Simultaneous Determination of Glyphosate, Glufosinate, and Aminomethylphosphonic Acid by CE

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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.。

注射液中酚磺乙胺及焦亚硫酸钠含量的同时测定

第28卷第11期2016年11月化学研究与应用^Chemical Research and Application Vol.28,No. 11 Nov. ,2016文章编号：1004-1656(2016) 11-1610-04注射液中酚磺乙胺及焦亚硫酸钠含量的同时测定杨凤珍％袁华，范小振，张文育(沧州师范学院化学与化工学院，河北沧州061001)摘要：©磺乙胺及焦亚硫酸钠均在酸性的吐温80-罗丹明6G体系中产生化学发光，但具体发光条件不同，因此利用化学发光分析法，在同一体系中根据不同条件可对针剂注射液中酚磺乙胺及焦亚硫酸钠的含量进行同时测定。

酚磺乙胺的质量浓度在〇.〇5 ~3.(Vg• m T1范围内与光信号呈现良好的线性关系，检出限为0. 01网• m l/1，相对标准偏差小于3.5% 〇 = 5)。

测定焦亚硫酸钠的工作曲线线性范围为0. 07 ~ 5. 0叫•m l/1,检出限为0.02叫•m l/1,相对标准偏差小于4.2%〇= 5)。

该测定方法可用于针剂药物中酚磺乙胺及焦亚硫酸钠的含量测定，结果令人满意。

关键词:化学发光;酸磺乙胺;焦亚硫酸钠;吐温80;罗丹明6G中图分类号=0657. 3 文献标志码:ASimultaneous determination of etamsylate and sodium pyrosulfite in injectionY A N G Feng-z h e n*，Y U A N H u a，F A N X ia o-z h e n，Z H A N G W e n-yu(College of Chemistry and Chemical engineering,Cangzhou Normal University,Cangzhou 061001,China) Abstract：The chemiluminescence emission was generated by mixing etamsylate or sodium pyrosulfite with acidic tween80-rhoda- mine6G. The specific luminescent conditions were different, so the content of etamsylate and sodium pyrosulfite could be determined in the same chemiluminescence system according to the different conditions. The quantitation range was from 0. 05 to 3. Ojjig • mL 1with a detection limit of 0. 01 jjig • mL 1 ,and a relative standard deviation of less than 3. 5% (n = 5)for etamsylate. The working curve liner range was 0. 07 to 5. Ojjig *mL4with a detection limit of 0. 02 |jig *mL 1 , and arelative standard deviation of less than 4. 2% (n = 5)for the determination of sodium pyrosulfite. The proposed method was applied to the quantitation of etamsylate and sodium pyrosulfite in injection with satisfactory results.Key words ：chemiluminescence ；etamsylate ； sodium pyrosulfite ；tween80；rhodamine 6G酚磺乙胺又名止血敏。

HPLC同时测定草珊瑚中9个有效成分的含量

HPLC同时测定草珊瑚中9个有效成分的含量*潘馨，衷林清，周楚楚，蒙昱如，凌秀芳，廖华军**（福建中医药大学药学院，福州350122）摘要　目的：建立HPLC法同时测定草珊瑚中落新妇苷、新绿原酸、绿原酸、隐绿原酸、咖啡酸、槲皮素-3-O-β-D-葡萄糖醛酸苷、异嗪皮啶、山柰酚-3-O-β-D-葡萄糖醛酸苷和迷迭香酸的含量。

方法：采用Diamonsil 十八烷基硅烷键合硅胶（C18）色谱柱（250 mm×4.6 mm，5 μm），流动相为乙腈（A）-0.2%磷酸水溶液（B），梯度洗脱（0~20 min，2% A→15% A；20~23 min，15% A→12.5% A；23~42 min，12.5% A；42~70 min，12.5% A→30% A），流速1.0 mL·min-1，检测波长为290 nm（落新妇苷）、344 nm（新绿原酸、绿原酸、隐绿原酸、咖啡酸、槲皮素-3-O-β-D-葡萄糖醛酸苷、异嗪皮啶、山柰酚-3-O-β-D-葡萄糖醛酸苷和迷迭香酸），柱温35 ℃。

结果：落新妇苷、新绿原酸、绿原酸、隐绿原酸、咖啡酸、槲皮素-3-O-β-D-葡萄糖醛酸苷、异嗪皮啶、山柰酚-3-O-β-D-葡萄糖醛酸苷和迷迭香酸分别在0.041~2.050、0.036 0~1.780、0.046~2.282、0.047~2.330、0.040~2.010、0.021~1.060、0.047~2.335、0.024~1.220、0.047~2.368 μg范围内与峰面积呈现良好的线性关系（r=0.999 8~1.000），平均加样回收率（n=6）在98.4%~101.7%之间，RSD为1.1%~2.7%。

17批草珊瑚样品中落新妇苷、新绿原酸、绿原酸、隐绿原酸、咖啡酸、槲皮素-3-O-β-D-葡萄糖醛酸苷、异嗪皮啶、山柰酚-3-O-β-D-葡萄糖醛酸苷和迷迭香酸的含量（n=3）测定结果分别为0.29~1.09、0.03~0.29、0.62~1.37、0.38~1.32、0.06~0.42、0.03~0.51、0.17~0.68、0.05~0.59、0.44~2.22 mg·g-1。

11、HPLC同时测定麦冬药材中3种黄酮类成分的含量_贾诚

基金项目：“国家重大新药创制”科技重大专项（2010ZX09502－004）；科技创新专项资金（08FDZDSH01404）作者简介：贾诚，男，硕士研究生研究方向：中药新药研发*通讯作者：叶正良，男，研究员研究方向：中药新药研发Tel ：（022）HPLC 同时测定麦冬药材中3种黄酮类成分的含量贾诚1，2，叶正良2*，姜秀晶1，周大铮2，李德坤2（1.天津中医药大学中药学院，天津300193；2.天津天士力之骄药业有限公司，天津300402）摘要：目的测定麦冬药材中甲基麦冬黄烷酮A 、甲基麦冬黄烷酮B 和6-醛基异麦冬黄烷酮A 的含量，为麦冬药材的质量控制提供科学依据。

方法采用Waters Symmetry C 18柱（4.6mm ˑ250mm ，5μm ），乙腈-0.05%磷酸梯度洗脱，流速为1.0mL ·min －1，检测波长296nm ，柱温30ħ。

结果甲基麦冬黄烷酮A 、甲基麦冬黄烷酮B 和6-醛基异麦冬黄烷酮A 分别在9.6896.8、4.96 49.6、4.18 41.8μg ·mL －1内具有良好的线性关系，平均回收率（n =6）分别为100.1%（RSD 1.03%），99.5%（RSD 1.35%）和100.2%（RSD 1.16%）。

结论该方法快速、准确，可用于麦冬药材中甲基麦冬黄烷酮A 、甲基麦冬黄烷酮B和6-醛基异麦冬黄烷酮A 的含量测定。

关键词：麦冬；甲基麦冬黄烷酮A ；甲基麦冬黄烷酮B ；6-醛基异麦冬黄烷酮A ；HPLC 中图分类号：R283文献标志码：A文章编号：1001－2494（2011）15－1209－03Simultaneous Determination of Contents of Three Flavonoid Ingredients in Radix Ophiopogonis by HPLCJIA Cheng 1，2，YE Zheng-liang 2*，JIANG Xiu-jing 1，ZHOU Da-zheng 2，LI De-kun 2（1.Tianjin University of TraditionalChinese Medicine ，Tianjin 300193，China ；2.Tianjin Tasly Pride Pharmaceutical Company Limited ，Tianjin 300402，China ）ABSTRACT ：OBJECTIVETo determine the contents of methylophiopogonanone A ，methylophiopogonanone B and 6-aldehydo-isoophiopogonone A in Radix Ophiopogonis.METHODSWaters Symmetry C 18column （4.6mm ˑ250mm ，5μm ）was used withphosphoric acid （0.05%，v /v ）and acetonitrile as the mobile phase.Stepwise gradient elution was adopted.The flow rate was 1.0mL ·min －1，the detection wavelength was 296nm ，and the column temperature was maintained at 30ħ.RESULTSThe calibrationcurves showed good linearity within the range of 9.68－96.8，4.96－49.6and 4.18－41.8μg ·mL －1for methylophiopogonanone A ，methylophiopogonanone B and 6-aldehydo-isoophiopogonone A ，respectively.The mean recoveries （n =6）were 100.1%（RSD 1.03%），99.5%（RSD 1.35%）and 100.2%（RSD 1.16%），respectively.C ONCLUSION This method is simple and accu-rate.And it can be used for the determination of methylophiopogonanone A ，methylophiopogonanone B and 6-aldehydo-isoophio-pogonone A in Radix Ophiopogonis .KEY WORDS ：Radix ophiopogonis ；methylophiopogonanone A ；methylophiopogonanone B ；6-aldehydo-isoophiopogonone A ；HPLC麦冬来源于百合科植物麦冬［Ophiopogon ja-ponicus （Thunb.）Ker-Gawl.］的干燥块根。

