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用液质法检测水和土中的草甘膦及草铵膦

Journal of Chromatography A,1081(2005)145–155Residue determination of glyphosate,glufosinate and aminomethylphosphonic acid in water and soil samples by liquidchromatography coupled to electrospray tandem mass spectrometryMar´ıa Ib´a˜n ez,´Oscar J.Pozo,Juan V.Sancho,Francisco J.L´o pez,F´e lix Hern´a ndez∗Research Institute for Pesticides and Water,University Jaume I,E-12071Castell´o n,SpainReceived11February2005;received in revised form3May2005;accepted17May2005AbstractThis paper describes a method for the sensitive and selective determination of glyphosate,glufosinate and aminomethylphosphonic acid (AMPA)residues in water and soil samples.The method involves a derivatization step with9-ﬂuorenylmethylchloroformate(FMOC)in borate buffer and detection based on liquid chromatography coupled to electrospray tandem mass spectrometry(LC–ESI-MS/MS).In the case of water samples a volume of10mL was derivatized and then4.3mL of the derivatized mixture was directly injected in an on-line solid phase extraction(SPE)–LC–MS/MS system using an OASIS HLB cartridge column and a Discovery chromatographic column.Soil samples were ﬁrstly extracted with potassium hydroxide.After that,the aqueous extract was10-fold diluted with water and2mL were derivatized.Then, 50␮L of the derivatized10-fold diluted extract were injected into the LC–MS/MS system without pre-concentration into the SPE cartridge. The method has been validated in both ground and surface water by recovery studies with samples spiked at50and500ng/L,and also in soil samples,spiked at0.05and0.5mg/kg.In water samples,the mean recovery values ranged from89to106%for glyphosate(RSD<9%),from 97to116%for AMPA(RSD<10%),and from72to88%in the case of glufosinate(RSD<12%).Regarding soil samples,the mean recovery values ranged from90to92%for glyphosate(RSD<7%),from88to89%for AMPA(RSD<5%)and from83to86%for glufosinate (RSD<6%).Limits of quantiﬁcation for all the three compounds were50ng/L and0.05mg/kg in water and soil,respectively,with limits of detection as low as5ng/L,in water,and5␮g/kg,in soil.The use of labelled glyphosate as internal standard allowed improving the recovery and precision for glyphosate and AMPA,while it was not efﬁcient for glufosinate,that was quantiﬁed by external standards calibration.The method developed has been applied to the determination of these compounds in real water and soil samples from different areas.All the detections were conﬁrmed by acquiring two transitions for each compound.©2005Elsevier B.V.All rights reserved.Keywords:Glyphosate;Glufosinate;AMPA;Water;Soil;Liquid chromatography;Electrospray interface;Tandem mass spectrometry;Derivatization1.IntroductionGlyphosate[N-(phosphonomethyl)glycine]and glufos-inate[ammonium dl-homoalanin-4-(methyl)phosphinate] are broad spectrum,nonselective,post-emergence herbicides extensively used in various applications for weed control in aquatic systems and vegetation control in non-crop areas. Aminomethylphosphonic acid(AMPA)is the major degra-dation product of glyphosate found in plants,water and soil ∗Corresponding author.Tel.:+34964728100;fax:+34964728066.E-mail address:hernandf@exp.uji.es(F.Hern´a ndez).[1].Chemical structures of these phosphorus-containing her-bicides are given in Fig.1.Due to the extensive worldwide use of these compounds and the restrictive regulations for water in the European Union,very sensitive methods for the determination of pes-ticide residues are required.However,the determination of these two herbicides at the sub␮g/L level is difﬁcult due to their ionic character,low volatility,low mass and lack of chemical groups that could facilitate their detection.Even more difﬁcult can result the residue determination in soil at low concentration levels(e.g.below0.1mg/kg),due to the complexity of this matrix sample.Most methods developed0021-9673/$–see front matter©2005Elsevier B.V.All rights reserved. doi:10.1016/j.chroma.2005.05.041146M.Ib´a ˜n ez et al./J.Chromatogr.A 1081(2005)145–155Fig.1.Chemical structures of glyphosate,AMPA and glufosinate,and derivatization reaction with FMOC.R:H or alkyl group.until now require derivatization procedures to enable analy-sis by gas chromatography (GC)or high-performance liquidchromatography (HPLC).GC/MS methods involved deriva-tization with different reagents [2–8]to confer volatility to the analytes.Normally,there is quite a lot of sample manip-ulation,and the methods are time-consuming and tedious.Physicochemical characteristics of these compounds ﬁt better with LC analysis,although the lack of adequate chem-ical groups (e.g.chromophores,UV absorption,ﬂuorogenics)hamper their measurement by conventional detectors.For these reasons,both pre-column and post-column derivatiza-tion procedures have been employed.Pre-column procedures are based mainly on derivatization with 9-ﬂuorenylmethyl chloroformate (FMOC)[9–15]to form ﬂuorescent deriva-tives (improve detection)and/or to reduce the polar character of the analytes facilitating the chromatographic retention.In post-column procedures,the most common reaction is with o-phthalaldehyde (OPA)and mercaptoethanol [16]or with OPA and N ,N -dimethyl-2-mercaptoethylamine [17].Nor-mally,HPLC has been used in combination with ﬂuorescence detection after derivatization [11–17],although in a few cases glyphosate has been determined directly by ion chromatogra-phy (IC)with UV detection [18]or suppressed conductivity detection [19],but with limited sensitivity.The potential of capillary electrophoresis combined with mass spectrometry [20]and with indirect ﬂuorescence detection [21]has also been explored,although the lack of sensitivity and/or selec-tivity of these techniques together with the difﬁculty for preconcentrating the analytes,limited their application in the ﬁeld of residues.In our research group,we have developed efﬁcient and selective methods based on the use of coupled-column liquid chromatography (LC–LC),which was proved to be an excel-lent way of minimizing sample treatment and improving sen-sitivity in a variety of sample matrices,as water,soil,fruit and vegetables [11,13–15,22].However,the use of conventional ﬂuorescent detection limited the sensitivity required in pesti-cide residue analysis,and also hampered the unequivocal con-ﬁrmation of the residues detected,which nowadays is widely accepted that has to be reached by MS techniques.Searching a method that could satisfy the requirements of sensitivity and selectivity,and unequivocal conﬁrmation of glyphosate in water,the use of MS spectrometric techniques in combina-tion with LC has been investigated by several groups.Thus,IC has been applied,due the ionic character of this analyte,coupled to MS with electrospray interface [23],while RPLC has been used in combination with ICP-MS with P detection [24].However,the sensitivity reached with these techniques was not sufﬁcient.Lee et al [9]obtained better results with the combination LC–MS.In this case,the molecular ions of the derivatized glyphosate,AMPA and glufosinate,as well as a fragment ion of each compound,were monitored in negative ionisation mode obtaining detection limits around 0.1␮g/L.The use of isotope-labelled glyphosate as inter-nal standard minimised derivatization variations and matrix effects.However,although MS based methods could be con-sidered as highly selective methods,the occurrence of false positives might be still possible mainly in the analysis of rel-atively dirty samples,as some interferences can share the same MS properties as the analyte.This may also occur in water sample analysis as it has been reported in some papers,producing constructive discussions on this subject [25].The improved sensitivity and selectivity of tandem MS make this technique ideal for the trace level determination of polar and/or ionic pesticides in water by LC–MS/MS meth-ods,as it has been proved in our laboratory [26–27].This tech-nique was also applied several years ago to the determination of glyphosate and AMPA in water [10],although considerable variation was observed caused by irreproducibility in deriva-tization and fragmentation.4-mL volume was passed through the SPE cartridge,claiming detection limits for glyphosate and AMPA around 0.03␮g/L.When dealing with more complex matrices,such as soil samples,an important loss in the sensitivity can occur a con-sequence of the ionisation suppression from the co-extracted components of the matrix,hampering correct quantiﬁcation.This matrix-effect depends on the analyte-sample combina-tion.Different approaches have been used either to minimize or to correct the matrix effect,such as increasing the sample pretreatment,performing matrix-matched calibration,using an isotope labelled standard or simply diluting the sample [28].Thus,the labeled glyphosate has been used as internal standard for the LC–MS determination of this herbicide [9].Conﬁrmation of the identity of residues in unknown sam-ples is of utmost importance in order to avoid reporting falseM.Ib´a˜n ez et al./J.Chromatogr.A1081(2005)145–155147positives.Recently,the European Union has adopted the con-cept of identiﬁcation points(IPs)as quality criterium for the conﬁrmation of contaminant residues[29].For compounds with an established MRL,a minimum of three IPs is required for satisfactory conﬁrmation of the compound identity.When LC–MS/MS technique is used,the monitoring of two MS/MS transitions,ing one precursor ion and two product ions, allows to earn four IPs,fulﬁlling the requirements of this cri-terium[25].The aim of this paper is to develop a rapid and robust method for the determination of low concentrations of glyphosate,its principal degradation product,AMPA,and glufosinate in water and soil by SPE–LC–ESI-MS/MS,that fulﬁl the requirements of excellent sensitivity and unequiv-ocal conﬁrmation of the residues detected according to the European Union guidelines.Following the most widely accepted criteria,four IPs will be achieved,thus avoiding the possibility of reporting false positives.2.Experimental2.1.ChemicalsGlyphosate(98%),glufosinate(99%)and AMPA(99%) reference standards were purchased from Dr Ehrenstorfer (Augsburg,Germany),Riedel-de-H¨a en(Seelze,Germany) and Sigma(St Louis,MO,USA),respectively.Isotope-labeled glyphosate(1,2-13C,15N),used as surrogate inter-nal standard(IS),was purchased from Dr Ehrenstorfer. Analytical reagent-grade disodium tetraborate decahydrate was obtained from Scharlab(Barcelona,Spain)and9-ﬂuorenylmethylchloroformate(FMOC-Cl)was purchased from Sigma.Reagent-grade hydrochloric acid,formic acid, potassium hydroxide(KOH),acetic acid(HAc)and ammo-nium acetate(NH4Ac)as well as LC-grade acetonitrile were purchased from Scharlab.LC-grade water was obtained by purifying demineralised water in a Nanopure II system(Barn-stead Newton,MA,USA).Standard stock solutions were prepared dissolving approx-imately50mg powder,accurately weighted,in100mL of water obtaining aﬁnal concentration of approximately 500mg/L.A50-mg/L composite standard was prepared in water by mixing and diluting the individual standard stock solutions.Standard working solutions for the LC–MS/MS analysis and for fortiﬁcation of samples were prepared by dilution of the50-mg/L composite standard with water.All standard solutions were stored in nonsilanized glass.The isotope-labeled glyphosate was purchased as1.1mL of100-␮g/mL stock solution in water.A11-␮g/mL stan-dard solution was prepared by dissolving1.1mL of the stock solution in10mL of water.Standard working solutions were prepared by diluting the intermediate standard solution with water.Solutions of5%borate buffer(pH approximately9)in HPLC-grade water and solutions containing12,000mg/L of FMOC-Cl in acetonitrile were used for the derivatization step prior to the analysis.2.2.InstrumentationFor the analysis of water samples,the mass spectrometer was interfaced to a LC system based on a233XL autosam-pler with a loop of4.3mL(Gilson,Villiers-le-Bel,France) and2pumps:an Agilent1100(Agilent,Waldbron,Germany) binary pump used to condition and wash the cartridge(P-1) and a Waters Alliance2695(Waters,Milford,MA,USA)qua-ternary pump used for the chomatographic separation(P-2), as can be seen elsewhere[24].The SPE preconcentration was performed using an Oasis HLB cartridge,20mm×2.1mm i.d.(Waters),as C-1.For the LC separation,a Discovery col-umn C18,5␮m50×2.0mm i.d.(Supelco,Bellefonte,PA, USA),was used as C-2.Mobile phase consisted of water pH 2.5(adjusted with formic acid)in P-1,and mixtures of aque-ous5mM acetic acid/ammonium acetate(pH4.8)water and acetonitrile in P-2.For the analysis of soil samples,the mass spectrometer was directly interfaced to the Waters Alliance2695(Waters) quaternary pump.The mobile phases and the column used were the same as in the case of water samples.A Quattro LC(quadrupole-hexapole-quadrupole)mass spectrometer(Micromass,Manchester,UK)with an orthog-onal Z-spray-electrospray interface was used.Drying gas as well as nebulising gas was nitrogen,generated from pressur-ized air in a NG-7nitrogen generator(Aquilo,Etten-Leur, NL).The nebuliser gasﬂow was set to approximately80L/h and the desolvation gasﬂow to800–900L/h.Datastation operating software was MassLynx v4.0.For operation in MS/MS mode,collision gas was Argon 99.995%(Carburos Metalicos,Valencia,Spain)with a pres-sure of approximately1×10−3mbar in the collision cell. Capillary voltage of3.5kV was used in positive ionization mode.The interface temperature was set to350◦C and the source temperature to120◦C.Dwell times of0.17s/scan were chosen.2.3.SPE procedureThe conditioning of the Oasis cartridge was performed with LC-grade water at pH2.5at aﬂow-rate of1mL/min for7min.An aliquot of4.3mL of water sample was pre-concentrated(1mL/min)into the cartridge and washed with acidiﬁed LC-grade water during4min.After washing,the sample was transferred in backﬂush mode to the C-2column and a gradient in P-2started.2.4.LC procedureTo perform the chromatographic separation,the gra-dient used in P-2was water5mM HAc/NH4Ac(pH 4.8)–acetonitrile,where the percentage of organic modiﬁer was changed as follows:0min,10%;5min,10%;5.1min,148M.Ib´a˜n ez et al./J.Chromatogr.A1081(2005)145–15590%;9min,90%;9.1min,10%;14min,10%.The chro-matographic separations were completed within20min. 2.5.Sample procedureThe derivatization procedure was based on Sancho et al. [14,15](see Fig.1),with slight modiﬁcations.2.5.1.Water samplesGround and surface water samples were collected in plas-tic bottles from different sites of the Valencian Mediterranean region and stored in a freezer at−18◦C until analysis.Ten millilitre of water sample was introduced into a glass tube together with100␮L of isotope-labeled glyphosate standard (110␮g/L).Samples were derivatised by adding0.6mL of 5%borate buffer(pH9)followed by0.6mL of FMOC-Cl reagent(12000mg/L),and allowing the reaction to take place overnight at room temperature.After that,samples wereﬁltered through a0.45␮m syringeﬁlter and acidiﬁed with hydrochloric acid until pH1.5.Finally,4.3mL of the acidiﬁed derivatized samples were directly injected into the SPE–LC–ESI-MS/MS system.Fortiﬁcation of surface or ground waters for recovery experiments was performed by adding1mL of5or50ng/mL mixture solutions to100mL of blank water sample in order to yield fortiﬁcation levels of50or500ng/L,respec-tively.2.5.2.Soil samplesSoil samples was collected from a public garden,sus-pected to have been contaminated by glyphosate.Air-dried soil samples were homogenized and5.0g subsamples were transferred to centrifuge tubes(50mL).Samples were extracted by shaking with0.6M KOH(10mL)on a mechan-ical shaker for30min,and then centrifuged at3500rpm for 30min.The alkaline sample extracted was separated and neu-tralized by adding drops of HCl6M and0.6M until pH7, approximately.After that,the neutralized supernatant was 10-fold diluted with HPLC-grade water.The derivatization step was performed as follows:2-mL of the10-fold diluted supernatant was pipetted into a glass tube together with 120␮L of the labelled internal standard(1.10mg/L),120␮L of5%borate buffer(pH9)and120␮L of FMOC-Cl reagent (12000mg/L).The tube was swirled and left overnight at room temperature.After that,samples wereﬁltered through a0.45␮m syringeﬁlter and acidiﬁed with hydrochloric acid until pH1.5.Finally,50␮L of the acidiﬁed deriva-tized extract was directly injected into the LC–ESI-MS/MS system.Fortiﬁcation of soil samples for recovery experiments was performed by adding1mL of250ng/mL or2500ng/mL mix-ture solutions to5.0g of blank soil sample in order to yield fortiﬁcation levels of0.05mg/kg or0.5mg/kg,respectively. Samples were equilibrated for1h prior to extraction.AMPA and glyphosate were quantiﬁed using isotope labelled glyphosate as internal standard,in both water and soil samples.In the case of glufosinate,quantiﬁcation was performed with external calibration.2.6.Validation studyLinearity of the method was evaluated analysing eight standard solutions by duplicate,in the range25–5000ng/L for water samples,and in the range1–500␮g/L for soil extracts.Precision(repeatability,expressed as relative standard deviation,in%)and recoveries were determined within day by analysing fortiﬁed blank samples in quintupli-cate.This experiment was performed at two spiking lev-els:50and500ng/L in water,and0.05and0.5mg/kg in soil.The limits of detection(LOD),deﬁned as the lowest concentration that the analytical process can reliably dif-ferentiate from background levels,were obtained when the signal was three times the average of background noise in the chromatogram at the lowest analyte concentration assayed.The limits of quantiﬁcation(LOQ)were estab-lished as the lowest concentration assayed and validated, which gave satisfactory recovery(70–120%)and precision (<15%RSD).The speciﬁcity of the method was evaluated by analysing a blank procedure,a processed blank sample,and a blank sam-ple spiked at the lowest fortiﬁcation level assayed(LOQ),i.e. 50ng/L in water and0.05mg/kg in soil.Under these condi-tions,the response obtained for both the blank procedure and the blank samples should not exceed30%of the response corresponding to the LOQ.2.7.Data evaluationTo ensure the quality of the analysis when processing real-world samples,blank samples fortiﬁed at the LOQ and10×LOQ concentration levels(50and500ng/L for waters,and 0.05and0.5mg/kg for soils)were used as quality controls (QC)distributed along the batch of samples every three-four injections.The quantiﬁcation of the sample batch was con-sidered satisfactory if the QC recoveries were in the range of 70–120%.The values found in real samples were conﬁrmed by means of the two transitions selected for each compound. In this way,quantiﬁcation was carried out independently with each transition(see MS Optimisation),accepting a deviation of±20%in the concentrations obtained with both transi-tions.3.Results and discussion3.1.MS optimisationFull-scan MS spectra and product-ion MS/MS spectra of the FMOC derivatives of glyphosate,glufosinate and AMPA were recorded in both positive and negative ionisation modes.M.Ib´a ˜n ez et al./J.Chromatogr.A 1081(2005)145–155149Fig.2.The positive ion electrospray full scan mass spectrum (top)and product ion spectra (bottom)of (a)AMPA-FMOC,(b)glyphosate-FMOC and (c)glufosinate-FMOC,obtained from the chromatographic peak of 10mg/L standard solution of each compound,previously derivatizated.Spectra were obtained from the chromatographic peak of 10mg/L standard solution of each compound,previously derivatized.Although these compounds have been traditionally recorded in negative ion mode [9,10],in our work the sensi-tivity in positive ion mode was found to be approximately two times higher.Moreover,the product ions observed in negative ion mode were due to neutral unspeciﬁc losses of FMOC,or FMOC plus water.Thus,any isobaric compound that could have been derivatized with FMOC and also presented a water loss,would show the same product ions in its MS/MS spec-tra,being therefore not very selective.For all these reasons,positive ion mode was selected.The positive-ion electrospray full scan spectrum of AMPA-FMOC at a cone of 30V showed a base peak at m /z 334corresponding to the protonated derivatized molecule [M +H]+.The MS/MS spectra showed three abundant frag-ments at m /z 179,156and 112(Fig.2a).As can be seen in Fig.3a,fragments at m /z 179,m /z 156(M-178)and m /z 112(M-222)would appear in any isobaric amine that could have been derivatized with FMOC.As there were not signiﬁcant differences in the selectivity of these transitions,the criterium applied for their selection was the sensitivity,choosing the two most sensitive ones.The positive-ion electrospray full scan spectrum of glyphosate-FMOC at a cone of 30V showed a peak at m /z 392corresponding to the protonated derivatized molecule [M +H]+.The MS/MS spectra showed abundant fragments at m /z 214,179,170and 88(Fig.2b).The fragments at m /z 179and the fragments at m /z 214(M-178)and m /z 170(M-222)would appear in any isobaric amine that could have been derivatized with FMOC (Fig.3a).Thus,the selected reac-tion monitoring (SRM)transitions chosen were 392→88for quantiﬁcation as the most selective (see Fig.3b)andTable 1Optimised MS/MS parameters for the FMOC derivatives of glyphosate,AMPA,glufosinate and internal standard,selected for the residue analysis of water and soil Compound Cone voltage (V)Precursor ion (m /z )Product ion (m /z )a Collision energy (eV)Glyphosate-FMOC 30392.0Q 88.120q 214.110Glufosinate-FMOC 30404.0Q 136.125q 208.210AMPA-FMOC30334.0Q 179.120q 112.115Isotope-labeled glyphosate-FMOC30395.0Q 91.120q 217.110aQ ,Transition used for quantiﬁcation;q :transition used for conﬁrmation.150M.Ib´a ˜n ez et al./J.Chromatogr.A 1081(2005)145–155Fig.3.(a)Common fragmentation pathway for the three derivatised compounds;(b)speciﬁc fragmentation pathway for glyphosate and glufosinate.392→214for conﬁrmation as it was the most sensitiveamong the less selective.In the case of glufosinate,the positive-ion electrospray full scan spectrum showed a peak at m /z 404corresponding to the protonated molecule of glufosinate-FMOC.The MS/MS spectrum showed four abundant fragments at m /z 208,182(M-222),179and m /z 136(Fig.2c).We choose the most selective transitions:404→208and 404→136(see Fig.3b)despite their lower sensitivity.The selected reaction monitoring (SRM)transitions cho-sen for the residue determination of the three compounds,as well as the optimised MS/MS parameters,are shown in Table 1.3.2.Method optimisationFirstly,several attempts were carried out in order to deter-mine these compounds directly,i.e.without any previousM.Ib´a˜n ez et al./J.Chromatogr.A1081(2005)145–155151derivatization.For this purpose we checked Hydrophilic Interaction Chromatography using an Atlantis TM HILIC 5␮m Silica Column(100mm×2.1mm i.d.,Waters).This column offers superior retention for very polar compounds that are not well retained under reversed-phase conditions. Although the retention obtained with this column at acidic pH was satisfactory,we observed poor sensitivity,making necessary a preconcentration step.We did not try to perform such a preconcentration because this step is difﬁcult for sub-ppb levels of glyphosate and forces one to a higher sample manipulation.Additionally,the conditions to obtain satisfac-tory retention and peak shape were very speciﬁc and changed drastically when changing either pH of the sample or modiﬁer concentration in the mobile phase,decreasing the robustness of the method.For these reasons,a derivatisation procedure was carried out in order to increase the retention of analytes in the most common RPLC cartridges and to work under no so strict conditions.Derivatization procedures with FMOC-Cl have already been reported in the literature[9–15].Due to the low sol-ubility and stability of FMOC-Cl in water,this reagent is usually prepared in acetonitrile.Normally the high con-centration of FMOC required for the derivatization,makes that the derivatized sample presents a high percentage of acetonitrile.Thus,a dilution step with water is necessary to reduce the organic percentage[14],with the subse-quent loss of sensitivity,to retain glyphosate,glufosinate and AMPA in the cartridge due to the high polar charac-ter of these compounds,even derivatized.In this paper,we decreased the volume of the FMOC solution used but increas-ing its concentration and also the volume of water sample derivatized with the aim of minimizing the dilution factor. The effect of adding different FMOC concentrations with different reaction times was studied.The best results for both,water and soil samples,were obtained after perform-ing the reaction overnight with a FMOC concentration of 12,000mg/L.On the other hand,as the borate solution could not buffer properly the alkaline sample extract,a neutralizing step was necessary before the derivatization.Any attempt ofﬁxing the volume of HCl necessary to neutralize the KOH excess failed due to the different nature of the soils.Therefore,this step was done manually adding drops of HCl6M and0.6M until pH around7.Once the derivatization reaction took place overnight, hydrochloric acid was added to stop the reaction,by low-ering the pH.In soil samples,after direct injection of50␮L of the derivatized acidiﬁed extract,recoveries around25%with RSD up to80%were obtained for the three analytes,showing a severe matrix effect in both the MS instrument and/or the derivatization procedure.Among the solutions described to solve this problem(see Section1),the increase of the sample treatment was not considered as the best strategy for monitor-ing programs where rapid methods are preferred.Moreover, the use of matrix-matched standards calibration is not a robust approach when environmental samples are analysed,due to their different origin and composition,making the selection of a blank matrix difﬁcult.Thus,the use of internal stan-dards(IS)was tested,but only isotope-labelled glyphosate was commercially available.As expected,the use of this IS improved accuracy and pre-cision for glyphosate as it compensated the matrix effects,due to the similar chemical behaviour of analyte and IS.However, still ionization inhibition occurred lowering the sensitivity of the overall analytical procedure.In the case of AMPA and glufosinate,although better recoveries were obtained(around 116–127%),the RSDs were still unacceptable(higher than 15%).Therefore,the dilution of soil extracts with LC grade water was assayed as a fast and simple way to minimize matrix interferences.Thus,ﬁve soil samples of different origins were fortiﬁed at the0.5mg/kg and their extracts derivatized and,10-fold and20-fold diluted with water.According to our results(see Table2),10-and20-fold dilution would be adequate for accurate quantiﬁcation,even without internal standard.However,the use of internal standard improved the RSDs,especially for glyphosate.In the case of glufosinate, quantiﬁcation with labelled glyphosate IS did not improve the results.A similar situation has been previously reported in literature,when using analogues IS,demonstrating the dif-ﬁculty of selecting an adequate IS when the labelled analyte is not available[28].Finally,glyphosate and AMPA were quantiﬁed using internal standard meanwhile glufosinate was quantiﬁed with external standard calibration.A10-fold dilu-tion of the extract was chosen as it led to the best LODs.In regard to water samples,after injection of4.3mL of the derivatized sample into the SPE–LC–MS/MS,recoveriesTable2Effect of dilution of soil extracts previously to the derivatization step on the recovery and reproducibility of the method(n=5)a Compound Without dilution10-Fold dilution20-Fold dilution%Recovery b (%RSD)%Recovery c(%RSD)%Recovery b(%RSD)%Recovery c(%RSD)%Recovery b(%RSD)%Recovery c(%RSD)Glyphosate25(79)97(6)83(24)98(3)83(23)91(11) AMPA28(46)127(27)87(9)98(11)89(8)98(10) Glufosinate27(56)116(18)94(8)118(19)92(8)107(9)a Five different soil samples,spiked at0.5mg/kg each.b Quantiﬁcation without internal standard.c Quantiﬁcation with internal standard.。

人体结构学 Human Structure学习通章节答案期末考试题库2023年

人体结构学 Human Structure学习通超星课后章节答案期末考试题库2023年1.Which bones belong to the shoulder girdle?答案:Scapula###Clavicle2.The paired cerebral bones are答案:parietal bone###temporal bone3.Shoulder joint is formed by答案:head of humerus###glenoid cavity of scapula4.Please deseribe the location and openings of the paranasal sinuses答案:答5.Please describe the formation, main structures and communications of the middle Cranial fossa.答案:答6.Please describe the joints of the vertebral bodies.答案:答7.Please describe the joints of the vertebral arches.答案:答8.Please describe the composition, characteristics and movements of theshoulder joint.答案:答9.Which bone belongs to the long bone?答案:Femur10.Which bones belong to the irregular bone?答案:Vertebra###Sphenoid bone11.The blood- testis barrier does NOT include the答案:gap junction between adjacent spermatogomia12.Which of the following description is true about the primordial follicles答案:The primordial follicle consists of a primary oocyte and a layer of flat follicle cells.13.(英文答题,第一空填1个单词,第二空3个单词)The axial bone contains fromup downwards _____ and_____.答案:skull###bonesoftrunk14.About the component of nephron, the correct option is答案:renal corpuscle, proximal tubules, distal tubules and thin segment15.About the features of proximal tubule, the WRONG option is答案:The cytoplasm of epithelial cell is weakly basophilic.16. A patient presents in your office after having a positive result on a homepregnancy test. Her menstrual cycle has always been the classic 28-day cycle discussed in textbooks, with ovulation occurring on the 14th day following the start of menstruation. Her menstrual period began on August 19.2019.You estimate her EDD to be答案:on May 26, 202017.Which of the following is NOT considered one of the fetal membranes答案:buccopharyngeal membrane18.Which bone does not form the anterior cranial fossa?答案:Temporal bone19.Which bone forms both the middle and posterior cranial fossa?答案:Temporal bone20.(英文答题,第一空填1个单词,第二空1个单词,第三空2个单词)Thesternum consists from up downwards of_____ , _______ and ______ .答案:manubrium###body###xiphoidprocess21.Of the following statements about epididymis, the WRONG option is答案:The ductus epididymis is lined with a simple columnar epithelium22.Of the following statements about trachea, the WRONG option is答案:The adventitia is constructed of the elastic cartilage rings.23.The interalveolar septum does NOT contain答案:ciliated cell24.All the following cells are included in the spermatogenic epithelium, EXCEPTthe答案:Leydig cells25.Please describe the general features of the vertebrae.答案:答26.Which bone forms the posteroinferior part of the bony nasal septum?答案:Vomer27.Drawing pictures of Thoracic vertebra from anterior and lateral view.答案:答28.Which bone does not form the thoracic Cage?答案:Sacrum29.About the scapula, which of the statements is not true?答案:It has three borders, three angles and three surfaces.30.About the component of the renal corpuscle, the WRONG option is答案:At the vascular pole, the efferent arteriole enters the glomerulus.31.Of the following statements about podocyte, the correct option is答案:They form the visceral layer of the Bowman's capsule.32.An infant is born with a sacrococcygeal teratoma. Biopsy(组织活检) andhistologic analysis reveal that it contains intestinal epithelia, cardiac muscle, cartilage, and integument tissue. You counsel the mother that the tumor is benign（良性的）and recommend surgical removal. This tumor was caused by which developmental anomaly?答案:Failure of primitive streak regression33.Which of the following structure is NOT included in the secondary follicle?答案:secondary oocyte34.All the following are from mesoderm EXCEPT the答案:spinal cord35.Which of the following descriptions is NOT true about the corpus luteum?答案:The corpus luteum continues to produce estrogen and progesterone during the whole process of pregnancy.36.Of the following statements about the alveolus of lung, the WRONG option is答案:It opens on the wall of terminal bronchioles.37.Which of the following descriptions is NOT true about the secretory phase ofa menstrual cycle?答案:The basal layer of endometrium becomes thicker .38.Of the following statements about Leydig cells, the correct option is答案:It secretes testosterone.39.Of the following statements about macula densa, the WRONG option is答案:It is derived from smooth muscle fibers of afferent arteriole.40.Of the following statements concerning terminal bronchioles, the WRONGoption is答案:They have some mixed gland.41.Of the following options, the blood-air barrier does NOT contain答案:typeⅡalveolar cells42.All the following cells are included in the spermatogenic cells, EXCEPT答案:Sertoli cells43.Which of the following does not belong to the joints of the vertebral arches?答案:Anterior longitudinal ligament44.Human chorionic gonadotropin is produced by the答案:syncytiotrophoblast45.Which of the followings is not enclosed in the articular capsule of shoulderjoint ?答案:Tendon of the short head of the biceps46.The pathway connecting the infratemporal fossa with the orbit is答案:inferior orbital fissure47.When does ovulation occur in a menstrual cycle?答案:the 14th day。

