsequent trimethylsilylation with N-methyl-N-(trimethylsilyl)-trifluoroacetamide[14,15].A C12,C15, C19,C22,C28,C32,and C36n-alkanes mixture was used for the deter-mination of retention time indices[16].For the quality control(QC)samples,an aliquot(40␮l)of all prepared sample extracts,regardless of extraction procedure,was mixed in an Eppendorf tube in cold conditions.Due to visible con-tamination,extract D3was excluded from both the QC preparation and the subsequent LC–MS and GC–MS analysis.The quality con-trol pool was subsequently divided over several vials and analyzed regularly throughout the whole analysis batch,in both GC–MS and LC–MS.The quality control approach for GC–MS and LC–MS metabolomics has been adopted from Sangster et al.[13].All extracts and QCs were analyzed in parallel on both the LC–MS and GC–MS platform in a1-day time window.2.3.Liquid chromatography–mass spectrometryFor the liquid chromatography part,an Alliance2690LC sys-tem(Waters,Milford,MA,USA)was used.The LC column used was an Atlantis dC18column2.1mm×150mm;3␮m(Waters, Milford,MA,USA).The LC mobile phase consisted of(A)water containing0.1%(v/v)formic acid;(B)90/10acetonitrile/water containing0.1%(v/v)formic acid.Both eluents(A)and(B)werefil-tered through a0.45␮m membranefilter(Alltech Associates,Inc., Lokeren,Belgium)and degassed for5min in an ultrasonic bath (Branson,Danbury,CT,USA)prior to use.Gradient elution chro-matography was always performed starting with100%solvent A. Within a20min time interval,%B composition was increased to 40%,followed by a%B increase up to100%within5min.This com-position was then maintained for5final minutes after which the whole system was allowed to re-equilibrate at initial conditions. This generic gradient had separately been optimized[17].MS experiments were performed using a Q-TOF micro quadrupole orthogonal acceleration time-of-flight mass spectrom-eter(Waters,Milford,MA,USA)equipped with a dual sprayer orthogonal electrospray source(Z-spray®,LockSpray®).The instru-ment was operated in positive ion electrospray mode.ESI capillary voltage was optimized to3000V and cone voltage was set to30V. Full scan spectra were acquired over an m/z100–1000range at a scan accumulation rate of2scan/s and an interscan delay of0.1s. All spectra were collected in continuum,single MS mode.Leucine-enkephalin was used as the lockmass solution(m/z556.2771; 2␮g/ml in50/50AcN/H2O)and infused at a constantflow rate of5␮l/min using a Gilson307pump(Gilson,Middleton,WI), equipped with a LC Packingsflow splitter(Dionex Corporation, Sunnyvale,CA,USA).2.4.Gas chromatography–mass spectrometrySamples were analyzed using an Agilent7683B Series Injec-tor(Agilent,Santa Clara,CA)coupled to an Agilent HP6890Series gas chromatograph system and a5973Mass Selective Detector (Agilent,Santa Clara,CA),i.e.a quadrupole type GC–MS sys-tem.A Varian factorFOUR capillary column VF-5ms(5%phenyl 95%dimethylpolysiloxane,30m×0.25mm ID,df=0.25␮m)con-nected to a10m EZ-guard column was used.A constant column flow of1ml/min helium was applied.The injector was kept at 230◦C.Samples were splitless injected(1␮l)during1.5min using a totalflow of39ml/min which was reduced to24ml/min after 2min.The temperature programmed separation started at70◦C for5min,and then ramped by5◦C/min to325◦C within51min. After1min at325◦C,the oven was cooled to the initial tempera-ture of70◦C within5.10min.A temperature equilibration phase of 5min was allowed before the next injection.The transferline and EI source temperature were250and200◦C.EI spectra were acquired between60and600Da.The electron multiplier voltage was set on 1700V.2.5.Data handlingRaw LC–MS data were processed using QuanLynx®and MarkerLynx®(Waters,Milford,MA),a data processing tool for metabolomics applications.MarkerLynx uses ApexTrack©peak integration to detect chromatographic peaks.The track peak parameters were set as follows:peak width at5%height 20s,automatic peak-to-peak baseline noise,intensity thresh-old50,mass window0.5amu,retention time window1.0min, noise elimination level 4.0and mass tolerance0.10amu.The XCMS package[18]in R version 2.6.1was applied to align the GC–MS chromatograms with the following argument values: xcmsSet(fwhm=7.5,max=300,snthresh=2,step=0.1,steps=2, mzdiff=0.5),group(bw=7.5,max=300).All GC–MS data were further normalized using ribitol as an internal standard.AMDIS [19]was used to identify compounds out of the GC–MS chro-matograms.Quantifier ions for all compounds(e.g.,substrates and end products of invertase and phosphatase)were adopted from the Golm Metabolome Database(http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/msri/gmd smq.html).SIMCA-P(Umet-rics,Umea,Sweden)was used for some aspects of the multivariate data processing.All acquired raw data were subjected to mean-centering,normalization and pareto scaling before multivariate analysis.3.Results and discussion3.1.Quality comparison of the different extraction proceduresTo evaluate and compare quality,a quality control sample was analyzed regularly throughout both the LC–MS and GC–MS analy-sis sequences.This sample is prepared from aliquots of all extracts, providing a representative“mean”of the investigated samples.It is not to contain any variability originating from the extraction step and can thus be considered