沙利度胺的传说

The International Journal of Biochemistry&Cell Biology39(2007)1489–1499ReviewThe thalidomide sagaMagda Melchert a,∗,Alan List ba Mofﬁtt Cancer Center and Research Institute,12902Magnolia Drive,SRB-4,Tampa,FL33612,USAb12902Magnolia Drive,SRB-4,Tampa,FL33612,USAAvailable online30January2007AbstractOver the past50years,thalidomide has been a target of active investigation in both malignant and inﬂammatory conditions. Although initially developed for its sedative properties,decades of investigation have identiﬁed a multitude of biological effects that led to its classiﬁcation as an immunomodulatory drug(IMiD).In addition to suppression of tumor necrosis factor-alpha(TNF-␣), thalidomide effects the generation and elaboration of a cascade of pro-inﬂammatory cytokines that activate cytotoxic T-cells even in the absence of co-stimulatory signals.Furthermore,vascular endothelial growth factor(VEGF)and betaﬁbroblast growth factor (bFGF)secretion and cellular response are suppressed by thalidomide,thus antagonizing neoangiogenesis and altering the bone marrow stromal microenvironment in hematologic malignancies.The thalidomide analogs,lenalidomide(CC-5013;Revlimid)and CC-4047(Actimid),have enhanced potency as inhibitors of TNF-␣and other inﬂammatory cytokines,as well as greater capacity to promote T-cell activation and suppress angiogenesis.Both thalidomide and lenalidomide are effective in the treatment of multiple myeloma and myelodysplastic syndromes for which the Food and Drug Administration granted recent approval.Nonetheless,each of these IMiDs remains the subject of active investigation in solid tumors,hematologic malignancies,and other inﬂammatory conditions.This review will explore the pharmacokinetic and biologic effects of thalidomide and its progeny compounds.©2007Elsevier Ltd.All rights reserved.Keywords:Thalidomide;Immunomodulatory agent;Angiogenesis;Lenalidomide;CC-4047Contents1.Introduction (1490)2.Pharmacokinetic properties (1490)3.Pharmacodynamic effects (1491)3.1.Immunomodulation (1491)3.2.T-cell activation (1492)3.3.Angiogenesis (1492)3.4.Bone marrow microenvironment (1493)3.5.Anti-tumor and apoptotic mechanisms (1493)4.Role of IMiDs in cancer therapy (1493)4.1.Multiple myeloma (1493)4.2.Myelodysplastic syndromes (1493)5.Conclusion (1496)References (1496)∗Corresponding author.Tel.:+18137453163;fax:+18137453071.E-mail addresses:Melcheme@mofﬁ(M.Melchert),ListAF@mofﬁ(A.List).1357-2725/$–see front matter©2007Elsevier Ltd.All rights reserved.doi:10.1016/j.biocel.2007.01.0221490M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991.IntroductionThalidomide(Thalomid TM,Celgene Corp,Warren, NJ)is a synthetic glutamic acid derivative which was ini-tially developed in the early1950s as an anticonvulsant for the treatment of epilepsy.Following a lack of sufﬁ-cient efﬁcacy as an anti-epileptic,it was eventually mar-keted as a sleep aid and was also widely used as an anti-emetic during pregnancy(Randall,1990).Prior to its use in gravid women,the teratogenic effects of thalidomide in animals or humans were unknown.However,by1962, two published reports by William McBride in Australia and Widukind Lenz in Germany emerged describing limb and bowel malformations in children born to moth-ers who were exposed to thalidomide during pregnancy (Lenz,1962;McBride,1961).Use of this agent dur-ing theﬁrst trimester of gestation led to alarming rates of phocomelia,defects in long bones,absence of auri-cles,cleft lip,and cardiac and gastrointestinal anomalies. While thalidomide had not received approval by the Food and Drug Administration(FDA)in the United States (US)at this time due to concerns of neurotoxicity,the widespread international use of this drug as a sedative was eventually halted during the early1960s.The pharmacological effects of thalidomide extended beyond its neurosedative effects,and for that reason, it was subsequently investigated in a number of der-matologic,rheumatologic,and malignant diseases.In 1965,theﬁrst report of clinical activity in erythema nodosum leprosum(ENL)was serendipitously discov-ered by a dermatologist from Israel,Jacob Sheskin (Sheskin,1965).Thalidomide was found to be effec-tive in reducing fevers,night sweats,and improving skin lesions in a patient with ENL.However,it was not until1998that thalidomideﬁrst received approval by the FDA for the treatment of ENL(Pearson&Vedagiri, 1969;Sheskin,1965;Waters,1971).More recently, thalidomide was approved for the treatment of multi-ple myeloma in May2006,and has reported efﬁcacy in a wide spectrum of malignant and non-malignant dis-eases.Access to this agent in the US is restricted,and requires participation in the System for Thalidomide Education and Prescription Safety(STEPS)program to prevent adverse outcomes related to teratogenicity (Zeldis,Williams,Thomas,&Elsaved,1999).Thalidomide and its analogs are immunomodula-tory drugs(IMiDs)that exhibit a multitude of biologic effects on cytokine and cell-mediated responses.These effects are largely responsible for the clinical efﬁcacy seen in conditions such as lupus erythematosis,apt-hous ulcers that occur in human immunodeﬁciency virus and Behcet’s disease,ENL,graft versus host dis-ease,and cancer(Hamuryudan et al.,1998;Jacobson et al.,1997;Kumar,Witzig,&Rajkumar,2004;Parker et al.,1995;Pearson&Vedagiri,1969).Thalidomide is the most widely studied of the IMiDs;however, the newer structural analogs,CC-5013(lenalidomide, Revlimid TM,Celgene Corp.,Warren,NJ)and CC-4047 (Actimid TM),promise even greater activity for clinical development.These two analogs of thalidomide were developed in the mid-1990s with enhanced potency and a relatively favorable toxicity proﬁle.This review will summarize the pharmacokinetic and biologic properties of thalidomide and its derivatives.2.Pharmacokinetic propertiesThalidomide(␣[N=Phthalimido]glutarimide)con-tains a glutarimide moiety with a single chiral center and is formulated as a racemic mixture of two active enantiomers,S(−)and R(+)(see Fig.1).Initially,the S(−)isoform was thought to be the enantiomer pri-marily responsible for the teratogenic effects and the R(−)isoform for the sedative properties.Puriﬁcation of the R(−)enantiomer was attempted in order to optimize its clinical application,but was eventually not found to be technically feasible secondary to the rapid interconversion of isomers under physiologic con-ditions.Furthermore,both forms were found to be teratogenic in the New Zealand rabbit model,and thus it remains a racemic mixture(Eriksson,Bjorkman,Roth,& Fig.1.Adapted with permission from Macmillan Publishers Ltd:Nature Reviews Cancer,2004(Bartlett et al.,2004);(a)the chemical structure of thalidomide;(b)alteration of the structure of thalidomideby adding an amino(NH2−)group at the4position of the phthaloyl ring to generate the IMiDs CC-5013and CC-4047.For CC-5013,one of the carbonyls(C O)of the4-amino-substituted phthaloyl ring has been removed.M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991491Hoglund,2000;Fabro,Smith,&Williams,1967).Poor water solubility led to the development of thalidomide exclusively as an oral agent,with peak plasma con-centrations of the drug detected at3–6h(Chen et al., 1989).Upon ingestion,thalidomide undergoes sponta-neous non-enzymatic hydrolytic cleavage into more than 12different metabolites.Thalidomide and its metabo-lites are quickly eliminated in the urine,with a mean elimination half-life of approximately5h.The pharma-cokinetic properties of thalidomide in the presence of renal or hepatic dysfunction are largely unknown.Dos-ing with thalidomide is disease-dependent,with daily oral schedules of50–800mg,demonstrating efﬁcacy in a variety of disease processes.Over the last decade,second-generation IMiD com-pounds have been developed by chemical modiﬁcation of the structural backbone of thalidomide in order to enhance immunomodulatory potency and minimize the dose-limiting neurotoxic effects.Both lenalidomide and CC-4047are4-amino-gultaramide derivatives of thalidomide in which an amino group was added to the fourth carbon of the phthaloyl ring of the parent com-pound(see Fig.1)(Bartlett,Dredge,&Dalgleish,2004; Teo et al.,2003).Both agents also exist as a racemic mixture of the active S(−)and R(+)forms.Like thalido-mide,they are both administered in daily oral dosing every21–28days of monthly cycles.Renal elimination predominates,and caution is recommended in dosing of patients with impaired creatinine clearance.The ter-minal half-life of lenalidomide and CC-4047after oral administration are reported as3and7h,respectively (Bartlett et al.,2004;Schey et al.,2004).In contrast to thalidomide in which somnolence,constipation,and peripheral neuropathy are common dose-limiting toxic-ities,both lenalidomide and CC-4047lack signiﬁcant neurosedative toxicity.Dose-limiting neutropenia and thrombocytopenia are the most common toxic sequelae, while venous thromboembolism has been reported with use of all three agents.3.Pharmacodynamic effects3.1.ImmunomodulationThalidomide and its derivatives are potent immunomodulators with biologic effects on both cytokine stimulation and cell-mediated immunity(see Fig.2).One of the key mediators responsible for the anti-inﬂammatory responses seen with the use of IMiD agents is tumor necrosis factor-alpha(TNF-␣). Thalidomide enhances degradation of TNF-␣mRNA, thus suppressing this pro-inﬂammatory cytokinerelease Fig.2.Reproduced with permission from Journal of Clinical Oncol-ogy,2004(Kumar et al.,2004).Proposed mechanism of action of thalidomide in cancer illustrated using myeloma as an example. Thalidomide inhibits angiogenesis,enhances effects of the immune system,inhibits binding of tumor cells to stroma,and inhibits various cytokines.Thaliomide may also have direct effects on the tumor.NK: natural killer;TNF:tumor necrosis factor;IL-6:interleukin-6;VEGF: vascular endothelial growth factor;NF-kB:nuclear factor kappa B. from endotoxin-stimulated monocytes and macrophages (Moreira et al.,1993).The effect on TNF-␣is felt to be largely responsible for the clinical beneﬁt seen in patients with inﬂammatory conditions such as ENL,who have high endogenous TNF-␣production that is suppressed following thalidomide treatment (Sampaio et al.,1993).In addition,both lenalidomide and CC-4047have up to a50,000-fold higher potency than thalidomide as inhibitors of cytokine generation, including suppression of endotoxin-induced TNF-␣secretion(Bartlett et al.,2004;Corral et al.,1999a). Activation of the transcription factor nuclear factor kappa B(NF-kB),a key regulator of TNF-␣and inter-leukin(IL)-8production,is blocked after thalidomide exposure through inhibition of the inhibitor of kappa B (IkB)kinase(Keifer,Guttridge,Ashburner,&Baldwin, 2001).However,the cellular response to IMiD agents is quite complex and lineage and ligand speciﬁc,with evidence that TNF-␣generation is actually enhanced in the setting of T-cell activation.This is demonstrable in1492M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–1499vitro with upregulation of TNF-␣production by CD4+ and CD8+T-lymphocytes stimulated by anti-CD3 (Marriott et al.,2002).Furthermore,an increase in TNF-␣serum concentration has been reported upon exposure to IMiD agents in early-phase trials involving solid tumors and inﬂammatory dermatologic diseases such as toxic epidermal necrolysis(Bartlett et al.,2004; Wolkenstein et al.,1998).While the biologic effects of TNF-␣modulation by the IMiD agents may be central to the therapeutic bene-ﬁts seen in a variety of inﬂammatory conditions,there are several other cytokines that are similarly affected by this class of agents that may play a signiﬁcant role in immune modulation.Generation of pro-inﬂammatory enzymes and cytokines,such as cyclooxygenase-2(COX-2), interleukin-1beta(IL-1␤),transforming growth fac-tor(TGF)-␤,and IL-6are suppressed upon exposure to IMiDs,and contribute to the activation of the T-cell receptor(TCR),particularly in the T H1subset (Bartlett et al.,2004;Corral et al.,1999a;Musto,2004). Analogous to TNF-␣modulation,IL-12secretion is sup-pressed by IMiDs when monocytes are stimulated by lipopolysaccharide and is enhanced in the setting of T-cell stimulation(Corral et al.,1999a,1999b).Expansion of both T-cells and NK-cells is promoted by IL-12secre-tion and thus IMiD agents have the potential to be useful adjuncts in the development of cancer vaccines and other immunotherapuetic approaches(Trinchieri,1998).Fur-thermore,IL-12stimulates interferon-␥(IFN-␥)produc-tion and both cytokines have demonstrable anti-tumor activity and anti-angiogenic-activity(Beatty&Paterson, 2001;Qin&Blankenstein,2000;Trinchieri,1998). 3.2.T-cell activationThe immune response to foreign antigens is a highly regulated process requiring the presentation of major histocompatability(MHC)-bound peptides on antigen presenting cells(APCs)to the TCR.Acces-sory molecules,such as B7on APC and CD28on the T-cell surface,provide secondary signals that are vital to the promotion of T-cell responsiveness.These interactions lead to the subsequent activation and pro-liferation of T-cells followed by a cascade of cytokine and cellular responses.Thalidomide and its deriva-tives are able to enhance the CD8+T-cell response in the absence of these secondary co-stimulatory sig-nals(Haslett,Corral,Albert,&Kaplan,1998;Mueller, Jenkins,&Schwartz,1989).In vitro data of primary human T-cells suggest that thalidomide enhances IL-2 mediated T-cell proliferation and IFN-␥production via the TCR complex.The effect of thalidomide on T-cell expansion is dose-dependent and occurs even at low lev-els of CD3stimulation.Furthermore,thalidomide affects the balance between T-helper(Th)-1and-2subsets at least in part through cytokine modulation of IL-4,IL-5,and IFN-␥(McHugh et al.,1995).In the presence of thalidomide,mitogen-and antigen-stimulated human peripheral blood mononuclear cells preferentially induce the Th-2subtype,which corresponds with maximum enhancement of IL-4production.Thalidomide derivatives are also potent co-stimulators of T-cells that enhance activation of CD8+ T-cells in vitro(Stirling,2001).Upon exposure to CMV and inﬂuenza virus matrix proteins,both CC-5013and thalidomide were shown to enhance CD8+cytokine production and cytotoxic activity(Haslett,Hanekom, Muller,&Kaplan,2003).CC-4047has demonstrated durable,tumor-speciﬁc Th-1type immunity in mice tumor xenograft models(Dredge,Marriott,Todryk et al.,2002).These preclinical investigations have suggested a potential role for the use of IMiD agents in the development of tumor vaccine adjuncts and as modulators of immune response in the setting of defective CD4+mediated immunity such as HIV and graft versus host disease.3.3.AngiogenesisIn the early1990s,thalidomide wasﬁrst reported to exhibit potent anti-angiogenic properties that were thought to contribute to the teratogenic effects of limb bud malformations observed in offspring of thalidomide exposed gravid mothers(D’Amato,Loughnan,Flynn, &Folkman,1994).Eventually,the effect of IMiDs on vasculogenesis was recognized as a mechanism of poten-tial clinical beneﬁt in the treatment of malignancies in which neoangiogenesis is a conserved feature of the malignant phenotype.Vascular endothelial growth fac-tor(VEGF)and betaﬁbroblast growth factor(bFGF)are potent stromal mitogens that are produced in excess in a variety of malignancies,including multiple myeloma and myeloid disorders.Paracrine and autocrine secre-tion of VEGF causes proliferation of multiple myeloma cell lines and has also been shown to promote self-renewal of leukemia progenitors(Bellamy et al.,2001; Gupta et al.,2001).Secretion of both VEGF and bFGF from tumor and bone marrow stromal cells is sup-pressed upon exposure to IMiDs,resulting in reduced endothelial cell migration and adhesion(D’Amato et al.,1994;Dredge,Marriott,Macdonald et al.,2002; Lentzsch et al.,2003).Both thalidomide and CC-4047 have been shown to suppress the induction of VEGF in co-cultures of multiple myeloma cell lines and boneM.