美国安全数据表300000001169页1 - 产品说明书

USA SAFETY DATA SHEET1. CHEMICAL PRODUCT AND COMPANY IDENTIFICATIONProduct name:CHEMLOK AP-133Product Use/Class: AdhesiveLORD Corporation111 LORD DriveCary, NC 27511-7923 USATelephone: 814 868-3180Non-Transportation Emergency: 814 763-2345Chemtrec 24 Hr Transportation Emergency No.800 424-9300 (Outside Continental U.S. 703 527-3887)EFFECTIVE DATE: 05/07/20202. HAZARDS IDENTIFICATIONGHS CLASSIFICATION:Flammable liquids Category 2Acute toxicity Inhalation - Vapours Category 4 - 10.9% of the mixture consists of ingredient(s) of unknown toxicity.Acute toxicity Inhalation - Dust and Mist Category 4 - 10.9% of the mixture consists of ingredient(s) of unknown toxicity.Serious eye damage/eye irritation Category 2ACarcinogenicity Category 2Reproductive toxicity Category 2Specific target organ systemic toxicity (single exposure) Category 1 Central nervous system, Eyes, SystemictoxicitySpecific target organ systemic toxicity (single exposure) Category 3Specific target organ systemic toxicity (repeated exposure) Category 1 Liver, Central nervous system, EyesAspiration hazard Category 1Hazardous to the aquatic environment - acute hazard Category 2Hazardous to the aquatic environment - chronic hazard Category 2GHS LABEL ELEMENTS:Symbol(s)Signal WordD ANGERHazard StatementsHighly flammable liquid and vapor.Harmful if inhaled.Causes serious eye irritation.Suspected of causing cancer.Suspected of damaging fertility or the unborn child.May cause harm to breast-fed children.Causes damage to organs.(Central nervous system, Eyes, Systemic toxicity)May cause respiratory irritation.May cause drowsiness or dizziness.Causes damage to organs through prolonged or repeated exposure.(Liver, Central nervous system, Eyes)May be fatal if swallowed and enters airways.Toxic to aquatic life.Toxic to aquatic life with long lasting effects.300000001169Precautionary StatementsPreventionKeep away from heat/sparks/open flames/hot surfaces. - No smoking.Ground/Bond container and receiving equipment.Use explosion-proof electrical/ventilating/lighting equipment.Use only non-sparking tools.Take precautionary measures against static discharge.Obtain special instructions before use.Do not handle until all safety precautions have been read and understood.Wear protective gloves/protective clothing/eye protection/face protection.Use personal protective equipment as required.Do not breathe dust/fume/gas/mist/vapors/spray.Wash thoroughly after handling.Do not eat, drink or smoke when using this product.Use only outdoors or in a well-ventilated area.Avoid release to the environment.ResponseIn case of fire: refer to section 5 of SDS for extinguishing media.Call a POISON CENTER or doctor/physician if you feel unwell.IF exposed: Call a POISON CENTER or doctor/physician.Specific treatment (see supplemental first aid instructions on this label).IF INHALED: Remove to fresh air and keep at rest in a position comfortable for breathing.IF ON SKIN (or hair): Remove/Take off immediately all contaminated clothing. Rinse skin with water/shower.IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.Continue rinsing.IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician.Do NOT induce vomiting.Collect spillage.StorageStore in a well-ventilated place. Keep cool.Store in a well-ventilated place. Keep container tightly closed.Store locked up.Disposal:Dispose of contents/container in accordance with waste/disposal laws and regulations of your country or particular locality.Other Hazards:This product contains component(s) which have the following warnings; however based on the GHS classification criteria of your country or locale, the product mixture may be outside the respective category(s).Acute: Vapor harmful; may affect the brain or nervous system causing dizziness, headache or nausea. May cause central nervous system depression characterized by the following progressive steps: headache, dizziness, staggering gait, confusion, unconsciousness or coma. May be absorbed through the skin in harmful amounts. May cause skin irritation. Possible irritation of the respiratory system can occur causing a variety of symptoms such as dryness of the throat, tightness of the chest, and shortness of breath. Contains methanol; may be harmful or fatal if swallowed;ingestion of methanol may cause blindness or permanent eye damage. Cannot be made non-poisonous. The silane material in this product can hydrolyze to form ethanol. Ethanol can cause moderate eye irritation, moderate skin irritation; and can be absorbed through the skin causing headache, nausea, and general discomfort. Ethanol is toxic by inhalation. If swallowed, the silane material in this product can hydrolyze in the stomach to form ethanol. Refer to the ethanol warnings on this (M)SDS. Ingestion is not an expected route of entry in industrial or commercial uses.Chronic: May cause kidney damage. May affect the gastrointestinal system. Prolonged or repeated contact may result in dermatitis. IARC has designated Methyl isobutyl ketone to be in Group 2B - possibly carcinogenic tohumans. ACGIH considers Ethyl alcohol to be an A3 carcinogen (confirmed animal carcinogen with unknownrelevance in humans).withheld.FIRST AID - EYE CONTACT: Flush eyes immediately with large amount of water for at least 15 minutes holding eyelids open while flushing. Get prompt medical attention.FIRST AID - SKIN CONTACT: Flush contaminated skin with large amounts of water while removing contaminated clothing. Wash affected skin areas with soap and water. Get medical attention if symptoms occur.FIRST AID - INHALATION: Move person to fresh air. Restore and support continued breathing. If breathing is difficult, give oxygen. Get immediate medical attention.FIRST AID - INGESTION: If swallowed, do not induce vomiting. Call a physician or poison control center immediately for further instructions. Never give anything by mouth if victim is rapidly losing consciousness, unconscious or convulsing.SUITABLE EXTINGUISHING MEDIA: Carbon Dioxide, Dry Chemical, Foam, Water FogUNSUITABLE EXTINGUISHING MEDIA: Not determined for this product.SPECIFIC HAZARDS POSSIBLY ARISING FROM THE CHEMICAL: Flammable liquid and vapor. Keep containers tightly closed. Isolate from heat, electrical equipment, sparks, open flame, and other sources of ignition. Closed containers may rupture when exposed to extreme heat. Use water spray to keep fire exposed containers cool. 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Refer to OSHA 29CFR Part 1910.106 "Flammable and Combustible Liquids" for specific storage requirements.INCOMPATIBILITY: Strong acids, bases, and strong oxidizers.Engineering controls: Sufficient ventilation in pattern and volume should be provided in order to maintain air contaminant levels below recommended exposure limits. Caution: Solvent vapors are heavier than air and collect in lower levels of the work area. Sufficient ventilation (using explosion-proof equipment) should be provided to prevent flammable vapor/air mixtures from accumulating.PERSONAL PROTECTION MEASURES/EQUIPMENT:RESPIRATORY PROTECTION: Use a NIOSH approved chemical/mechanical filter respirator designed toremove a combination of particulates and organic vapor if occupational limits are exceeded. For emergencysituations, confined space use, or other conditions where exposure limits may be greatly exceeded, use an approved air-supplied respirator. For respirator use observe OSHA regulations (29CFR 1910.134) or use in accordance with applicable laws and regulations of your country or particular locality.SKIN PROTECTION: Use neoprene, nitrile, or rubber gloves to prevent skin contact.EYE PROTECTION: Use safety eyewear including safety glasses with side shields and chemical goggles where splashing may occur.OTHER PROTECTIVE EQUIPMENT: Use disposable or impervious clothing if work clothing contamination is likely. Remove and wash contaminated clothing before reuse.HYGIENIC PRACTICES: Wash hands before eating, smoking, or using toilet facility. Do not smoke in anychemical handling or storage area. Food or beverages should not be consumed anywhere this product is handled or stored. Wash thoroughly after handling.Typical values, not to be used for specification purposes.ODOR: Solvent VAPOR PRESSURE: N.D.APPEARANCE: Clear VAPOR DENSITY: Heavier than Air PHYSICAL STATE: Liquid LOWER EXPLOSIVE LIMIT: 1.2 %(V)UPPER EXPLOSIVE LIMIT: 36.5 %(V)FLASH POINT: 57 °F, 14 °C SetaflashClosed CupBOILING RANGE: 65 - 100 °C EVAPORATION RATE: Slower than n-butyl-acetate AUTOIGNITION TEMPERATURE:N.D.DENSITY: 0.82 g/cm3 - 6.82 lb/gal DECOMPOSITION TEMPERATURE:N.D. VISCOSITY, DYNAMIC: ≥0.82 mPa.s @ 25 °C ODOR THRESHOLD: N.D.VISCOSITY, KINEMATIC: ≥1 mm2/s @ 25 °CSOLUBILITY IN H2O: Insoluble VOLATILE BY WEIGHT: 90.72 %pH: N.A.VOLATILE BY VOLUME: 92.96 %FREEZE POINT: N.D. VOC CALCULATED: 6.11 lb/gal, 731 g/lCOEFFICIENT OF WATER/OILN.D.DISTRIBUTION:LEGEND: N.A. - Not Applicable, N.E. - Not Established, N.D. - Not DeterminedHAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur under normal conditions.STABILITY: Product is stable under normal storage conditions.CONDITIONS TO AVOID: High temperatures. Sources of ignition.INCOMPATIBILITY: Strong acids, bases, and strong oxidizers.HAZARDOUS DECOMPOSITION PRODUCTS: Carbon monoxide, carbon dioxide, Oxides of nitrogen, Oxides of siliconEXPOSURE PATH: Refer to section 2 of this SDS.SYMPTOMS:Refer to section 2 of this SDS.TOXICITY MEASURES:Germ cell mutagenicity: No classification proposedCarcinogenicity: Category 2 - Suspected of causing cancer.Components contributing to classification: Methyl isobutyl ketone.Reproductive toxicity: Category 2 - Suspected of damaging fertility or the unborn child. May cause harm to breast-fed children.Components contributing to classification: Methanol. Toluene.PERSISTENCE AND DEGRADABILITY:Not determined for this product.BIOACCUMULATIVE: Not determined for this product.MOBILITY IN SOIL: Not determined for this product.OTHER ADVERSE EFFECTS: Not determined for this product.DISPOSAL METHOD: Disposal should be done in accordance with Federal (40CFR Part 261), state and local environmental control regulations. If waste is determined to be hazardous, use licensed hazardous waste transporter and disposal facility.US DOT RoadProper Shipping Name: AdhesivesHazard Class: 3SECONDARY HAZARD: NoneUN/NA Number: 1133Packing Group: IIEmergency Response Guide Number: 128IATA CargoPROPER SHIPPING NAME: AdhesivesHazard Class: 3HAZARD CLASS: NoneUN NUMBER: 1133PACKING GROUP: IIEMS: 3LIMDGPROPER SHIPPING NAME: AdhesivesHazard Class: 3HAZARD CLASS: NoneUN NUMBER: 1133PACKING GROUP: IIEMS: F-EThe listed transportation classification applies to non-bulk shipments. It does not address regulatory variations due to changes in package size, mode of shipment or other regulatory descriptors. For the most accurate shipping information, refer to your transportation/compliance department.U.S. FEDERAL REGULATIONS: AS FOLLOWS:SARA SECTION 313This product contains the following substances subject to the reporting requirements of Section 313 of Title III of the Superfund Amendment and Reauthorization Act of 1986 and 40 CFR part 372.:Chemical Name CAS Number Weight % Less ThanMethanol67-56-115.0%Toluene108-88-3 1.0%Methyl isobutyl ketone108-10-10.9%TOXIC SUBSTANCES CONTROL ACT:INVENTORY STATUSThe chemical substances in this product are on the TSCA Section 8 Inventory.EXPORT NOTIFICATIONThis product contains the following chemical substances subject to the reporting requirements of TSCA 12(B) if exported from the United States:NoneUnder HazCom 2012 it is optional to continue using the HMIS rating system. It is important to ensure employees have been trained to recognize the different numeric ratings associated with the HazCom 2012 and HMIS schemes.HMIS RATINGS - HEALTH: 2* FLAMMABILITY: 3 PHYSICAL HAZARD: 0* - Indicates a chronic hazard; see Section 2Revision: Company LogoEffective Date: 05/07/2020The information contained herein is, to the best of our knowledge and belief, accurate. However, since the conditions of handling and use are beyond our control, we make no guarantee of results, and assume no liability for damages incurred by use of this material. It is the responsibility of the user to comply with all applicable federal, state and local laws and regulations.。

Atmosphere-Corrected Phase-Referencing

a rXiv:as tr o-ph/39575v122Se p23Future Directions in High Resolution Astronomy:The 10th Anniversary of the VLBA ASP Conference Series,Vol.***,2003J.D.Romney &M.J.Reid (eds.)Atmosphere-Corrected Phase-Referencing Andreas Brunthaler Max-Planck-Institut f¨u r Radioastronomie,Auf dem H¨u gel 69,53121Bonn,Germany Mark J.Reid Harvard-Smithsonian Center for Astrophysics,60Garden Street,MS 42,Cambridge,MA 02138,USA Heino Falcke ASTRON,P.O.Box 2,7990AA Dwingeloo,The Netherlands Max-Planck-Institut f¨u r Radioastronomie,Auf dem H¨u gel 69,53121Bonn,Germany Abstract.One major problem of phase-referencing VLBI observations are phase errors due to the unknown tropospheric zenith delay at each antenna.These errors degrade the quality of the phase-referenced image and limit the achievable astrometric accuracy.We will present and com-pare two independent methods to estimate the zenith delay oﬀset at each antenna.The zenith delay oﬀsets can then be used to correct the data.These corrections improve the quality of the phase-referenced image and an astrometric accuracy of 10µas can be achieved.With this accuracy,measurements of proper motions in the Local Group become feasible.1.IntroductionPhase-referencing has become a standard technique in Very Long Baseline Inter-ferometry (VLBI)to measure accurate relative positions of radio sources.The concept of phase-referencing is based on the assumption that the phase errors of two sources with a small angular separation on the sky are similar.One observes a target source between two scans on a calibrator.Then one can interpolate the calibration between the two scans and apply this interpolated phase corrections to the phase of the target source.The phase-referenced diﬀerence phase contains information only about the target source structure and its position relative to the calibrator and contains noise from interpolation errors.One major problem of phase-referencing VLBI observations is the unknown tropospheric zenith delay at each antenna.The VLBA correlator model uses a seasonally averaged and latitude-dependent atmospheric model (Niell 1996),which can misestimate the zenith delay by a few centimeters.The excess path length caused by the dry troposphere to a radio signal from a source with zenith12Author&Co-authorangle Z is given bycml≈0.228A C∆α[µas]∆δ[µas]∆α[µas]∆δ[µas] -411213101 2002/01/17-8125Mean-5±4114±11APS Conf.Ser.Style3 using system temperature measurements and standard gain curves.A fringeﬁt was performed on J0027+5958and the solutions were applied to IC10.Then we modeled the phase data of IC10as described above.The position oﬀset for the two observations are given in Table1(A).The positions obtained from the two observations are consistent and have an rms of≈10µas.The zenith delay oﬀsets are typically of the order of a few centimeters.The values of theﬁrst observation on2002January12are given in the second column of Table2.2.2.Phase correctionFigure1.Phased-referenced image of a H2O maser spot in IC10without correction a),with atmosphere correction from theﬁt to thephases b),with corrections form the geodetic-like observations c)andafter phase self-calibration d).The contour levels start at25mJy and√increase by a factor of4Author&Co-authorOPCODE=’ATMO’.Fig.1shows the phase-referenced images of a strong H2O maser component in IC10with(b)and without(a)the corrections.The noise in the image is reduced while the peakﬂux increased in the corrected image. The peak-to-noise ratio in the corrected image is101compared to77in the uncorrected image.The positions of the maser feature in the corrected maps for the two observations are given in Table1(B)and agree within10µas with the positions derived from theﬁt to the phases.The positions obtained from theﬁt to the phases and from the corrected maps are not independent and the agreement is not surprising.However,it is a consistency check for the method.2.3.Geodetic-like observationsA second,independent method to estimate the zenith delay oﬀset is to use geodetic-like observations.Here,a number of bright quasars with positions known to better than1mas were observed for45minutes before and after the phase-referencing observation.The quasars were observed at diﬀerent eleva-tions with a frequency setup of8IFs spread over450MHz.A fringe-ﬁt on the quasars yielded then a multi-band delay and a rate for each baseline and source. These multi-band delays and rates were thenﬁt with a model that consisted of a zenith delay oﬀset and drift at all antennas as well as a clock oﬀset and drift at all antennas except the reference antenna.The parameters of the best model-ﬁt are given in Table2.The diﬀerences between the observed multi-band delay and the model are less than0.1ns.The zenith delay oﬀsets obtained from theﬁt to the phases and from the geodetic-like observations are in general comparable although they can be dif-ferent for some antennas.The diﬀerences between the two methods are not surprising,since the model in the geodetic-like observations includes not only a zenith delay oﬀset but also a drift,a clock oﬀset and a clock drift.Table2.Fit parameters for the observation on2002January12:Zenith delay oﬀset(τ0,p)from theﬁt to the phase data,zenith delayoﬀset(τ0)and rate(˙τ0)and clock oﬀset(∆t)and rate(˙∆t)from thegeodetic-like observations.The errors are the formal errors of the least-squaresﬁt.Antennaτ0[cm]˙τ0[cm h−1]∆t[ns]˙∆t[ns d−1]2.5±0.64.4±0.37.0±0.21.0±0.3-0.4±0.4-3.3±0.19.4±0.44.7±0.4-0.2±0.4APS Conf.Ser.Style5 3.ConclusionsWe have presented a technique to correct phase-referencing observations for inaccurate zenith delays in the VLBA correlator model.The quality of the atmosphere-corrected phase-referenced images can be improved by this tech-nique.The peak-to-noise ratio in the corrected images increases by≈30%.By using this technique,an astrometric accuracy of10µas can be achieved.Two independent methods were presented to estimate the zenith delay oﬀ-sets at each antenna.The images obtained from the correction with the zenith delay oﬀsets from theﬁt to the phases(Fig.1(b))and from the geodetic-like observations(Fig.1(c))are very similar and the positions measured from these images are consistent.This is a proof of concept for our technique.The astrometric accuracy of this technique enables us to detect signiﬁcant proper motions of galaxies in the Local Group out to a distance of800kpc within a few years.One needs at least three epochs of observations to make reliable statements about measured proper motions.Further observations are underway and the results will be presented elsewhere.ReferencesBeasley,A.,&Conway,J.E.1995,VLBI Phase-Referencing,in ASP Conf.Ser.82:Very Long Baseline Interferometry and the VLBA,eds.J.A.Zensus, P.J.Diamond&P.J.Napier(San Francisco:ASP),327 Brunthaler,A.,Reid,M.J.,Falcke,H.,Greenhill,L.J.,&Henkel,C.2002 in Prodeedings of the6th European VLBI Network Symposium,E.Ros, R.W.Porcas,A.P.Lobanov,and J.A.Zensus(eds.),MPIfR Bonn, Germany,p.189.Niell,A.1996,J.Geophys.Res.,101,3227Reid,M.J.,Readhead,A.C.S.,Vermeulen,R.C.,&Treuhaft,R.N.1999, ApJ,524,816Thompson,A.R.,Moran,J.M.,&Swenson,G.W.2001,Interferometry and Synthesis in Radio Astronomy(Second ed.).Wiley&Sons,New York。

鲜核桃致病菌分离鉴定及二氧化氯对其抑制效应研究

孟文彦，万杨卓群，张东莉，等. 鲜核桃致病菌分离鉴定及二氧化氯对其抑制效应研究[J]. 食品工业科技，2023，44（20）：370−379.doi: 10.13386/j.issn1002-0306.2023010142MENG Wenyan, WAN Yangzhuoqun, ZHANG Dongli, et al. Isolation and Identification of Pathogen from Fresh Walnut and the Inhibition Effect of Chlorine Dioxide[J]. Science and Technology of Food Industry, 2023, 44(20): 370−379. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023010142· 贮运保鲜 ·鲜核桃致病菌分离鉴定及二氧化氯对其抑制效应研究孟文彦，万杨卓群，张东莉，刘方玥，马惠玲*（西北农林科技大学生命科学学院，陕西杨凌 712100）摘　要：鲜核桃贮存期易发生霉变，货架寿命短，极大地限制了其流通量和核桃产业的经济效益。

本文以‘清香’核桃为试材，通过致霉病原菌分离和回接验证、形态与分子鉴定，离体和活体抑菌试验，及处理后鲜核桃品质评价等，研究了二氧化氯（Chlorine dioxide ，ClO 2）用于该霉变控制的效应。

结果表明，鲜核桃霉变区分离纯化出的病原菌主要有两种，为青霉（Penicillium sp ）和塔宾曲霉（Aspergillus tabinus ）；鲜核桃发粘区分离出的病原菌主要也是两种，为桉树假单胞菌（Pseudomonas eucalyptus ）和黄色微球菌（Micrococcus flavus ）。

在离体条件下，0~40 mg/L ClO 2熏蒸对这4种病原菌抑制作用均具有明显量效关系，且作用的IC 50值在7~20 mg/L 之间，16、30、40 mg/L ClO 2分别100%抑制了青霉、塔宾曲霉和两种细菌在离体条件下的繁殖和生长。