as a mass spectrometric replicate.As a result,monitoring this kind of QC data may be used to evaluate the analytical performance during the analysis sequence on both mass spectrometric tools[20].Fig.1displays thefirst principal component t1of the principal component analysis for all samples versus the samples in run order.The QC runs are included,iden-tified as squares.This principal component is a newly calculated latent variable that explains as much of the present variation as possible in the original dataset.It accounted for55.1%and47.0% of the total variability in the GC–MS and LC–MS dataset,respec-tively.The QCs clearly showed limited variation throughout both analysis approaches.We thus conclude that both analysis meth-ods provide measurement stability for the duration of the analysis sequence.Consequently,the differences observed between the var-ious extraction approaches are to be ascribed to variations in the extraction set-up and not to the variability inherent to the analyti-cal platform.Fig.1also clearly indicates extract B3as an outlier in the GC–MS analysis.PCA on respectively the GC–MS and the LC–MS data was con-ducted to study the differences or trends between all extraction procedures(Fig.2for LC–MS and Fig.3for GC–MS).Strong out-liers(samples outside the Hotelling’s T295%tolerance ellipse)were excluded from further data processing(i.e.sample B3for GC–MS analysis).The PCA score plot in Fig.2clearly distinguishes three groups within the procedures as analyzed by LC–MS:the extrac-R.t’Kindt et al./J.Chromatogr.B877 (2009) 3572–35803575Fig.1.Time series plot of thefirst PCA component for GC–MS analysis(upper pane) and LC–MS analysis(lower pane).The2 and3 limits are also shown.QCs are shown in squares.Extract B3is spotted as an outlier in the GC–MS analysis.tion step performed with80/20methanol/H2O without heating of the Thermomixer(procedures A and B),the extraction step per-formed with20/60/20chloroform/methanol/H2O without heating of the Thermomixer(procedures D and E)andfinally the extraction procedures with the Thermomixer heated up to70◦C(procedures C and F).Not only do the procedures including heating during extraction separate themselves form the other procedures,heating moreover clearly increases the analytical variability.The samples of procedures C and F do show a larger scatter in the PCA score plot compared to the other procedures.PCA also separates the different approaches when using the GC–MS data(Fig.3),albeit less con-clusive.In addition,procedures B and D cannot be distinguished based on those metabolites detected in the GC–MS analysis.The corresponding loadings plots allow pinning down those metabo-lites responsible for the classification of the extraction procedures. In a metabolomics application,identification of this relevant sub-set of metabolites is the next step.The reliable identification of metabolites using GC–MS libraries is a major advantage within this platform.On the contrary,identification still is the bottleneck in current LC–MS analysis,as no comprehensive metabolite libraries are available for this metabolomics platform.In spite of this,some metabolites could be annotated using accurate mass measurement with a LockSpray®device[17]and subsequent database search(see Fig.2).If,at the other hand,we compare the number of detected m/z-tR pairs between the different procedures for the LC–MS analysis (Fig.4),it is clear that procedure A produces the highest number of m/z-tR pairs(3773;n=3).Extraction with the three-phase sys-tem(i.e.chloroform/methanol/H2O or procedure D,E and F)yields a significantly reduced number of m/z-retention time pairs com-pared with the two-phase system(i.e.methanol/H2O or procedure A,B and C).As an example,the observed difference in extracted signals between procedure A and D amounts up to43%(3773ver-sus2144m/z-tR signals).Heat maps,plotting LC–MS m/z-value against retention time,unravel clear regions of contrast between the different extraction procedures,emphasizing this observation (Fig.5).The retention time zone ranging from17to27min shows the largest between profile dissimilarity.The procedures based on extraction with methanol/H2O(procedures A,B and C)show a higher density profile within this region compared to the extrac-tions based on chloroform/methanol/H2O(procedures D,E and F). Clearly,the addition of CHCl3to the extraction solvent has a signifi-cant influence on the solubility of the secondary metabolites eluting in the time range from17to27min.Heating up to70◦C is obviously also less favourable(LC–MS analysis results),independently of the extraction solvent combination used.Procedure C,e.g.,detects29% less m/z-tR pairs than procedure A,although the same extraction solvent combination is used(3773versus2687m/z-tR signals). Extraction procedures using a heated Thermomixer at70◦C(pro-Fig.2.Score(left)and loadings plot(right)obtained from PCA of the entire LC–MS dataset;t1and t2of the score plot accounted for47.0%and12.8%of the total variability in the pareto scaled data of all extraction procedures,respectively.Some metabolites responsible for the different extraction set-up could be annotated based on accurate mass measurement using lockmass calibration:1.glutathione(m/z308.0907;ppm−2.92);2.deoxyadenosine(m/z252.1078;ppm−7.42);3.