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991493marrow stromal cells(Gupta et al.,2001).Additionally, thalidomide inhibits bFGF-induced angiogenesis in vivo using a rabbit cornea micropocket assay(D’Amato et al.,1994).In murine lymphoma and rectal carcinoma cell line xenografts,IMiDs decrease tumor microvessel formation(Dredge,Marriott,Macdonald et al.,2002; Lentzsch et al.,2003).The latter anti-angiogenic effect is independent of TNF-␣suppression or endothelial cell proliferation,and appears to be dose-or concentration-dependent.Furthermore,lenalidomide has been shown to attenuate AKT-dependent phosphorylation,resulting in a reduction of bFGF-induced endothelial cell migra-tion(Dredge,Marriott,Macdonald et al.,2002;Dredge et al.,2005).3.4.Bone marrow microenvironmentThe pro-inﬂammatory responses within the bone marrow microenvironment are thought to play a sup-portive or tumor nurturing role in many hematologic diseases,including multiple myeloma and myeloid dis-orders.TNF-␣induces expression of IL-6secretion by bone marrow stromal cells and is markedly upregulated upon adhesion of multiple myeloma cells to bone mar-row stromal cells(Gupta et al.,2001;Witzig,1999). The adhesion molecules,ICAM-1,LFA-1,and VCAM-1,are similarly affected in the bone marrow milieu (Miyamoto et al.,1995).IL-6promotes the prolifera-tion of multiple myeloma cell lines and inhibits Fas-and dexamethasone-induced apoptosis in vitro.Both thalidomide and CC-4047decrease upregulation of IL-6in co-cultures of bone marrow stromal and multiple myeloma cells,suggesting an additional mechanism of therapeutic beneﬁt in multiple myeloma.Lenalido-mide induces activation of cell adhesion molecules and increases␤1integrin-mediated adhesion to extracel-lular matrix proteinﬁbronectin(Dredge et al.,2005; Miyamoto et al.,1995).3.5.Anti-tumor and apoptotic mechanismsIndependent of the immune-modulating activities, IMiDs have direct anti-proliferative activity in hema-tologic malignancies.The IMiDs,as a class,induce concentration-dependent inhibition of proliferation in multiple myeloma cell lines and primary myeloma cells that are resistant to standard chemotherapy(Lentzsch et al.,2003).Effects on apoptosis are evident at multiple levels of death receptor signaling,including potentia-tion of TNF-related apoptosis-inducing ligand(TRAIL), inhibition of apoptosis protein-2,increased sensitivity to Fas induction,and upregulation of caspase-8activa-tion(Keifer et al.,2001;Mitsiades et al.,2002).G0/G1 cell cycle arrest or apoptosis in leukemia cell lines and other hematologic malignancies occurs with lenalido-mide exposure and is mediated,at least in part,by A-dependent mechanisms(Dredge et al.,2005).Further-more,lenalidomide has preferential anti-proliferative activity against a5q mutant cell line(MUTZ-1)with corresponding induction of expression of genes encoded at the5q locus(Gandhi et al.,2004).These preclinical ﬁndings have been conﬁrmed in clinical trials involving patients with myelodysplastic syndromes(MDS).4.Role of IMiDs in cancer therapyThalidomide and its analogs have therapeutic potential in a wide spectrum of diseases given their multifaceted pharmacologic effects.The known immunomodulatory and anti-angiogenic properties of IMiDs provided the impetus to investigate these agents in the treatment of both hematologic malignancies and in solid tumors.Numerous early-phase trials in solid tumors have shown activity in prostate cancer,breast cancer,Kaposi’s sarcoma,renal cell cancer,melanoma, neuroendocrine tumors,hepatocellular carcinoma,lung cancer,and gliomas(Kumar et al.,2004).Response rates have been promising in some;however,further studies are needed to elucidate the true magnitude of thera-peutic beneﬁt in solid tumors.In addition,the IMiDs are attractive agents for the treatment of both myeloid and lymphoid malignancies with reported activity in non-Hodgkin’s lymphoma,acute myeloid leukemia,and myeloﬁbrosis with myeloid metaplasia(Kumar et al., 2004).However,the earliest focus of clinical investiga-tions involved multiple myeloma and MDS and thus will be discussed in further detail.4.1.Multiple myelomaThe use of IMiDs in the treatment of multiple myeloma has recently emerged as the standard of care following multiple reports of efﬁcacy in both front-line therapy and in relapsed or refractory dis-ease(Glasmacher et al.,2006;Rajkumar,Blood,Vesole, Fonseca,&Greipp,2006;Singhal et al.,1999;Tosi, Zamagni,&Cellini,2004).In May2006,thalidomide was approved by the US FDA for the treatment of newly diagnosed multiple myeloma in combination with dex-amethasone.The approval was based on a phase III multicenter trial performed by the Eastern Coopera-tive Oncology Group(ECOG)in which patients were randomized to four cycles of dexamethasone40mg on days1–4,9–12,17–20of28-day cycles either1494M.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–1499Table1Summary of MM-009and MM-010,phase III trials of CC-5013in multiple myelomaMM-009MM-010North American trial International trialCC-5013+Dex Dex alone CC-5013+Dex Dex alone TTP(weeks)60.120.753.420.6 Overall RR(%)61.222.85821.7CR(%)26.5 4.113.6 4.0 Dex:dexamethasone;TTP:time to progression;RR:response rate;CR:complete response.alone or with200mg daily of thalidomide(Table1) (Rajkumar et al.,2006).Paraprotein responses in serum and urine monoclonal protein levels were observed in63%of patients treated on the combination arm compared to41%of patient receiving dexamethasone alone(p=.0017).Toxicities were signiﬁcant,with17% of patients developing deep venous thrombosis which prompted the recommendation for anticoagulation dur-ing combination therapy.Other adverse events included rash,bradycardia,and neuropathy.As a consequence,the thalidomide/dexamethasone combination is a common induction regimen for use as initial therapy in multiple myeloma and prior to autologous transplant.Thalidomide has also enjoyed success in the salvage setting,with multiple phase I and II trials demon-strating efﬁcacy either alone with response rates of 25–35%,or in combination with dexamethasone with response rates of50–60%(Glasmacher et al.,2006; Tosi et al.,2004).Combination chemotherapy trials with thalidomide have yielded variable rates of suc-cess in relapsed and refractory disease when applied in conjunction with dexamethasone,cisplatin,adriam-cyin,cyclophosphamide,etoposide,clarithramycin,and melphalan(Coleman et al.,2002;Kyriakou et al.,2005; Lee et al.,2003;Srkalovic,Elson,Trebisky,Karam, &Hussein,2002).Finally,thalidomide has also shown beneﬁt in the maintenance setting.The Intergroupe Fran-cophone du Myelome(IFM)recently reported the results of trial involving597patients who were randomized after autologous stem cell transplantation to three of the treatment strategies,i.e.,either(A)no maintenance, (B)pamidronate maintenance,or(C)thalidomide and pamidronate maintenance treatment until disease pro-gression(Attal et al.,2006).A complete or very good partial response was achieved in55%of patients in arm A,57%in arm B,and67%in arm C(p=0.03).The 3-year post-randomization probability of event-free sur-vival was improved with the thalidomide combination maintenance with36%of patients in arm A,37%in arm B,and52%in arm C remaining without evidence of disease progression(p<.009).The4-year probabil-ity of survival from the date of diagnosis was improved in the thalidomide/pamidronate maintenance arm(77% versus74%versus87%;p<.04).The initial dose of thalidomide was400mg daily,with39%of patients dis-continuing treatment secondary to adverse effects at a median of8months.Thus,while toxicities remain sub-stantial,thalidomide is an effective agent in the treatment of myeloma in all stages of the disease.Of particular importance,maintenance therapy with thalidomide in this trial did not increase the risk of thromboembolic complications,suggesting that thrombogenic potential may be highest when thalidomide is administered dur-ing induction therapy when tumor burden is high or when combined with agents that have intrinsic thrombogenic potential.Lenalidomide was subsequently approved by the FDA in June2006for combination treatment with dex-amethasone for relapsed or refractory multiple myeloma. Based on encouraging data from several phase I and II trials,two phase III randomized,multicenter trials (MM-009,US and MM-010,Europe)were initiated comparing lenalidomide and dexamethasone combi-nation treatment with dexamethasone and placebo in patients with multiple myeloma who had received at least one prior therapy(Weber et al.,2006).Interim results were reported at the American Society of Clinical Oncology Meeting in2006.Lenalidomide was adminis-tered in a daily25mg dose for21days in combination with dexamethasone pulse dose treatment of40mg on days1–4,9–12,and17–20in a28-day cycle.Major reductions in serum paraprotein concentration were reported in51–53%of the lenalidomide/dexamethasone group and were statistically superior to those seen in patients receiving dexamethasone alone(OR 5.5 [3.9,9.1];p<.0001).The median time to progression (TTP)in MM-009compared favorably:37weeks in the lenalidomide/dexamethasone arm versus19.9weeks for dexamethasone alone(HR=0.356[0.257,0.494]; p>.0001).Toxicities were manageable;however,grade 3/4neutropenia and thrombocytopenia were experienced in27%and17%of patients,respectively,with a7.8% incidence of deep vein thrombosis.Importantly,the adverse events commonly seen with thalidomide,such asM.Melchert,A.List/The International Journal of Biochemistry&Cell Biology39(2007)1489–14991495neuropathy,constipation,and sedation,were infrequent in these trials.Other toxicities include included rash, gastrointestinal symptoms,myalgias,and pulmonary embolism.Among patients receiving the lenalidomide combination in the MM-009study,thrombo-embolic events were reported in20of87patients(23%)who received concomitant recombinant erythropoietin ther-apy and in4of83patients who did not receive erythropoietin(5%)(Knight,DeLap,&Zeldis,2006). In the group that received placebo and dexametha-sone,thrombosis occurred in5of67patients(7%) who received concomitant erythropoietin and in1of 103patients(1%)who did not receive erythropoietin. Multivariate analysis of both studies combined showed an independent correlation between the development thrombotic events and treatment with the combination of lenalidomide and high-dose dexamethasone or treatment with concomitant erythropoietin.Of particular interest, thrombotic events were not reported among23patients who received aspirin or salicylates during theﬁrst month of treatment,as compared with52of668patients who did not receive aspirin or salicylates.A recent study showed a reduction in the thrombosis rate with the administration of aspirin in myeloma patients receiving a combination of thalidomide and anthracycline,sug-gesting that thrombotic risk with the IMiDs may be reduced when used in combination therapy by avoid-ance of recombinant erythropoietins that have inherent thrombotic potential,or prophylaxis with aspirin or alter-nate strategies(Baz et al.,2005).The latter approach is a subject of current investigation in an ECOG study.Dis-tribution of lenalidomide,like its parent compound,is also highly regulated given concerns for possible ter-atogenic effects.All patients prescribed this drug must participate in the Rev-Assist program prior to receiving medication.A phase I,dose-escalation study of CC-4047in multiple myeloma has also been reported with demon-strated safety and efﬁcacy(Schey et al.,2004).The drug was well tolerated,with the main dose-limiting toxic-ities being neutropenia and venous thrombosis with a maximum tolerated dose of2mg/day.A25%or more reduction in paraprotein was seen in67%of patients,and 54%achieved greater than a50%reduction.In support of T-cell activation as possible mechanistic role,increased serum IL-2receptor and IL-12levels were detected in patients receiving CC-4047.4.2.Myelodysplastic syndromesIMiDs were initially targeted as potential agents for the treatment of MDS based on their immunomodula-tory and anti-angiogenic features.Theﬁrst report of the use of thalidomide in MDS involved a trial of83 patients and included biologic correlates designed to assess angiogenic and inﬂammatory responses(Raza et al.,2001).Thalidomide was initiated at100mg daily and titrated upwards to a dose of400mg daily as tolerated. At12weeks of treatment,only61%of patients were able to complete the planned course of treatment sec-ondary to dose-limiting toxicities.Only13%of patients experienced a major erythroid response,and platelet or neutrophil improvements were uncommon.How-ever,erythroid responses were durable,with a median duration of306days.Thalidomide has since been inves-tigated in multiple trials at doses of200–1000mg daily in the treatment of both low-and high-risk MDS,with either hematologic improvement or partial responses reported in approximately20–60%of patients(Bouscary et al.,2005;Moreno-Aspitia et al.,2006;Zorat et al.,2001).At these doses,tolerance of thalidomide is discouraging,with a high frequency of dose-limiting fatigue,constipation,neuropathy,and sedation.For this reason,enthusiasm for development of thalidomide in the treatment of MDS has been tempered.In contrast,lenalidomide has shown remarkable activ-ity in the treatment of MDS with toxicities that compare favorably to thalidomide.The initial report on the safety and efﬁcacy of lenalidomide in MDS included 43patients with symptomatic anemia who had either failed treatment with erythropoietin(EPO)or were poor candidates for beneﬁt from EPO therapy(frequent transfusions(>2per month)and/or high endogenous ery-thropoietin serum concentration(>500mU/ml))(List et al.,2005).Patients were randomized to one of three dos-ing schedules:25mg daily,10mg daily,or10mg/day for21of ing the International Work-ing Group(IWG)criteria,56%of patients experienced durable erythroid responses and20of32patients who previously required RBC support became transfusion independent.Furthermore,10of12patients with an isolated interstitial deletion of chromosome5(5q31.1) experienced an erythroid response compared to57%of patients with a normal karyotype or12%of patients with other chromosomal abnormalities.Patients with 5q31.1had particular beneﬁt with a longer duration of transfusion independence.Both cytogenetic responses and decreased medullary myeloblast percentage were noted in patients responding to lenalidomide,and were more pronounced in patients with chromosome5q31.1 deletion.The dose-limiting toxicities were related to myelosuppression,with grade3or greater neutrope-nia(58%)and thrombocytopenia(50%)necessitating dose reductions or treatment interruption in47–77%of。