Theoretical X-Ray Absorption Debye-Waller Factors

a r X i v :c o n d -m a t /0702397v 1 [c o n d -m a t .m t r l -s c i ] 16 F eb 2007Theoretical X-Ray Absorption Debye-Waller FactorsFernando D.Vila and J.J.RehrDepartment of Physics,University of Washington,Seattle,WA 98195H.H.Rossner and H.J.KrappeHahn-Meitner-Institut Berlin,Glienicker Strasse 100,D-14109Berlin,Germany(Dated:February 6,2008)An approach is presented for theoretical calculations of the Debye-Waller factors in x-ray ab-sorption spectra.These factors are represented in terms of the cumulant expansion up to third order.They account respectively for the net thermal expansion σ(1)(T ),the mean-square relative displacements σ2(T ),and the asymmetry of the pair distribution function σ(3)(T ).Similarly,we ob-tain Debye-Waller factors for x-ray and neutron scattering in terms of the mean-square vibrational amplitudes u 2(T ).Our method is based on density functional theory calculations of the dynamical matrix,together with an eﬃcient Lanczos algorithm for projected phonon spectra within the quasi-harmonic approximation.Due to anharmonicity in the interatomic forces,the results are highly sensitive to variations in the equilibrium lattice constants,and hence to the choice of exchange-correlation potential.In order to treat this sensitivity,we introduce two prescriptions:one based on the local density approximation,and a second based on a modiﬁed generalized gradient approxima-tion.Illustrative results for the leading cumulants are presented for several materials and compared with experiment and with correlated Einstein and Debye models.We also obtain Born-von Karman parameters and corrections due to perpendicular vibrations.I.INTRODUCTIONThermal vibrations and disorder in x-ray absorption spectra (XAS)give rise to Debye-Waller (DW)factors varying as exp[−W (T )],where W (T )≈2k 2σ2(T )and σ2(T )is the mean square relative displacement (MSRD)of a given multiple-scattering (MS)path.1These Debye-Waller factors damp the spectra with respect to increas-ing temperature T and wave number k (or energy),and account for the observation that the x-ray absorption ﬁne structure (XAFS),“melts”with increasing temperature.2The XAFS DW factor is analogous to that for x-ray and neutron diﬀraction or the M¨o ßbauer eﬀect,whereW (T )=(1/2)k 2u 2(T ).The diﬀerence is that the XAFS DW factor refers to correlated averages over relative dis-placements,e.g.,σ2= [(u R −u 0)·ˆR]2 for the MSRD,while that for x-ray and neutron diﬀraction refers to themean-square displacements u 2(T )= (u ·ˆR)2 of a given atom.Due to their exponential damping,accurate DW factors are crucial to a quantitative treatment of x-ray absorption spectra.Consequently,the lack of precise Debye-Waller factors has been one of the biggest limi-tations to accurate structure determinations (e.g.,coor-dination number and interatomic distances)from XAFS experiment.Due to the diﬃculty of calculating the vibrational dis-tribution function from ﬁrst principles,XAFS Debye-Waller factors have,heretofore,been ﬁtted to experimen-tal data or estimated semi-empirically,e.g.,from cor-related Einstein and Debye models.3,4However,these ad hoc approaches are unsatisfactory for several reasons.First,there are often many more DW factors in the MS path expansion than can be ﬁt reliably.Second,semi-empirical models typically ignore anisotropic contribu-tions and hence do not capture the detailed structure ofthe phonon spectra.To address these problems,we introduce ﬁrst principles procedures for calculations of the Debye-Waller factors in XAS and related spectra.Our approach is based primar-ily on density functional theory (DFT)calculations of the dynamical matrix,together with an eﬃcient Lanc-zos algorithm for the projected phonon spectra.5,6DFT calculations of crystallographic Debye-Waller factors and other thermodynamic quantities have been carried out previously using modern electronic structure codes,7,8,9and our work here builds on these developments,with particular emphasis on applications to XAS.Due to intrinsic anharmonicity in the interatomic forces,the behavior of the DW factors is extremely sen-sitive to the equilibrium lattice constant a .For exam-ple,we ﬁnd that σ2varies approximately as a 6γ,where γ=−d ln ¯ω/d ln V is the mean Gr¨u neisen parameter which is typically about 2for fcc metals,and ¯ωrefersto the mean phonon frequency.Consequently σ2is also very sensitive to the choice of the exchange-correlation potential in the DFT,since a 1%error in lattice constant yields an error of 6γ≈10%in σ2.As a result,relatively small errors in the lattice constant predicted by the local density approximation (LDA)which tends to overbind,or the generalized gradient approximation (GGA)which tends to underbind,become greatly magniﬁed 10in DW calculations.In order to treat this sensitivity we have developed two ad hoc prescriptions for ab initio calculations of Debye-Waller factors based on DFT calculations with I)the con-ventional LDA and II)a modiﬁed-GGA (termed hGGA)described below.For comparison we also present se-lected results with a conventional GGA,with the corre-lated Einstein and Debye models,and with an empirical model based on the Born-von Karman parameters ob-2tained from ﬁts to phonon spectra.Detailed results are presented for a number of fcc and diamond structures.II.FORMALISM A.CumulantsIn this section we outline the formalism used in our approach.Physically,the DW factors in XAS arise from a thermal and conﬁgurational average of the XAS spec-tra µ(E ) over the pair (or MS path length)distribution function,where µ(E )is the x-ray absorption coeﬃcient in the absence of disorder.The eﬀects of disorder and vi-brations are additive,but since the factors due to conﬁg-urational disorder are dependent on sample history and preparation,in this paper we focus only on the thermal contribution.The eﬀect of the DW factors on the XAFS χ(k )is dominated by the average over the oscillatory be-havior of each path in the multiple-scattering (MS)path expansion χR (k )∝sin(2kR +Φ).If the disorder is not too large the average is conveniently expressed in terms of the cumulant expansion,1,11ei 2kr≡e 2ikR 0e −W (T ),(1)W (T )=−∞ n =1(2ik )n2µR∞12ρR (ω)dω,(5)where µR is the reduced mass associated with the path,β=1/k B T ,and ρR (ω)is the vibrational density of states projected on R .In the following,the path index subscript R is suppressed unless needed for clarity.The ﬁrst cumulant σ(1)is generally path-dependent and reﬂects the anharmonic behavior of a system.For monoatomic systems,this quantity is directly propor-tional to the net thermal expansion ∆a =a (T )−a 0,which can be obtained by minimizing the vibrational free energy F (a,T ).Within the quasi-harmonic approxima-tion,F (a,T )is given by a sum over the internal energy E (a )and the vibrational free energy per atom F (a,T )=E (a )+3k B T∞dωln 2sinhβ¯h ω2R 0σ2⊥.(7)3from DFT calculations using the LDA with our prescriptionI;the hGGA(see text)with our prescription II;and fromexperiment12.We have shown(see Appendix)thatσ2⊥andσ2are closelyrelated,and hence thatσ2⊥can be estimated in terms ofσ2.Second,the position dependent XAFS amplitude fac-tors exp(−2R/λ)/R2give rise to an eﬀective radial dis-tribution function g(R)→g(R)exp(−2R/λ)/R2whichshifts thermal expansion observed in XAFS by an addi-tional correction∆σ(1)=−2λ σ2.(8)This second correction is often included in XAFS analysis routines and has been taken into account in the experi-mental results presented here.14Note that the corrections in Eq.(7)and(8)are both of the same order of magni-tude and partially cancel.nczos AlgorithmThe VDOSρR(ω)has often been approximated by means of Einstein and Debye models based on empiri-cal data.Although these models are quite useful,espe-cially for isotropic systems such as metals without highly directional bonds,their limitations are well known.13,15 To overcome some of these limitations Poiarkova and Rehr5,13proposed a method in which the VDOS is cal-culated from the imaginary part of the lattice dynamical Green’s functionρR(ω)=−2ωω2−D+iǫ 0 .(9)Here|0 is a Lanczos seed vector representing a nor-malized,mass-weighted initial displacement of the atoms along the multiple-scattering path R,and D is the dy-namical matrix of force constantsD jlα,j′l′β=(M j M j′)−1/2∂2E(a)for the nearest neighbor single scattering path in Cu.The experimental diﬀerence values 14were shifted to match the LDA (I)results at 0K.C.Correlated Einstein ModelAlthough the cumulants other than the second are of-ten negligible for small anharmonicity,their calculation using the apparatus of anharmonic lattice dynamics is computationally demanding.On the other hand,it has been shown that these cumulants can be approximated to reasonable accuracy using a correlated anharmonic Ein-stein model for each MS path,4,18and this is the method adopted here.In this approach an Einstein model is constructed for each MS path keeping only cubic anhar-monicity,yielding the eﬀective one-dimensional potentialV (x )=1kσ2.(14)Note that this relation diﬀers from that in Refs.[4,18]in that it contains an extra multiplicative factor η=1/ ω−2 ¯ω2,as discussed in the Appendix.for the nearest neighbor single scattering path in Ge.The experimental diﬀerence values 20were shifted to match the LDA (I)results at 0K.III.DFT CALCULATIONS A.Computational StrategyAs noted above,one of the main aims of this paper is tocalculate the force constants within the quasi-harmonic approximation using DFT and an appropriate choice of exchange-correlation functional.Due to the extreme sen-sitivity of the phonon spectra to the interatomic dis-tances,as discussed above,the most important parame-ters entering the calculation of the dynamical matrix are the lattice constant and the geometry of the system.A typical example of the eﬀect of expansion is illustrated in Fig.1,which shows the variation of the ﬁrst moment of the VDOS (i.e.the average frequency ¯ν)projected along the nearest-neighbor single-scattering path of Cu.For comparison Fig.1also shows ¯νobtained with a model based on the Born-von Karman parameters at 298K.As expected,when the system expands the vibrational fre-quencies are red-shifted due to the weakening of the inter-atomic interactions.From the common slopes in Fig.1.,we see that all of the functionals have similar Gr¨u neisen parameters γ≈2.2at the experimental lattice constant 3.61˚A ,in accord with the experimental value 192.0±0.2.Note that although at a given lattice constant the GGA functional always produces a stiﬀer model than LDA,i.e.,with higher mean frequencies,the results at the equilib-rium GGA lattice constant tend to be softer than at the equilibrium LDA lattice constant.Moreover,when com-pared with the experimental value,the LDA and GGA functionals respectively underestimate and overestimate the mean frequency by about 5%.This translates into a 20-25%error in the DW factors calculated with these methods.This margin of error is too large to make the DW factors of signiﬁcant value in quantitative EXAFS analysis.Based on the above considerations,we there-5nearest neighbor single scattering path in Cu,with and with-out the perpendicular correction from Eq.7,and obtained either from the minimization of the free energy(FE)or from the correlated Einstein model(EM).Both experimental dif-ference values with14and without21the perpendicular motion correction were shifted to match the LDA(I)results at0K. fore propose two alternative prescriptions to stabilize our DW factor calculations:I.Ourﬁrst prescription is based on DFT calculations using the LDA exchange-correlation functional at the cal-culated equilibrium lattice constants a(T)at a given tem-perature.Note however that the errors in the LDA esti-mates of the lattice constant are often larger than those obtained inﬁts to XAFS experiment.II.Our second prescription is based on DFT calcula-tions using a modiﬁed GGA exchange-correlation func-tional termed hGGA(with half-LDA and half-GGA)at the experimentally determined lattice constant a(T)at a given temperature.As described below,this functional is constructed on the assumption that the“true”func-tional lies somewhere between pure LDA and GGA.This second prescription may be useful,for example,during ﬁts of XAFS data to experiment,during which the inter-atomic distance is reﬁned.Clearly,the use of experimental structural parameters limits prescription II,since it requires the knowledge of the crystal structure at each of the temperatures of in-terest.Such information is only available a priori for a handful of systems,although it could be introduced as part of theﬁt procedure.B.Exchange-Correlation FunctionalsIn the course of this work,we investigated a num-ber of exchange-correlation functionals.Generally,the exchange-correlation functional is attractive and hence strongly aﬀects the overall strength and curvature of the interatomic potential.On the other hand it is well known that LDA functionals tend to overbind,yielding latticeI:Born-von Karman parameters D m ij(N/m)fromscattering compared with ab initio calculations from work.m ij LDA GGA hGGA Expt110xx10.466.0312.9810.71(17)[22]zz−3.28−1.65−4.111.75(20) K xy12.487.2715.9412.32(32)200xx1.060.701.340.06(29)yy−0.03−0.08−0.05−0.23(19)110xx29.1723.6729.9425.681(168)[23] Pt zz−7.60−6.67−8.77−7.703(251)90K xy31.4426.0233.5030.830(303) 200xx4.984.675.185.604(329)yy−1.56−1.33−1.28−1.337(194) constants smaller than experiment typically by about 1%.In contrast,GGA functionals tend to underbind10 by about the same amount.These errors are conﬁrmed by our calculations,which show that for Cu the LDA yields a lattice constant of3.57˚A at0K and3.58˚A at 298K,while the GGA yields3.69˚A and3.70˚A respec-tively,experiment being3.61˚A.Moreover,the eﬀect of the functionals on the phonon structure is even larger. For example,Narasimhan and de Gironcoli10show that the thermal expansion is about10%high with LDA and 10%low with GGA.Although signiﬁcant eﬀort has been put into so-called meta-GGA functionals24,25that address these issues, they have not yet been widely implemented.Therefore, to be consistent with various results10and to preserve the advantages of the LDA and GGA functionals we have devised a modiﬁed functional termed hGGA which is a mixture of50%LDA and50%GGA,i.e.,with a50%re-duction in both the additional exchange and correlation terms in the GGA.The motivation for the50-50mixture stems from the observation that the experimental values for many quantities are roughly bracketed by the LDA and GGA predictions.To simplify the development,we chose the closely related Perdew-Wang9226(LDA)and Perdew-Burke-Ernzerhof27(GGA)functionals.For this case the equal parts mixing can be achieved with two simple changes:First,theκparameter in the exchange energy term in PBE is reduced by half.This change preserves all the conditions on which PBE was founded, except the Lieb-Oxford bound.Second,the gradient con-tribution H to the correlation energy is also reduced by half.Similar modiﬁed functionals for solids have been proposed by Perdew et al,28suggesting that modiﬁca-6 TABLE II:Debye-Waller factorsσ2n(T)(in10−3˚A2)for thesingle scattering path to the n th shell of some fcc lattice met-als.The experimental diﬀerence values were shifted to matchthe LDA(I)at80K and the experimental error(in parenthe-ses)indicates the error in the least signiﬁcant digits.n CD LDA(I)GGA hGGA(II)ExptCu19.048.2210.458.567.99(16)[29] 300K211.1611.4414.3112.0311.2(5) 311.5010.7613.5311.289.7(6)411.6610.7013.4911.2011.4(10)Pt15.414.986.084.904.83(05)[29] 300K26.697.238.717.346.8(5)36.916.928.406.896.7(6)47.017.178.717.167.0(6)Ge15.113.423.983.763.5(1)[29] 295K27.4310.3811.919.709.6(8)37.6413.0915.0311.84GaAs15.153.964.593.864.2(1)[29] 295K27.2010.8011.6910.199.6(11) (As Edge)37.6814.8316.6614.0020shells,the full force constant matrix for these clusters must be built by replicating the3×3D jlα,j′l′βblocks obtained for each jl,j′l′pair.ttice and Force ConstantsThe temperature-dependent latticeconstant a(T)is obtained by minimizing F(a,T)in Eq.(6)with respect to a at a given temperature T.Within the electronic structure code ABINIT,the total VDOSρa(ω)is calcu-lated with histogram sampling in q-space.However,we ﬁnd it more convenient here to use a Lanczos algorithm in real space,similar to the approach used for the MSRD. This can be done by modifying the initial normalized dis-placement state|0 in Eq.(9)to that for a single atomic displacement,rather than the displacement along a given MS path.If more than one atom is present in the unit cell the contributions from each atom must be calculated and added.Similarly for anisotropic systems one must trace over three orthogonal initial displacements.Fig.2 shows a typical VDOS generated using the Lanczos al-gorithm.Weﬁnd the free energies calculated with this approach deviate from the q-space histogram method by less then2meV,i.e.,to within1%.To minimize F(a,T)eﬃciently we proceed as follows: First,the lattice constant is optimized with respect to the internal energy E(a)and a potential energy surface (PES)for the cell expansion is built around the mini-mum.Second,the ab initio force constants are computed at each point of the PES to obtain the vibrational com-ponent of F(a,T).Since this is the most time-consuming part of the calculation,we have taken advantage of the approximately linear behavior for small variations as il-lustrated in Fig. 1.Then,each element of the force constants matrix is interpolated according toD jlα,j′l′β=A jlα,j′l′β+B jlα,j′l′β∆a(16) from just two ab initio force constant calculations with slightly diﬀerent lattice parameters.This interpolation7ponentσ2⊥of the Debye-Waller factor for the nearest neigh-bor single scattering path in Cu.For comparison we also plot 2.5σ2to show the correlationσ2⊥≈γ⊥σ2,together with the values extracted from experiment.14scheme allows us to reduce the computational cost of a typical calculation by a factor of2/3,while introducing an error of less than2%in the average frequencies.Once the values of F(a,T)on the PES are obtained,we de-termine the minimum a(T)byﬁtting F(a,T)to a Morse potentialF(a,T)=D0 e−2β(a−a(T))−2e−β(a−a(T)) .(17)We have estimated that the numerical error in this mini-mization is of order5×10−4˚A or less byﬁtting only the internal energy component E(a)and comparing with the minima obtained using conjugate gradient optimization.putational DetailsAll the ABINIT calculations reported here use Troullier-Martins scheme—Fritz-Haber-Institut pseu-dopotentials.We found that an8×8×8Monkhorst-Pack k-point grid and an energy cutoﬀof60au(12au for Ge)were suﬃcient to achieve convergence with respect to the DW factors.In all cases where the geometries were varied,an energy cutoﬀsmearing of5%was included to avoid problems induced by the change in the num-ber of plane wave basis sets.For metallic systems,the occupation numbers were smeared with the Methfessel and Paxton33scheme with broadening parameter0.025. Results are presented for LDA(Perdew-Wang9226)and GGA(Perdew-Burke-Ernzerhof27)functionals,as well as for our mixed hGGA functional.IV.RESULTSA.Born-von Karman parametersdispersion curves are often parametrized in of so-called Born-von Karman(BvK)coupling con-These parameters are essentially the Cartesianof the real space dynamical matrix deﬁned in(10).The main diﬀerence between the Born-vonparameters and force constants obtained within quasi-harmonic approximation is that the former are at speciﬁc temperatures while the temperatureof the quasi-harmonic model arises implicitly the dependence of the lattice parameters on ther-expansion.The dominant BvK coupling constants to the second neighbor)are presented in Table I. Weﬁnd that both the LDA with prescription I and the hGGA with prescription II generally give force constants that are within a few percent of experiment.Typically the LDA force constants with prescription I are slightly higher than those from the hGGA with prescription II. Also,note that the transverse components of the BvK pa-rameters tend to be overestimated.We have also consid-ered the pure PBE GGA functionals,butﬁnd that they produce force constants that are signiﬁcantly weaker due to their larger equilibrium lattice constants(Fig.1).B.Mean-square Relative Displacements Calculations of the MSRD for the dominantﬁrst near neighbor path for fcc Cu are shown in Fig.3,and de-tailed results for various scattering paths are presented in Table II.Both of our prescriptions I and II yield re-sults in good agreement with experiment.For Cu even the correlated Debye model is quite accurate.Note also a slight deviation from linearity in temperature T due to the variation in the dynamical matrix with tempera-ture is visible both in the experimental curve and in the calculation using prescription I.Similarly,calculations of the MSRD for theﬁrst neigh-bor path in Ge are shown in Fig.4,and detailed re-sults for various scattering paths are given in Table III. Again,both of our prescriptions yield results in good agreement with experiment,with the LDA prescription being slightly better.For this case,however,the corre-lated Debye model is signiﬁcantly in error;this is not unexpected given the strong anisotropy of the diamond lattice.Tables II and III also include similar results for Ag,Pt and GaAs.C.Thermal ExpansionThe thermal expansion can now be calculated in two ways.First,by minimizing the free energy of the sys-tem using Eq.(6)one can obtain the overall thermal expansion corresponding to the expansion of the lattice8the nearest neighbor single scattering path in Cu.The exper-imental diﬀerence values14were shifted to match the LDA(I)results at0K.constant a(T).For monoatomic systems the thermal ex-pansion of any MS path is simply proportional to thelattice constant.More generally,the expansion is MSpath dependent,and canbeestimated usingthe corre-lated Einstein model of II C and the Appendix.From Eq.(14)and the Einstein model Gr¨u neisen parameter γ=−k3R/k,this model predicts that theﬁrst cumu-lantσ(1)has a temperature dependence proportional to σ2/R,σ(1)=3γη5≈2.24attemperature.We also show that for fcc structures the due to perpendicular motion is smaller than crystallographic contribution by a factor ofγ⊥/6γ,for Cu is about20%.To illustrate this correlation, 6shows the perpendicular motion contributionσ2⊥both by the Lanczos procedure and with acorrelation factorγ⊥=2.5.We have carried out similar calculations ofσ2⊥for the of diamond lattices.Due to the strongly directional in diamond structures,and non-negligible bondforces,the calculations are more complicated for fcc materials.Our ab initio calculations using LDA with prescription I yield a ratio that varies from to7.2between0and600K,in reasonable agreement experiment whereγ⊥varies between3.5±0.6and ±0.5in the same range.34In contrast our single near neighbor spring model(Appendix)gives a smaller high temperature valueγ⊥=3.5and the addition of a sin-gle bond-bending parameter does not improve the agree-ment.E.Third CumulantAs for theﬁrst cumulant,the third cumulant can be estimated from the correlated Einstein model,and the relationσ(3)=η 2−4σ2 2 σ(1)σ2.(19)Again an additional scaling factorηis needed to correct the original Einstein model relations whenσ(1)andσ2 are replaced by the full results from our LDA calcula-tions.Also the presence of this factorηgives another correction to the relationσ(1)σ2/σ(3)≈2given by clas-sical models35or the correlated Einstein model at high temperatures.F.Crystallographic Debye-Waller FactorsFinally,we present results for the x-ray and neu-tron crystallographic Debye Waller factors W(T)= (1/2)k2u2(T),where the mean-square displacements u2(T)= (u·ˆR)2 are given by Eq.(5),withρa(ω)given by the total vibrational density of states per site,as cal-culated by our Lanczos algorithm with an appropriate seed state.17For this case good agreement is obtained for both of our DFT prescriptions at low temperature, although the errors become more signiﬁcant at higher temperatures.Also,weﬁnd that the convergence of the Lanczos algorithm is slower than for the path dependent Debye-Waller factors,requiring approximately16itera-tions to achieve convergence to1%.9Debye-Waller factor for the nearest neighbor single scatter-ing path in Cu,and compared to experimental values36.V.DISCUSSION AND CONCLUSIONSWe have developed aﬁrst principles approach for cal-culations of the Debye-Waller factors in various x-ray spectroscopies,based on DFT calculations of the dynam-ical matrix and phonon spectra for a given system.We ﬁnd that the results depend strongly on the choice of exchange-correlation potential in the DFT,but we have developed two prescriptions that yield stable results,one based on the LDA and one based on a modiﬁed GGA termed hGGA.Calculations for the crystalline systems presented here show that our LDA prescription yields good agreement with experiment for all quantities,typ-ically within about±10%.Second,if the lattice con-stant is known a priori,our hGGA prescription also pro-vides an accurate procedure to estimate the MSRD.An-harmonic corrections and estimates of the contribution from perpendicular vibrations are estimated using a cor-related Einstein model.For these anharmonic quantities, however,we have found that the comparative softness of the lattice dynamics with the GGA and hGGA func-tionals leads to results which are somewhat less accu-rate than those for the LDA.Finally we have also calcu-lated the crystallographic Debye Waller factors.Our ap-proach also yields good results for calculations of DW fac-tors in anisotropic systems,as illustrated for for Ge and GaAs.All of these results demonstrate that the prescrip-tions developed herein can yield quantitative estimates of Debye-Waller factors including anharmonic eﬀects in var-ious crystalline systems,and generally improve on phe-nomenological models.Extensions to molecular systems are in progress.Acknowledgmentsthank S.Baroni,K.Burke,A.Frenkel,X.Gonze, Van Hung,P.Fornasini,M.Newville,and J.Perdew many comments and suggestions.This work is sup-in part by the DOE Grant DE-FG03-97ER45623and DE-FG02-04ER1599(FDV),and was facili-by the DOE Computational Materials Science Net-APPENDIXthis Appendix we brieﬂy discuss the correlated Ein-model used in estimating anharmonic contributions the DW factors.The model is illustrated with an application to the correlated Einstein model for calcu-lating the mean-square radial displacement(MSRD)σ2 and mean square perpendicular displacement(MSPD)σ2⊥= |∆ u⊥|2 .The construction of Einstein models is not unique in that diﬀerent physical quantities reﬂect diﬀerent aver-ages over the VDOS.For example,the theoretical MSRD given by Eq.(5)reﬂects an average over a thermal weight factor varying as1/ω2at high temperatures.Thus the Einstein model parameters in our prescription are con-structed to preserve the correct high temperature behav-ior ofσ2.1,3Theﬁrst step in this construction is the cal-culation of¯ω2from the total potential energy for a net displacement x of a path along a particular seed displace-ment state|0 .Next this value is renormalized to give the correct MSRD at high temperatures.Thus we deﬁnek0=ηµ 0|D|0 (A.1) where¯ω2is given from Eq.(12)and the factorη= 1/ ω−2 ¯ω2,where ω−2 is the inverse second moment of the projected VDOS.The cubic coupling k3is then deﬁned to be consistent with the variation in k given by the Gr¨u neisen parameterγ=d ln¯ωk.(A.2) and hence must be is similarly renormalizedk3=ηdR 0|D(R)|0 .(A.3) Then the Einstein frequencyωE in the quasi-harmonic approximation is obtained from the relation18k=k0+6k3¯x=µω2E.(A.4) whereµis the reduced mass.For Cu using the LDA (I)prescription for the dynamical matrix,this procedure yieldsη=0.73,k0=54.7N/m,k3=−48.4N/m˚A,and k=51.1N/m.The scaling factorηthus forces the relationσ2(T)→σ2E(T)=k B T/µω2E at high temperature,whereσ2(T)in。

An increasing threat in hospitals_ multidrug-resistant Acinetobacter baumannii.