[sinapate−H2O+H]+(m/z 207.0652;ppm−2.37);4.1-caffeoyl-4-deoxyquinic acid(m/z339.1072;ppm−2.30);5.[1-caffeoyl-4-deoxyquinic acid−H2O+H]+(m/z321.0965;ppm−2.80);6.adenosine (m/z268.1038;ppm−3.13);7.5-methylsufinylpentyl nitrile(m/z146.0633;ppm−4.93);8.[sinapate−H2O+H]+(m/z207.0657;ppm−2.41);9.Background ions originating from the purified water in eluent A.3576R.t’Kindt et al./J.Chromatogr.B877 (2009) 3572–3580Fig.3.Score (left)and loadings plot (right)obtained from PCA of the entire GC–MS dataset;t1and t2of the score plot accounted for 51.0%and 23.0%of the total variability in the pareto scaled data of all extraction procedures,respectively.The metabolites most responsible for the classification of the different extraction set-ups are marked:1.glucose;2.galactose;3.fructose;4.phosphoric acid;5.myo-inositol;6.ethylglucopyranoside;7.pyroglutamic acid;8.sucrose;9.galactonic acid;10.glycerol;11.glycerol-3-phosphate.cedures C and F)show an overall less dense heat map compared to the cold extraction procedures.Five of the most intense ions in the region from 17to 27min were extracted and their peak areas were compared between the different extraction approaches as a proof of concept (Fig.6).Addition of CHCl 3clearly decreases the solubility of these metabolites in the extraction solvent,whereas heating to 70◦C during extraction removes substances from the final extract most probably because of enhanced chemical degradation.Several authors discussed the temperature sensitivity of phenolic com-pounds already.Temperatures above 40◦C resulted in a decreased extraction yield of polyphenolic compounds due to degradation,caused by hydrolysis,internal redox reactions and polymerisa-tions [21–24].Because the secondary metabolites eluting in the region from 17to 27min are missed in the extractions based on CHCl 3/MeOH/H 2O 20/60/20(v/v/v),background ions originating from the purified water in eluent A show up more pronounced in these mass chromatograms.This explains the presence of these contaminant peaks in the PCA loadings plot of the LC–MS data in Fig.2.This presence of the background ions is instrumental in clas-sifying these samples away from the other extraction procedures in the resulting PCA score plot.The number of masses detected in the GC–MS analysis shows a somewhat opposite tendency compared to the LC–MS analysis.Heating up the Thermomixer to 70◦C (procedures C and F)yields a slightly higher portion of detectable m /z -tR pairs compared toaFig.4.Count of m /z -tR pairs or peaks detected in the different extraction approaches in both GC–MS as LC–MS analysis (n =3;n =2for procedure D).The dotted line represents the sum of m /z -tR pairs for a specific extraction procedure.reduced temperature extraction (4◦C)using the Thermomixer (dif-ferences from minimum 4%to maximum 9%more m /z -tR pairs).The range of metabolites that is detected in the GC–MS analysis (i.e.mainly primary metabolites)seems to dissolve better when the temperature of the extraction solvent is raised,as in proce-dure C and F.Opposite to the LC–MS data,heat maps plotting m /z -value against retention time for the GC–MS data unravel no distinct regions of contrast between the profiles of the different extraction procedures (data not shown).We conclude that in any of the extraction procedures none of the metabolite classes are likely to be missed in the GC–MS analysis.In the light of obtaining an opti-mized extraction procedure for a comprehensive metabolomics approach on both GC–MS and LC–MS,the number of peaks detected in both platforms was summed up for each extraction procedure (dotted line in Fig.4).As a result,procedure A and B yield a sig-nificantly higher number of metabolites compared to the other extraction set-ups.Overall,procedure A provides the best results in terms of variability and the number of detected m /z -tR pairs taking GC–MS and LC–MS data together.Aiming for a combined GC–MS and LC–MS approach inevitably entails accepting a ing procedure A as front-end is least at the expense of overall performance.Nevertheless,the results also indicate that no matter how distinctly a method pursues comprehensiveness in metabolite yield,100%full metabolite coverage is beyond reach.It is therefore a judicious choice for every metabolomics exper-iment whether a more focussed approach thus extraction,guided by the experimental aim,is followed or an untargeted one while aware of its inherent performance limitations.3.2.Enzymatic activity considerationsAs the homogenization step is carried out in liquid nitrogen,no enzymatic conversion is likely to occur in this stage of sample treatment.The Thermomixer based extraction using a 2:1ratio sol-vent volume to frozen tissue weight is,nevertheless,a critical step regarding enzyme reactivation.As mentioned in Section 1,both invertase and phosphatase activity can be monitored for evaluating residual enzymatic activity in plant extracts [1,11].Both substrates and end products of these enzymes can be detected using GC–MS analysis (quantifier ions were adopted from http://csbdb.mpimp-golm.mpg.de/csbdb/gmd/msri/gmd smq.html ).Phosphatase hydrolyses both glucose-6-phosphate (6TMS:m /z 387.1at tR 38.2)and fructose-6-phosphate (6TMS:m /z 315.0at tR 38.2)with the formation of phosphate (3TMS:m /z 314.1at tR。