葡萄糖脱氢酶法的英文

葡萄糖脱氢酶法的英文Glucose Dehydrogenase Method.The glucose dehydrogenase method is a spectrophotometric assay for determining the concentration of glucose in a sample. It is based on the enzymatic reaction catalyzed by glucose dehydrogenase (GDH), which oxidizes glucose to gluconic acid and reduces NAD+ to NADH. The amount of NADH produced is stoichiometrically equivalent to the amount of glucose in the sample, and can be measured by its absorbance at 340 nm.The reaction is carried out in a cuvette containing a buffer, GDH, NAD+, and the sample. The change in absorbance at 340 nm is monitored over time, and the rate of change is used to calculate the concentration of glucose in the sample.The glucose dehydrogenase method is a simple, rapid, and accurate method for determining the concentration ofglucose in a variety of samples, including blood, urine, and food products. It is commonly used in clinical chemistry laboratories and in food analysis.Principle of the Method.The glucose dehydrogenase method is based on the following enzymatic reaction:Glucose + NAD+ + H2O → Gluconic acid + NADH + H+。

酵母静息细胞催化丙酮酸乙酯不对称还原制(S)-乳酸乙酯

ＹＡＮＧｉｎＺＨＡＮＧｉＺｈｒｇ，ｏＪｅ，
ＬｅｃｎｅｏｌｅＳｃｕｎＵｉｒｔＣｅｇｕ６０６，ｉｕｎＣｉ／ｉｃｌｇ，ｉａｎｖｓｙｈｎｄ１０５Ｓｃａ，ｈａｆＳｅＣｅｈｅｉ，ｈｎ
ＡｂｔａｔＴｈｒｉｔｉｓｈｔｘｉｉｄｔｅｃｔｌｔｃｉｉｒｓｍｍｅｒｅｕｔｎｏｔｙｙｕａｅｔ一ｔｙｃａｅｗｅｅｉｌｔｄｓｒｃ：ｉｙｓｘｓａｎａｈｂｔｈａａｙｉａｔｔｆｙｔｒｔｅｅｃｖｙｏａｔｉｒｄｃｉｆｅｈｌｒｖｔｏ（ｅｈｌａｔｔｒｏａｅｃｏｐｌｓ
２ｐｒｅｔｆｉｍｄｉ，ｈｎｄｄｃｌｏｅｅＣｅｇｕ６０８，ｉｕｎＣｉａＤｅａｔｎｏＢｏｅｉｎＣｅｇｕｍｃｅＭｅｉｌｇ，ｈｎｄ１０３Ｓｃａ，ｈａＣｌｈｎ
ＤｐｒｅｔｆｄｃｌｘｍｎｔｎＣｅｇｕｄｃｌｏｌｅＣｅｇｕ１０３Ｓｈａ，ｈｎｅａｔｎＭｅｉａｉｉ，ｈｎｄｉｌｇ，ｈｎｄ０８，ｉｕｎＣｉｍｏａＥａｏＭｅａＣｅ６ｃａ
体湿度０１５ｇｍｌ内酮酸乙酯初始浓度６．７／，５ｍｍｏ／，３Ｃ反应４条什，酮酸乙酯转化率达９．％，物对映体过ｌ于２。Ｌ８ｈ的丙５５产
量值（ｅ值）为９．％．ｅ２１

超高效液相色谱法同时测定柑橘中主要酚酸和类黄酮物质_郑洁_赵其阳_张耀海_焦必宁

超⾼效液相⾊谱法同时测定柑橘中主要酚酸和类黄酮物质_郑洁_赵其阳_张耀海_焦必宁⽹络出版时间：2014-12-03 14:37⽹络出版地址：/doc/12dcefde846a561252d380eb6294dd88d1d23d63.html/kcms/detail/11.1328.S.20141205.1534.002.html中国农业科学 2014,47(23):4706-4717Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2014.23.015超⾼效液相⾊谱法同时测定柑橘中主要酚酸和类黄酮物质郑洁1,2，赵其阳2，张耀海2，焦必宁2,3（1西南⼤学园艺园林学院，重庆 400716；2西南⼤学柑桔研究所/农业部柑橘产品质量安全风险评估实验室（重庆），重庆400712；3国家柑桔⼯程技术研究中⼼，重庆 400712）摘要：【⽬的】建⽴超⾼效液相⾊谱快速、同时测定柑橘中主要酚酸和类黄酮组成及含量的⽅法，为柑橘中酚类物质的开发提供技术⽀撑。

【⽅法】⾸先对上机条件优化，检测波长选择基于全扫描（190—400 nm），选择所有物质都有最⼤吸收光谱的波长；柱温、流动相类型和流速优化参考相关⽂献，为了能使基线分离，采⽤梯度洗脱⽅式；再从提取剂类型、提取次数和时间进⾏单因素⽐较试验，对柑橘样品处理⽅法进⾏优化；柑橘样品经⼄酸⼄酯振荡提取30 min，果⽪和果⾁分别提取4次和3次，蒸发浓缩，甲醇定容上机进⾏测定。

【结果】以ACQUITYUPLC BEH C18液相⾊谱柱（2.1 mm×100 mm，1.7 µm）为分离柱，柱温为35℃，进样量为3.0 µL，流速为0.3 mL·min-1；梯度洗脱，以0.3%⼄酸⽔溶液（A）/甲醇（B）为流动相：95%—80%（0—3 min）A，80%—80%（3—8 min）A，80%—70%（8—12 min）A，70%—20%（12—17 min）A，20%—95%（17—20 min）A；定量波长为283 nm。

高效液相色谱法同时测定L_苏糖酸钙和柠檬酸的含量

快速、简便、准确的实验方法。

此方法已成功应用于静脉输注甘露醇开放血脑屏障对头孢三嗪透过血脑屏障影响的研究中。

参考文献:[1]　赵　侠,孙培红,周　颖,等.离子对反相高效液相色谱法测定人血清和尿液中的头孢曲松[J ].中国临床药理学杂志,2002,18(3):204.[2]　张　玉,王凯平,谭红艾,等.胆道手术病人中头孢曲松钠的药动学[J ].中国医院药学杂志,2001,21(9):5372539.[3]　G aillard JL ,Abadie V ,Cheron G ,et al .Concentrations of ceftriax 2one in cerebrospinal fluid of children with meningitis receiving dexamethasone therapy[J ].A nti micro A gents Chemother ,1994,38(5):120921210.[4]　Buke AC ,Cavusoglu C ,Karasulu E ,et al .Does dexamethasone af 2fect ceftriazone penetration into cerebrospinal fluid in adult bacte 2rial meningitis[J ].Int J A nti microb A gents ,2003,21:4522456.[5]　黄　英,梁茂植,余　勤,等.血浆头孢曲松HPLC 测定法及其在胸外术后患者的应用[J ].中国抗生素杂志,2000,25(2):109.[6]　葛庆华,吴卫泽,支晓瑾,等.头孢曲松(罗氏芬)在T 管胆汁中药动学研究[J ].中华肝胆外科杂志,2004,10(1):23225.(本文编辑:姜　黄)收稿日期:2005-04-20作者简介:高　革(1966-),男,河南新乡人,本科,实验师,研究方向:分析测试实验研究。

Simultaneous Determination of Folic Acid and Uric Acid

Simultaneous Determination of Folic Acid and Uric AcidFolic acid (FA) is water-soluble of vitamin B group (VB9). It exists in the kidneys and livers of animals, plants, mushrooms and algae. FA is an important substance for keeping activity and healthof living beings and is essential for cell growth and division in the human body. It participates in lots of bodily reactions and mainly in synthesis of nucleic acid and some important substances, and promotes the synthesis of protein from amino acid. Deficiency of FA causes high homotype cysteine acidemia, psychosis, devolution of mentality, and anaemia, and it is thought to increase the likelihood of heart attack and stroke. As a consequence, it is necessary to quantify FA with reliable results.As we known, uric acid (UA) is the primary final product of purine metabolism in the human body, and is excreted via urine. It is of biomedical significance, and plays determining roles not only in human metabolism but also in the central nervous and renal systems. Abnormal levels of UA are symptoms of several diseases such as gout, hyperuricaemia and Lesch-Nyhan syndrome. UA is also a risk factor for leukemia, pneumonia and cardiovascular disease. In addition, numerous reports have shown FA and UA usually co-exist biological systems and that they influence each other in their respective activities. Therefore, investigation of neurological behavior and also simultaneous determination of FA and UA is of great importance for the elucidation of their precise physiological functions. Due to interesting electrochemical properties of FA and UA, application of the modified electrodes is considered as the common routes in this area. However, ascorbic acid (AA) is always coexisting with FA and UA in the extra cellular fluid of the central nervous system and serum. Therefore, simultaneous determination of FA and UA in the presence of AA is an important issue not only in diagnostic and pathological research but also in the field of biomedical chemistry. The electrochemical response of AA will certainly interfere in the detection of FA and UA under coexistence of AA on conventional electrodes. To resolve this problem, various chemical modification of electrode surface have been made for the determination of FA and UA in the presence of AA.In the recent years, many of the studies focused on substituted polythiophene derivatives, and among others poly(3,4-ethylenedioxythiophene) (PEDOT) electrosynthesized either in organic or in aqueous media, has the advantage of providing very stable and highly conductive films. This polymer possesses many favourable properties such as high conductivity, good biocompatability, band gap width, excellent stability or optical transparency in its conducting state, as well as an environment-friendly feature. Owing to these properties, it exhibits promising potential applications in various fields, such as electro-chromic devices, anti-static coatings, capacitors and fuel cells. In addition, PEDOT has received particular attention for the electroanalytical application, e.g., detection of chemical and biomolecules such as NADH, ascorbic acid and cysteine, etc. due to retaining of its electroactivity for wide range of pH. Recently, the developments in nano-structured conducting polymers and polymer nanocomposites have large impact on electro- catalysis research and sensor applications. Thus interest has been shown on the synthesis of carbon-based nanomaterials/PEDOT composites that exhibit unique properties of both individual components in a synergistic manner. Carbon nanotubes (CNTs), categorized as multi-walled carbon nanotubes (MWCNTs) and single walled carbon nanotubes (SWCNT), have generated a considerable interest since being discovered due to their excellent electrical conductivity,chemical stability, high surface area and high mechanical strength. Consequently, CNTs have been used as an electrode material or modifier to promote electron transfer reactions between biomolecules and the underlying electrodes. For these reasons, CNTs have the ability to compound with PEDOT to increase the mechanical properties of polymer and enhance the electrical characteristics by facilitating the charge-transfer processes between the two components when used as electrodes for constructing of efficient electrochemical sensors. To the best of our knowledge, there is few article reported the simultaneous electroanalytical determination of FA and UA by using conducting polymer PEDOT nanocomposites.。