Intensive care units (ICUs) of hospitals harbour critically ill patients who are extremely vulnerable to infections. These units, and their patients, pro-vide a niche for opportunistic microorganisms that are generally harmless for healthy individuals but that are often highly resistant to antibiotics and can spread epidemically among patients. Infections by such organisms are difficult to treat and can lead to an increase in morbidity and mortality. Furthermore, their eradication from the hospital environment can require targeted measures, such as the isolation of patients and temporary closure or even reconstruction of wards. The presence of these organisms, therefore, poses both a medical and an organizational burden to health-care facilities.One important group of bacteria that is associated with these problems is the heterogeneous group of organisms that belong to the genus Acinetobacter. This genus has a complex taxonomic history. Since the 1980s, in parallel with the emergence of acinetobacters as noso-comial pathogens, the taxonomy of the genus has been refined; 17 named species have been recognized and 15 genomic species (gen.sp.) have been delineated by DNA–DNA hybridization, but these do not yet have valid names (TABLE 1). The species that is most commonly involved in hospital infection is Acinetobacter bauman-nii, which causes a wide range of infections, including pneumonia and blood-stream infections. Numerous studies have reported the occurrence of multidrug-resistant (MDR) A. baumannii in hospitals, and at some locations pandrug-resistant strains have been identified. Currently, A. baumannii ranks among the most important nosocomial pathogens. Additionally, the number of reports of community-acquired A. baumannii infection has been steadily increasing, although overall this type of infection remains rare. Despite the numerous publi-cations that have commented on the epidemic spread of A. baumannii, little is known about the mechanisms that have favoured the evolution of this organism to multi-drug resistance and epidemicity. In this Review, we dis-cuss the current state of knowledge of the epidemiology, antimicrobial resistance and clinical significance of acinetobacters, with an emphasis on A. baumannii. The reader is also referred to previous reviews of this organism that have been written by pioneers in the field1,2.Identification of Acinetobacter speciesIn 1986, a phenotypic system for the identification of Acinetobacter species was described3, which together with a subsequent simplified version4 has proven useful for the identification of most, but not all, Acinetobacter species. In particular, Acinetobacter calcoaceticus, A. baumannii, gen.sp. 3 and gen.sp. 13TU cannot be separated well by this system4. These species are also highly similar by DNA–DNA hybridization5 and it has therefore been proposed that they should be grouped together into the so-called A. calcoaceticus–A. baumannii (Acb) complex4. From a clinical perspective this might not be appropriate, as the complex combines three of the most clinically relevant species (A. baumannii, gen.sp. 3 and gen.sp. 13TU) with an environmental spe-cies (A. calcoaceticus). It is noteworthy that the perform-ance of commercial systems for species identification that are used in diagnostic microbiology is also unsatisfactory.*Department of Infectious Diseases C5‑P, Leiden University Medical Centre, Albinusdreef 2, P.O.BOX 9600, 2300 RC Leiden, the Netherlands.‡Centre of Epidemiology and Microbiology, National Institute of Public Health, Srobarova 48, 10042 Prague, Czech Republic.§Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Goldenfelsstrasse 19‑21, 50935 Cologne, Germany. Correspondence to L.D.e‑mail: l.dijkshoorn@lumc.nl doi:10.1038/nrmicro1789An increasing threat in hospitals: multidrug-resistant Acinetobacter baumanniiLenie Dijkshoorn*, Alexandr Nemec‡ and Harald Seifert§Abstract | Since the 1970s, the spread of multidrug-resistant (MDR) Acinetobacter strains among critically ill, hospitalized patients, and subsequent epidemics, have become an increasing cause of concern. Reports of community-acquired Acinetobacter infections have also increased over the past decade. A recent manifestation of MDR Acinetobacter that has attracted public attention is its association with infections in severely injured soldiers. Here, we present an overview of the current knowledge of the genus Acinetobacter, with the emphasis on the clinically most important species, Acinetobacter baumannii.DNA–DNA hybridizationDetermines the degree ofsimilarity between the genomicDNA of two bacterial strains;the gold standard to assesswhether organisms belong tothe same species.Pandrug-resistantIn this Review, refers toA. baumannii that are resistantto all available systemicanti-A. baumannii antimicrobialagents, except for polymyxins.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |939©2007Nature Publishing GroupIsolateA population of bacterial cells in pure culture that is derived from a single ing these systems, the clinically relevant species ofthe Acb complex are frequently uniformly identified asA. baumannii and many other species are not identified6–8.These problems have led to the development of genotypicmethods for Acinetobacter species identification, some ofwhich are discussed in BOX 1(also see fIg. 1). Currently,precise species identification is not feasible in most labora-tories, except for a few Acinetobacter reference laboratories.In light of the difficulties in distinguishing A. baumannii,gen.sp. 3 and gen.sp. 13TU, in this Review these specieswill be referred to as A. baumannii (in a broad sense)unless otherwise stated.Epidemiology of clinical acinetobactersThe natural habitat of Acinetobacter species.MostAcinetobacter species have been found in clinicalspecimens (TABLE 1), but not all are considered to beclinically significant. One important question is wheredoes A. baumannii come from? Furthermore, are thereenvironmental or community reservoirs? As mentionedearlier, A. baumannii, gen.sp. 3 and gen.sp. 13TU are themost frequent species that are found in human clinicalspecimens5,9,10. Of these, gen.sp. 3 was the most prevalentspecies among clinical isolates in a Swedish study5. In2 european studies, Acinetobacter lwoffii was the most Table 1 | Classification of the genusAcinetobacterto label these species by the initials of their respective authors, Tjernberg and Ursing (TU)5 or Bouvet and Jeanjean (BJ)108.940 | DeCeMbeR 2007 | vOlUMe 5 /reviews/micro©2007Nature Publishing GroupNature Reviews | Microbiology predominant species to be found on the skin of healthy individuals, with carrier rates of 29% and 58%, whereas other Acinetobacter species, including Acinetobacter junii , Acinetobacter johnsonii , Acinetobacter radioresistens and gen.sp. 15bJ, were detected at lower frequencies 11,12. The carrier rates for A. baumannii (including gen.sp. 13TU) in these studies ranged from 0.5 to 3%, whereas for gen.sp. 3 the rates ranged from 2 to 6%11,12. The faecal carriage of A. baumannii among non-hospitalized individuals in the United Kingdom and the Netherlands was 0.9%13. The most predominant species in faecal samples from the Netherlands were A. johnsonii (17.5%) and gen.sp. 11 (4%)13. A. baumannii was also recovered from the bodylice of homeless people 14 and it was proposed that the organisms were associated with transient bacteraemia in these individuals. In a study in Hong Kong, the car-rier rates of A. baumannii , gen.sp. 3 and gen.sp. 13TU on the skin of healthy individuals were 4, 32 and 14%, respectively 15. Thus, the carrier rates for gen.sp. 3 and gen.sp. 13TU in that study were strikingly higher than in the european studies. These findings indicate that, at least in europe, the carriage of A. baumannii in the community is relatively low. Apart from its occurrence in humans, A. baumannii has also been associated with infection and epidemic spread in animals at aveterinary clinic 16.NATURe RevIewS | microbiologyvOlUMe 5 | DeCeMbeR 2007 | 941© 2007Nature Publishing Group908070605040302010100Acinetobacter grimontiiGen.sp. 15TUAcinetobacter junii Acinetobacter haemolyticus Gen.sp. 14BJGen.sp. 13TUA. baumannii Gen.sp. 3Gen.sp.‘close to 13TU’Acinetobacter venetianusAcinetobacter calcoaceticusGen. sp.‘between 1 and 3’Acinetobacter tjernbergiaeAcinetobacter towneriAcinetobacter ursingiiGen.sp. 13BJ or 14TU Gen.sp. 15BJ Gen.sp. 17A. baylyiAcinetobacter lwoffiiAcinetobacter schindleriAcinetobacter bouvetiiAcinetobacter gerneriGen.sp. 10Gen.sp. 11Acinetobacter johnsoniiAcinetobacter radioresistens Acinetobacter parvusPearson correlation Species Gen.sp. 16Gen.sp. 6Nature Reviews | Microbiology Acinetobacter tandoii EndemicThe constant presence of aninfectious agent in a givengeographical area or hospital.There are few available data on the environmental occurrence of A. baumannii , gen.sp. 3 and gen.sp. 13TU,but these species have been found in varying percent-ages in vegetables, fish, meat and soil 17,18. A. baumannii has also recently been found in aquacultures of fish and shrimp farms in Southeast Asia 19. However, it is not yet clear to what extent these findings are attributable to an environmental niche or to contact with humans or animals. A. baumannii has been described as a soil organism, but without the support of appropriate references 20. It was probably assumed that the wide occurrence of unspeciated acinetobacters in soil and water 21 is also applicable to A. baumannii . However, in fact, there is little evidence that A. baumannii is a typical soil resident. Taken together, the existing data indicate that A. baumannii has a low prevalence in the community and that its occurrence in the environment is rare.A. baumannii in hospitals. The most striking mani-festation of A. baumannii is the endemic and epidemic occurrence of MDR strains in hospitals. The closely related gen.sp. 3 and gen.sp. 13TU might have a similar role 22–24, and their involvement could have been under-estimated as these species are phenotypically difficult to discriminate from A. baumannii . Most investigations ofA. baumannii in hospitals have been ad hoc studies thatwere triggered by an outbreak. More in-depth studies of the prevalence of this species in hospitals, including antibiotic-resistant and antibiotic-susceptible strains, are required to better understand its true importance.Depending on the local circumstances, and the strain in question, the pattern of an outbreak can vary. Therecan be a common source or multiple sources and somestrains have a greater tendency for epidemic spread than others. epidemiological typing — mostly by genotypic methods, such as amplified fragment length polymor-phism (AFlP) analysis (BOX 1) — is an important tool that can distinguish an outbreak strain from other, concurrent strains, and assess the sources and mode oftransmission of the outbreak strain.A scheme that depicts the dynamics of epidemic A. baumannii on a hospital ward is provided in fIg. 2. An epidemic strain is most commonly introduced by apatient who is colonized. Once on a ward, the strain can then spread to other patients and their environment. A. baumannii can survive in dry conditions 25 and during outbreaks has been recovered from various sites in thepatients’ environment, including bed curtains, furnitureand hospital equipment 26. These observations, and thesuccess that cleaning and disinfecting patients’ rooms has had in halting outbreaks, emphasize the role of thehospital environment as a reservoir for A. baumanniiduring outbreaks. The bacteria can be spread throughthe air over short distances in water droplets and inscales of skin from patients who are colonized 27, but the most common mode of transmission is from the handsof hospital staff. Patients who are colonized or infected by a particular A. baumannii strain can carry this strain at different body sites for periods of days to weeks 28, and colonization can go unnoticed if the epidemic strain isnot detected in clinical specimens 2,29.Population studies of A. baumannii . Comparative typ-ing of epidemic strains from different hospitals has indicated that there can be spread between hospitals. For example, during a period of outbreaks in the Netherlands that involved eight hospitals, one common strain was found in three of these hospitals and another common strain was found in two others 26. Similar observations of interhospital spread of MDR strains in particular geographical areas have been made in the Czech Republic 30, the United Kingdom 31, Portugal 32and the United States 33. Highly similar, but distinguishable, strains have been found at different locations and at different time points,Figure 1 | Amplified fragment length polymorphism (AFlP) analysis of Acinetobacter strains. A condensed dendrogram of the AFLP (described in BOX 1)fingerprints of 267 Acinetobacter reference strains of 32 described genomic species. Allspecies are well separated at the 50% cluster-cut-off level, which emphasizes the powerof this method for the delineation and identification of Acinetobacter species.942 | DeCeMbeR 2007 | vOlUMe 5/reviews/micro© 2007Nature Publishing GroupNature Reviews |MicrobiologyClonesA group of bacteria that wereisolated independently fromdifferent sources in time andspace, but share so manyidentical traits that it is likelythat they evolved from acommon ancestor.T yping methodA tool that differentiatesbacterial strains below the species level.RibotypingA typing method in which chromosomal DNA is digested by restriction enzymes, fragments are separated by electrophoresis and, finally, particular fragments are detected by labelled rRNA probes to generate DNA-banding patterns, which allows the differentiation of bacterial isolates.without a direct epidemiological link. It is assumed thatthese strains represent particular lineages of descent(clones). examples are european clones I–III34–36, whichhave been delineated by a range of genotypic typing meth-ods, such as AFlP analysis (BOX 1a; fIg. 1), ribotyping,macrorestriction analysis by pulsed-field gel electrophore-sis and, most recently, multilocus sequence typing (seeboth MlST systems in Further information). Strainsthat belong to these clones are usually highly resistantto antibiotics, although within a clone there can bevariation in antibiotic susceptibility. Apparently, theseclones are genetically stable strains that are particularlysuccessful in the hospital environment and evolveslowly during their spread. whether these strains haveparticular virulence attributes or an enhanced ability tocolonize particular patients (discussed below) remainsto be established. Their wide spread might be explainedby the transfer of patients between hospitals and regionsover the course of time, although in many cases there isno evidence for this. It is also possible that they circulateat low rates in the community and are able to expand inhospitals under selective pressure from antibiotics. Sofar, their resistance to antimicrobial agents is the onlyknown selectively advantageous trait.Figure 2 | overview of the dynamics between patients, bacteria and the hospital environment. The possible modes of Acinetobacter baumannii entry into a ward are shown. Entrance through a colonized patient is the most likely mode. However, introduction through contaminated materials (such as pillows104) has also been documented. Notably, introduction by healthy carriers is also conceivable, although it is not known whether the rare strains that circulate inthe community have epidemic potential. Once on a ward, A. baumannii can spread from the colonized patient to the environment and other susceptible patients. The direct environment of the patient can become contaminated by excreta, air droplets and scales of skin. Interestingly, A. baumannii can survive well in the dry environment25, a feature it shares with staphylococci. Hence, the contaminated environment can become a reservoir from which the organism can spread. The acquisition of A. baumannii by susceptible patients can occur through various routes, of which the hands of hospital staff are thought to be the most common, although the precise mode of transmission is usually difficult to assess.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |943©2007Nature Publishing GroupMacrorestriction analysisA typing method in which chromosomal DNA is digested with rare-cutting enzymes, so creating large fragments that are separated in an alternating electric field (pulsed-field electrophoresis) according to their size.OsteomyelitisAn infection of bone or bone marrow.Clinical impact of Acinetobacter infectionsNosocomial infections. Acinetobacters are opportun-istic pathogens that have been implicated in variousinfections that mainly affect critically ill patients inICUs. Hospital-acquired Acinetobacter spp.infectionsinclude: ventilator-associated pneumonia; skin andsoft-tissue infections; wound infections; urinary-tractinfections; secondary meningitis; and bloodstreaminfections. These infections are mainly attributed toA. baumannii, although gen.sp. 3 and gen.sp. 13TUhave also been implicated. Nosocomial infectionsthat are caused by other Acinetobacter species, suchas A. johnsonii, A. junii, A. lwoffii, Acinetobacterparvus, A. radioresistens, Acinetobacter schindleriand Acinetobacter ursingii, are rare and are mainlyrestricted to catheter-related bloodstream infec-tions8,37–40. These infections cause minimal mortalityand their clinical course is usually benign, althoughlife-threatening sepsis has been observed occasion-ally41. The rare outbreaks of some of these species (forexample, A. junii) have been found to be related tocontaminated infusion fluids41.The risk factors that predispose individuals to theacquisition of, and infection with, A. baumannii aresimilar to those that have been identified for otherMDR organisms. These include: host factors suchas major surgery, major trauma (in particular, burntrauma) and prematurity in newborns; exposure-related factors such as a previous stay in an ICU, thelength of stay in a hospital or ICU, residence in a unitin which A. baumannii is endemic and exposure tocontaminated medical equipment; and factors thatare related to medical treatment such as mechani-cal ventilation, the presence of indwelling devices(such as intravascular catheters, urinary cathetersand drainage tubes), the number of invasive proce-dures that are performed and previous antimicrobialtherapy42. Risk factors that are specific for a par-ticular setting have also been identified, such as thehydrotherapy that is used to treat burn patients andthe pulsatile lavage treatment that is used for wounddébridement43,44.The most frequent clinical manifestations of noso-comial A. baumannii infection are ventilator-associatedpneumonia and bloodstream infection, both of whichare associated with considerable morbidity and mor-tality, which can be as high as 52%45,46. Risk factorsfor a fatal outcome are severity-of-illness markers, anultimately fatal underlying disease and septic shockat the onset of infection. bacteraemic A. baumanniipneumonia has a particularly poor prognosis46. Acharacteristic clinical manifestation is cerebrospinal-shunt-related meningitis, caused by A. baumannii inpatients who have had neurosurgery47. wound infec-tions have been reported mainly in patients who havesevere burns or trauma, for example, soldiers who havebeen injured during military operations43,48. Urinary-tract infections related to indwelling urinary-tractcatheters usually run a more benign clinical courseand are more frequent in rehabilitation centres thanin ICUs49.The clinical impact of nosocomial A. baumanniiinfection has been a matter of continuing debate. Manystudies report high overall mortality rates in patientsthat have A. baumannii bacteraemia or pneumonia45,46.However, A. baumannii mainly affects patients withsevere underlying disease and a poor prognosis. It hastherefore been argued that the mortality that is observedin patients with A. baumannii infections is caused bytheir underlying disease, rather than as a consequenceof A. baumannii infection. In a case-control study, blotand colleagues50 addressed whether A. baumannii con-tributes independently to mortality and concluded thatA. baumannii bacteraemia is not associated with a sig-nificant increase in attributable mortality. Similar find-ings for A. baumannii pneumonia have been reportedby Garnacho and colleagues51. by contrast, in recentreviews of matched cohort and case-control studies,Falagas and colleagues52,53 concluded that A. baumanniiinfection was associated with an increase in attributablemortality, ranging from 7.8 to 23%. These contradictoryconclusions show that the debate on the clinical impactof A. baumannii is still ongoing.Community-acquired infections.A. baumannii isincreasingly recognized as an uncommon but impor-tant cause of community-acquired pneumonia. Mostof the reported cases have been associated withunderlying conditions, such as alcoholism, smoking,chronic obstructive pulmonary disease and diabetesmellitus. Community-acquired A. baumannii pneu-monia appears to be a unique clinical entity thathas a high incidence of bacteraemia, a fulminantclinical course and a high mortality that ranges from40 to 64%. It has been observed almost exclusivelyin tropical climates, in particular in Southeast Asiaand tropical Australia54,55. It is currently unclear, how-ever, if host factors or particular virulence factors areresponsible for these severe infections. Multidrugresistance in these organisms is uncommon55. Othermanifestations of community-acquired A. baumanniiinfections are rare.Infections associated with natural disasters and warcasualties. A characteristic manifestation of nosoco-mial A. baumannii is wound infection that is associ-ated with natural or man-made disasters, such as theMarmara earthquake that occurred in 1999 in Turkey,the 2002 bali bombing and military operations48,56,57.A strikingly high number of deep-wound infections,burn-wound infections and osteomyelitis cases havebeen reported to be associated with repatriated casual-ties of the Iraq conflict48. Isolates often had multidrugresistance. based on the common misconception thatA. baumannii is ubiquitous, it has been argued that theorganism might have been inoculated at the time ofinjury, either from previously colonized skin or fromcontaminated soil. However, recent data clearly indi-cate that contamination of the environment of fieldhospitals and infection transmission in health-carefacilities have had a major role in the acquisition ofA. baumannii58.944 | DeCeMbeR 2007 | vOlUMe 5 /reviews/micro©2007Nature Publishing GroupNature Reviews |MicrobiologyQuorum sensingThe phenomenon whereby the accumulation of signalling molecules enables a single cell to sense the number of bacteria (cell density) that are present, which allows bacteria to coordinate certain behaviours or actions.Epidemicity and pathogenicityThe fact that colonization with A. baumannii is morecommon than infection, even in susceptible patients,emphasizes that the pathogenicity of this species is gen-erally low. However, once an infection develops, it canbe severe. Studies on the epidemicity and pathogenicityfactors of A. baumannii are still at an elementary stage.A number of putative mechanisms that might have a rolein colonization, infection and epidemic spread are sum-marized in fIg. 3. Genetic, molecular and experimentalstudies are required to elucidate these mechanisms inmore detail.Recent DNA sequencing of a single A. baumanniistrain identified 16 genomic islands that carry putativevirulence genes that are associated with, for example,cell-envelope biogenesis, antibiotic resistance, autoin-ducer production, pilus biogenesis and lipid metabo-lism59. Resistance to desiccation, disinfectants25,60 andantibiotics is important for environmental survival. Theextraordinary metabolic versatility3 of A. baumanniicould contribute to its proliferation on a ward and inpatients. Pilus-mediated biofilm formation on glass andplastics has been demonstrated61. If formed on medicaldevices, such as endotracheal tubes or intravascularcatheters, these biofilms would probably provide a nichefor the bacteria, from which they might colonize patientsand give rise to respiratory-tract or bloodstream infec-tions. electron microscopy studies have demonstratedthat pili on the surface of acinetobacters interact withhuman epithelial cells62. In addition, thread-like connec-tions between these bacteria were suggestive of an earlyphase of biofilm formation. The pili and hydrophobicsugars in the O-side-chain moiety of lipopolysaccharide(lPS)63 might promote adherence to host cells as a firststep in the colonization of patients. Quorum sensing, thepresence of which has been inferred from the detectionof a gene that is involved in autoinducer production59,could control the various metabolic processes, includingbiofilm formation.Resistance to antibiotics, as well as the protectiveconditions of the skin (such as dryness, low pH, theresident normal flora and toxic lipids) and those ofthe mucous membranes (such as the presence of mucus,lactoferrin and lactoperoxidase and the sloughing ofcells) are prerequisites for bacterial survival in a host thatis receiving antibiotics. In vitro and animal experimentshave identified various factors that could have a rolein A. baumannii infection. For example, A. baumanniiouter membrane protein A (AbOmpA, previouslycalled Omp38) has been associated with the inductionof cytotoxicity64. Iron-acquisition mechanisms65 andserum resistance66 are attributes that enable the organ-ism to survive in the bloodstream. The lPS and lipid Aof one strain, at the time named A. calcoaceticus, hadbiological activities in animals that were similar to thoseof other enterobacteria67. These included lethal toxicity,pyrogenicity and mitogenicity for mouse-spleen b cells.More recently, A. baumannii lPS was found to be themajor immunostimulatory component that leads toa proinflammatory response during A. baumanniipneumonia68 in a mouse model.Taken together, the chain of events from environmen-tal presence to the colonization and infection of patientsdemonstrates the extraordinary ability of A. baumannii toadapt to variable conditions. This ability suggests that theorganism must possess, in addition to other factors, effec-tive stress-response mechanisms. Together with its resist-ance to antibiotics, these mechanisms might explain thesuccess of particular A. baumannii strains in hospitals.Figure 3 | The factors that contribute to Acinetobacter baumannii environmentalpersistence and host infection and colonization. Adherence to host cells, asdemonstrated in an in vitro model using bronchial epithelial cells62, is considered to be afirst step in the colonization process. Survival and growth on host skin and mucosalsurfaces require that the organisms can resist antibiotics and inhibitory agents and theconditions that are exerted by these surfaces. Outgrowth on mucosal surfaces andmedical devices, such as intravascular catheters and endotracheal tubes61, can result inbiofilm formation, which enhances the risk of infection of the bloodstream and airways.Quorum sensing59 might have a regulatory role in biofilm formation. Experimentalstudies have identified various factors that could have a role in A. baumannii infection, forexample, lipopolysaccharide has been shown to elicit a proinflammatory response inanimal models67,68. Furthermore, the A. baumannii outer membrane protein A has beendemonstrated to cause cell death in vitro64. Iron-acquisition mechanisms65 and resistanceto the bactericidal activity of human serum66 are considered to be important for survivalin the blood during bloodstream infections. Environmental survival and growth requireattributes such as resistance to desiccation25,60, versatility in growth requirements3, biofilm-forming capacity61 and, probably, quorum-sensing activity59. Finally, adequate stress-response mechanisms are thought to be required for adaptation to different conditions.NATURe RevIewS |microbiology vOlUMe 5 | DeCeMbeR 2007 |945©2007Nature Publishing Group。

(英文)合成,与DNA和两个新的铜(II)的抗增殖活性相互作用配合甲斑蝥素和苯并咪唑衍生物

Synthesis,interaction with DNA and antiproliferative activities of two novel Cu(II)complexes with norcantharidin and benzimidazolederivativesWen-Ji Song a ,b ,Qiu-Yue Lin a ,b ,⇑,Wen-Jiao Jiang b ,Fang-Yuan Du b ,Qing-Yuan Qi b ,⇑,Qiong Wei ba Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces,Zhejiang Normal University,321004,PR China bCollege of Chemical and Life Science,Zhejiang Normal University,321004,PR Chinah i g h l i g h t sTwo novel Cu(II)complexes withnorcantharidin derivatives have been synthesized.Complexes structure was determined by X-ray diffraction.The complexes and ligands bound DNA moderately via partial intercalation modes.Complexes could cleave plasmid DNA via hydroxyl radical mechanism. Complex(1)has strongest activity against human hepatoma cells.g r a p h i c a l a b s t r a c tTwo novel complexes [Cu(L)2(Ac)2]Á3H 2O(1)(L =N-2-methyl benzimidazole demethylcantharate imide,C 15H 13N 2O 3,Ac =acetate,C 2H 3O 2)and [Cu(bimz)2(DCA)](2)(bimz =benzimidazole,C 7H 6N 2;DCA =dem-ethylcantharate,C 8H 8O 5)were synthesized and characterized.The DNA-binding properties of complexes were investigated by electronic absorption spectra,ﬂuorescence spectra,viscosity measurements and agarose gel electrophoresis.The interaction between the complexes and bovine serum albumin (BSA)was investigated by ﬂuorescence spectra.The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721)were tested in vitro .a r t i c l e i n f o Article history:Received 23June 2014Received in revised form 17August 2014Accepted 23August 2014Available online 1September 2014Keywords:NorcantharidinBenzimidazole derivatives Copper complex DNA bindingAntiproliferative activitya b s t r a c tTwo novel complexes [Cu(L)2(Ac)2]Á3H 2O (1)(L =N-2-methyl benzimidazole demethylcantharate imide,C 16H 15N 3O 3,Ac =acetate,C 2H 3O 2)and [Cu(bimz)2(DCA)](2)(bimz =benzimidazole,C 7H 6N 2;DCA =dem-ethylcantharate,C 8H 8O 5)were synthesized and characterized by elemental analysis,infrared spectra and X-ray diffraction techniques.Cu(II)ion was four-coordinated in complex 1,Cu(II)ion was ﬁve-coordi-nated in complex 2.A large amount of intermolecular hydrogen-bonding and p –p stacking interactions were observed in these complex structures.The DNA-binding properties ofthese complexes were inves-tigated using electronic absorption spectra,ﬂuorescence spectra,viscosity measurements and agarose gel electrophoresis.The interactions between the complexes and bovine serum albumin (BSA)were investi-gated by ﬂuorescence spectra.The antiproliferative activities of the complexes against human hepatoma cells (SMMC7721)were tested in vitro .And the results showed that these complexes could bind to DNA in moderate intensity via partial intercalation,and complexes 1and 2could cleave plasmid DNA through hydroxyl radical mechanism.Title complexes could effectively quench the ﬂuorescence of BSA through/10.1016/j.saa.2014.08.0691386-1425/Ó2014Elsevier B.V.All rights reserved.⇑Corresponding authors.Address:Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces,Zhejiang Normal University,321004,PR China (Q.Y.Lin).Tel.:+8657982283353;fax:+8657982282269.E-mail addresses:sky51@ (Q.-Y.Lin),Qingyuanqi@ (Q.-Y.Qi).static quenching.Meanwhile,title complexes had stronger antiproliferative effect compared to L and Na2(DCA)within the tested concentration range.And complex1possessed more antiproliferative active than complex2.Ó2014Elsevier B.V.All rights reserved.IntroductionIn recent years,the interactions of Cu(II)complexes with DNA and protein molecules drew more and more scholars’attention [1,2].Copper(II)complexes are well suited for DNA hydrolysis due to the strong Lewis acid properties of the cupric ion.Several copper(II)complexes have been developed as artiﬁcial nucleases, and showed versatile DNA cleavage properties in the absence or presence of a redox agent[3,4].Planar heterocyclic based complexes have received consider-able interest in nucleic-acid chemistry because of their diverse chemical reactivity,unusual electronic properties,and peculiar structure,which results in non-covalent interactions with DNA [5].Benzimidazole derivatives possess a variety of biological activ-ities and pharmacological effects.Several compounds containing benzimidazole group,have been reported to exhibit antimicrobial, anticancer,antifungal and anti-inﬂammatory activities[6]. Especially,the combinations of the pharmaceutical agents with some metal ions can further improve their biological activity.Demethylcantharidin(NCTD,7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylc acid anhydride)and disodium demethylcantharate (Na2(DCA)),as the derivatives of cantharidin,have been applied in clinical use[7].Meanwhile,demethylcantharate(DCA)could inhibit the activities of protein phosphatases1(PP1)and2A(PP2A)effec-tively[8,9].A range of amines were applied to react with norcantha-ridin,and results showed high level of cytotoxicity[10,11].Based on our previous investigations and as a continuation of our research program on complexes containing demethylcanthari-din[12,13],we synthesized two novel Cu(II)complexes containing demethylcantharidin.The interactions of these complexes with DNA and bovine serum albumin(BSA)were investigated.In addi-tion,antiproliferative activities against human hepatoma cells (SMMC-7721)were tested in vitro.Experimental sectionsMaterials and instrumentsAll reagents and chemicals were obtained from commercial sources.Demethylcantharidin(NCTD,C8H8O4)was obtained from Nanjing Zelang Medical Technology Co.Ltd.;Na2(DCA)was prepared in accordance with the literature described technique[14];2-ami-nomethylbenzimidazole dihydrochloride(ambiÁ2HCl)was pre-pared using the literature technique[15];benzimidazole(bimz, C7H6N2)and ct-DNA were obtained from Sinopharm Chemical Reagent Co.Ltd.;ct-DNA(q=200l g mLÀ1,c=3.72Â10À4mol LÀ1), with A260/A280=1.8–2.0,was prepared using50mmol LÀ1NaCl; Plasmid DNA(pDsRed2-C1)was purchased from Clontech Co.Ltd. America;Bovine Serum Albumin(BSA)was purchased from Beijing BioDee BioTech Co.Ltd.and was stored at4°C;BSA(q= 500l g mLÀ1,c=7.47Â10À6mol LÀ1)was prepared using 5mmol LÀ1NaCl solution;MTT(methyl thiazolyl tetrazolium)was purchased from the Sigma Company;Human hepatoma cells (SMMC-7721)was purchased from Shanghai Institute of Cell Bank. Other chemical reagents in analytical reagent grade were used with-out further puriﬁcation.Elemental analyses of C,H and N were carried out in Vario EL III elemental analyzer.Infrared spectra were obtained using the KBr disc method by NEXUS-670FT-IR spectrometer in the spectral range of4000–400cmÀ1.Diffraction intensities of the complexes were collected at293K on Bruker SMART APEX II CCD diffractom-eter.Electronic absorption spectra were obtained using UV-2501 PC spectrophotometer.Viscosity experiments were carried on Ubbelodhe viscometer.Fluorescence emission spectra were obtained by Perkin–Elmer LS-55spectroﬂuorometer.Agarose gel electrophoresis was performed on PowerPac Basic electrophoresis apparatus(BIO-RAD).Gel image formation were obtained on UNI-VERSAL HOOD11-S.N.(BIO-RAD Laboratories).Synthesis of LN-2-methyl benzimidazole demethylcantharate imide (L=C16H15N3O3)was prepared in accordance to the literature tech-niques[16].Mixture of1mmol norcantharidin(NCTD),1mmol2-aminomethylbenzimidazole dihydrochloride,1mmol cadmium acetate,and10mL distilled water was sealed in a25mL Teﬂon-lined stainless vessel and heated at433K for3d,then cooled slowly to room temperature.The solution was thenﬁltered and was allowed to stand still for3weeks until forming colorless crystals.Anal.Calcd. (%)for C16H15N3O3:C,64.65;H,5.05;N,14.14.Found(%):C,64.62;H, 5.03;N,14.16.IR(KBr pellet,cmÀ1):1617,1392(t(C@O));1446 (t(C@N));1258,1033,1001(t(C A O A C)).Synthesis of the complexesSynthesis of the complex1In a20mL weighing bottle,Cu(Ac)2ÁH2O(0.06g,0.3mmol)was dissolved in water(2mL).The L(0.089g,0.3mmol)solution in mixed solvents of water and ethanol(2:1,v/v)(10mL)was then added dropwisely with stirring under room temperature.The mix-ture solution wasﬁltered after two hours.One week after,blue crystals with suitable size for single-crystal X-ray diffraction were obtained.Anal.Calcd.(%)for C36H42N6O13Cu(1):C,52.05;H,5.06; N,10.12.Found(%):C,52.01;H,5.03;N,10.29.IR(KBr pellet, cmÀ1):3445(t(OH));1572,1395(t(C@O));1464(t(C@N));1254, 1057,984(t(C A O A C)).Synthesis of the complex2A mixture of Cu(Ac)2ÁH2O(0.5mmol)and Na2DCA(0.5mmol) was dissolved in water.And1.0mmol benzimidazole(bimz)in ethanol was added dropwisely into the mixed solution and stirring at room temperature.The solution wasﬁltered after two hours. One week later,blue crystals with suitable size for single-crystal X-ray diffraction were obtained.Anal.Calcd.(%)for Cu(C22H20 N4O5)(2):C,54.55;H,4.13;N,11.57.Found(%):C,54.25;H, 4.01;N,11.78.IR(KBr pellet,cmÀ1):3432(t(OH));1635,1396 (t(C@O));1432(t(C@N));1251,1032,981(t(C A O A C)).DNA bindingElectronic absorption spectraElectronic absorption spectra were collected at25°C byﬁxing the concentrations of the complexes,with DNA concentration ranging from0to7.44Â10À5mol LÀ1.Absorption spectra mea-surements were carried out at200–400nm,and DNA in Tris–HCl buffer solution(pH=7.4)was used as reference.W.-J.Song et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy137(2015)122–128123Fluorescence spectraFluorescence quenching experiments were carried out by add-ing DNA solutions(0–7.44Â10À4mol LÀ1)to the samples contain-ing2.00Â10À5mol LÀ1complexes.The mixture were diluted by Tris–HCl buffer solution(pH=7.4).Fluorescence for1was recorded at excitation wavelength(k ex)of248nm and emission wavelength(k em)between250nm and500nm.Fluorescence for 2was recorded at244nm excitation wavelength(k ex)and emis-sion wavelength(k em)between255nm and450nm(k em).Viscosity measurementViscosity measurements were pounds were added to DNA solution(3.72Â10À4mol LÀ1)with microsyringes. The concentration of the compounds were controlled within the range of0–3.33Â10À6mol LÀ1.The relative viscosities g were cal-culated using equation[17]:g=(t–t0)/t0,where t0and t represent theﬂow time of DNA solution through the capillary in the absence and presence of complex.The average values of three replicated measurements were used to evaluate the viscosity of the samples. Data were presented as(g/g0)1/3versus the ratio of the concentra-tion of compounds to DNA,where g was the viscosity of DNA in the presence of compound and g0was the viscosity of DNA. Interaction with pDsRed2-C1plasmid DNAInteractions between the complexes and pDsRed2-C1plasmid DNA were studied using agarose gel electrophoresis.The samples were incubated at37°C for3h,followed by addition of0.25% bromo-phenol blue and1mmol LÀ1EDTA.The DNA cleavage prod-ucts were submitted to electrophoresis in1.0%agarose gel contain-ing0.5l g mLÀ1ethidium bromides.The bands were photographed.Interaction with BSAFluorescence spectraThe complexes(0–26.7Â10À9mol LÀ1)were added to solution containing4.98Â10À7mol LÀ1BSA and Tris–HCl buffer(pH=7.4). Fluorescence spectra were obtained by recording the emission spectra(285–480nm)at excitation wavelength of280nm.Antiproliferative activity evaluationThe antiproliferative activities of the compounds(1,2,L and Na2(DCA))were evaluated by human hepatoma cells(SMMC-7721).The MTT assay was applied to measure the antiproliferative activities[18].The compounds were dissolved in DMSO as 100mmol LÀ1stock solutions,and diluted in culture medium before using.The target concentration of DMSO in the medium was less than0.1%,and it did not interfere with the tested bioactiv-ity results[19].Cells were seeded for24h before adding com-pounds,and incubated for72h.Then100l L MTT(1mg mLÀ1, dissolved in DMEM nutrient solution)was added into each well and incubated for4h(37°C).The absorbance was measured by microplate reader at570nm.The inhibition rate was calculated accordingly.The errors quoted were standard deviations,which three replicates were involved in the calculation[20].Crystal structure determinationSingle crystals,sized0.345mmÂ0.279mmÂ0.214mm(1) and0.345mmÂ0.287mmÂ0.156mm(2),were used for X-ray diffraction analysis.The structures were solved by direct methods and reﬁned by full-matrix least-squares techniques using the SHEL-XTL-97program package[21,22].All non-hydrogen atoms were reﬁned anisotropically.Besides the hydrogen atoms on oxygen atoms,which were located from the difference Fourier maps,other hydrogen atoms were generated geometrically.Crystal data and experimental details for structural analyses are listed in Table1. Results and discussionStructural description of complexesTwo novel complexes have been characterized by X-ray single crystal diffraction.The spectral results indicated that the space groups of the complexes were C2/C(1)and Pna21(2).Selected bond lengths and angles of complexes1,2were listed in Tables2and3. Hydrogen bond lengths and angles of complex1,2were listed in Tables S1and S2.Molecular structures of the title complexes were shown in Fig.1.The packing diagram was shown in Fig.S1.In complex1,the Cu(II)ion was four-coordinated.Each Cu(II) coordinated with two imine nitrogen N(2)(or N(2A))from ligand (L),and two oxygen atoms of different carboxyl groups from acetate ions,forming electrically neutral complex.This molecule was cen-trally symmetric,with the symcenter at the centre of CuN2O2.The bond angles of O(1)A Cu(1)A O(1)#1,O(1)A Cu(1)A N(2),O(1)#1A Cu(1)A N(2)#1and N(2)A Cu(1)A N(2)#1are88.38(14)°,90.23 (10)°,90.23(10)°and97.25(14)°,respectively,all of which are close 90°.Thus,a slightly distorted quadrangle was formed around Cu(1) by N(1),N(2),O(1),and O(3).The composition of the complex was [Cu(L)2(Ac)2]Á3H2O(1).Fig.S1showed that the hydrogen-bonding formed due to the presence of the nitrogen atoms and the oxygen atoms from the imide(L)and acetate ligands,and crystallization water molecules.The complex is rich in intramolecular and intermo-lecular hydrogen bonds,such as N(1)A H(1A)...O(1W);O(3W)A H(3WA)...O(4);O(2W)A H(2WA)...O(2).These hydrogen-bonding stabilized this crystal structure.In complex2,Cu(II)ion wasﬁve-coordinated.Each Cu(II)coor-dinated with two azomethine nitrogen N(1)(or N(3))from two bimz,two carboxylate oxygen atoms O2and O3in two different carboxylate groups,and one bridge oxygen atoms O1from dem-ethylcantharate,forming a distorted tetragonal pyramid structure. The composition of the complex was[Cu(bimz)2(DCA)](2).Fig.S1 showed that the hydrogen-bonding formed due to the presence of the nitrogen atom from the bimz and the oxygen atoms from demethylcantharate,such as N(2)A H(2A)...O(5)#1,N(4)A H(4A)... O(3)#2.Meanwhile,complexes1and2contain the benzimidazole group,resulting k–k stacking effects among the complexes.There-fore,we concluded that the synergistic effect,including p–p stack-ing and hydrogen-bonding interactions,existed between the complexes and biomacromolecule,which could be the fundamen-tal cause of the biological activity change found in macromolecules [23].DNA binding studiesElectronic absorption spectraThe application of electronic absorption spectroscopy is one of the most useful techniques in DNA-binding studies[24].Changes observed in the UV spectra upon titration can provide evidence for the intercalative interaction mode pattern,since hypochro-mism would occur from p–p stacking interactions[25].To further investigate the possible binding modes and to obtain the binding constants(K b)of complex to DNA,we also studied the effect of DNA titration to the title complexes by electronic absorption spec-tra at298K.Results are shown in Fig.2((a):1;(b):2).The intrinsic binding constant(K b)was determined by the equa-tion:[DNA]/(e A–e F)=[DNA]/(e B–e F)+1/[K b(e B–e F)],where[DNA] was the concentration of DNA,e A,e F and e B corresponded to the apparent extinction coefﬁcient,the extinction coefﬁcient for the124W.-J.Song et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy137(2015)122–128W.-J.Song et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy137(2015)122–128125Table1Crystal data of complex1and2.Complex12Chemical formula CuC36H42N6O13CuC22H20N4O5 Formula weight794.28483.97Crystal system Monoclinic Orthorhombic Space group C2/C Pna21a(Å)21.572(4)15.8667(3) b(Å)11.3687(19)9.9975(2)c(Å)17.371(4)12.5228(2) a(°)90.0090.00b(°)111.528(18)90.00c(°)90.0090.00Volume(Å3)3963.0(13)1986.46(6) Z44Crystal size(mm)0.345Â0.279Â0.2140.345Â0.287Â0.156 Shape Block BlockFig. beled ORTEP diagrams of complex1(a)and2(b)with30%thermalprobability ellipsoids shown.complexes1and2are quenched in presence of DNA.The plexes showed strong emission bands at around297nm(1 nm(2),as shown in Fig.3.According to the Stern–Volmer equation:F0/F=1+K sv[Q],F0and F represent theﬂuorescence intensities in the absence and presence of quencher,respectively [Q]is the quencher concentration and K sv is the Stern–Volmer constant,K sv were calculated as 4.26Â103mol LÀ1(1)Â103mol LÀ1(2).The binding intensity of complex1 stronger than complex2,this is consistent to the results found electronic absorption spectra.Viscosity measurementsfurther study the binding mode of the compounds interact-with DNA,DNA viscosity at25°C was investigated(Fig.experimental data showed that the relative viscosity of steadily decreased after adding complexes and L,and it increase after adding benzimidazole.But there was no signiﬁcant viscosity change occurred after adding Na2DCA.The possible expla-nation is that the complexes and L were partially inserted to DNA base pairs and resulting in a kink in the DNA helix,therefore decreased the DNA effective length[29].Because of the planar benzimidazole ring could also insert to the DNA base pair,and the steric hindrances of complexes were enhanced due to the non-planar structure of demethylcantharate(DCA).From Fig.4, the interactions of complex(1)with DNA is signiﬁcantly stronger than complex(2).The result agrees with the electronic absorption spectra andﬂuorescence spectra conclusion.Interaction with pDsRed2-C1plasmid DNAThe cleavage reaction on pDsRed2-C1plasmid DNA can be mon-itored by agarose gel electrophoresis.When pDsRed2-C1plasmid DNA is subjected to electrophoresis,different migration speeds were observed[30].Relatively fast migrations were observed at the intact supercoil form(Form I).If scission occurs on one strand (nicking),the supercoil will partially relax to generate a slow moving open-circular form(Form II)[31].Absorption spectra of the complex1(a)and2(b)in the presence of increasing amount of DNA.[complex]=3.00Â10À6mol LÀ1,from(1)to(5):Â105=0,0.74,1.48,2.24and2.98mol LÀ1,respectively.(b).[DNA]Â103.72,5.58and7.44mol LÀ1,respectively.3.Fluorescence spectra of the complex1(a)and2(b)in the absence presence of increasing the amount of DNA;insert in Figs.3–5:ﬂuorescence quenching curve of the complex by DNA.k ex=248nm(1),k ex=244nm [complex]=2Â10À5mol LÀ1;[DNA]/(10À4mol LÀ1),from1to5:0,1.86,3.72,7.44,respectively.4.Effect of increasing amounts of the compounds on the relative viscosity[DNA]=3.72Â10À4mol LÀ1;[complex]/10À6=0,0.67,1.33,2.00,2.67mol LÀ1,respectively.5gave the electrophoretograms of the interactionRed2-C1plasmid DNA with increasing concentrations of complexes. Complexes are capable of cleaving plasmid DNA when the concen-of complexes was greater than500l M.When the concentra-complexes increased,the amount of Form I diminished gradually,and Form II paring channel4–6, cleavage ability of complexes was enhanced by adding ascorbic order to investigate the reaction mechanism,dimethylsulfox-(DMSO)was introduced to the experimental design.DMSO scavenger could inhibit the cleavage ability of complexes signiﬁcantly in channel5and7.With increasing amount of acid(V c),Cu(II)complex was reduced to Cu(I)complex.complex then reacts with dissolved oxygen generating superoxide anion(O2À),hydrogen peroxide(H2O2)and hydroxyl (ÅOH).Finally,the ROS attacks the plasmid DNA leading single and double DNA strand breaks.So the cleavage hydroxyl radical mechanism[32].Interaction with BSAFluorescence spectra and quenching mechanismresults of title complexes quenching the BSAﬂuorescence ing,which generated via intense interaction[36].Binding constants and binding sitesAssuming there were n identical and independent binding sites in protein,the binding constant K A can be calculated using equation[37]:lg(F0–F)/F=lg K A+n lg[Q].The values of K A were 1.59Â106L molÀ1(1), 5.4Â104L molÀ1(2),and 2.78Â104L molÀ1(Na2DCA).The values of n were0.88(1),0.68(2)and 0.66(Na2DCA).The results indicated that strong bindingElectrophoretic separation of pDsRed2-C1DNA induced by complexesLane1:DNA alone;lane2:DNA+complex(250l M);lane3:DNA(500l M);lane4:DNA+complex(750l M);lane5:DNA++DMSO(750l M);lane6:DNA+complex(750l M)+V c(750complex(750l M)+V c(750l M)+DMSO(750l M).[DNA]=3.06.Fluorescence spectra of BSA in the absence and the presence of complex2(b)Inset:Stern–Volmer plots of theﬂuorescence titration data ofcomplexes.[BSA]=4.98Â10À7mol LÀ1;[complex]Â109=0,6.67,13.3,20.0,mol LÀ1,from(1)to(5),respectively(a):complex1,(b):complex2.7.Inhibition effects of compounds on SMMC-7721cell growth.Data representmean+S.D.and all assays were performed in triplicate for three independentexperiments.interaction existed between the complexes and BSA.The binding intensity of complexes was stronger than Na2DCA,and the binding site of complexes was one.Antiproliferative activity evaluationAs shown in Fig.7,the antiproliferative activity of complex1, complex2,L and Na2DCA at the given concentration showed a dose-dependent manner against human hepatoma cells(SMMC-7721)in vitro.The inhibition ratios tested revealed that complex1and2had strong antiproliferative activities against human hepatoma cells (SMMC-7721)lines in vitro compare to L and Na2DCA.The inhibi-tion rates of complex(1)against SMMC-7721lines(IC50=24.55±0.48l mol LÀ1)is much higher than that of L(IC50=116.63±2.66 l mol LÀ1)[38].The inhibition rates of complex(1)against SMMC-7721lines is much higher than complex(2)(IC50=41.82±3.90 l mol LÀ1).The inhibition rates of two novel complexes were higher than that of the transition metal complexes of demethyl-cantharate and thiazole derivatives[12,13]against SMMC-7721 cells.which suggests that various compositions and structures of complexes would lead to different antiproliferative activities,and this can be important in designing and synthesizing novel anti-cancer drugs[39].It is clear that the strong interaction found between complexes and biomacromolecules(DNA or BSA)is directly correlated to the antiproliﬁferative activity of complexes.ConclusionsTwo novel Cu(II)complexes[Cu(L)2(Ac)2]Á3H2O(1)(L=N-2-methyl benzimidazole demethylcantharate imide,C16H15N3O3, Ac=acetate,C2H3O2)and[Cu(bimz)2(DCA)](2)(bimz=benzimid-azole,C7H6N2;DCA=demethylcantharate,C8H8O5)were synthe-sized and characterized.The crystal structure of complex1and2 were determined by X-ray diffraction.These complexes had strong DNA and BSA binding intensity and high inhibition rates against human hepatoma cells(SMMC-7721)in plex(1)had intense antiproliferative activities against the human hepatoma cells(SMMC-7721)in vitro,which had the potential to develop as an anti-cancer drug in the future.AcknowledgmentWe thank Institute of Zhejiang Academy of Medical Science for helping with antiproliferative activity test.Appendix A.Supplementary materialCrystallographic data for the structure reported in this article has been deposited with the Cambridge Crystallographic Data Center CCDC909444(1),918105(2).Copies of the data can be obtained free of charge on application to the CCDC,12Union Road,Cambridge CB21EZ,UK(deposit@).The packing diagrams of complexes were shown in Fig.S1.Hydrogen bond lengths and angles of complex1,2were listed in Tables S1and S2.Supplementary data associated with this article can be found,in the online version,at 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生物等效性研究指导原则英文版

Technique Guideline for Human Bioavailability and BioequivalenceStudies on Chemical Drug ProductsContents(Ⅰ) Establishment and Validation for Biological Sample Analysis Methods (2)1. Common Analysis Methods (2)2. Method Validation (2)2.1 Specificity (2)2.2 Calibration Curve and Quantitative Scale (3)2.3 Lower Limit of Quantitation (LLOQ) (3)2.4 Precision and Accuracy (4)2.5 Sample Stability (4)2.6 Percent recovery of Extraction (4)2.7 Method Validation with microbiology and immunology (4)3. Methodology Quality Control (5)(Ⅱ) Design and Conduct of Studies (5)1. Cross-over Design (5)2. Selection of Subjects (6)2.1 Inclusion Criteria of Subjects: (6)2.2 Cases of Subjects (7)2.3 Division into Groups of the Subjects (7)3. Test and Reference Product, T and R (8)4. Sampling (8)(Ⅲ) Result Evaluation (9)(Ⅳ) Submission of the Contents of Clinical Study Reports (9)Technique Guideline for Human Bioavailability and BioequivalenceStudies on Chemical Drug ProductsSpecific Requirements for BA and BE Studies(Ⅰ) Establishment and Validation for Biological Sample Analysis MethodsBiological samples generally come from the whole blood, serum, plasma, urine or other tissues. These samples have the characteristics such as little quantity for sampling, low drug concentration, much interference from endogenous substances, and great discrepancies between individuals. Therefore, according to the structure, biological medium and prospective concentration scale of the analytes, it is necessary to establish the proper quantitative assay methods for biological samples and to validate such methods.1. Common Analysis MethodsCommonly used analysis methods at present are as follows: (1) Chromatography: Gas Chromatography(GS), High Performance Liquid Chromatography (HPLC), Chromatography－mass Spectrometry (LC-MS, LC-MS-MS, GC-MS, GC-MS-MS), and so on. All the methods above can be used in detecting most of drugs; (2) Immunology methods: radiate immune analysis, enzyme immune analysis, fluorescent immune analysis and so on, all these can detect protein and polypeptide; (3) Microbiological methods: used in detecting antibiotic drug.Feasible and sensitive methods should be selected for biologic sample analysis as far as possible.2. Method ValidationEstablishment of reliable and reproducible quantitative assay methods is one of the keys to bioequivalence study. In order to ensure the method reliable, it is necessary to validate the method entirely and the following aspects should be generally inspected:2.1 SpecificityIt is the ability that the analysis method has to detect the analytes exactly and exclusively, when interference ingredient exists in the sample. Evidences should be provided that the analytes are the primary forms or specific active metabolites of the test drugs. Endogenous instances, the relevant metabolites and degradation products in biologic samples should not interfere with the detection of samples. If there are several analytes, each should be ensured not to be interfered, and the optimal detecting conditions of the analysis method should be maintained. As for chromatography, at least 6 samples from different subjects, which include chromatogram of blank biological samples, chromatogram of blank biologic samples added control substance (concentration labeled) and chromatogram of biologic samples after the administration should beexamined to reflect the specificity of the analytical procedure. As for mass spectra (LC-MS andLC-MS-MS) based on soft ionization, the medium effect such as ion suppression should be considered during analytic process.2.2 Calibration Curve and Quantitative ScaleCalibration curve reflects the relationship between the analyte concentration and the equipment response value and it is usually evaluated by the regression equation obtained from regression analysis (such as the weighted least squares method). The linear equation and correlation coefficient of the calibration curve should be provided to illustrate the degree of their linear correlation. The concentration scale of calibration curves is the quantitative scale. The examined results of concentration in the quantitative scale should reach the required precision and accuracy in the experiment.Dispensing calibration samples should use the same biological medium as that for analyte, and the respective calibration curve should be prepared for different biological samples. The number of calibration concentration points for establishing calibration curve lies on the possible concentration scale of the analyte and on the properties of relationship of analyte/response value. At least 6 concentration points should be used to establish calibration curve, more concentration points are needed as for non-linear correlation. The quantitative scale should cover the whole concentration scale of biological samples and should not use extrapolation out of the quantitative scale to calculate concentrations of the analyte. Calibration curve establishment should be accompanied with blank biologic samples. But this point is only for evaluating interference and not used for calculating. When the warp* between the measured value and the labeled value of each concentration point on the calibration curve is within the acceptable scale, the curve is determined to be eligible. The acceptable scale is usually prescribed that the warp of minimum concentration point is within ±20% while others within ±15%. Only the eligible calibration curve can be carried out for the quantitative calculation of clinical samples. When linear scale is somewhat broad, the weighted method is recommended to calculate the calibration curve in order to obtain a more exact value for low concentration points. ( *: warp=[(measured value － labeled value)/labeled value]×100%)2.3 Lower Limit of Quantitation (LLOQ)Lower limit of quntitation is the lowest concentration point on the calibration curve, indicating the lowest drug concentration in the tested sample, which meets the requirements of accuracy and precision. LLOQ should be able to detect drug concentrations of samples in 3~5 eliminationhalf-life or detect the drug concentration which is 1/10~/20 of the C max. The accuracy of the detection should be within 80~120% of the real concentration and its RSD should be less than 20%. The conclusions should be validated by the results from at least 5 standard samples.2.4 Precision and AccuracyPrecision is, under the specific analysis conditions, the dispersive degree of a series of the detection data from the samples with the same concentration and in the same medium. Usually, the RSD from inter- or intra- batches of the quality control samples is applied to examine the precision of the method. Generally speaking, the RSD should be less than 15% and that around LLOQ should be less than 20%. Accuracy is the contiguous degree between the tested and the real concentrations of the biological samples (namely, the warp between the tested and the real concentrations of the quality-controlled samples). The accuracy can be obtained by repeatedly detecting the analysis samples of known concentration which should be within 85~115% and which around LLOQ should be within 80~120%.Generally, 3 quality-control samples with high, middle and low concentrations are selected for validating the precision and accuracy of the method. The low concentration is chosen within three times of LLOQ, the high one is close to the upper limit of the calibration curve, and the middle concentration is within the low and the high ones. When the precision of the intra-batches is detected, each concentration should be prepared and detected at least 5 samples. In order to obtain the precision of inter-batches, at least 3 qualified analytical batches, 45 samples should be consecutively prepared and detected in different days.2.5 Sample StabilityAccording to specific instances, as for biological samples containing drugs, their stabilities should be examined under different conditions such as the room temperature, freezing, thaw and at different preservation time, in order to ensure the suitable store conditions and preservation times. Another thing that should be paid attention to is that the stabilities of the stock solution and the analyte in the solution after being treated with, should also be examined to ensure the accuracy and reproducibility of the test results.2.6 Percent recovery of ExtractionThe recovery of extraction is the ratio between the responsive value of the analytes recovered from the biological samples and that of the standard, which has the same meaning as the ratio of the analytes extracted from the biologic samples to be analyzed. The recovery of extraction of the 3 concentrations at high, middle and low should be examined and their results should be precise and reproduceable.2.7 Method Validation with microbiology and immunologyThe analysis method validation above mainly aims at chromatography, with many parameters and principles also applicable for microbiological and immunological analysis. However, some special aspects should be considered in the method validation. The calibration curve of the microbiological and immunological analysis is non-linear essentially, so more concentration pointsshould be used to construct the calibration curve than the chemical analysis. The accuracy of the results is the key factor and if repetitive detection can improve the accuracy, the same procedures should be taken in the method validation and the unknown sample detection.3. Methodology Quality ControlThe unknown samples are detected only after the method validation for analysis of biological samples has been completed. The quality control should be carried out during the concentration detection of the biological samples in order to ensure the reliability of the method in the practical application. It is recommended to assess the method by preparing quality-control samples of different concentrations by isolated individuals.Each unknown sample is usually detected for only one time and redetected if necessary. In the bioequivalence experiments, biological samples from the same individual had better to be detected in the same batch. The new calibration curve should be established when detecting biological samples of each analysis batch and high, middle and low concentrations of the quality-control samples should be detected at the same time. Each concentration should at least have two samples and should be equally distributed in the detection sequence of the unknown samples. When there are a large number of unknown samples in one analysis batch, the number of the quality-control samples at different concentrations should be increased to make the quality-control samples exceed 5% of the unknown sample population. The warp of detection result from the quality-control samples should usually be less than 15%, while the warp of the low concentration point should be less than 20% and at most 1/3 results of the quality-control samples at different concentrations are allowed to exceed the limit. If the detection results of the quality-control samples do not accord with the above requirements, the detection results of the samples in this analysis batch should be blanked out.The samples with concentrations higher than the upper quantitation limit should be detected once more using corresponding diluted blank medium. As for those samples with concentrations lower than the lower quantitation limit, during pharmacokinetics analysis, those sampled before reaching C max should be calculated as zero while those after C max should be calculated as ND (Not detectable), so as to decrease the effect of the zero value on the AUG calculation.(Ⅱ) Design and Conduct of Studies1. Cross-over DesignCurrently, the crossover design is the most wildly applied method in the BE study. As for the drug absorption and clearance, there is a transparent variation among individuals. Therefore, the coefficient of variability among individuals is far greater than that of the individual himself. That is why the bioequivalence study is generally required to be designed on the principle of self crossover control. Subjects are randomly divided into several groups and treated in sequence, of whichsubjects in one group take the test products first and then the reference product, while subjects in the other take the reference products first and then the test products. A long enough interval is essential between the two sequences, which is called Wash-out period. In this way, every subject has been treated twice or more times sequentially, which is equal to self-control. Therefore, the influence of drug products on drug absorption can be discriminated from the others, and the effect of various test periods and individual difference on the results can be eliminated.Two-sequence crossover design, three-sequence crossover design are adopted respectively according to the amount of the test product. If two varieties of drug products are to be compared, the two-treatment, two-period or two-sequence crossover design will be a preferable choice. When three varieties of products (two test products and one reference product) are included, thethree-formulation, three-period and double 3×3 Latin square design will be the suitable choice. And a long enough wash-out period is required among respective periods.Wash-out period is set on purpose to eliminate the mutual disturbance of the two varieties of drug products and avoid the treatment in the prior period from affecting that of the next period. And the wash-out period is generally longer than or equal to 7 elimination half lives.While the half-lives of some drugs or their active metabolites are too long, it is not suitable to apply the crossover design. Under this circumstance, parallel design is adopted, but the sample size should be enlarged.However, as for some highly variable drugs, except for increase of the subjects, repetitive cross-over design can be applied, to test possibly existing difference in individual when receive the same preparation twice.2. Selection of Subjects2.1 Inclusion Criteria of Subjects:The difference among individuals of the subjects should be minimized so that the difference of the drug products can be detected. The inclusion criteria and exclusion criteria should be noted in the trial scheme.Male healthy subjects are recruited generally. And as to the drugs of special purpose, proper subjects are recruited according to specific conditions. If female healthy subjects are recruited, the possibility of gestation should be avoided. If the drugs to be tested have some known adverse effects, which may do harm to the subjects, patients can also be included as the subjects.Age: 18~ 40 years old generally. The difference in age of the subjects in one batch should not be more than 10 years.Body weight: not less than 50kg as to normal subjects. Body Mass Index (BMI), which is equal to body weight (kg)/ body height 2 (m2), is generally required to be in the range of standard body weight. For the subjects in one batch, the taken dosage is the same, the range of the bodyweight, therefore, should not have great disparity.The subjects should receive the overall physical examination and be proved healthy. There is not medical history of heart, kidney, digestive tract, nervous system, mental anomaly, metabolism dysfunction, and so on. The physical examination has revealed normal blood pressure, heart rate, electrocardiogram, and respiratory rate. Laboratory data have revealed normal hepatic function, renal function and blood function. Those examinations are essential to prevent the metabolism of drugs in vivo from being interfered by the diseases. According to the classification and safety of drugs, special items examinations are required before, during and after the test, such as the blood glucose examination, which is required in the drug trial of hypoglyceimic agents.In order to avoid the interference by other drugs, no administration of other drugs is allowed from two weeks before and till the end of the test. Moreover, the cigarette, wine,beverage with caffeine, or some fruit juice that may affect the metabolism of the drug, is forbidden during the trial period also. The subjects had better have no appetite of cigarette and wine. Possible effects of the cigarette-addicted history should not be neglected in the discussion of results.Due to the metabolism variance resulted by known genetic polymorphism of drugs, the safety factor which may be effected by the slow metabolism speed of drugs should be considered.2.2 Cases of SubjectsThe cases of the subjects should meet the statistic requirement. And according to the current statistical methods, 18~24 cases are enough for most drugs to meet the requirement of sample size. But as to some drugs of high variability, more cases may be required correspondingly.The cases of a clinic trial are determined by three fundamental factors: （1）Significance level: namely, the value of α, for which value 0.05 or 5% is often adopted;（2）Power of a test: namely, the value of 1-β. β is the index that represents the probability of the type error, which is also theⅡprobability of misjudging the actually efficacy drugs as inefficient drugs, and value not less than 80% is commonly stated; （3）Coefficient of variance（CV%）and Difference（θ）: In the equivalence test of two drugs, the greater CV% and θ of the test indexes are, the more cases are required. The CV% and θ are unknown before the trial and can only be estimated by the above parameters of the owned reference products or running the preliminary test. Moreover, when a BA test has been finished, the value of N can be calculated according to the parameters such as θ, CV% and 1-β and then compared with the cases adopted in the finished BA test to determine whether the cases are reasonable or not.2.3 Division into Groups of the SubjectsThe subjects should be randomly divided into different comparable groups. The cases of the two groups should guarantee the best comparability.3. Test and Reference Product, T and RThe quality of the reference products directly affect the results reliability of BE trial. Generally, the domestic innovator products of the same dosage form which has been approval to be on sale are commonly selected. If it failed in acquiring the innovator products, the key product on the market can also be chosen as the reference product and the related quality certifications (such as the test results of the assay and dissolution) and the reasons for option should be provided. When it comes to the drug study of specific purpose, other on-sale dosage forms which are of the same kind and similar with pharmaceutics properties are selected as the reference products and those reference products should be already on sale and qualified in quality. The difference in assay between the test product and reference product should not exceed 5%.The test product should be the scale-up product or manufacture scale product, which is consistent with the quality standards for clinical application. And the indexes such as the in vitro dissolution, stability, content or valence assay, consistency reports between batches should be provided to the test unit for reference. As for some drugs, the data of polymorphs and optical isomers should be offered additionally. The test and reference product should be noted with the advanced development unit, batch number, specification, storage conditions and expiry date.For future reference, the test and reference product should be kept long enough after the trialtill the product is approved to be on sale.4. SamplingThere is a significant sense in designing the sampling point to guarantee both the reliability of the trial results and the rationality of calculating the pharmacokinetics parameters. Commonly, there should be preliminary tests or the pharmacokinetics literatures at home and abroad served as the evidences of designing the reasonable sampling points. When the blood-drug concentration assay is performed, the absorption phase, balance phase and clearance phase should be considered overall. There must be enough sampling points in every phase of the C-T curve and around the T max. The concentration curve, therefore, can fully reflect the entire procedure of the drugs distribution in vivo. And the blank blood samples are taken before the administration. Then at least 2~3 points are sampled in the absorption phase, at least 3points are sampled near the C max and 3-5 points in the clearance phase. Try to avoid that the first point gets the C max, and running the preliminary test may avoid this. When the continuously-sampling results show that the drugs’ primary forms or the active metabolites are at the point of 3～5 half- lives or the blood drug concentration is 1/10～1/20 ofC max, the values of AUC0-t/AUC0-∞are generally bigger than 80% .For the terminal clearance item doesn’t affect the evaluation of the products’ absorption process much, as to the long half-life drugs, the sampling periods should be continued long enough, so that the whole absorption process can be compared and analyzed. In the multiple administration study, the BA of some drugs is known to beaffected by the circadian rhythm, samples of which should be taken 24 hours continuously if possible.When the BA of the test drugs can’t be determined by detecting the blood-drug concentration, if the primary forms and the active metabolites of the test drugs are mainly be excreted in urine (more than 70% of the dosage), the BA assay may be performed by detecting the urine drug concentration, which is the test of the accumulated excretion quantity of drugs in urine to reflect the intake of drugs. The test products and trial scheme should accord with the demands of BA assay. The urine samples should be collected at intervals, and the collection frequency and intervals of which should meet the demands of evaluating the excretion degree of the primary forms and the active metabolites of the test products in urine. However this method cannot reflect the absorption speed of the drugs and gets many error factors, it is not recommended generally.Some drugs metabolize so rapidly in vivo that it is impossible to detect the primary forms in biological samples. Under these circumstances, the method determining the concentration of corresponding active metabolites in biological samples is adopted to perform the BA and BE studies.(Ⅲ) Result EvaluationAt present, the weighting function of AUC on drug absorption degree is comparatively affirmed, while C max and T max sometimes are not sensitive and seemly enough for weighting the absorption speed due to their dependence on the arrangement of sampling time, and they are therefore not suitable for drug products with multi-peak phenomena and for experiments with large individual variation. During the evaluation, if there are some special instances of inequivalence, a specific analysis should be performed for specific problems.As for AUC，the 90% confidence interval is generally required within the scope of 80%~125%. As for the drugs with narrow treatment spectrum, the above scope should likely be appropriately reduced. While in a few instances, having been validated to be reasonable, the scope can also be increased. So does C max. And as for T max, statistical evaluation is required only when its release speed is closely correlated to clinical therapeutic effects and safety, the equivalence scope of which can be ascertained according to the clinical requirements.When bioavailability ratio of test products is higher than that of reference products, which is called suprabioavailability, the following two instances can be considered: 1). Whether the reference product itself is a product with low bioavailability, which results in the improvement of the test drug's bioavailability; 2). The quality of the reference product meets the requirement, and the test drug really has higher bioavailability.(Ⅳ) Submission of the Contents of Clinical Study ReportsIn order to satisfy the demand of evaluation, a clinical report of bioequivalence study shouldinclude the following contents: (1)Experiment subjective;(2) Establishment of analysis methods for bioavailability samples and data of inspection, as well as provision of the essential chromatograms;(3) Detailed experiment design and operation methods , including data of all the subjects，sample cases，reference products，given dosage，usage and arrangement of sampling time;(4) All data about original measurement of unknown sample concentrations，pharmacokinetics parameters and drug-time curve of each subjects;(5) Data handling procedure and statistical analysis methods as well as detailed procedure and results of statistics;(6) Observation results of clinical adverse reactions after taking medicine，midway exit and out of record of subjects and the reasons;(7) Result analysis and necessary discussion on bioavailability or bioequivalence; (8) References. A brief abstract is required before the main body; at the end of the main body, names of the experiment unit, chief persons of the study and experiment personnel should be signed to take the responsibility for the results of the study.。

Berry phase

Intrinsic Hall conductivity (Kubo Formula)
Thouless, Kohmoto, Nightingale, den Nijs (1982) Kohmoto (1985)
xy
e2 h

n,k
nF En (k ) Bnz (k )

A (k ): field strength; n : band index Bn (k ) k n
mx x kx
• Ohmic conductivity is dissipative: under T, electric field is even e2 2 J j E j where k F l charge current is odd h (only states close to the Fermi energy contribute!) • Charge supercurrent and Hall current are non-dissipative:

d p ε tr[ μ S F (p) ν S F (p) λ S F (p)]
3 μνλ
1
1
1
• Hall Conductance in terms of momentum space topology
Intrinsic spin Hall effect in p-type semiconductors
Topological Aspects of the Spin Hall Effect
Yong-Shi Wu Dept. of Physics, University of Utah Collaborators: Xiao-Liang Qi and Shou-Cheng Zhang