高效液相色谱法同时测定党参中3种指标性成分含量

doi ：4. 3969/j. Rsc. 406 - 493 0 202 0 03. 04高效液相色谱法同时测定党参中3种指标性成分含量**基金项目：陕西省重点研发计划［2015SF-258］；陕西省中医药研究院科研课题［2013 - 07］ ;2018年中医药公共卫生服务补助资金资助项目［财社〔203〕43号］。

第一作者：陈娟，女，博士研究生，助理研究员，研究方向为药物制剂的质量标准与活性成分，(电子信箱)chenjuan 』007jie@ 47. com 。

△通信作者：张红，女，博士研究生，研究员，研究方向为中药药效物质基础与质量控制，(电子信箱)zhabghoog9393@ 。

陈娟,陈瑞明，孟雪,杨智峰，王娣，鲁文静,蒙麦侠，张红人(陕西省中医药研究院，陕西西安7(0061)摘要：目的建立同时测定党参中紫丁香苷、党参块苷和苍术内酯川含量的高效液相色谱法。

方法色谱柱为Kromasil Ci3柱(250 mm X 40 mm, 5 pm ),流动相为乙腈(A) - 0. 1%磷酸水溶液(B),梯度洗脱(0-20 min 时 4%A,22〜30 min 时 4% A 〜30%A, 30〜50 min 时30%A -YO/A ),流速为0. 8 mL/mic,柱温为32 C ，检测波长为220 cm,进样量为4 pL 。

结果紫丁香苷、党参块苷和苍术内酯川在各自的质量浓度范围内线性关系良好(厂M 0.998 9, n=6),平均回收率分别为4098% ,40 87% ,9905% , RSD 分别为0. 70% ,0. 54%,O 20%(n=6)o 结论该方法分离效果和重复性均良好，回收率高，可用于党参药材的质量控制。

关键词：党参；高效液相色谱法;紫丁香苷；党参块苷;苍术内酯川；含量测定中图分类号：R932;R284 1;R286・0 文献标志码：A 文章编号：1006 -4931(2021)05 -0069-04Simultaneaus Determination of Three Iodeacs Componeats in Codonopsic Pilosula by HPLCCHEN Juan, CHEN RuUmg , MENG Xa, YANG Zhtfeng, WANG Di, LU Wenjing. MENG Maixin, ZHANG Hong(Shaanxi Academy of Traainonal Chines p Memcine , XC aa, SSaaaxi , China 712061)Abstract : Objective Ta estab/sh a high - performance /quid chromamgraphy(HPLC) method far the simuOaneons determiqatioo of syringing, todetyoOn, atracmenoCUv 皿 in Co P opop s p pilosula. Methods The chromamgraphP column was Kromasil C o coCmc (250 mm X4. 2 mm , 3 pm), the moCilo phase was acemoiRile (A) - 0. 1 % phosphoric acid apueons soluXoo(B) with grapienl eCXon((1 - 20 min ,4%A ；20 -30 min 10%A - 30%A ；30 - 50 min,30% A - 70% A, , the iow rate was 0. 8 mL/min,thv column temperature was 30 C ,the demcXon wavelength was 220 cm , a np the injecUon volume was 4 pL. Respite The syringing , to Oem o On , atracmCnoOdo 皿 showedgood /near reCtmoships within their own range G M 0098 9, n=6), the averapo recoveries were 40 98% , 40 87% anp 99.65%with RSDo of 0. 90% , 0. 84% and 0 20% ( n = 6). Conclusion The method has a good separation eUect , repeatabUUy anp high recov ery ,which can be used far the quality control of Codonopsp pilosula.Key word ：: Codoaopsis pilosula ; HPLC ; syringin ; ChemoCn ; atracmCnoOdo 皿;content determinatioo党参为桔梗科植物党参CoPonopsppilosulo (Franch. 5Nannf.、素花党参 CoPonopsp pilosulo Nannf . var. modesta(Nannf. ) L. T. Sken 或川党参 CoPonopsp iangsUen Oliv .的干燥根，具有补中益气、健脾益肺功效4］。

211126682_在线固相萃取-二维液相色谱法同时测定儿童配方奶粉中维生素A、D、E

黄燕，林灵超，蒋婷婷，等. 在线固相萃取-二维液相色谱法同时测定儿童配方奶粉中维生素A 、D 、E[J]. 食品工业科技，2023，44（9）：317−323. doi: 10.13386/j.issn1002-0306.2022060239HUANG Yan, LIN Lingchao, JIANG Tingting, et al. Simultaneous Determination of Vitamins A, D and E in Children's Formula Milk Powder by Combining Online Solid Phase Extraction and Two-Dimensional Liquid Chromatography[J]. Science and Technology of Food Industry, 2023, 44(9): 317−323. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022060239· 分析检测 ·在线固相萃取-二维液相色谱法同时测定儿童配方奶粉中维生素A 、D 、E黄　燕，林灵超，蒋婷婷，周雅雯，周小靖*（浙江福立分析仪器股份有限公司，浙江台州 317500）摘　要：建立在线固相萃取（SPE ）-二维液相色谱法同时测定儿童配方奶粉中脂溶性维生素A 、D 2、D 3和α-、β-、γ-、δ-维生素E 含量的方法。

样品经皂化后直接进样，以聚合物为填料的PLRP-S 色谱柱为在线固相萃取柱净化皂化液，采用SPE 泵反向洗脱目标组分，并通过引入在线稀释将目标组分聚焦在第一维色谱柱柱头。

以NuovaSil TM PFP （4.6 mm×150 mm ，3 μm ）为第一维色谱柱，甲醇-0.1%甲酸水溶液为流动相进行梯度洗脱，分离维生素A 和E 的4 种异构体。