Ch27 ANALYSIS OF BIOLOGICAL MATERIALS AND XENOBIOTICS

Manahan, Stanley E. "ANALYSIS OF BIOLOGICAL MATERIALS AND XENOBIOTICS" Environmental ChemistryBoca Raton: CRC Press LLC,200027ANALYSIS OF BIOLOGICALMATERIALS AND XENOBIOTICS__________________________27.1.INTRODUCTIONAs defined in Chapter 22 a xenobiotic species is one that is foreign to living systems. Common examples include heavy metals, such as lead, which serve no physiologic function, and synthetic organic compounds that are not made in nature. Exposure of organisms to xenobiotic materials is a very important consideration in environmental and toxicological chemistry. Therefore, the determination of exposure by various analytical techniques is one of the more crucial aspects of environmental chemistry.This chapter deals with the determination of xenobiotic substances in biological materials. Although such substances can be measured in a variety of tissues, the greatest concern is their presence in human tissues and other samples of human origin. Therefore, the methods described in this chapter apply primarily to exposed human subjects. They are essentially identical to methods used on other animals and, in fact, most were developed through animal studies. Significantly different tech-niques may be required for plant or microbiological samples.The measurement of xenobiotic substances and their metabolites in blood, urine, breath, and other samples of biological origin to determine exposure to toxic substances is called biological monitoring. Comparison of the levels of analytes measured with the degree and type of exposure to foreign substances is a crucial aspect of toxicological chemistry. It is an area in which rapid advances are being made. For current information regarding this area, the reader is referred to excellent reviews of the topic,1,2 and several books on biological monitoring such as those by Angerer, Draper, Baselt, and Kneip and coauthors as listed in the back of this chapter under “Supplementary References,” are available as well.The two main approaches to workplace monitoring of toxic chemicals are workplace monitoring, using samplers that sample xenobiotic substances from workplace air, and biological monitoring. Although the analyses are generally much more difficult, biological monitoring is a much better indicator of exposure becauseit measures exposure to all routes—oral and dermal as well as inhalation—and it gives an integrated value of exposure. Furthermore, biological monitoring is very useful in determining the effectiveness of measures taken to prevent exposure, such as protective clothing and hygienic measures.27.2.INDICATORS OF EXPOSURE TO XENOBIOTICSThe two major considerations in determining exposure to xenobiotics are the type of sample and the type of analyte. Both of these are influenced by what happens to a xenobiotic material when it gets into the body. For some exposures, the entry site composes the sample. This is the case, for example, in exposure to asbestos fibers in the air, which is manifested by lesions to the lung. More commonly, the analyte may appear at some distance from the site of exposure, such as lead in bone that was originally taken in by the respiratory route. In other cases the original xenobiotic is not even present in the analyte. An example of this is methemoglobin in blood, the result of exposure to aniline absorbed through the skin.The two major kinds of samples analyzed for xenobiotics exposure are blood and urine. Both of these kinds of samples are analyzed for systemic xenobiotics, which are those that are transported in the body and metabolized in various tissues. Xeno-biotic substances, their metabolites, and their adducts are absorbed into the body and transported through it in the bloodstream. Therefore, blood is of unique importance as a sample for biological monitoring. Blood is not a simple sample to process, and subjects often object to the process of taking it. Upon collection, blood may be treated with an anticoagulant, usually a salt of ethylenediaminetetraacetic acid (EDTA), and processed for analysis as whole blood. It may also be allowed to clot and be centrifuged to remove solids; the liquid remaining is blood serum.Recall from Chapter 22 that as the result of Phase 1 and Phase 2 reactions, xenobiotics tend to be converted to more polar and water soluble metabolites. These are eliminated with the urine, making urine a good sample to analyze as evidence of exposure to xenobiotic substances. Urine has the advantage of being a simpler matrix than blood and one that subjects more readily give for analysis. Other kinds of samples that may be analyzed include breath (for volatile xenobiotics and volatile metabolites), air or nails (for trace elements, such as selenium), adipose tissue (fat), and milk (obviously limited to lactating females). Various kinds of organ tissue can be analyzed in cadavers, which can be useful in trying to determine cause of death by poisoning.The choice of the analyte actually measured varies with the xenobiotic substance to which the subject has been exposed. Therefore, it is convenient to divide xenobiotic analysis on the basis of the type of chemical species determined. The most straightforward analyte is, of course, the xenobiotic itself. This applies to elemental xenobiotics, especially metals, which are almost always determined in the elemental form. In a few cases organic xenobiotics can also be determined as the parent compound. However, organic xenobiotics are commonly metabolized to other products by Phase 1 and Phase 2 reactions. Commonly, the Phase 1 reaction product is measured, often after it is hydrolyzed from the Phase 2 conjugate, using enzymes or acid hydrolysis procedures. Thus, for example, trans,trans-muconic acid can be measured as evidence of exposure to the parent compound benzene. In other cases aPhase 2 reaction product is measured, for example, hippuric acid determined as evidence of exposure to toluene. Some xenobiotics or their metabolites form adducts with endogenous materials in the body, which are then measured as evidence of exposure. A simple example is the adduct formed between carbon monoxide and hemoglobin, carboxyhemoglobin. More complicated examples are the adducts formed by the carcinogenic Phase 1 reaction products of polycyclic aromatic hydro-carbons with DNA or hemoglobin. Another class of analytes consists of endogenous substances produced upon exposure to a xenobiotic material. Methemoglobin formed as a result of exposure to nitrobenzene, aniline, and related compounds is an example of such a substance which does not contain any of the original xenobiotic material. Another class of substance causes measurable alterations in enzyme activity. The most common example of this is the inhibition of acetylcholinesterase enzyme by organophosphates or carbamate insecticides.27.3.DETERMINATION OF METALSDirect Analysis of MetalsSeveral biologically important metals can be determined directly in body fluids, especially urine, by atomic absorption. In the simplest cases the urine is diluted with water or with acid and a portion analyzed directly by graphite furnace atomic absorption, taking advantage of the very high sensitivity of that technique for some metals. Metals that can be determined directly in urine by this approach include chromium, copper, lead, lithium, and zinc. Very low levels of metals can be measured using a graphite furnace atomic absorption technique, and Zeeman back-ground correction with a graphite furnace enables measurement of metals in samples that contain enough biological material to cause significant amounts of “smoke”during the atomization process, so that ashing the samples is less necessary.A method has been published for the determination of a variety of metals in diluted blood and serum using inductively coupled plasma atomization with mass spectrometric detection.3 Blood was diluted 10-fold and serum 5-fold with a solution containing ammonia, Triton X-100 surfactant, and EDTA. Detection limits adequate for measurement in blood or serum were found for cadmium, cobalt, copper, lead, rubidium, and zinc.Metals in Wet-Ashed Blood and UrineSeveral toxicologically important metals are readily determined from wet-ashed blood or urine using atomic spectroscopic techniques. The ashing procedure may vary, but always entails heating the sample with strong acid and oxidant to dryness and redissolving the residue in acid. A typical procedure is digestion of blood or urine for cadmium analysis, which consists of mixing the sample with a comparable volume of concentrated nitric acid, heating to a reduced volume, adding 30% hydrogen peroxide oxidant, heating to dryness, and dissolving in nitric acid prior to measurement by atomic absorption or emission. Mixtures of nitric, sulfuric, and perchloric acid are effective though somewhat hazardous media for digesting blood, urine, or tissue. Wet ashing followed by atomic absorption analysis can be used forthe determination in blood or urine of cadmium, chromium, copper, lead, manganese, and zinc, among other metals. Although atomic absorption, especially highly sensitive graphite furnace atomic absorption, has long been favored for measuring metals in biological samples, the multielement capability and other advantages of inductively coupled plasma atomic spectroscopy has led to its use for determining metals in blood and urine samples.4Extraction of Metals for Atomic Absorption AnalysisA number of procedures for the determination of metals and biological samples call for the extraction of the metal with an organic chelating agent in order to remove interferences and concentrate the metal to enable detection of low levels. The urine or blood sample may be first subjected to wet ashing to enable extraction of the metal. Beryllium from an acid-digested blood or urine sample may be extracted by acetylacetone into methylisobutyl ketone prior to atomic absorption analysis. Virtually all of the common metals can be determined by this approach using appropriate extractants.The availability of strongly chelating extracts for a number of metals has lead to the development of procedures in which the metal is extracted from minimally treated blood or urine, then quantified by atomic absorption analysis. The metals for which such extractions can be used include cobalt, lead, and thallium extracted into organic solvent as the dithiocarbamate chelate, and nickel extracted into methyl-isobutyl ketone as a chelate formed with ammonium pyrrolidinedithiocarbamate.Methods for several metals or metalloids involve conversion to a volatile form. Arsenic, antimony, and selenium can be reduced to their volatile hydrides, AsH3, SbH3, and H2Se, repectively, which can be determined by atomic absorption or other means. Mercury is reduced to volatile mercury metal, which is evolved from solution and measured by cold vapor atomic absorption.27.4.DETERMINATION OF NONMETALS AND INORGANICCOMPOUNDSRelatively few nonmetals require determination in biological samples. One important example is fluoride, which occurs in biological fluids as the fluoride ion, F-. In some cases of occupational exposure or exposure through food or drinking water, excessive levels of fluoride in the body can be a health concern. Fluoride is readily determined potentiometrically with a fluoride ion-selective electrode. The sample is diluted with an appropriate buffer and the potential of the fluoride electrode measured very accurately vs. a reference electrode, with the concentration calculated from a calibration plot. Even more accurate values can be obtained by the use of standard addition in which the potential of the electrode system in a known volume of sample is read, a measured amount of standard fluoride is added, and the shift in potential is used to calculate the unknown concentration of fluoride.Another nometal for which a method of determining biological exposure would be useful is white phosphorus, the most common and relatively toxic elemental form. Unfortunately, there is not a chemical method suitable for the determination of exposure to white phosphorus that would distinguish such exposure from relativelyhigh background levels of organic and inorganic phosphorus in body fluids and tissues.Toxic cyanide can be isolated in a special device called a Conway microdiffus-ion cell by treatment with acid, followed by collection of the weakly acidic HCN gas that is evolved in a base solution. The cyanide released can be measured spectropho-tometrically by formation of a colored species.Carbon monoxide is readily determined in blood by virtue of the colored car-boxyhemoglogin that it forms with hemoglobin. The procedure consists of measur-ing the absorbances at wavelengths of 414, 421, and 428 nm of the blood sample, a sample through which oxygen has been bubbled to change all the hemoglobin to the oxyhemoglobin form, and a sample through which carbon monoxide has been bubbled to change all the hemoglobin to carboxyhemoglobin. With the appropriate calculations, a percentage conversion to carboxyhemoglobin can be obtained.27.5.DETERMINATION OF PARENT ORGANIC COMPOUNDSA number of organic compounds can be measured as the unmetabolized com-pound in blood, urine, and breath. In some cases the sample can be injected along with its water content directly into a gas chromatograph. Direct injection is used for the measurement of acetone, n-butanol, dimethylformamide, cyclopropane, halo-thane, methoxyflurane, diethyl ether, isopropanol, methanol, methyl n-butyl ketone, methyl chloride, methylethyl ketone, toluene, trichloroethane, and trichloroethylene.For the determination of volatile compounds in blood or urine, a straightforward approach is to liberate the analyte at an elevated temperature allowing the volatile compound to accumulate in headspace above the sample followed by direct injection of headspace gas into a gas chromatograph. A reagent such as perchloric acid may be added to deproteinize the blood or urine sample and facilitate release of the volatile xenobiotic compound. Among the compounds determined by this approach are acetaldehyde, dichloromethane, chloroform, carbon tetrachloride, benzene, trichloroethylene, toluene, cyclohexane, and ethylene oxide. The use of multiple detectors for the gas chromatographic determination of analytes in headspace increases the versatility of this technique and enables the determination of a variety of physiologically important volatile organic compounds.5Purge-and-trap techniques in which volatile analytes are evolved from blood or urine in a gas stream and collected on a trap for subsequent chromatographic analysis have been developed. Such a technique employing gas chromatographic separation and Fourier transform infrared detection has been described for a number of volatile organic compounds in blood.627.6.MEASUREMENT OF PHASE 1 AND PHASE 2 REACTIONPRODUCTSPhase 1 Reaction ProductsFor a number of organic compounds the most accurate indication of exposure is to be obtained by determining their Phase 1 reaction products. This is because many compounds are metabolized in the body and don't show up as the parent compound.And those fractions of volatile organic compounds that are not metabolized may be readily eliminated with expired air from the lungs and may thus be missed. In cases where a significant fraction of the xenobiotic compound has undergone a Phase 2reaction, the Phase 1 product may be regenerated by acid hydrolysis.One of the compounds commonly determined as its Phase 1 metabolite is benzene,7 which undergoes the following reactions in the body (see Chapter 23,Section 23.4):OHH Benzene epoxide Benzene oxepin O H HOH + {O}Enzymatic OPhenolNonenzymatic rearrangement(27.6.1)Therefore, exposure to benzene can be determined by analysis of urine for phenol.Although a very sensitive colorimetric method for phenol involving diazotized p -nitroaniline has long been available, gas chromatographic analysis is now favored.The urine sample is treated with perchloric acid to hydrolyze phenol conjugates and the phenol is extracted into diisopropyl ether for chromatographic analysis. Two other metabolic products of benzene, trans ,trans -muconic acid 8 and S-phenyl mercapturic acid .9 are now commonly measured as more specific biomarkers of benzene exposure.Trans ,trans -muconic acid C C C C C C OH OHO H H Insecticidal carbaryl undergoes the following metabolic reaction: O C N CH 3H O Enzymatic processes OH Carbaryl 1-Naphthol+ other products(27.6.2)Therefore, the analysis of 1-naphthol in urine indicates exposure to carbaryl. The 1-naphthol that is conjugated by a Phase 2 reaction is liberated by acid hydrolysis, then determined spectrophotometrically or by chromatographyIn addition to the examples discussed above, a number of other xenobiotics are measured by their phase one reaction products. These compounds and their metabo-lites are listed in Table 27.1. These methods are for metabolites in urine. Normally the urine sample is acidified to release the Phase 1 metabolites from Phase 2conjugates that they might have formed and, except where direct sample injection isemployed, the analyte is collected as vapor or extracted into an organic solvent. In some cases the analyte is reacted with a reagent that produces a volatile derivative that is readily separated and detected by gas chromatography.Phase 2 Reaction ProductsHippuric acids, which are formed as Phase 2 metabolic products from toluene,the xylenes, benzoic acid, ethylbenzene, and closely related compounds, can be determined as biological markers of exposure. The formation of hippuric acid from toluene is shown in Chapter 23, Figure 23.2, and the formation of 4-methylhippuric acid from p -xylene is shown below:C H HH H 3C{O}C H H OH H 3C C OOH H 3C C O N H C H C O OH H 3C 2oxidation , Phase I +(27.6.3)Other metabolites that may be formed from aryl solvent precursors include mandelic acid and phenylgloxylic acid.Exposure to toluene can be detected by extracting hippuric acid from acidified urine into diethyl ether/isopropanol and direct ultraviolet absorbance measurement of the extracted acid at 230 nm. When the analysis is designed to detect the xylenes,ethylbenzene, and related compounds, several metabolites related to hippuric acid may be formed and the ultraviolet spectrometric method does not give the required specificity. However, the various acids produced from these compounds can be extracted from acidified urine into ethyl acetate, derivatized to produce volatile species, and quantified by gas chromatography.A disadvantage to measuring toluene exposure by hippuric acid is the production of this metabolite from natural sources, and the determination of tolulylmercapturic acid is now favored as a biomarker of toluene exposure.10 An interesting sidelight is that dietary habits can cause uncertainties in the measurement of xenobiotic metabolites. An example of this is the measurement of worker exposure to 3-chloropropene by the production of allylmercapturic acid.11 This metabolite is also produced by garlic, and garlic consumption by workers was found to be a confound-ing factor in the method. Thiocyanate monitored as evidence of exposure to cyanide is increased markedly by the consumption of cooked cassava!MercapturatesMercapturates are proving to be very useful Phase 2 reaction products for meas-uring exposure to xenobiotics, especially because of the sensitive determination ofTable 27.1. Phase 1 Reaction Products of Xenobiotics DeterminedParent compound Metabolite Method of analysisCyclohexane Cyclohexanol Extraction of acidified, hydrolyzed urinewith dichloromethane followed by gaschromatographyDiazinone Organic phos-Colorimetric determination of phosphatesphatesp-Dichloro-2,5-Dichloro-Extraction into benzene, gas chromato-benzene phenol graphic analysisDimethylform-Methylformamide Gas chromatography with direct sample amide introductionDioxaneβ-hydroxyethoxy-Formation of volatile methyl ester, gasacetic acid chromatographyEthybenzene Mandelic acid and Extraction of acids, formation of volatilerelated aryl acids derivatives, gas chromatography Ethylene glycol Methoxyacetic Extracted with dichloromethane, converted monomethyl acid to volatile methyl derivative, gas chrom-ether atographyFormaldehyde Formic acid Gas chromatography of volatile formicacid deriviativeHexane2,5-Hexanedione Gas chromatography after extractionwith dichloromethanen-heptane2-Heptanone, val-Measurement in urine by GC/MSerolactone, 2,5-heptanedioneIsopropanol Acetone Gas chromatography following extractionwith methylethyl ketoneMalathion Organic phos-Colorimetric determination of phosphatesphatesMethanol Formic acid Gas chromatography of volatile formic acidderivativeMethyl bromide Bromide ion Formation of volatile organobromine com-pounds, gas chromatography Nitrobenzene p-Nitrophenol Gas chromatography of volatile derivative Parathion p-Nitrophenol Gas chromatography of volatile derivative Polycyclic aryl1-Hydroxy-HPLC of urinehydrocarbons pyreneTable 27.1. (Cont.)StyreneMandelic acid Extraction of acids, formation of volatile derivatives, gas chromatography Tetrachloro-Trichloroacetic Extracted into Pyridine and measured color-ethyleneacid imetrically Trichloroethane TrichloroaceticExtracted into Pyridine and measured color-acid imetricallyTrichloro-Trichloroacetic Extracted into Pyridine and measured color-ethylene acid imetrically these substances by HPLC separation, and fluorescence detection of their o -phthal-dialdehyde derivatives. In addition to toluene mentioned above, the xenobiotics for which mercapturates may be monitored include styrene, structurally similar to toluene; acrylonitrile; allyl chloride; atrazine; butadiene; and epichlorohydrin.The formation of mercapturates or mercapturic acid derivatives by metabolism of xenobiotics is the result of a Phase 2 conjugation by glutathione. Glutathione (commonly abbreviated GSH) is a crucial conjugating agent in the body. This compound is a tripeptide, meaning that it is composed of three amino acids linked together. These amino acids and their abbreviations are glutamic acid (Glu), cysteine (Cys), and glycine (Gly). The formula of glutathione may be represented as illustrated in Figure 27.1, where the SH is shown specifically because of its crucialHX R Readily excreted mer-capturic acid conjugateCysteine conjugate R X S CC C O OH HH CH 3H C R X S C C C O OH H H H H NH Loss of glutamyl and glycinyl Glutathione conjugate excretion Xenobiotic Glutathione Glutathione transferase Glu Cys GlySX R+ Glu Cys Gly Figure 27.1. Glutathione conjugate of a xenobiotic species (HX-R) followed by formation of glutathione and cysteine conjugate intermediates (which may be excreted in bile) and acetylation to form readily excreted mercapturic acid conjugate.role in forming the covalent link to a xenobiotic compound. Glutathione conjugate may be excreted directly, although this is rare. More commonly, the GSH conjugate undergoes further biochemical reactions that produce mercapturic acids (compounds with N-acetylcysteine attached) or other species. The specific mercapturic acids can be monitored as biological markers of exposure to the xenobiotic species that result in their formation. The overall process for the production of mercapturic acids as applied to a generic xenobiotic species, HX-R (see previous discussion), is illus-trated in Figure 27.1.27.7.DETERMINATION OF ADDUCTSDetermination of adducts is often a useful and elegant means of measuring exposure to xenobiotics. Adducts, as the name implies, are substances produced when xenobiotic substances add to endogenous chemical species. The measurement of carbon monoxide from its hemoglobin adduct was discussed in Section 27.4. In general, adducts are produced when a relatively simple xenobiotic molecule adds to a large macromolecular biomolecule that is naturally present in the body. The fact that adduct formation is a mode of toxic action, such as occurs in the methylation of DNA during carcinogenesis (Chapter 22, Section 22.8), makes adduct measurement as a means of biological monitoring even more pertinent.Adducts to hemoglobin are perhaps the most useful means of biological monitor-ing by adduct formation. Hemoglobin is, of course, present in blood, which is the most accurate type of sample for biological monitoring. Adducts to blood plasma albumin are also useful monitors and have been applied to the determination of exposure to toluene diisocyanate, benzo(a)pyrene, styrene, styrene oxide, and aflatoxin B1. The DNA adduct of styrene oxide has been measured to indicate exposure to carcinogenic styrene oxide.13One disadvantage of biological monitoring by adduct formation can be the relatively complicated prodedures and expensive, specialized instruments requried. Lysing red blood cells may be required to release the hemoglobin adducts, derivatization may be necessary, and the measurements of the final analyte species can require relatively sophisticated instrumental techniques. Despite these complex-ities, the measurement of hemoglobin adducts is emerging as a method of choice for a number of xenobiotics including acrylamide, acrylonitrile, 1,3-butadiene, 3,3' dichlorobenzidine, ethylene oxide, and hexahydrophthalic anhydride.27.8.THE PROMISE OF IMMUNOLOGICAL METHODSAs discussed in Chapter 25, Section 25.5, immunoassay methods offer distinct advantages in specificity, selectivity, simplicity, and costs. Although used in simple test kits for blood glucose and pregnancy testing, immunoassay methods have been limited in biological monitoring of xenobiotics, in part because of interferences in complex biological systems. Because of their inherent advantages, however, it can be anticipated that immunoassays will grow in importance for biological monitoring of xenobiotics.14 As an example of such an application, polychlorinated biphenyls (PCBs) have been measured in blood plasma by immunoassay.15In addition to immunoassay measurement of xenobiotics and their metabolites, immunological techniques can be used for the separation of analytes from complexbiological samples employing immobilized antibodies. This approach has been used to isolate aflatoxicol from urine and enable its determination along with aflatoxins B1, B2, G1, G2, M1, and Q1 using high-performance liquid chromatography and post-column derivatization/fluorescence detection.16 A monoclonal antibody reactive with S-phenylmercapturic acid, an important Phase 2 reaction product of benzene resulting from glutathione conjugation, has been generated from an appropriate hapten-protein conjugate. The immobilized antibody has been used in a column to enrich S-phenylmercapturic acid from the urine of workers exposed to benzene.17 Many more such applications can be anticipated in future years.LITERATURE CITED1.Draper, William M., Kevin Ashley, Clifford R. Glowacki, and Paul R. Michael,“Industrial Hygiene Chemistry: Keeping Pace with Rapid Change in the Workplace,” Analytical Chemistry, 71, 33R-60R (1999). A comprehensive review of this topic is published every two years in Analytical Chemistry.2.Atio, A., “Special Issue: Biological Monitoring in Occupational andEnvironmental Health,” Science of the Total Environment, 199, 1-226 (1997). 3.Barany, Ebba, Ingvar A. Bergdahl, Andrejs Schutz, Staffan Skerfving, andAgneta Oskarsson, “Inductively Coupled Plasma Mass Spectrometry for Direct Multielement Analysis of Diluted Human Blood and Serum,” Journal of Analytical Atomic Spectroscopy, 12, 1005-1009 (1997).4.Paschal, Daniel C., Bill G. Ting, John C. Morrow, James L. Pirkle, Richard J.Jackson, Eric J. Sampson, Dayton T. Miller, and Kathleen L. Caldwell, “Trace Metals in Urine of United States Residents: Reference Range Concentrations,”Environmental Research, 76, 53-59 (1998).5.Schroers, H.-J. and E. Jermann, “Determination of Physiological Levels ofVolatile Organic Compounds in Blood Using Static Headspace Capillary Gas Chromatography with Serial Triple Detection, Analyst, 123, 715-720 (1998).6.Ojanpera, Ilkka, Katja Pihlainen, and Erkki Vuori, “Identification Limits forVolatile Organic Compounds in the Blood by Purge-and-Trap GC-FTIR,”Journal of Analytical Toxicology, 22, 290-295 (1998).7.Agency for Toxic Substances and Disease Registry, U. S. Department of Healthand Human Services, Toxicological Profile for Benzene, CD/ROM Version, CRC Press/Lewis Publishers, Boca Raton, FL, 1999.8.Scherer, Gerhard, Thomas Renner, and Michael Meger, “Analysis and Evaluationof Trans,trans-muconic Acid as a Biomarker for Benzene Exposure,” Journal of Chromatography, B: Biomedical Science Applications, 717, 179-199 (1998).9.Boogaard, Pieter J. and Nico J. Van Sittert, “Suitability of S-phenyl MercapturicAcid and Trans-trans-muconic acid as Biomarkers for Exposure to Low Concentrations of Benzene,” Environmental Health Perspectives Supplement, 104, 1151-1157 (1996).。

Anthocyanins Exist in the Circulation Primarily as Metabolites in Adult

Human Nutrition and MetabolismAnthocyanins Exist in the Circulation Primarily as Metabolites in Adult Men 1Colin D.Kay,*†2Giuseppe (Joe)Mazza,†and Bruce J.Holub**Department of Human Biology and Nutritional Sciences,University of Guelph,ON,Canada and †Agriculture and Agri-Food Canada,Paciﬁc Agri-Food Research Centre,Summerland,BC,CanadaABSTRACT Anthocyanins are reported to have many “health promoting”properties;however,despite numerous reports of their bioactivities,their absorption and metabolism in humans are poorly understood.The objective of this research was to detail the pharmacokinetic parameters of anthocyanins after the administration of a 721-mg oral dose of cyanidin 3-glycosides from chokeberry extract to human subjects.Solid-phase extraction,prepara-tive-HPLC,preparative-TLC,HPLC-diode array detection,HPLC-MS,and NMR were utilized to isolate,identity,and quantify anthocyanins in 0-to 7-h (0,1,2,3,4,5,6,7h)serum and 0-to 24-h urine samples (total individual urine voids over 24h).The cumulative concentration of total anthocyanins (parent and metabolites)detected in the serum (0–7h)was 376.65Ϯ16.20(nmol ⅐h)/L (area under the concentration time curve),reaching a maximum concentration (C max ϭ96.08Ϯ6.04nmol/L)within 2.8h.The parent anthocyanins represented only 32.0%[120.63Ϯ2.85(nmol ⅐h)/L]of the total anthocyanins detected with 68.0%[256.02Ϯ5.23(nmol ⅐h)identiﬁed as conjugated metabolites.Additionally,the total urinary excretion of anthocyanins over 24h was 1071.54Ϯ375.46␮g,reaching a maximal rate of excretion (R max ϭ202.74Ϯ85.06␮g/h)at 3.72Ϯ0.83h.Parallel to the serum data,only 32.5%(347.85Ϯ60.61␮g)of the anthocyanins excreted in the urine (total 24h)were the parent compounds with 67.5%(723.69Ϯ92.59␮g)occurring as conjugated metabolites.The metabolites were identiﬁed as glucuronidated and methylated derivatives of the parent cyanidin 3-glycosides.The above results indicate that cyanidin 3-glycosides are rapidly absorbed and metabolized extensively following a moderate-to-high oral dose in humans.J.Nutr.135:2582–2588,2005.KEY WORDS:●anthocyanins●cyanidin 3-glycosides●pharmacokinetics●metabolitesWithin the last decade,many studies have focused on the potential biological activities or health effects of anthocyanins in humans (1–3).Although there is a great deal of evidence indicating the bioactivity of anthocyanins,very little progress has been made in establishing the pharmacokinetics of these compounds,with aspects such as absorption and metabolism left essentially unstudied.Previously,it was reported that an-thocyanins were poorly absorbed and circulated in the blood exclusively as unmetabolized parent glycosides (4–6).It is only recently that researchers have begun to suggest that anthocyanins are metabolized;however,the identiﬁcation of derived metabolites has been limited as a result of their diver-sity and low concentrations in the blood.In our previous investigation focusing on identifying an-thocyanin metabolites in human serum and urine (7),subjects were fed ϳ1.2g of cyanidin 3-glycosides from chokeberries,which resulted in the identiﬁcation of glucuronide and methyl derivatives.The aim of the present investigation was to de-termine the pharmacokinetics of the cyanidin 3-glycosides in humans as well as to establish the extent of their metabolic fate after a lower,more realistic anthocyanin dose (721mg).Speciﬁcally,the chokeberry extract was chosen because itcontained exclusively cyanidin 3-glycosides,thereby permit-ting the monitoring of its metabolites and their pharmacoki-netics.Subsequent investigations will be required to identify the biological activity of these metabolites.SUBJECTS AND METHODSSubjects.Healthy male volunteers (n ϭ3;40Ϯ14.2y old)participated in the cyanidin 3-glycoside consumption intervention.Subjects had a mean BMI of 28.3Ϯ1.6kg/m 2and had no clinical disease as determined using a medical history questionnaire.Subjects were instructed to consume an essentially anthocyanin-free diet (no fruit or vegetables,including foods colored with red or blue dyes)for 2d before the study;they were also asked to avoid taking aspirin or anti-inﬂammatory medications,and antioxidant or herbal supple-ments for 2wk before the investigation.The major constituents of the anthocyanin-free washout diet were milk,tuna,white bread,chicken,and white pliance with the anthocyanin-free diet was monitored using food diaries and conﬁrmed in baseline samples via reverse-phase HPLC (RP-HPLC)3with diode array detection1Supported by the Heart &Stroke Foundation of Ontario (HSFO)and a Natural Sciences and Engineering Research Council of Canada (NSERC)post-doctoral fellowship (C.D.K.).2To whom correspondence should be addressed.E-mail:cdk14@.3Abbreviations used:C-3-ara,cyanidin 3-arabinoside;C-3-gal,cyanidin 3-galactoside;C-3-glu,cyanidin 3-glucoside;C-3-xyl,cyanidin 3-xyloside;CD 3OD,Methanol-d4;CF 3COOD,triﬂuoroacetic acid-d;C max ,maximum concen-tration;DAD,diode array detector;E 440/E max ,ratio of the absorbance intensity at 440nm vs.the maximum absorbance intensity;ESI-MS,electrospray ionization MS;m /z,mass to charge ratio;P,peak;PCA,perchloric acid;P-3-gal,peonidin 3-galactoside;Prep-HPLC,preparative HPLC;Rf,reference value;R max ,maxi-mum rate of urinary excretion;RP-HPLC,reverse phase HPLC;Rt,retention time;0022-3166/05$8.00©2005American Society for Nutrition.Manuscript received 31March 2005.Initial review completed 2May 2005.Revision accepted 27August 2005.2582by guest on December 14, 2010 Downloaded from(DAD).The diets met recommended dietary allowances for macro-nutrients,and no energy restrictions were imposed.All subjects gave written informed consent before the commencement of the investi-gation and experimental procedures followed were in accordance with the Helsinki Declaration of 1975as revised in 1983.Study design.Subjects were admitted to the clinic (Okanagan Clinical Laboratory;Penticton,BC)on the morning of the study dates after fasting (12h,24h no alcohol).Baseline urine samples (ﬁrst void,t ϭ0)were taken in the morning of each study date along with individual urine voids (total volume)over the next 24h (t ϭ0,2,4,6,8,10,12,and 24h).Immediately after baseline (t ϭ0)blood sampling,the volunteers consumed 7.1g of encapsulated (gel caps)chokeberry extract with 250mL of water.The extract contained 4cyanidin 3-glycosides (721.4mg):491.0mg cyanidin 3-galactoside (C-3-gal),175.3mg cyanidin 3-arabinoside (C-3-ara),27.8mg cya-nidin 3-xyloside (C-3-xyl),and 27.3mg cyanidin 3-glucuoside (C-3-glu),as determined by HPLC-DAD (Table 1;Fig.1).Subsequent blood samples were taken at t ϭ1,2,and 3h postconsumption of the extract.The experiment was repeated at a later date (30d wash-out)following the protocol outlined above with one variation.On d 2,blood samples were taken at t ϭ0,3,4,5h and on d 3,at t ϭ0,5,6,7h (n ϭ3;levels ϭ0,1,2,3,4,5,6,7).The sampling regimen was necessary to acquire the volume of blood needed for the analysis.Individual urine samples were also collected over 24h (levels ϭ0,2,4,6,8,10,12,24)at each visit (n ϭ3ϫ3repetitions;y ϭ9replicates).After consumption of the extract,subjects were instructed to consume 250mL of water every hour for 5h with subsequent ad libitum consumption.An anthocyanin-free lunch and dinner was provided for the subjects at 4and 8h postconsumption of the extract.Blood samples (ϳ20mL)were drawn by venipuncture from a brachial vein into 10-mL evacuated glass tubes (2tubes/time point)(Vacutainer;Becton Dickinson).The blood samples were allowed to clot at room temperature for 30min.Samples were then immediately centrifuged (1000ϫg)for 15min at 5°C to recover the serum.Urine samples were acidiﬁed with 20␮L of 12mol/L HCl/mL urine uponSPE,solid phase extraction;t ϭ0,baseline;t 1/2,elimination half-life;t 1/2a ,absorption half-life;t max ,time point at which maximal serum concentration oc-curs;t maxR ,time point at which maximal rate of urinary excretion occurs;TFA,triﬂuoroacetic acid;UV-vis,UV-visible.TABLE 1Identiﬁcation of anthocyanins and anthocyanin metabolites in human urine and serum after the consumptionof 721mg of cyanidin 3-glycosides 1–3Peak Anthocyanin/identityRtMWparent/daughterfragmentAbsorption spectraTLC (Rf)␭max E 440/E max (as %)1Cyanidin 3-galactoside 23.8449/287280,517310.362Cyanidin glucuronide 27.6463/287280,517310.383Cyanidin 3-arabinoside 29.6419/287280,517310.454Peonidin 3-galactoside33.4463/301280,517310.425Methylated cyanidin glucuronide 36.3477/301280,514370.396Methylated cyanidin glucuronide 37.0477/301280,515320.44Characteristics of anthocyanin standardsCyanidin 3-galactoside 4,523.8449/287280,517310.36Cyanidin 3-arabionside 429.6419/287280,517310.45Cyanidin 3-xyloside 439.0419/287280,517290.48Cyanidin 3-glucoside 4,527.3449/287280,517370.40Cyanidin 543.6287280,526230.80Peonidin 550.8301280,528260.841Identiﬁcation (retention time and absorption spectra)based on Agilent HPLC-DAD.2Identiﬁcation (MW)based on Waters microcapillary HPLC-MS.3Comparisons made between serum and urinary samples using HPLC-DAD (Fig.1,C and D )indicated that the serum peaks matched both retention time and UV-vis spectrum with the peaks identiﬁed as anthocyanins in the urine,and were therefore regarded as the same compounds.4Data based on analysis of puriﬁed anthocyanin from chokeberry extract.5Data based on analysis of purchased standard(Extrasynthese).FIGURE 1Chromatograms of anthocyanins in baseline human urine sample (A ),chokeberry extract (B ),typical serum sample (C ),and typical urine sample (D ).HPLC analysis as outlined in the methods.The identiﬁcation of each peak represented above (peaks 1–6)is given in Table 1.Urine and serum pharmacokinetic data of each compound represented by peaks 1–6are given in Tables 2and 3,respectively.PHARMACOKINETICS OF CYANIDIN 3-GLYCOSIDES 2583by guest on December 14, 2010Downloaded fromcollection.The serum and urine were stored at Ϫ80°C after removal/collection.Materials/reagents.The chokeberry extract (no.74190,lot L18010)was purchased from Artemis International.The anthocya-nin standards,cyanidin 3-glucoside chloride,cyanidin 3-galactoside chloride (ideain chloride),peonidin 3-glucoside chloride,cyanidin chloride,and peonidin chloride were purchased from Extrasynthese.Formic acid (Fisher Scientiﬁc),hydrochloric acid (HCl),triﬂuoro-acetic acid (TFA),and glacial acetic acid (DH)were all reagent grade;all solvents used for HPLC analysis were HPLC grade.Anthocyanin analysis.The chokeberry extract,serum,and urine anthocyanins were quantiﬁed via HPLC-DAD.Individual serum sam-ples were collected every hour for 7h (n ϭ3)and individual urine voids were collected (separately)over a 24-h period (n ϭ3ϫ3repetitions)as detailed in the study design.Every serum and urine sample was analyzed and quantiﬁed individually via HPLC-DAD before pooling for identiﬁcation purposes.After quantiﬁcation,urine samples were pooled for puriﬁcation (XAD adsorption chromatogra-phy),isolation [preparative (prep)-HPLC and prep-TLC],and iden-tiﬁcation (HPLC-MS;HPLC-DAD;TLC;NMR).Identiﬁcation of the anthocyanins was based on the matching of molecular weight (parent and daughter fragments),retention time (Rt),␭maxvis ,E 440/E max ,and reference (Rf)values with those of available anthocyanin standards as well as isolated chokeberry anthocyanins (Table 1).TLC data (post-acid hydrolysis)were utilized for additional conﬁrmation when considered necessary.Peaks lacking absorption maxima in the 280and 520nm range were not considered anthocyanin metabolites,and no attempt was made to identify these unknown compounds.Selected pharmacokinetic variables were determined for the identi-ﬁed compounds from the initial HPLC quantitative results (Table 2and 3;Figs.2and 3).Methods of extraction [C 18solid-phase extraction (SPE)]were modiﬁed from Kay et al.(7)and Tsuda et al.(8).The anthocyanins were extracted from biological ﬂuids before HPLC analysis using disposable SPE C 18cartridges (Supelclean ENVI-186mL 2000mg;Sigma;lot #SP2419C).Unﬁltered blood serum (4mL)or 2–10mL of unﬁltered acidiﬁed urine (2mL,t ϭ0–9h;or 10mL,t ϭ10–24h)were utilized for the extraction.Individual blood and urine samples (nonpooled)were extracted in duplicate,and each extract was in-jected into the HPLC column in duplicate (total of 4injections/sample)for quantitative HPLC analysis.Puriﬁcation of anthocyanins in pooled human urine samples (postquantitative HPLC analysis)was performed using Amberlite XAD-7polymeric adsorbent (Sigma;Lot #77H0157)before isolation of individual anthocyanin/metabolite peaks using prep-HPLC.The procedure was based on general methods as described by Markham (9).The column (50ϫ3.0cm)was ﬁlled with presoaked (24h EtOH:H 2O v:v)XAD-7resin (volume of 212cm 3)and loaded with 1L of unﬁltered acidiﬁed urine (pH Ϸ2.5).The column was drained under gravity,then washed with 500mL acidiﬁed H 2O (0.1%TFA),followed by 500mL MeOH:H 2O (30:70;0.1%TFA;ﬂow rate 10mL/min).Finally,the anthocyanin extract was eluted with 500mL of MeOH:H 2O (75:25;0.1%TFA;ﬂow rate 10mL/min)and evapo-rated.The anthocyanin-rich urine extract was then further puriﬁed via prep-HPLC.Analytical HPLC analysis was performed on an Agilent 1100series HPLC (Agilent Technologies)using a Zorbax SB C 18RP column (5␮m,4.6ϫ250mm)with a Supelguard LC-18guardTABLE 3Pharmacokinetic parameters of cyanidin 3-glycosides and corresponding metabolites in human serumafter the consumption of 721mg of cyanidin 3-glycosides 1PeakAnthocyanin/identityC max T max 2AUC t 1/2a 3nmol/Lh (nmol ⅐h)/L h 1Cyanidin 3-galactoside 23.36Ϯ 2.33 2.5(2–3)66.64Ϯ 3.27Ͻ1.352Cyanidin glucuronide 14.51Ϯ 4.04 2.0(2)90.93Ϯ 4.083Cyanidin 3-arabinoside 8.85Ϯ0.50 3.5(3–4)53.99Ϯ 2.43Ͻ1.674Peonidin 3-galactoside3.76Ϯ0.784.0(4)30.67Ϯ 1.125Methylated cyanidin glucuronide 12.81Ϯ0.42 2.5(2–3)34.42Ϯ 2.776Methylated cyanidin glucuronide 32.79Ϯ10.882.5(2–3)100.00Ϯ12.851Pharmacokinetic data are means ϮSD,n ϭ3,over 7h as quantiﬁed via HPLC-DAD (Table 1).2Values are medians (range).3t 1/2a :precise absorption half-lives for 2of the 3subjects could not be determined because there was only one serum sample (t ϭ1h)during the absorption phase.Therefore,as a result of the rapid absorption phase (Ͻ1h)for 2of the 3subjects,SDs cannot be speciﬁed.The absorption half-life values above are from 1subject only and it can be assumed that the mean absorption half-life for the 3subjects would be Ͻ1h.For a more accurate estimate of absorption additional half-life samples are required before the end of h 1.The t 1/2a value was determined using the method of residuals (14).TABLE 2Pharmacokinetic parameters of cyanidin 3-glycosides and corresponding metabolites in human urineafter the consumption of 721mg of cyanidin 3-glycosides 1PeakAnthocyanin/identityQuantityR maxt maxR t 1/2␮g␮g/h h h 1Cyanidin 3-galactoside 267.55Ϯ90.8248.74Ϯ14.80 3.39Ϯ1.14 3.72Ϯ0.422Cyanidin glucuronide 173.25Ϯ86.4639.47Ϯ20.77 3.17Ϯ0.86 3.63Ϯ0.563Cyanidin 3-arabinoside 80.30Ϯ31.3013.17Ϯ 4.68 2.94Ϯ1.39 4.05Ϯ0.824Peonidin 3-galactoside103.88Ϯ35.6319.27Ϯ7.74 3.39Ϯ1.14 4.28Ϯ0.595Methylated cyanidin glucuronide 111.06Ϯ60.6119.61Ϯ10.51 4.05Ϯ1.21 4.53Ϯ0.926Methylated cyanidin glucuronide 335.50Ϯ187.6666.63Ϯ37.134.05Ϯ1.214.39Ϯ0.831Pharmacokinetic data are means ϮSD,n ϭ9,over 24h as quantiﬁed via HPLC-DAD (Table 1).KAY ET AL.2584 by guest on December 14, 2010Downloaded fromcolumn (C 185␮m,4.6ϫ20mm;Supelco,Sigma-Aldrich).The following procedure was modiﬁed from previously published methods (7,10).Prep-HPLC separation of individual anthocyanins from pooled urine samples was performed on a Waters Chromatographic system (Waters)comprised of 3Model 510pumps,and a Model 490pro-grammable multiwavelength detector set at 525nm.The preparative column system (Waters PrepPak)consisted of 2Nova-Pak HR C 18radial compression cartridges (25ϫ100mm;6␮m,60Å;PrepPak Cartridge;Waters)with a Nova-Pak HR C 18guard insert (Waters).Injections were carried out on a manual injection port (Rheodyne)equipped with a 500-␮L injection loop.The column and injector were kept at ambient temperature,with an injection volume of 250–500␮L.The mobile phase consisted of 0.1%TFA in water (solvent A)and 100%MeOH (solvent B).The ﬂow rate was 15mL/min with an isocratic run of 80%A and 20%B.Peaks on the chromatogram corresponding to anthocyanins,as identiﬁed by spec-tral analysis (peaks detected at 525nm with ␭max 250–300and 500–550nm),were collected manually from the prep-HPLC column and concentrated using a rotary evaporator.The remaining H 2O was removed in a freeze-dryer and samples were sealed under nitrogen gas and stored at Ϫ80°C until further analysis.TLC procedures were based on methods described by Wagner and Bladt (11).Normal phase prep-TLC was utilized for the ﬁnal puriﬁ-cation of individual anthocyanins separated from the pooled urine samples (20ϫ20250-␮m silica gel Redi/plates;Analtech).The solvent system consisted of ethyl acetate,glacial acetic acid,formic acid,and H 2O (100:11:11:26).After the plates were developed,the anthocyanin bands were removed and dissolved in 5mL of MeOH containing 0.1%formic acid.The ﬁnal solution was ﬁltered through a 0.45Ϫ␮m polyvinylidene ﬂuoride syringe ﬁlter,evaporated in arotary evaporator,and brought to dryness in a freeze-dryer.The remaining extract was sealed under nitrogen gas and stored at Ϫ80°C until further analysis.For the postacid hydrolysis of anthocyanins for veriﬁcation of aglycones,0.20-mm silica gel 60analytical TLC plates (Macherey-Nagel;Batch 901/021)containing a ﬂuorescent indicator (UV 254)were used.Acid hydrolysis of the anthocyanin glycosides was achieved by dissolving a portion of the dry anthocyanin extracts in 200␮L of 2mol/L HCl.The solution was then sealed under nitrogen gas and heated to 100°C for 1.5h.The samples were then cooled immediately in an ice bath and plated using the above solvent system.MS identiﬁcation of individual compounds was conducted postseparation via prep-HPLC and prep-TLC (as outlined above).The analysis was carried out on a Waters Alliance 2695HPLC coupled serially with a Waters 2996photodiode array detector and a Waters ZQ 2000quadrupole analyzer utilizing the electrospray ionization interface (ESI-MS)(Waters).The chromatographic separation was performed on a 250ϫ 2.0mm Synergi 4-␮m Max-RP 80Åcolumn (Phenomenex)with a 4ϫ2mm Phenome-nex Max RP guard cartridge (Phenomenex).Injection volumes were 2␮L.The mobile phase consisted of an acidiﬁed (0.18%v:v acetic acid)water:acetonitrile mixture (95:5)(solvent A)and 100%acetonitrile (solvent B).The ﬂow rate was 130␮L/min;the solvent gradient program used 100%A at 0–2min and was ramped to 100%B at 60min.The instrument was operated in electrospray positive ion mode (ES ϩ).Micromass ZQ single quadrupole MS with electrospray interface and MassLynx 4.0software (Micro-mass)was used for data acquisition.The MS parameters were loosely based on methods previously published by Felgines et al.(12)and Garcı´a-Beneytez et al.(13).NMR spectra were obtained on a Bruker Avance DRX 500MHz spectrometer (Bruker Biospin),equipped with a cryoprobe,at 300K.For 1H (500MHz)NMR,a solvent mixture of methanol-d4to triﬂuoroacetic acid-d (CD 3OD:CF 3COOD)(98:2,v:v,200␮L)was used and ␦values were referenced to CD 3OD (CHD 2OD at 3.30ppm).Analysis of the 1H NMR spectra was based on the comparison of the chemical shift and relative intensity of the signals with those of standard compounds.Statistical analysis.The primary analyses were performed on urinary values (urinary values only)using the mixed models proce-dure (PROC MIXED)in SAS (version 9.1;SAS Institute).The data are presented as means ϮSD unless otherwise stated.The distribu-tions of anthocyanins were corrected with natural logtransforma-FIGURE 2Time course of total,parent,and metabolized antho-cyanins in human urine (A )and serum (B )of subjects after the con-sumption of 721mg of cyanidin 3-glycosides.For urinary (A ;n ϭ9replicates)and serum (B ;n ϭ3)data,values are means ϮSD as represented by vertical bars.(A )Different letters for the points indicate that the concentrations of total urinary anthocyanins differed across time,P Ͻ0.05.Identiﬁcation of each peak as labeled in the legend (as peaks 1–6)is given in Table 1.Urinary and serum pharmacokinetic data of each compound represented by peaks 1–6,is given in Tables 2and 3,respectively.FIGURE 3Cumulative time course of individual anthocyanins excreted in human urine after the consumption of 721mg of cyanidin 3-glycosides.Values are means ϮSD (n ϭ9replicates)as represented by vertical bars.Different letters for the lines indicate that the concen-trations differed at the level of the individual anthocyanin,P Ͻ0.05.For peak identities,refer to Table 1.For urinary and serum pharmacokinetic data of each compound represented by peaks 1–6,refer to Tables 2and 3,respectively.PHARMACOKINETICS OF CYANIDIN 3-GLYCOSIDES 2585by guest on December 14, 2010Downloaded fromtions,and unadjusted values are reported.The total level of antho-cyanins as well as the levels of individual anthocyanin species were evaluated over time.The model included time (levels ϭ0,2,4,6,8,10,12,and 24h)and anthocyanin species (levels ϭ1,2,3,4,5,6)as ﬁxed effects,and subjects (n ϭ3)and their replicate treatments (3replicates)were treated as random variables (y ϭ9replicates).For all analyses,the signiﬁcant main effects (P Յ0.05)were investigated using the Tukey-Kramer test.In addition,noncompartmental pharmacokinetic evaluation of urine and serum parent compounds,and their metabolites was per-formed on untransformed data according to standard methods (14).For urinary variables,there was no effect of subject (P ϭ0.3);therefore,subject (n ϭ3)and their replicate treatments (3replicates)were collapsed (y ϭ9replicates)for the determination of means (ϮSD)in the pharmacokinetic analysis.The analyses of serum variables included 3subjects with no replications and are presented as means ϮSD (n ϭ3;no replicates).Calculation of the area under the plasma concentration time curve was based on the mean serum concentra-tions of individual subjects using the trapezoidal rule.Absorption half-lives were determined graphically (SPSS SigmaPlot,IL)using the method of residuals.The geometric means of the elimination half-lives were determined graphically from the renal excretion rates of individual subjects.RESULTSSerum and urine samples collected before the administra-tion of the chokeberry extract (baseline,t ϭ0)contained no detectable anthocyanins (Fig.1A ).Postconsumption,both cyanidin 3-galactoside and cyanidin 3-arabinoside [peak (P)1,3;Fig.1]were present in the serum and urine.Both glucuronidated (loss m /z ϭ176upon fragmentation)and methylated (m ϩ14)derivatives of cyanidin were also present.In total,4derivatives/metabolites were isolated from the urine in sufﬁcient quantities for structural identiﬁcation.One metabolite (P2;Fig.1C and D )was identiﬁed as a cyanidin glucuronide,as indicated by its molecular ion at m /z 463and fragment at m /z 287,indicating a loss of m /z 176upon fragmentation (representing a glucuronide residue;m /z ϭ176;Table 1).Additionally,the hydrolysis of this compound re-sulted in an aglycone with the same HPLC Rt,UV-vis spectral data,and TLC Rf value as the purchased cyanidin standard.There was insufﬁcient evidence to determine the exact posi-tion of the glucuronide residue because the NMR spectra were uninterruptible (result of insufﬁcient quantity and poor solu-bility).The second identiﬁed metabolite (P4;Fig.1C and D )had the chemical characteristics of a methylated derivative of C-3-gal,having a parent ion of m /z 463,daughter fragment of m /z 301(consistent with methylation;m /z 287ϩ14ϭ301),and loss of m /z 162upon fragmentation (indicative of a hexose sugar;m /z ϭ162;Table 1).The hydrolysis of the compound resulted in an aglycone with similar HPLC Rt,UV-vis spectral data,and TLC Rf value as the purchased peonidin standard.There was insufﬁcient evidence to determine the exact posi-tion of the methylation and glucuronidation because the NMR spectra were uninterruptible (result of insufﬁcient quantity and poor solubility).Two other metabolites (P5,6;Fig.1,C and D)were identiﬁed as methylated derivatives of cyanidin glucuro-nide,having parent ions of m /z 477and daughter fragments of m /z 301,indicating a loss of m /z 176upon fragmentation (consistent with glucuronic acid residue;m /z ϭ176;Table 1).The derivatives were dissimilar to peonidin,having different HPLC and TLC parisons made between serum and urinary samples using HPLC-DAD (Fig.1,C and D;Table 1)indicated that the serum peaks matched both reten-tion time and UV-vis spectrum of the peaks identiﬁed as anthocyanins in the urine,and were therefore regarded as the same compounds.As a result of the sampling regimen,the mixed models procedure was performed on urinary variables only.There was a signiﬁcant main effect of time (P Ͻ0.0001)for the total (P1–6)and individual level of anthocyanins (P1,2,3,4,5,6),as well as a signiﬁcant interaction between time and anthocyanin species (P ϭ0.0016).Additionally,the level of total antho-cyanins did not differ among the 3subjects (P ϭ0.30).Serum variables were utilized only for pharmacokinetic analyses.The pharmacokinetic analysis of urine and serum variables (Tables 2and 3)and their graphical representations (Figures 2and 3)utilized untransformed data as was previously described (14).The results of both urinary (Table 2)and serum (Table 3)analyses indicated that parent compounds and their metabo-lites had similar pharmacokinetic proﬁles (T max and T 1/2).DISCUSSIONIn previous investigations,it was questioned whether the concentration of anthocyanins observed in the blood was sufﬁcient to yield biological activity.We hypothesized that unidentiﬁed anthocyanin metabolites may contribute to the reported effects of anthocyanins.A previous investigation by our group identiﬁed anthocyanin metabolites in human serum and urine after the consumption of cyanidin 3-glycosides in chokeberries (7).Subjects were fed ϳ1.2g of cyanidin 3-gly-cosides,leading to the identiﬁcation of glucuronide and meth-ylated derivatives in the serum and urine.The aim of the present investigation was to identify metabolites and their time course (pharmacokinetics)after a lower,more realistic anthocyanin dose.The present investigation involved a 721-mg oral dose of cyanidin 3-glycosides with the subsequent collection of serum over 7h and urine over 24h.This dose is equivalent to ϳ120–230g of whole berries (fresh weight)(15).Additionally,a 721-mg dose is roughly the median dose of 12reviewed anthocyanin human consumption trials in the literature (788Ϯ883mg)(5,10,12,16–24).The chokeberry extract as utilized in the present investigation was chosen for its simplistic anthocyanin proﬁle,consisting of only cyanidin 3-glycosides.The use of a fruit extract containing only one anthocyanidin species (cyanidin)was crucial for establishing the origin of methylated cyanidin derivatives.Further inves-tigations are required to identify the biological activity of these metabolites.In the present investigation,no anthocyanins were identi-ﬁed in the serum or urine of fasting subjects suggesting that the washout phase and prestudy dietary exclusion of anthocyanins was sufﬁcient.Glucuronidation was the major metabolic path-way observed for anthocyanin metabolism in the present in-vestigation,representing 59.8and 57.8%of the total antho-cyanins detected in the blood and urine,respectively.Methylation was the second most commonly observed meta-bolic transformation for anthocyanins,representing 43.8and 51.4%of the total anthocyanins detected in the serum and urine,respectively.Even though only a few researchers have reported glucuronidated and methylated anthocyanins in the urine and blood of humans and animals (7,8,12),methylated and glucuronidated derivatives of the ﬂavonoid quercetin are well documented (18,25,26).Although some recent investi-gations described the detection of anthocyanin metabolites in urine,this is the ﬁrst study to give detailed pharmacokinetic parameters for anthocyanin metabolites.In the present investigation,no attempt was made to iden-tify HPLC peaks lacking characteristic anthocyanin proﬁles (maxima in the 280and 520nm range).There are likely other anthocyanin metabolites (breakdown products)present in the serum and urine with absorbance outside the 240-to 525-nmKAY ET AL.2586 by guest on December 14, 2010Downloaded from。

葡萄汁酵母NOT5基因生物信息学分析

丁淑金，杨彦萍，邓茹友，等. 葡萄汁酵母NOT5基因生物信息学分析[J]. 食品工业科技，2022，43（18）：145−151. doi:10.13386/j.issn1002-0306.2021120309DING Shujin, YANG Yanping, DENG Ruyou, et al. Bioinformatics Analysis of the NOT5 Gene in Saccharomyces uvarum [J]. Science and Technology of Food Industry, 2022, 43(18): 145−151. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021120309· 生物工程 ·葡萄汁酵母NOT5基因生物信息学分析丁淑金，杨彦萍，邓茹友，马福仙，尹　拓，张汉尧*（西南林业大学林学院，西南地区生物多样性保育国家林业局重点实验室，云南昆明 650224）摘　要：目的：从转录组分析数据中得到葡萄汁酵母NOT5基因，并对其进行生物学信息分析，为后期研究该基因的作用打下基础。

方法：使用在线分析工具COILS Server 、SOPMA 、Alpha Fold 等分析预测NOT5基因编码蛋白质的一级结构、二级结构、三级结构及其结构域。

结果：葡萄汁酵母NOT5基因核苷酸序列的开放阅读框为1446 bp ，可编码481个氨基酸，位于XVI 染色体690107~691789，在系统发育树中与真贝酵母（Saccharomyces eubayanus NOT5 like protein XP018219088.1）NOT5基因的亲缘关系最近；其编码的蛋白质是不稳定的亲水蛋白，分子式为C 2493H 3848N 654O 795S 19，分子质量为56311.02，不含信号肽，无跨膜区域，存在卷曲螺旋区域，亚细胞定位于细胞质中的线粒体上；蛋白质的二级结构以随机卷曲为主；预测存在Pfam Not3和Pfam NOT2_3_5结构域；以Yeast Not1-Not2-Not5 (4by6.1.C)为模板构建NOT5蛋白的三级结构，两者的序列一致性可以达到89.88%。

2024年教师资格(初级中学)-英语知识与教学能力(高中)考试历年真题摘选附带答案

2024年教师资格（初级中学）-英语知识与教学能力（高中）考试历年真题摘选附带答案第1卷一.全考点押密题库(共100题)1.(单项选择题)(每题2.00 分)His___________in alcohol mined his whole life.A. indulgenceB. habitC. engagementD. addiction2.(单项选择题)(每题 2.00 分)Mr. Woods, I am here just in case anything out of the ordinary__________.A. happensB. happenC. would happenD. will happen3.(单项选择题)(每题 2.00 分) Which of the following activities is the most suitable for group work?______.A. guessing gameB. story tellingC. information-gapD. drama performance4.(单项选择题)(每题 2.00 分) Which of the following activities can be used to get the main idea of a passage?A. Reading the passage in detail.B. Reading to sequence the events.C. Reading to fill in the charts.D. Reading the first and last sentences of the passage and the paragraphs.5.(单项选择题)(每题 2.00 分) To our surprise, the stranger______to be an old friend of my mother’s.A. turned outB. turned upC. set outD. setup6.(单项选择题)(每题 2.00 分) new buildings will be built in my hometown.A. A great deals ofB. A lots ofC. A plenty ofD. A great number of7.(单项选择题)(每题 2.00 分) 请阅读Passage2,完成下列小题.Passage 2We had been wanting to expand our children‘s horizons by taking them to a place that was unlike anything wed been exposed to during our travels in Europe and the United States. In thinking about what was possible from Geneva,where we are based, we decided on a trip to Istanbul a two-hour plane ride from Zurich.We envisioned the trip as a prelude to more exotic ones, perhaps to New Delhi or Bangkok later this year, but thought our 11- and 13-year-old needed a first step away from manicured boulevards and pristine monuments.What we didn’t foresee was the reaction of friends, who warned that we were putting our children “in danger",referring vaguely, and most incorrectly, to disease, terrorism or just the unknown. To help us get acquainted with the peculiarities of Istanbul and to give our children a chance to choose what they were particularly interested in seeing, we bought anexcellent guidebook and read it thoroughly before leaving. Friendly warnings didn’t change our planning:although we might have more prudently checked with the U.S. Department’s list of troublespots. We didn’t see a lot of children among the foreign visitors during our six-day stay in Istanbul, but we found the tourist areas quite safe, veryinteresting and varied enough even to suit our son, whose oft-repeated request is that we not see “every single” church and museum in a given city.Vaccinations werent needed for the city, but we were concerned about adapting to the water for a short stay. So we used bottled water for drinking and brushing our teeth, a precaution that may seem excessive, but we all stayed healthy.Taking the advice of a friend, we booked a hotel a 20-minute walk from most of Istanbul’s major tourist sites. This not only got us some morning exercise, strolling over the Karakoy Bridge, but took us past a colorful assortment of fishermen, vendors and shoe shiners. From a teenager and preteens view. Istanbul street life is fascinating since almost everything can be bought outdoors. They were at a good age to spend time wandering the labyrinth of the Spice Bazaar, where shops display mounds of pungent herbs in sacks. Doing this with younger children would be harder simply because the streets are so packed with people; it would be easy to get lost.For →our two←, whose buying experience consisted of department stores and shopping mall boutiques, it was amazing to discover that you could bargain over price and perhaps end up with two of something for the price of one. They also learned to figure out the relative value of the Turkish lira, not a small matter with its many zeros.Being exposed to Islam was an important part of our trip. Visiting the mosques, especially the enormous Blue Mosque, was our first glimpse into how this major religion is practiced. Our children’s curiosity already had been piqued by the five daily calls to prayer over loudspeakers in every comer of the city, and the scarves covering the heads of many women. Navigating meals can be troublesome with children, but a kebab, bought on the street or in restaurants, was unfailingly popular. Since we had decided this trip was not for gourmets, kebabs spared us th e agony of trying to find a restaurant each day that would suit the adults’ desire to try something new amid children’s insistence that the food be served immediately. Gradually, we branched out to try some other Turkish specialties. Although our son had studied Islam briefly, it is impossible to be prepared for every awkward question that might come up, such as during our visits to the Topkapi Sarayi, the Ottoman Sultans9 palace, no guides were available so it was do-it-yourself, using our guidebook, which cheated us of a lot of interesting history and anecdotes that a professional guide could provide. Next time, we resolved to make such arrangements in advance.On this trip, we wandered through the magnificent complex, with its imperial treasures, its courtyards and its harem. The last required a bit of explanation that we would have happily left to a learned third party.Whom does“our two”in PARAGRAPH 5 refer to? ______ .A. the coupleB. the kidsC. the gourmetsD. local-style markets8.(单项选择题)(每题 2.00 分) Which of the following statements is NOT a way of presenting new vocabulary?____.A. definingB. using real objectsC. writing a passage by using new wordsD. giving explanations9.(单项选择题)(每题 2.00 分) Exceptional children are different in some significant ways from others of the same age, for the same age, for these children to develop to their full adult potential; their education must be adapted to those differences.Although we focus on the needs of exceptional children, we find ourselves describing their environment as well. While the leading actor on the stage captures our attention, we are aware of the importance of the supporting players and the scenery of the play itself. Both the family and the society in which exceptional children live are often the key to their growth and development. And it is in the public schools that we find the full expression of society5s understanding the knowledge, hopes, and fears that are passed on to the next generation.Education in any society is a mirror of that society. In that mirror we can see the strengths, the weaknesses, the hopes, the prejudices, and the central values of the culture itself. The great interest in exceptional children shown in public education over the past three decades indicates the strong feeling in our society that all citizens, whatever their special conditions, deserve the opportunity to fully develop their capabilities.“All men are created equal.” We’ve heard it many times, but it still has important meaning for education in a democratic society. Although the phrase was used by this countrys founders to denote equality before the law, it has also been interpreted to mean equality of opportunity. That concept implies educational opportunity for all children—the right of each child to receive help in learning to the limits of his or her capacity, whether that capacity be small or great. Recent court decisions have confirmed the right of all children—disabled or not —to an appropriate education, and have ordered that public schools take the necessary steps to provide that education. In response, schools are modifying their programs, adapting instruction to children who are exceptional to those who cannot profit substantially from regular programs.From its passage we learn that the educational concern for exceptional children______.A. is now enjoying legal supportB. disagrees with the tradition of the countryC. was clearly stated by the country’s foundersD. will exert great influence over court decisions10.(单项选择题)(每题 2.00 分)What stage can the following grammar activity be used at?The teacher asked students to arrange the words of sentences into different columns marked subject, predicate, object, object complement, adverbial and so on.A. Presentation.B. Practice.C. Production.D. Preparation.11.(单项选择题)(每题 2.00 分)According to Britain linguist F. Palmer, there are no real synonyms. Though"cast" and"throw" are considered synonyms, they are different in__________.A. styleB. collocationC. emotive meaningD. regions where they are used12.(单项选择题)(每题 2.00 分)What advantage do the new generation Latino writers have over LatinAmericanwriters according to the passage?A. The former are able to write in two different languages.B. The former can translate their works into different languages.C. The former are able to express ideas from a bi-cultural perspective.D. The former can travel freely across the border between two countries.13.(单项选择题)(每题 2.00 分) It is not so much the language______the cultural background that makes the book difficult to understanD.A. asB. butC. likeD. nor14.(单项选择题)(每题 2.00 分)Which inference in the brackets of the following sentences is apresupposition?A. Ede caught a trout. (Edecaught a fish.)B. Don' t sit on Carol' s bed. (Carol has a bed.)C. This blimp is over the house. (The house is under the blimp.)D. Coffee would keep me awake all night. (I don' t want coffee.)15.(单项选择题)(每题 2.00 分) There are many different ways of presenting grammar in the classroom. Among them, three are most frequently used and discusseD. Which one does not belong to them?______.A. deductive methodB. inductive methodC. guided discovery methodD. productive method16.(单项选择题)(每题 2.00 分) When a teacher teaches young learners English pronunciation, he should______.A. listen as much as possibleB. input regardless of students5 abilityC. tolerate small errors in continuous speechD. read more English materials17.(单项选择题)(每题 2.00 分) Which of the following activities is the best for training detailed reading?A. drawing a diagram to show the text structureB. giving the text an appropriate titleC. transforming information from the text to a diagramD. finding out all the unfamiliar words18.(单项选择题)(每题 2.00 分) Which of the following teacher‘s instruction could serve the purpose of eliciting ideas?A. Shall we move on?B. Read after me, everyoneC. What can you see in this picture?D. What does the world "quickly" mean?19.(单项选择题)(每题 2.00 分)Which of the following is suggested in the last paragraph?A. The quality of writing is of primary importance.B. Common humanity is central to news reporting.C. Moral awareness matters in editing a newspaper.D. Journalists need stricter industrial regulations.20.(单项选择题)(每题 2.00 分)What is the best title for this passage?A. Creativity. and InsightsB. Insights and Problem SolvingC. Where Do Insight Moments Come?D. Where Do Creativity Moments Come?21.(单项选择题)(每题 2.00 分) —Where did you find the wallet?—It was at the stadium______I played football.A. thatB. whereC. whichD. there22.(单项选择题)(每题 2.00 分) Water problems in the future will become more intense and more complex. Our increasing population will tremendously increase urban wastes, primarily sewage. On the other hand, increasing demands for water will decrease substantially the amount of water available for diluting wastes. Rapidly expanding industries which involve more and more complex chemical processes will produce large volumes of liquid wastes, and many of these will contain chemicals which are poisonous. To feed our rapidly expanding population, agriculture will have to be intensifieD. This will involve even increasing quantities of agriculture chemicals. From this, it is apparent that drastic steps must be taken immediately to develop corrective measures for the pollution problem.There are two ways by which this pollution problem can be lesseneD. The first relates to the treatment of wastes to decrease their pollution hazarD. This involves the processing of solid wastes prior to disposal and the treatment of liquid wastes, or effluents, to permit the reuse of the water or best reduce pollution upon final disposal.A second approach is to develop an economic use for all or a part of the wastes. Farm manure is spread in fields as a nutrient or organic supplement. Effluents from sewage disposal plants are used in some areas both for irrigation and for the nutrients containeD. Effluents from other processing plants may also be used as a supplemental source of water. Many industries,such as meat and poultry processing plants, are currently converting former waste production into marketable byproducts. Other industries have potential economic uses for their waste products.The phrase “prior to”（ParA. 2） probably means______.A. afterB. duringC. beforeD. beyond23.(单项选择题)(每题 2.00 分) 请阅读passage2，完成下列小题。