甘氨酸在高岭土表面的吸附和热缩合反应

[Article]物理化学学报(Wuli Huaxue Xuebao )Acta Phys.鄄Chim.Sin .,2007,23(1)：32-36January甘氨酸在高岭土表面的吸附和热缩合反应孟明∗夏凌燕郭丽红(天津大学化工学院催化科学与工程系,天津300072)摘要：采用TG/DTA 、FT ⁃IR 和in situ DRIFT 等技术对甘氨酸在高岭土表面的吸附和热缩合反应进行了表征,考察了甘氨酸平衡浓度和溶液pH 值对吸附行为的影响,同时探讨了原位条件下甘氨酸的缩合反应历程.结果表明,溶液呈弱酸性时,甘氨酸在高岭土上的吸附量最大,但吸附等温线不符合Langmuir 模型.在强酸性、弱酸性和碱性溶液中,吸附态的甘氨酸分别主要以阳离子、两性离子和阴离子形式存在.弱酸性溶液中,甘氨酸的—NH +3基团与高岭土表面的≡S —O -(S 为Si 或Al)基团之间的氢键作用是吸附的主要驱动力,而强酸性溶液中,≡S —O -基团的质子化,以及碱性溶液中—NH +3向—NH 2的转化,是导致吸附量下降的主要原因.In situ DRIFT 结果表明,在110-160℃温区,有明显的线式二肽形成；随着温度升高至210℃时,二肽进一步脱水,形成环化缩合产物哌嗪二酮(DKP).没有检测到硅酯类或铝酯类中间体的特征峰,反应可能按氢键促进下的自缩合机理进行,高岭土的存在使缩合反应温度有明显降低.关键词：甘氨酸;高岭土;吸附;热缩合;甘氨酸二肽;哌嗪二酮中图分类号：O643Adsorption and Thermal Condensation ofGlycine on KaoliniteMENG Ming ∗XIA Ling ⁃Yan GUO Li ⁃Hong(Department of Catalysis Science and Technology,School of Chemical Engineering and Technology,Tianjin University ,Tianjin 300072,P.R.China )Abstract ：Glycine was adsorbed on the surface of kaolinite from aqueous solution with variable concentrations and pH values.The TG/DTA,FT ⁃IR and in situ DRIFT techniques were employed to characterize the adsorption and thermal condensation performances of glycine on kaolinite.The results of the adsorption experiment indicated that the weakly acidic solution favored the adsorption greatly,and gave the largest adsorption amount,however,the adsorption isotherm did not meet the Langmuir model.FT ⁃IR results showed that in the strongly acidic,weakly acidic,and basic solutions,the glycine exists in the form of cation,zwitterion,and anion,respectively.In the weakly acidic solution,thehydrogen bond interaction between —NH +3and ≡S —O -groups should be the driving force of the adsorption,the hydroxylation of ≡S —O -group in strongly acidic solution and the conversion of —NH +3to —NH 2in the basic solution were the main reasons for the decrease of adsorption amount.The in situ DRIFT results revealed that linear dimer of glycine was formed during 110-160℃region.Up to 210℃,the dimer underwent further intra ⁃molecular condensation to form the cyclodimer diketopiperazine (DKP).During the reaction,no any evidence of the formation of R —CO —O —S (S=Si or Al)intermediates from the esterification reaction was found.The reaction should proceed by the self ⁃condensation mechanism promoted by the hydrogen bond interaction between glycine and kaolinite.The presence of the kaolinite has remarkably decreased the temperature of the condensation reaction.Key Words ：Glycine;Kaolinite ；Adsorption;Thermal condensation;Glycylglycine;DiketopiperazineReceived:July 17,2006;Revised:August 4,2006.∗Corresponding author.Email:mengm@;Tel:+8622⁃27892275.教育部高等学校博士学科点专项科研基金(20040056028)资助项目ⒸEditorial office of Acta Physico ⁃Chimica Sinica32No.1孟明等：甘氨酸在高岭土表面的吸附和热缩合反应从简单氨基酸分子到多肽的生命进化过程中,原始地壳中的SiO2、Al2O3及粘土可能起到了重要的催化作用[1-5].模拟实验发现,有无机氧化物催化剂存在的反应中肽键的形成速率提高了40-2000倍[2].另外,氨基酸分子在金属或氧化物表面的吸附在固相多肽合成[6]、有机质谱[7]、医学移植和生物传感器[8,9]等方面有着广泛的应用前景.但是到目前为止,对氨基酸分子在无机氧化物载体上的吸附和热缩合反应的研究多数采用的是宏观研究方法,如考察浓度、温度和pH值等对吸附量和吸附等温线的影响[10-12]；而对微观个体的吸附机制和反应机理的认识仍停留在假设阶段,如White等[13]提出的表面硅酯键和铝酯键的反应机理,至今仍缺乏分子水平上的证据.因此,开展氨基酸分子的吸附和热缩合反应的研究有助于从分子水平上了解SiO2、Al2O3和粘土等在早期生命的化学进化过程中(如肽键和多肽的形成)的作用本质,同时在氨基酸的分离、固相多肽的合成等应用方面也有重要指导意义.我们[14-16]曾系统研究了不同氨基酸分子在SiO2和Al2O3表面吸附和热缩合反应的机制,但都没有获得线式二肽或多肽形成的证据.本文详细考察了甘氨酸分子在高岭土表面的吸附和热缩合行为,用原位漫反射红外光谱技术对热缩合反应的历程进行了表征,并获得了线式二肽存在的谱学证据.1实验部分1.1吸附实验将2g经550℃焙烧的高岭土(化学式为Al2O3·2SiO2·2H2O)浸入50mL一定浓度和pH值的甘氨酸溶液中,在室温下搅拌吸附2h.真空抽滤后,用50mL 去离子水冲洗样品表面,最后室温干燥12h.用2 mol·L-1的NaOH和w(HCl)=10%的盐酸调节甘氨酸水溶液的pH值.样品记作Gly/Kao.1.2热重/差热分析(TG/DTA)在Perkin Elmer公司生产的Diamond TG/DTA 仪器上对吸附样品进行热分析实验.载气为空气或氮气,流量100mL·min-1,升温速率10℃·min-1,样品用量10-14mg.实验前用载气吹扫1h,除去样品表面部分物理吸附水.1.3红外光谱(FT⁃IR和in situ DRIFT)在Thermo Nicolet公司生产的Nexus FT⁃IR仪器上(配备原位池)测定样品的透射红外光谱,采用KBr压片测试.扫描次数为32,分辨率为4cm-1,扫描范围为4000-400cm-1.原位漫反射(in situ DRIFT)实验在可通气氛和可加热的原位池中进行,采用Thermo Spectra⁃Tech控温仪,测温范围为室温至510℃,每50℃为一台阶,每台阶恒温10min,以获得稳定的光谱数据.2结果与讨论2.1TG/DTA结果在空气气氛中,随温度升高,高岭土表面吸附的甘氨酸会被逐步氧化而去除,失重曲线显示,600℃后,样品不再有质量的变化,表明甘氨酸已被完全氧化脱除.图1和图2分别为不同平衡浓度和不同pH 值下制得的样品的热重微分(DTG)曲线.由图1和2可以看出,样品的第一个DTG峰位于130-250℃,可能对应甘氨酸的脱水缩合过程,将气氛更换为纯N2时,该峰的位置几乎不变,进一步说明此峰源于吸附态甘氨酸自身的变化,而非氧化所致.第二个DTG峰位于250-550℃,是一放热过程,因而归属为吸附态甘氨酸物种的氧化.图1中曲线(1)和(2)虽然显示出一个非常宽化的峰,但它可能包含了甘氨酸脱水缩合和氧化两个过程,因为这两个样品对甘氨酸的吸附量很低,缩合脱出的水很少,对应的DTG峰易被掩盖.与纯甘氨酸相比,第一个峰的温度明显降低,表明高岭土对脱水缩合反应有明显的催化作用.通过对DTG曲线中峰面积的积分可精确计算出甘氨酸在高岭土表面的吸附量(Q ad),表1给出了不同甘氨酸平衡浓度及pH值时的吸附量.由表1中图1不同甘氨酸平衡浓度制得的样品Gly/Kao的DTG曲线(pH=6.1)Fig.1DTG profiles of the samples Gly/Kao prepared at different equilibrium concentrations ofglycine(pH=6.1)(1)0.48,(2)0.78,(3)0.98,(4)1.17,(5)1.47mol·L-1,(6)pure glycine33Acta Phys.鄄Chim.Sin.,2007Vol.23数据可知,随平衡浓度升高,高岭土对甘氨酸的吸附量逐渐增加,其吸附等温线如图3所示,由于1/Q ad 对1/c eq 不呈线性,表明甘氨酸在高岭土上的吸附不符合Langmuir 吸附模型.由表1中数据还可以看出,甘氨酸水溶液的pH 值对吸附有显著影响,当溶液为弱酸性时(pH 为4-6),吸附量最大;而在强酸性或碱性溶液中,吸附都受到明显抑制.这与不同pH 值时,甘氨酸在水溶液中的存在形式及高岭土表面的电性密切相关.根据甘氨酸的p K a 值和高岭土的零电荷点(PZC)可以识别不同pH 值时甘氨酸的存在形式及高岭土表面的带电情况,参见示意图1.由示意图1可知,在弱酸性溶液中(pH 为4-6),甘氨酸主要以两性离子(+H 3N —CH 2—COO -)的形式存在,—NH +3基团与高岭土表面的≡S —O -(S 为Si 或Al)基团可以产生较强的氢键作用,因而吸附甘氨酸能力较强；而在较强的酸性溶液中(pH 约3.7),高岭土表面的≡S —O -基团易质子化形成≡S —OH或≡S —OH 2+基团,使—NH +3与高岭土表面的氢键作用削弱,吸附能力降低；碱性溶液中,虽然高岭土表面的≡S —O -基团数量会有所增加,但此时甘氨酸主要呈阴离子(H 2N —CH 2—COO -)形式,高岭土表面的≡S —O -基团与甘氨酸的—NH 2基团的氢键作用较之与—NH +3基团弱得多(氢键的本质为静电相互作用),因而甘氨酸的吸附量会显著下降.2.2FT 鄄IR 结果在相同pH 值,不同平衡浓度下制得的样品的红外谱图示于图4,其中,图4(7)为纯α甘氨酸晶体(以两性离子+H 3N —CH 2—COO -存在)的红外谱图,其主要特征峰的归属[14,15,17-19]列于表2.由图4可知,平衡浓度较低时,因吸附量太低,红外特征峰很不明显,但随着平衡浓度的增加,峰强度逐渐增加,并且位置越来越接近纯甘氨酸晶体.由于受水的特征峰(1632cm -1)影响,—NH +3的反对称变形振动和—COO -的反对称伸缩振动峰难以形成独立的峰.考虑到样品的—NH +3对称变形振动峰(1503cm -1,1526cm -1)、—COO -基团的对称伸缩振动峰(1414cm -1)以及—CH 2基团的特征峰(1336,1443cm -1)均与纯甘氨酸相差不大,说明吸附强度并不大,推测图2不同pH 值制得的样品Gly/Kao 的DTG 曲线Fig.2DTG profiles of the samples Gly/Kao preparedat different pH values(1)pH=2.3,(2)pH=4.0,(3)pH=6.1,(4)pH=9.6,(5)pH=11.0;initial concentration:1.5mol ·L -1c eq /(mol ·L -1)0.480.780.981.171.471.76Q total /(mmol ·g -1)0.480.530.600.660.780.93pH Q total /(mmol ·g -1)2.30.614.00.676.10.719.60.3411.00.12表1不同平衡浓度和pH 值下甘氨酸在高岭土上的吸附量Table 1Effect of equilibrium concentrations andpH values on the total adsorption amounts of glycineon kaolinite (Q total)图3室温下甘氨酸在高岭土上的吸附等温线Fig.3Adsorption isotherm of glycine on kaolinite atroomtemperature示意图1溶液pH 值对甘氨酸物种形成和高岭土表面电荷的影响Scheme 1Effect of pH value of the solution on glycine speciation and kaolinite surface chargePZC:point of zero charge34No.1孟明等：甘氨酸在高岭土表面的吸附和热缩合反应可能是静电吸引产生的氢键相互作用.根据示意图1可知,在pH=6.1时,甘氨酸主要以两性离子形式存在,而高岭土的表面带负电性.因而,甘氨酸两性离子的—NH +3基团与高岭土表面的≡S —O -基团产生氢键作用的可能性很大.在相同初始浓度、不同pH 值制得样品的红外谱图示于图5.由此图可知,当pH=2.3时,可检测到—COOH 基团中C ‗O 伸缩振动峰(1725cm -1),—NH +3对称变形振动峰(1503cm -1),以及—COO -的对称伸缩振动峰(1408cm -1),表明样品中同时存在甘氨酸阳离子和两性离子.随pH 值提高至6.1,C ‗O 伸缩振动峰消失,—COO -的对称伸缩振动峰(1413cm -1)和—NH +3对称变形振动峰(1526cm -1)均显著增强,振动频率越来越接近纯甘氨酸；并且在1632cm -1处的水峰明显向低波数偏移,这主要是受—COO -的反对称伸缩振动峰和—NH +3基团的反对称变形振动峰影响,这些结果表明,pH=6.1时,甘氨酸主要以两性离子存在于高岭土表面.当pH 值进一步提高至9.6时,水峰又回复到1632cm -1处,这主要是因为碱性溶液中,不存在—NH +3基团,同时由于吸附量的大幅下降,—COO -的反对称伸缩振动峰不明显,因而对水峰的影响很小；值得一提的是,随着—NH +3对称变形振动峰(1526cm -1)的消失,出现了一个宽化的弱峰(约1480cm -1),归属为—NH 2基团的对称变形振动,由此可见,碱性溶液中,甘氨酸主要以阴离子形式被吸附.2.3In situ DRIFT 结果无机氧化物催化氨基酸缩合形成肽键和肽链的反应机理目前仍存在多种解释,如硅酯类或铝酯类中间体机理[13]、氢键促进的自缩合机理[15]等.本文采用原位漫反射红外光谱方法考察了高岭土上甘氨酸缩合反应的产物及历程,试图对反应机理作进一步探索.图6示出了样品Gly/Kao (pH=6.1,初始浓度为1.5mol ·L -1)的原位漫反射红外光谱.由此图可知,图4不同甘氨酸平衡浓度制得的样品Gly/Kao 的红外谱图Fig.4FT ⁃IR spectra of the samples Gly/Kao preparedat different glycine equilibriumconcentrationsc eq /(mol ·L -1):(1)0.18,(2)0.27,(3)0.38,(4)0.48,(5)0.78,(6)0.98;(7)pure glycine;pH =6.1图6空气气氛下样品Gly/Kao 的原位漫反射红外光谱Fig.6In situ DRIFT spectra of the sampleGly/Kao under air flow(1)room temperature,(2)110℃,(3)160℃,(4)210℃,(5)260℃,(6)310℃,(7)510℃表2纯α甘氨酸的特征峰归属Table 2Assignments of main glycine bands in the1000-1800cm -1regionν:stretching,δas/s :asymmetric/symmetric deformation,ρw/t/r :wagging/twisting/rockingBand (cm -1)161015931507,15271443Assignmentsδas (NH +3)νas (COO -)δs (NH +3)δs (CH 2)Band (cm -1)Assignments 1413νs (COO -)1333ρw (CH 2)1132ρr (NH +3)1034ν(C-N)图5不同pH 值制得的样品Gly/Kao 的红外谱图Fig.5FT ⁃IR spectra of the samples Gly/Kaoprepared at different pH values(1)pH=2.3,(2)pH=6.1,(3)pH=9.6;initial concentration of 1.5mol ·L -135Acta Phys.鄄Chim.Sin.,2007Vol.23加热到110℃时,样品的红外谱图发生了明显变化, 1632cm-1处水峰基本消失,在1596cm-1和1658 cm-1处现出了两个新峰,分别归属为—NH+3基团的反对称变形振动和—C‗O伸缩振动.由于甘氨酸两性离子(+H3N—CH2—COO-)没有—C‗O特征峰, 1658cm-1处的特征峰必然来源于甘氨酸的缩合产物,而环化缩合产物哌嗪二酮(DKP)中—C‗O伸缩振动频率较高(约1678cm-1)[14,15],加之纯甘氨酸二肽在1659cm-1处有羰基特征峰(未示出),因此,可以将1658cm-1处的特征峰归属为线式缩合产物,即甘氨酸二肽,它是两个甘氨酸分子间脱去一分子水形成的线式二聚体.随着温度升高至210℃,谱图又发生了明显变化,即—C‗O伸缩振动峰位移至1678 cm-1,同时在1467cm-1处出现了一个新峰,它们分别为DKP中的—C‗O伸缩振动和环伸缩振动(ring stretching)特征峰,该结果表明,此时有环化缩合产物DKP形成；进一步加热至260℃和310℃, 1467cm-1特征峰继续增强,同时伴随—COO-对称伸缩振动峰的明显减弱,说明环化缩合反应进行的程度增大.至510℃时,红外峰的强度均显著降低,多数峰几乎消失,这主要是由于缩合产物逐渐被氧化去除所致.值得一提的是,本实验中没有检测到文献[13]提到的硅酯类、铝酯类物种R—CO—O—X (X=Si或Al)的特征峰.在高岭土上,甘氨酸的缩合反应可能更符合氢键促进的自缩合机理,即吸附的甘氨酸主要以氢键形式与高岭土表面作用,这种作用能够使甘氨酸分子得以活化,从而促进缩合反应温度的下降(DTG结果).我们前期的研究[14-16]表明,甘氨酸或谷氨酸在SiO2或Al2O3上可以产生多层吸附并直至晶化,但晶化后的甘氨酸或谷氨酸均由α晶型转化为β晶型,这种转变表明,多层吸附的氨基酸分子或晶化的氨基酸晶体中,氢键构型发生了明显变化,从而使缩合反应更容易进行.在本文中,由于在高岭土上甘氨酸的吸附量较小,没有检测到其晶相,因而难以判断晶型的转变与否.但考虑到高岭土的Si—O四面体层和Al—O(H)八面体层结构基元与SiO2和Al2O3的类似性,甘氨酸在高岭土上缩合反应温度的降低可能也源于表面氢键作用而产生的诱导活化.3结论(1)无机化合物高岭土可以有效地吸附有机甘氨酸分子,溶液浓度和pH值是影响甘氨酸在高岭土上吸附的两个主要因素.当溶液为弱酸性时(pH 为4-6),吸附量最大,强酸性和碱性溶液中吸附受到明显抑制.甘氨酸在高岭土表面的吸附驱动力主要为氢键,吸附等温线不符合Langmuir吸附模型.(2)FT⁃IR结果表明,在强酸性、弱酸性和碱性溶液中,吸附态的甘氨酸分别主要以阳离子、两性离子和阴离子形式存在.In situ DRIFT结果表明,在110-160℃温区,有明显的线式二肽的形成；随着温度升高至210℃时,二肽进一步脱水,形成环化缩合产物哌嗪二酮(DKP);310℃以上,缩合产物才被逐渐氧化分解.在整个反应温区,没有检测到硅酯类或铝酯类物种的特征峰,缩合反应可能按氢键促进下的自缩合机理进行,高岭土使缩合反应温度的降低可能源于表面氢键的诱导作用.References1Lahav,N.;White,D.;Chang,S.Science,1978,201(4350):672White,D.H.;Erickson J.C.J.Mol.Evol.,1980,16(3/4):2793Bujdák,J.;Rode,B.M.J.Mol.Evol.,1997,45(5):4574Son,H.L.;Suwannachot,Y.;Bujdák,J.;Rode,B.M.Inorganica Chimica Acta,1998,272(1/2):895Aquino,A.J.A.;Tunega,D.;Gerzabek,M.H.;Lischka,H.J.Phys.Chem B,2004,108(28):101206Merrifield,R.B.Angew.Chem.Int.Ed.Engl.,1985,24(10):799 7Bennninghoven,A.;Kempken,M.;Kluesener,P.Surf.Sci.,1988, 206(3):L9278Thull,R.Med.Prog.Technol.,1982,9(2/3):1199Lundström,I.;Salaneck,W.R.J.Colloid&Interface Sci.,1985, 108(1):28810Zhao,Z.G.Chem.Res.&Appl.,2001,13(6):599[赵振国.化学研究与应用,2001,13(6):599]11Liu,X.L.;Zhang,Z.B.Marine Sciences,2002,26(9):2 [刘效兰,张正斌.海洋科学,2002,26(9):2]12Ikhsan,J.;Johnson,B.B.;Wells,J.D.;Angove,M.J.J.Colloid.&Interface Sci.,2004,273(1):113White,D.H.;Kennedy,R.M.;Macklin,J.Origins of Life,1984, 14(1/4):27314Meng,M.;Stievano,L.;Lambert,J.F.Chin.J.Catal.,2005,26(5):393[孟明;Stievano,L.;Lambert,J.F.催化学报,2005,26(5):393]15Meng,M.;Stievano,L.;Lambert,ngmuir,2004,20(3):914 16Guo,L.H.;Meng,M.;Zha,Y.Q.Chin.J.Catal.,2006,27(2):189 [郭丽红,孟明,查宇清.催化学报,2006,27(2):189]17Ihs,A.;Liedberg,B.;Uvdal,K.;Törnkvist,C.;Bodö,P.;Lundström,I.J.Colloid&Interface Sci.,1990,140(1):19218Uvdal,K.;Bodö,P.;Ihs,A.;Liedberg,B.;Salaneck,W.R.J.Colloid&Interface Sci.,1990,140(1):20719Laulicht,I.;Pinchas,S.;Samuel,D.;Wasserman,I.J.Phys.Chem., 1966,70(9):271936。