CURRENTS AND THEIR COUPLINGS IN THE WEAK SECTOR OF THE STANDARD MODEL

(4)
which is eﬀective four-fermion interaction with the Fermi constant given √the familiar 2 2 h ℓ by GF / 2 = g /(8mW ). Here Jµ and Jµ are known as the hadron and lepton currents, where
(2)
1 where t3L (i) is the weak isospin of fermion i ( + 1 2 for ui and νi ; − 2 for di and ℓi ) and qi is the charge of ψi in units of e. The three terms in the interaction Lagrangian of Eq. 1 represent the chargedcurrent weak interaction, the electromagnetic interaction and the neutral-current weak interaction respectively. Note that the Lorentz structure involves only vectors and axial vectors; there is no compelling experimental evidence for scalars, pseudoscalars or tensor constructions. The minimal Standard Model as described by Eq. 1 successfully3 explains W and Z decays, neutrino-hadron scattering, neutrino-electron scattering and parity-violating electron-hadron neutral-current experiments, providing that radiative corrections to order α, the ﬁne-structure constant, are applied. In this chapter we will focus our discussion on the charged-current weak interaction in semi-leptonic decays involving quarks and leptons from the ﬁrst family. As an example, consider the decay d → ue− ν e , for which the T -matrix is2

EMEA：生物等效性指南

European Medicines AgencyPre-Authorisation Evaluation of Medicines for Human Use7 Westferry Circus, Canary Wharf, London, E14 4HB, UKTel. (44-20) 74 18 84 00 Fax (44-20) 74 18 86 13E-mail: mail@emea.europa.eu http://www.emea.europa.euLondon, 24 July 2008 Doc. Ref. CPMP/EWP/QWP/1401/98 Rev. 1COMMITTEE FOR MEDICINAL PRODUCTS FOR HUMAN USE(CHMP)DRAFT GUIDELINE ON THE INVESTIGATION OF BIOEQUIVALENCEDRAFT AGREED BY THE EFFICACY WORKING PARTYJuly 2008 ADOPTION BY CHMP FOR RELEASE FOR CONSULTATION24 July 2008 END OF CONSULTATION (DEADLINE FOR COMMENTS) 31 January 2009This guideline will replace the “Note for guidance on the investigation of bioavailability and bioequivalence" CPMP/EWP/QWP/1401/98 and the related questions in the Q&A document (EMEA/CHMP/EWP/40326/2006). This guideline includes recommendations on BCS-based biowaivers.Comments should be provided to EWPSecretariat@emea.europa.eu using this templateKEYWORDS Bioequivalence, pharmacokinetics, biowaiver, in vitro dissolution, genericsGUIDELINE ON THE INVESTIGATION OF BIOEQUIVALENCETABLE OF CONTENTSEXECUTIVE SUMMARY (3)11.INTRODUCTION (BACKGROUND) (3)232.SCOPE (4)43.LEGAL BASIS (4)54.MAIN GUIDELINE TEXT (5)4.1D ESIGN, CONDUCT AND EVALUATION OF BIOEQUIVALENCE STUDIES (5)674.1.1Study design (5)84.1.2Reference and test product (6)94.1.3Subjects (7)104.1.4Study conduct (7)114.1.5Characteristics to be investigated (9)124.1.6Strength and dose to be investigated (10)134.1.7Chemical analysis (12)144.1.8Evaluation (12)154.1.9Narrow therapeutic index drugs (15)164.1.10Highly variable drugs or drug products (16)174.2I N-VITRO DISSOLUTION TESTS (16)184.2.1In-vitro dissolution tests complementary to bioequivalence studies (16)4.2.2In-vitro dissolution tests in support of biowaiver of strengths (16)19204.3V ARIATIONS (16)4.4S TUDY REPORT (17)2122DEFINITIONS (17)23APPENDIX I (19)24D ISSOLUTION TESTING (19)25APPENDIX II (21)B IOEQUIVALENCE STUDY REQUIREMENTS FOR DIFFERENT DOSAGE FORMS (21)2627APPENDIX III (24)BCS-BASED B IOWAIVER (24)2829APPENDIX IV (28)D ECISION TREE ON MEASUREMENT OF PARENT COMPOUND OR METABOLITE (28)3031APPENDIX V (29)D ECISION TREE ON SELECTION OF DOSE AND STRENGTH IN BIOEQUIVALENCE STUDIES (29)3233EXECUTIVE SUMMARY3334This guideline defines when bioequivalence studies are necessary and formulates requirements for 35their design, conduct, and evaluation. The guideline focuses primarily on bioequivalence forimmediate release dosage forms with systemic action.36(background)371. INTRODUCTION38Two medicinal products containing the same active substance are considered bioequivalent if their 39bioavailabilities (rate and extent) after administration in the same molar dose lie within acceptable 40predefined limits. These limits are set to ensure comparable in vivo performance, i.e. similarity interms of safety and efficacy.4142In bioequivalence studies, the plasma concentration time curve is used to assess the rate and extent of43absorption. Meaningful pharmacokinetic parameters and preset acceptance limits allow the final 44decision on bioequivalence of the tested products. AUC, the area under the concentration time curve, 45reflects the extent of exposure. C max, the maximum plasma concentration or peak exposure, and the46time to maximum plasma concentration, t max, are parameters that are influenced by absorption rate.It is the objective of this guideline to define when bioequivalence studies are necessary and to4748formulate requirements for their design, conduct, and evaluation. The possibility of using in vitro 49instead of in vivo studies is also addressed.The concept of bioequivalence forms the basis for approval of generic application, but it may also be5051applicable to hybrid application, extensions and variations applications, and to different formulations 52used during the development of a new medicinal product containing a new chemical entity.For generic applications, the purpose of establishing bioequivalence is to demonstrate equivalence in5354biopharmaceutic quality between the generic product and a reference medicinal product in order to 55allow bridging of clinical data associated with the reference medicinal product. The current definition56for generic products is found in Directive 2001/83/EC, Article 10(2)(b). In general, a generic product 57is a product which has the same qualitative and quantitative composition in active substances as the58reference medicinal product, the same pharmaceutical form as the reference medicinal product, and whose bioequivalence with the reference medicinal product has been demonstrated by appropriate 5960bioavailability studies. By definition it is considered that different salts, esters, ethers, isomers, 61mixtures of isomers, complexes or derivatives of an active substance are considered to be the same active substance, unless they differ significantly in properties with regard to safety and/or efficacy.6263Furthermore, various immediate-release oral pharmaceutical forms are considered to be one and the 64same pharmaceutical form. It is also stated in the Directive that bioavailability studies need not be65required if it can be demonstrated that the generic medicinal product meets the relevant criteria for a 66biowaiver.67Hybrid applications rely on the results of preclinical tests and clinical trials of an approved referencemedicinal product and include new data. These new data may include bioequivalence or comparative6869bioavailability data.70Also applications for extensions such as additional dosage forms, new strengths, new routes of 71administration often need support of bioequivalence in order to bridge data from the authorised 72reference medicinal product.73Variations for a change in composition or for significant manufacturing changes which may affect drug bioavailability may also require support of bioequivalence studies.7475During development of a new chemical entity, the principles of bioequivalence may be applied in 76order to bridge data between different formulations e.g. between a formulation used in the pivotal clinical studies and the to-be-marketed formulation. In such situations however, wider acceptance 7778limits may be acceptable if these are justified based on data provided with a complete application,adequately addressing the clinical relevance of the widening from both a safety and efficacy 79perspective. 802. SCOPE 81This guideline focuses on recommendations for bioequivalence studies for immediate release 82formulations with systemic action. 83Specific recommendations regarding bioequivalence studies for modified release products, 84transdermal products and orally inhaled products are given in other guidelines (see section 3). 85Recommendation for the comparison of biologicals to reference medicinal products can be found in 86guidelines on biosimilar products. Recommendations for pharmacokinetics of therapeutic proteins are 87also described in a specific guideline (CPMP/EWP/89249/04). 88In case bioequivalence cannot be demonstrated using drug plasma concentrations, in exceptional 89circumstances pharmacodynamic or clinical endpoints may be needed. This situation is outside the 90scope of this guideline and the reader is referred to therapeutic area specific guidelines. 91Furthermore, this guideline does not cover aspects related to generic substitution as this is subject to 92national legislation. 933. LEGAL BASIS 94This guideline applies to Marketing Authorisation Applications for human medicinal products 95submitted in accordance with the Directive 2001/83/EC as amended, under Art. 8(3) (full 96applications), Art 10b (fixed combination), Art. 10 (1) (generic applications), Art 10(3) (hybrid 97applications), and also for line extension and variation applications in accordance with Commission 98Regulations (EC) No 1084/2003 and 1085/2003. 99This guideline should be read in conjunction with the Annex I of Directive 2001/83/EC as amended, 100as well as European and ICH guidelines for conducting clinical trials, including those on: 101− General Considerations for Clinical Trials (ICH topic E8, CPMP/ICH/291/95) 102 − Guideline for Good Clinical Practice (ICH E6 (R1), CPMP/ICH/135/95) 103 − Structure and Content of Clinical Study Reports (ICH E3, CPMP/ICH/137/95) 104 − CHMP guidance for users of the centralised procedure for generics/hybrid applications 105 (EMEA/CHMP/225411/2006) 106− Modified Release Oral and Transdermal Dosage Forms: Section II (CPMP/EWP/280/96) 107 − Requirements for clinical documentation for orally inhaled products (OIP) including the 108 requirements for demonstration of therapeutic equivalence between two inhaled products for 109use in the treatment of Asthma and Chronic Obstructive Pulmonary Disease (COPD) 110(CPMP/EWP/4151/00 rev 1). 111− Fixed Combination Medicinal Products (CPMP/EWP/240/95) 112 − Clinical Requirements for Locally Applied, Locally Acting Products containing Known 113 Constituents (CPMP/EWP/239/95) 114− Good manufacturing practice (Eudralex volume 4). 115The guideline should also be read in conjunction with relevant guidelines on pharmaceutical quality. 116The test products used in the bioequivalence study must be prepared in accordance with GMP-117regulations. 118Bioequivalence trials should be conducted in accordance to Directive 2001/20/EC of the European 119parliament and of the Council. 120Companies may also apply for CHMP Scientific Advice, via the EMEA, for specific queries not 121covered by existing guidelines.1224. MAIN GUIDELINE TEXT1231244.1 Design, conduct and evaluation of bioequivalence studies125In the following sections, requirements for the design, conduct and evaluation of bioequivalencestudies investigating immediate release formulations with systemic action are described.126127The formulation and the characteristics of the active substance can affect the requirements for128bioequivalence studies. When the test product contains a different salt, ester, ether, isomer, mixture of 129isomers, complex or derivative of an active substance than the reference product, bioequivalence 130should be demonstrated in appropriate bioavailability studies. However, when the active substance in131test and reference products are identical or contain comparable salts, in vivo bioequivalence studies may, in some situations, not be required as described in APPENDIX II (bioequivalence study 132133requirements) and III (biowaiver).134The pharmacokinetic and physico-chemical properties of the substance affect the number of studies needed and the design of the studies. The choice of number of studies and study design should be 135136thoroughly justified based on the physico-chemical characteristics of the substance and its 137pharmacokinetic properties, discussing especially linearity in pharmacokinetics, activity of138metabolites, contribution of metabolites to the effect, the need for enantioselective analysis, and 139solubility of the active substance. In the context of this guideline, high solubility and low solubility is 140defined according to the Biopharmaceutics Classification System (BCS) definition of high and lowsolubility, as defined in APPENDIX III.141142The clinical overview of an application for marketing authorisation should list all studies carried out143with the product applied for. All bioequivalence studies comparing the product applied for with the reference product of interest must be submitted.144145design4.1.1 Study146The study should be designed in such a way that the formulation effect can be distinguished from 147other effects.148Standard design149If two formulations are going to be compared, a two-period, two-sequence single dose crossover150design is the design of choice. The treatment periods should be separated by an adequate wash out 151period.152Alternative designs153In general, single dose studies will suffice. However, in case of dose or time-dependent pharmacokinetics, resulting in markedly higher concentrations at steady state than expected from 154155single dose data, a potential difference in AUC between formulations may be larger at steady state 156than after single dose. Hence, a multiple dose study may be required in addition to the single dose 157study to ensure that the products are bioequivalent regarding AUC also at steady state. However, if the158single dose study indicates very similar PK profile for test and reference (the 90% confidence interval 159for AUC is within 90-111), the requirement for steady-state data may be waived.In certain cases when a single dose study cannot be conducted in healthy volunteers due to tolerability 160161reasons, and a single dose study is not feasible in patients, conduct of a multiple dose study in patients 162may be acceptable (see also section 4.1.6 Strength and Dose).A multiple dose study as an alternative to a single dose study may also be acceptable if problems of 163164sensitivity of the analytical method preclude sufficiently precise plasma concentration measurements165after single dose administration. As C max at steady state may be less sensitive to differences in the absorption rate than C max after single dose, bioequivalence should, if possible, be determined for C max 166167after the single dose administration (i.e. after the first dose of the multiple dose study) as a measure ofpeak exposure while extent of exposure can be based on demonstration of bioequivalence of AUC at 168169steady state.170In steady-state studies the administration scheme should preferably follow the highest usual dosagerecommendation (see also section 4.1.6 Strength and dose).171172Under certain circumstances, provided the study design and the statistical analyses are scientifically173sound, alternative well-established designs could be considered such as parallel design for substances 174with very long half-life and replicate designs e.g. for substances with highly variable pharmacokinetic 175characteristics (see section 4.1.10).1764.1.2 Reference and test productFor Article 10(1) and 10(3) applications the chosen reference medicinal product must be a medicinal 177178product authorised in the Community, on the basis of a complete dossier in accordance with the 179provisions of Article 8 of Directive 2001/83/EC, as amended. The product used as reference product in 180the bioequivalence study should be part of the global marketing authorisation of the reference medicinal 181product (as defined in Article 6(1) second subparagraph of Directive 2001/83/EC). The choice of the 182reference medicinal product should be justified by the applicant in Module 1.2, and Module 1, section 1831.5.2.184Test products in an application for a generic product are normally compared with the corresponding 185dosage form of a reference medicinal product.In an application for extension of a concerned medicinal product and when there are several dosage 186187forms of this medicinal on the market, the dosage form used for the initial approval of the concerned 188medicinal product (and which was used in clinical efficacy and safety studies) should be used ascomparative product, unless otherwise justified.189190For variations of a concerned medicinal product, the comparative medicinal product for use in191bioequivalence and dissolution studies is usually that authorised under the currently registered 192formulation, manufacturing process, packaging etc.193When variations to a generic product are made, the comparative medicinal product for the 194bioequivalence study should be the reference medicinal product.195The reference and test products should be packed in an individual way for each subject and period.196Packaging, which is a manufacturing operation, should be performed and documented in accordance 197with good manufacturing practice, including Annex 13 to the EU guide to GMP. It should be possible198to identify unequivocally the identity of the product administered to each subject at each trial period.Packaging and administration of the products to the subjects should therefore be documented in detail. 199200This documentation should include all precautions taken to avoid and identify potential dosing201mistakes.202Batch control results of the test and reference products should be reported. The assayed content of the203batch used as test product should not differ more than 5% from that of the batch used as reference 204product determined with the test procedure proposed for routine quality testing of the test product. In order to demonstrate that a representative batch of the reference product with regards to dissolution 205206and assay content has been selected, the applicant should present dissolution profiles and content 207analysis of at least 3 batches of the reference product, unless otherwise justified.The test product used in the study should be representative of the product to be marketed and this 208209should be justified by the applicant. In the case of oral solid forms for systemic action the test product 210should usually originate from a batch of at least 1/10 of production scale or 100,000 units, whicheveris greater, unless otherwise justified. The production of batches used should provide a high level of 211212assurance that the product and process will be feasible on an industrial scale. In case of a productionbatch smaller than 100,000 units, a full production batch will be required. If the product is subjected to 213214further scale-up, this should be properly validated.215Samples of the product from full production batches should be compared with those of the test batch, and should show similar in vitro dissolution profiles when employing suitable dissolution test 216217conditions (see Appendix I).218The study sponsor will have to retain a sufficient number of all investigational product samples in the 219study for one year in excess of the accepted shelf life or two years after completion of the trial or until 220approval whichever is longer to allow re-testing, if it is requested by the authorities.2214.1.3 SubjectsNumber of subjects222223The number of subjects to be included in the study should be based on an appropriate sample size 224calculation. The minimum number of subjects in a cross-over study should be 12.Selection of subjects225226The subject population for bioequivalence studies should be selected with the aim to permit detection 227of differences between pharmaceutical products. In order to reduce variability not related to 228differences between products, the studies should normally be performed in healthy volunteers unless 229the drug carries safety concerns that make this unethical. This model, in vivo healthy volunteers, is 230regarded adequate in most instances to detect formulation differences and the results will allow extrapolation to populations in which the reference product is approved (the elderly, children, patients 231232with renal or liver impairment, etc.).233The inclusion/exclusion criteria should be clearly stated in the protocol. In general, subjects should preferably be between 18 - 55 years old and of weight within the normal range according to accepted 234235normal values for the Body Mass Index. The subjects should be screened for suitability by means of 236clinical laboratory tests, an extensive review of medical history, and a comprehensive medical 237examination. Depending on the drug’s therapeutic class and safety profile, special medical 238investigations and precautions may have to be carried out before, during and after the completion of 239the study. Subjects could belong to either sex; however, the risk to women of childbearing potential 240should be considered on an individual basis. Subjects should preferably be non-smokers and without a 241history of alcohol or drug abuse. If moderate smokers are included (less than 10 cigarettes per day) 242they should be identified as such and the consequences for the results should be discussed. 243Phenotyping and/or genotyping of subjects may be considered for safety or pharmacokinetic reasons.In parallel design studies, the treatment groups should be comparable in all known prognostic 244245variables that affect the pharmacokinetics of the active substance (e.g. ethnic origin, smoking status, 246extensive/poor metabolic status). This is an essential pre-requisite to give validity to the study results. 247If the investigated active substance is known to have adverse effects and the pharmacological effects 248or risks are considered unacceptable for healthy volunteers, it may be necessary to use patients, under 249suitable precautions and supervision, instead. In such case the applicant should justify the alternative. 250conduct4.1.4 Study251StandardisationThe test conditions should be standardised in order to minimise the variability of all factors involved 252253except that of the products being tested. Therefore, it is recommended to standardise diet, fluid intake 254and exercise.The time of day for ingestion should be specified. As fluid intake may influence gastric passage for 255256oral administration forms, the test and reference products should be administered with a standardisedvolume of fluid (at least 150 ml). All meals and fluids taken after the treatment should also be 257258standardised in regard to composition and time of administration during the sampling period. As the 259bioavailability of an active moiety from a dosage form could be dependent upon gastrointestinaltransit times and regional blood flows, posture and physical activity may need to be standardised.260261The subjects should abstain from food and drinks, which may interact with circulatory,262gastrointestinal, hepatic or renal function (e.g. alcoholic or xanthine-containing beverages or 263grapefruit juice) during a suitable period before and during the study.264Subjects should not take any other concomitant medication (including herbal remedies) for an265appropriate interval before as well as during the study. In case concomitant medication is unavoidable and a subject is administered other drugs, for instance to treat adverse events like headache, the use 266267must be reported (dose and time of administration) and possible effects on the study outcome must be 268addressed.In case the study is to be performed under fasting conditions, subjects should fast during the night 269270prior to administration of the products, unless otherwise justified.271Sampling times272A sufficient number of samples to adequately describe the complete plasma concentration-time profile 273should be collected. The sampling schedule should include frequent sampling around C max to provide a 274reliable estimate of peak exposure. The sampling schedule should be planned to avoid C max being thefirst point of a concentration time curve. When partial AUC is to be determined, frequent early 275276sampling is recommended with preferably at least two quantifiable samples before expected t max. The277sampling schedule should also cover the plasma concentration time curve long enough to provide a reliable estimate of the extent of exposure which is achieved if AUC t is at least 80% of AUC∞. At least 278279three to four samples are needed during the terminal log-linear phase in order to reliably estimate the280terminal rate constant (which is needed for a reliable estimate of AUC∞).281A sampling period longer than 72 h is not considered necessary for any immediate release282formulation. Hence, for drugs with a long half-life, comparison of extent of exposure using truncated 283AUCs at 72 h is acceptable.Fasting or fed conditions284285The study should be conducted during fasting conditions unless the SPC recommends intake of the 286originator product only in the fed state. If the recommendation of food intake in the SPC is based onpharmacokinetic properties such as higher bioavailability, the bioequivalence study should be 287288conducted in the fed state. Also if the recommendation of food intake is intended to decrease adverse289events or to improve tolerability, it is recommended to conduct the bioequivalence study in fed state, although a bioequivalence study under fasting conditions could be acceptable if this has been 290291adequately justified.292For products with enhanced release characteristics differing from conventional immediate release 293formulations (e.g. microemulsions or solid dispersions), bioequivalence studies performed under both294fasted and fed conditions are required.295In cases where information is required in both the fed and fasted states, it is preferable to conduct a four-period single dose crossover design study (both products fed and fasted) rather than conducting 296297two separate bioequivalence studies in fed and fasted state, respectively. In a four-period crossover 298design study, the food effect on test and reference product can be evaluated which is not the case when299conducting two separate two-period, two-sequence single dose crossover design studies under fasting 300and fed conditions, respectively. In addition to the bioequivalence evaluation of test/reference in 301fasting and in fed state, the food effect can be presented for test and reference, i.e. the ratio302food/fasting and 90% confidence interval for test and reference, respectively.303In studies performed under fed conditions, the composition of the meal should be according to304recommendations in the SPC of the reference product. If no recommendation on the composition of305the meal is given in the reference product SPC, the meal should be a "standardized non high-fat meal" 306(about 650 kcal with about 30% of calories derived from fat). The composition of the meal should be 307described with regard to protein, carbohydrate and fat content (specified in grams, calories and relative 308caloric content (%)).3094.1.5 Characteristics to be investigated310Pharmacokinetic parameters311In studies to determine bioequivalence after a single dose, AUC t, AUC∞, C max and t max should be 312determined. Additional parameters that may be reported include the terminal rate constant, λz, and t1/2.For products where rapid absorption is of importance, partial AUCs can be used as a measure of early 313314exposure. The partial area can in most cases be truncated at the population median of t max values for 315the reference formulation. However, an alternative time point for truncating the partial AUC can be 316used when clinically relevant. The time point for truncating the partial AUC should be pre-specified 317and justified in the study protocol.318In studies to determine bioequivalence at steady state, AUCτ, C max,ss,C min,ss,t max,ss and fluctuation should be determined.319320Definitions of the pharmacokinetic parameters are given in section 6.321Additional parameters may be presented. The methods of estimating parameters should be specified.The use of compartmental methods for the estimation of parameters is not acceptable.322323Parent compound or metabolites324Recommendations for measuring parent compound and metabolite(s) depend on the contribution of parent compound and metabolite(s), respectively, to activity as detailed below and in Appendix IV. 325326In principle, evaluation of bioequivalence should be based upon measured concentrations of the parent compound. The reason for this is that C max of a parent compound is usually more sensitive to detect 327328differences between formulations in absorption rate than C max of a metabolite.329Also for inactive prodrugs, demonstration of bioequivalence for parent compound is the preferred 330option when the pharmacokinetics of pro-drug and active metabolite(s) is linear. In this situation, the 331active metabolite does not need to be measured. However, in case the pro-drug or active metabolites 332display non-linear pharmacokinetics (or it is difficult to conclude linear pharmacokinetics from 333available data), it is recommended to demonstrate bioequivalence for the main active metabolite. In 334such case, the parent compound does not need to be measured provided that it is inactive from efficacy 335and safety perspectives. Moreover, some pro-drugs may have low plasma concentrations, be quickly eliminated and have high variability, resulting in difficulties in demonstrating bioequivalence for 336337parent compound in a reasonably sized bioequivalence study. In this situation it is acceptable to 338demonstrate bioequivalence for the main active metabolite without measurement of parent compound. 339Furthermore, in situations where the pro-drug exposure is low and exposure to active metabolite is 340very much higher, it is acceptable to demonstrate bioequivalence for the main active metabolite 341without measurement of parent compound.The use of a metabolite as a surrogate for an active parent compound can only be considered if the 342343applicant presents convincing arguments demonstrating that it is not possible to reliably measure the 344parent compound after single dose administration or at steady state. However, as C max of the metabolite 345is usually less sensitive to differences in the absorption rate than C max of the parent drug, 346bioequivalence should, if possible, be determined for C max of the parent compound as a measure of 347peak exposure while extent of exposure can be based on demonstration of bioequivalence of AUC of metabolite. Furthermore, when using metabolite data as a substitute for parent drug concentrations, the 348349applicant should present any available data supporting the view that the parent drug exposure will be。

Effective SU(2)_L x U(1) theory and the Higgs boson mass

07000 M´ exico D.F., M´ exico
Since the standard electroweak theory is renormalizable, the ultraviolet (quadratic and logarithmic) divergencies appearing in loop calculations can be removed by proper redeﬁnitions of a small number of parameters (e.g., masses). The renormalization program allows to get ﬁnite predictions for physical quantities except for those few parameters which require redeﬁnitions. In this way, we renounce to give some physical content to the divergent behaviour of the theory at very high energies [1]. Instead of following the renormalization program, we can consider that SU (2)L ⊗ U (1) is an eﬀective theory with an ultraviolet cut oﬀ at the energy scale Λ and try to trigger some information on the model itself from the behaviour of divergent terms. In other words, we assume that a more complete theory than SU (2)L ⊗ U (1) introduces new physical eﬀects at high energy that cut oﬀ the ultraviolet divergencies. Below Λ, SU (2)L ⊗ U (1) is supposed to describe all the electroweak processes in a satisfactory way. In this letter we consider SU (2)L ⊗ U (1) as an eﬀective theory below Λ and assume the vanishing of the common divergent contributions (tadpoles) to the masses of all the particles. We ﬁnd that the electroweak theory becomes eﬀective even at cut oﬀ energies as small as Λ ∼ 0.7 TeV. This criterion for vacuum stability under radiative corrections allows to derive a mass for the Higgs boson that diﬀers from its asymptotic value (Λ → ∞) mH = at Λ ∼ 0.7 TeV. Let us start by considering the divergent one-loop tadpole contributions. These tadpoles give a universal contribution to self-mass corrections because the bare masses of all the particles are proportional to the vacuum expectation value v of the Higgs ﬁeld. The tadpoles are gauge-dependent and are given by [4] δvt = v α 16π