Simultaneous Determination of Perfluorinated Compounds in Edible Oil

ABSTRACT: A simple analytical method was developed for the simultaneous analysis of 18 perﬂuorinated compounds (PFCs) in edible oil. The target compounds were extracted by acetonitrile, puriﬁed by gel permeation chromatography (GPC) and dispersive solid-phase extraction (DSPE) using graphitized carbon black (GCB) and octadecyl (C18), and analyzed by liquid chromatography−electrospray ionization tandem mass spectrometry (LC-ES-MS/MS) in negative ion mode. Recovery studies were performed at three fortiﬁcation levels. The average recoveries of all target PFCs ranged from 60 to 129%, with an acceptable relative standard deviation (RSD) (1−20%, n = 3). The method detection limits (MDLs) ranged from 0.004 to 0.4 μg/kg, which was signiﬁcantly improved compared with the existing liquid−liquid extraction and cleanup method. The method was successfully applied for the analysis of all target PFCs in edible oil samples collected from markets in Beijing, China, and the results revealed that C6−C10 perﬂuorocarboxylic acid (PFCAs) and C7 perﬂuorosulfonic acid PFSAs were the major PFCs detected in oil samples. KEYWORDS: perﬂuorinated compounds, edible oil, gel permeation chromatography, dispersive solid-phase extraction, HPLC-MS/MS

高效液相色谱法同时测定化妆品中辣椒碱等9种热感剂

第42 卷第 5 期2023 年5 月Vol.42 No.5641~646分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）高效液相色谱法同时测定化妆品中辣椒碱等9种热感剂吴志珊1，2，吴姣娇1，2，尹伟成1，2，殷帅1，2，李卓芳2，姜成君1*（1．湖南省药品检验检测研究院，湖南长沙410001；2．岳阳市食品药品检验所，湖南岳阳414000）摘要：建立了测定化妆品中辣椒碱等9种热感剂的高效液相色谱法。

样品经80%甲醇超声提取后，过滤，以乙腈-0.5%甲酸水溶液为流动相，采用SymmetryShield TM RP18色谱柱（250 mm × 4.6 mm，5 μm）分离，液相色谱分离，二极管阵列检测器进行检测。

在优化实验条件下，9种热感剂的分离度好，专属性高，其质量浓度在0.2 ~ 100 μg/mL范围内与峰面积呈良好线性关系，相关系数（r2）均不小于0.999 5，检出限（LOD）为0.001 0 ~ 0.003 4 mg/kg。

以水基类、乳剂类、膏霜类3种阴性样品作为空白基质，9种热感剂在0.004、0.02、1 mg/kg 3种加标水平下的回收率为75.6% ~ 115%，相对标准偏差（RSD）为0.083% ~ 7.0%。

该方法专属性强、灵敏度高、准确度好，可用于化妆品中9种热感剂含量的测定。

关键词：热感剂；化妆品；高效液相色谱法（HPLC）中图分类号：O657.72；S641.3文献标识码：A 文章编号：1004-4957（2023）05-0641-06Simultaneous Determination of 9 Self-heating Compounds Including Capsaicin in Cosmetics by High PerformanceLiquid ChromatographyWU Zhi-shan1，2，WU Jiao-jiao1，2，YIN Wei-cheng1，2，YIN Shuai1，2，LI Zhuo-fang2，JIANG Cheng-jun1*（1．Hunan Institute for Drug Control，Changsha 410001，China；2．Food andDrug Inspection in Yueyang，Yueyang 414000，China）Abstract：A high performance liquid chromatographic（HPLC） method was established for the determi⁃nation of 9 self-heating compounds，including capsaicin in cosmetics．The samples were ultrasonically extracted with 80% methanol，and then filtered．The target compounds were separated on a Symme⁃tryShield TM RP18（250 mm × 4.6 mm，5 μm） with a mobile phase consisting of acetonitrile and 0.5% formic acid by gradient elution，and analyzed by HPLC with a PDA detector．Under the optimal condi⁃tions，good linear relationships were obtained for nine compounds in the concentration range of 0.2－100 μg/mL，with correlation coefficients（r2） not less than 0.999 5．The limits of detection（LODs）were in the range of 0.001 0－0.003 4 mg/kg．The spiked recoveries for water，lotion and cream ma⁃trixes at three levels of 0.004，0.02，1 mg/kg ranged from 75.6% to 115%，with relative standard de⁃viations（RSDs，n = 3） of 0.083%－7.0%．This method is accurate and sensitive，and is suitable for the qualitative and quantitative analysis of 9 self-heating compounds in cosmetics.Key words：self-heating compounds；cosmetics；high performance liquid chromatography（HPLC）近年来，随着人们审美要求的提高，消费者越来越注重自身形象和外表，具有健美功效的化妆品越来越受青睐。

离子色谱法同时测定饮用水中一碘乙酸、二碘乙酸

化学分析计量CHEMICAL ANALYSIS AND METERAGE第29卷，第4期2020年7月V ol. 29，No. 4Jul. 202062doi ：10.3969／j.issn.1008–6145.2020.04.014离子色谱法同时测定饮用水中一碘乙酸、二碘乙酸王存进1，代文彬1，钟志雄2（1.青岛睿谱分析仪器有限公司，青岛　266000；　2.广东省疾病预防控制中心，广州　510000）摘要　建立离子色谱法同时测定饮用水中一碘乙酸和二碘乙酸的方法。

样品经Na 柱、RP 柱和滤膜过滤后直接进样分析，采用Ionpac AS19（4.0 mm ×250 mm ，7.5 μm ）色谱柱，氢氧化钾梯度淋洗，抑制电导检测进行测定。

一碘乙酸、二碘乙酸的质量浓度在0.002～0.1 mg ／L 范围内与色谱峰面积线性关系良好，相关系数不小于0.999 0。

采用氢氧根淋洗液和动态量程电导检测器可以获得nS 级的噪声，检出限达到ng ／L 级。

自来水和黄河水中均未检出一碘乙酸、二碘乙酸，加标回收率在85.25%～115.00%，测定结果的相对标准偏差为0.49%～3.77%（n =6）。

该方法精确度高，回收率和重现性良好，是检测新型碘代酸消毒副产物的可靠方法。

关键词　一碘乙酸；二碘乙酸；离子色谱法；氢氧根梯度淋洗中图分类号：O657.7 文献标识码：A 文章编号：1008–6145(2020)04–0062–05Simultaneous determination of monoiodoacetic acid and diiodoacetic acid in drinking waterby ion chromatographyWANG Cunjin 1，DAI Wenbin 1，ZHONG Zhixiong 2（1. Qingdao Reepo Analytical Instrument Co.，Ltd.，Qingdao 266000，China;2. Guangdong province center for disease control and prevention ，Guangzhou 510000，China ）Abstract A method was developed for the simultaneous determination of monoiodoacetic acid and diiodoacetic acid in drinking water by ion chromatography. Sample was treated by Na column, RP Column and filtration for direct injection.The method use of Ionpac AS19 (4.0 mm ×250 mm, 7.5 μm) column and KOH gradient elution for separation and suppressed conductivity for detection. The mass concentration of monoiodoacetic acid and diiodoacetic acid showed good linear relationship with the peak area in the range of 0.002–0.1 mg ／L, and the correlation coef ﬁcient were not less than 0.999 0. The detection limits were down to ng ／L due to the use of hydroxide eluent and dynamic range conductivity detector with nS baseline noise. There was no monoiodoacetic acid or diiodoacetic acid in tap water and Yellow River water, the stangard addition recovery was 85.25%–115.00%, and the relative standard deviations of determination results were 0.49%–3.77%, respectively. The method has high accuracy, good recovery and reproducibility, and is a reliable method for the detection of new iodoacid disinfection by–products.Keywords monoiodoacetic acid; diiodoacetic acid; ion chromatography; hydroxide gradient elution随着城市化水平的不断提高和城市集中供水方式的发展，生活饮用水的水质问题逐渐引起人们的注意。

表面活性剂结合QuEChERS气相色谱串联质谱法同时测定大豆油中有机氯农药、多氯联苯及多环芳烃

第40卷第1期 2019年1月质谱学报Journal of Chinese Mass Spectrometry SocietyVol. 40 No. 1Jan. 2019表面活性剂结合QuEChERS-气相色谱-串联质谱法同时测定大豆油中有机氯农药、多氯联苯及多环芳烃李晓贝，刘福光，周昌艳，陈珊珊(上海市农业科学院农产品质量标准与检测技术研究所，农业农村部农产品质量安全风险评估实验室，上海201403)摘要：建立了气相色谱-串联质谱(G C-M S/M S)同时测定大豆油中34种有机氯农药（O C P s)、12种多氯联苯（PCBs)及A种多环芳烃（P A H s)残留量的检测方法。

首先使用表面活性剂作为乳化剂制备相对稳定的油/水乳状液，采用乙腈-二甲苯（：1，W V)为提取剂，以Q u E C h E R S前处理方法对其提取2次后净化，随后进行G C-M S/M S检测。

对=种表面活性剂及添加浓度进行优化，结果表明，表面活性剂的添加可显著提高大豆油中〇C P s、P C B s及P A H s的提取率，最佳添加方案为大豆油：水：Tween60 =1 t 3 : 0. 01(m/m/m)%该方法在1/2. 5/5/10〜400 #g/L浓度范围内的线性关系良好，相关系数记为0. 993 8〜0. 999 7%在5〜100 #g/k g添加浓度水平内，相对标准偏差为1. 59%〜20. 50%，34种O C P s在大豆油中的添加回收率为64. 35%〜120. 63%，12种P C B s的添加回收率为49. 65%〜97. 76%，6种P A H s的添加回收率为74. 05%〜101. 52%。

该方法操作简单、油脂去除效果良好，灵敏度、精密度及回收率均可满足国内外相关标准中大豆油中残留限量的要求％关键词：表面活性剂；大豆油；Q u E C h E R S;气相色谱-串联质谱（G C-M S/M S);有机氯农药；多氯联苯；多环芳烃中图分类号：0657. 63 文献标志码：A文章编号：1004-2997(2019)01-0060-14doi：10. 7538/zpxb. 2018. 0085Simultaneous Determination of OCPs，PCBs and PAHs in Soybean Oil Using Surfactant-Based QuEChERS Method Coupled with GC-MS/MSLI Xiao-bei，LIU Fu-guang，ZHOU Chang-yan，CHEN Shan-shan{Institute fo r Agri-Food Standards and Testing Technology，ShanghaiAcademy ofA gricultu ral Sciences，Shanghai 201403，China) Abstract:A method of gas chromatography-tandem mass spectrometry (GC-MS/M S)收稿日期=2018-07-02;修回日期：2018-10-25基金项目：国家食用菌产品质量安全风险评估重大专项(GJFP2018006);国家蔬菜产品质量安全风险评估重大专项(GJFP2018002)资助作者筒介:李晓贝（1990—），女（汉族），河南信阳人，研究员实习员，从事农药及有机污染物残留分析研究％E-mail；291112576@通信作者：陈珊珊（1983—），女（汉族），广西玉林人，副研究员，从事农药残留分析及农产品质量安全风险评估研究％E-mail；cssml00@163. com第1期李晓贝等:表面活性剂结合QuEChERS■气相色谱-串联质谱法同时测定大豆油中有机氯农药、多氯联苯及多环芳烃61was developed for determination of 34 kinds of organochlorine pesticides (OCPs), 12 kinds of polychlorinated biphenyls (PCBs)and 6kinds of polycyclic aromatic hydrocarbons (PAH s)in soybean oil.Firstly, 1g soybean oil and3 g purified water were emulsified with surfactant,then extracted with 5mL acetonitrile-xylene mixture (9:1，V/V) by QuEChERS method，and detected by GC-MS/MS.The optimal experiments of adding surfactants about species and concentrations showed that the adding of surfactants can significantly increase the extraction efficiency of OCPs，PCBs，PAHs in soybean oil，and the appropriate surfactant is Tween 60. The calibration curves show a good linearity for all the congeners，and the relative coefficient is 0. 993 8-0. 999 7.The relative standard deviations (RSDs)are in the range of 1.59%-20. 50 V，and the spiked recoveries of OCPs range from 64. 35%to 120. 63%，that of PCBs are in the range of49. 65 %-97. 76 %,and PAHs range from74. 05 %to 101. 52%. The developed analyticalmethod is quick and easy maneuverability with satisfactory effect on the removing of oil，which is suitable for the determination of OCPs，PCBs，PAHs in soybean oil.Keywo r d s:surfactant;soybean oil;QuEChERS；gas chromatography-triple quadrupole mass spectrometry (GC-M S/M S); organochlorine pesticides(OCPs); polychlorinated biphenyls(PCBs);aromatic hydrocarbons(PAH s)美国农业局及中国国家粮油信息中心统计结果显示，2015年全球大豆油消费量5 198万吨（占全球植物油消费量的29%，排名第二），其中，中国大豆油消费量为1 450万吨，在中国植物油消费总量中稳居第一[1]%有机氯类农药 (organochlorine pesticides，OCPs)、多氯联苯 (polychlorinated biphenyls，PCBs)及多环芳烃(polycyclic aromatic hydrocarbons，PAH s)均属于持续性有机污染物（persistent organic pollutants，POPs)，脂溶性极强且难以降解，易在环境及生物体中富集，尤其可在动物脂肪组织中富集并产生危害性更大的代谢产物，对人体具有致畸、致癌性[27]%因此，许多国家对植物中的OCPs、PCBs、P A H s制定了最大残留限量值（maximum residue limit，M RL)，如我国 GB 2763—2016规定大豆油中七氯的M R L为0.02 mg/kg，植物油中氯丹的M R L值为0.02 mg/kg[8];GB 2762—2017规定油脂及其制品中苯并M芘的M R L值为0.01 mg/kg[9];欧盟规定植物油中PCBs的M RL值为0.04mg/kg[10]%基于OCPs、PCBs、P A H s的强脂溶性及植物油基质的复杂性，使得植物油中该类化合物的提取存在较大困难，至今尚无相应的国家标准，仅有的同时检测植物油中多种农药残留的相关行业标准SN/T4428—2016《出口油料中多种农药残留量的测定液相色谱-质谱/质谱法011]也不包含此类化合物。

胶束电动色谱法测定五维他口服溶液中维生素B1、B2、B6、烟酰胺、泛酸钙与苯甲酸钠

第39卷第7期2722年9月分析测试学报FENXI CESHI XUEBAO (Joumat cf Instumextal Analysis )Vol. 39 Nc. 91155-1159doi : 10. 3969/j. issn. 1004 -4457. 2221. 09・ 15胶束电动色谱法测定五维他口服溶液中维生素B 1、B 2、B 6、烟酰胺、泛酸钙与苯甲酸钠李丹凤-,黄罗健4,朱健萍-,卢日刚1**收稿日期：2727 -75 -15；修回日期：2727 -06 -74基金项目：广西重点研发计划资助项目(桂科AB13201123) ； 2213年广西壮族自治区食品药品安全科研项目资助(桂食药科2213-4直属)*通讯作者：卢日刚，主任药师，研究方向：食品药品的检验与研究，E - mail : *****************(1.广西壮族自治区食品药品检验所，广西南宁530021 ； 2.桂林医学院药学院，广西桂林541100) 摘要：建立了胶束电动色谱(MEKC)法通过波长切换同时测定五维他口服溶液中的维生素B -、B 7、B 6、烟酰胺、泛酸钙及苯甲酸钠含量的方法，并优化了检测波长、十二烷基硫酸钠(SDS)的浓度、分离电压、缓冲液浓度及pH 值等实验条件。

最佳分离条件为以未涂渍的标准熔融石英毛细管柱(75 imx50cm ,有效长度 42 cm )为分离通道；44 mmol/P 硼酸+44 mmol/P 硼砂+20 mmol/P SDS(pH 9. 0)为运行缓冲液，分离电压20 kV ,柱温25 C ,检测波长为270 nm (维生素B -、B 7、B^烟酰胺及苯甲酸钠)和200 nm (泛酸钙),电动进样5kV 、进样时间5 so 结果显示，6种化合物可在13 mm 内实现基线分离，且在一定质量浓度范围内线性良好，相关系数J O 不小于0.993 9； 3个加标水平下的平均回收率为97. 4% - 103% , RSD 为0. 60% -1.8%。

气相色谱-质谱法测定温室大棚土壤中13种有机磷酸酯

第39卷第12期2020年12月分析测试学报FENXI CESHI XUEBAO(Journal of Instrumental Analysis)Vol.39No.121538-1543d o i: 10.3969/j.issn.1004 -4957. 2020.12. 016气相色谱-质谱法测定温室大棚土壤中13种有机磷酸酯桑永珠12,厉文辉2*，刘杰民2,曲琛2,赵研君2,蔡寒梅3,洪思程2(1.国标(北京)检验认证有限公司，北京100088; 2.北京科技大学化学与生物工程学院，北京100083; 3.中国建材检测认证集团有限公司，北京100024)摘要：该文建立了气相色谱-质谱法（GC- MS)检测温室大棚土壤中磷酸三乙酯、磷酸三丙酯、磷酸三正丁酯、磷酸三异丁酯、磷酸三丁氧乙酯、磷酸三(2-乙基己基）酯、磷酸三（2-氯乙基）酯、磷酸三（1-氯-2-丙基)酯、磷酸三（，3-二氯-2-丙基)酯、磷酸三苯酯、磷酸三甲苯酯、磷酸甲苯二苯酯、2-乙基己基二苯基磷酸酯13种有机磷酸酯(OPEs)的分析方法，并优化了提取溶剂、提取方式、提取时间、萃取柱种类和色谱条件等实验参数。

样品以正己烷-丙酮(体积比1 :1)为提取溶剂超声提取，经F l o i l固相萃取柱净化，再采用气相色谱-质谱法(选择离子监测模式)检测，内标法定量。

结果显示：13种化合物在1~500 ^;/1^质量浓度范围内线性良好，相关系数（2)为0.999 3~0.999 7，检出限（10)，5/^¥= 3)为0.04~2.31^;/1^，定量下限（LOQ，S/iV= 10)为0.13~7.69叫^，在3个加标浓度（1.0、10、50|xg/L)下的回收率为54.5%~130%，相对标准偏差（RSD)为1.7%~9.9%。

采用该方法对北京地区温室蔬菜大棚中41份土壤样品进行分析发现，除磷酸三丙酯（TPrP)外，其余12种O P E S^有检出，含量为1.90~632 ng/g(干重），平均值为87.8ng/g，中位值为39.7 ng/g。

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Simultaneous Determination of Glyphosate,Glufosinate,and Aminomethylphosphonic Acid by Capillary Electrophoresis After 9-Fluorenylmethyl Chloroformate DerivatizationSarah Y.Chang a,b*()and Miao-YiWei a()a Department of Applied Chemistry,Chaoyang University of Technology,Wufeng,Taichung County,Taiwan413,R.O.C.b Department of Chemistry,Tunghai University,Taichung,Taiwan407,R.O.C.A capillary electrophoresis(CE)with UV absorption detection method is described for the simultaneous determination of glufosinate,glyphosate,and aminomethylphosphoric acid.The9-fluorenylmethyl chloro-formate(FMOC-Cl)was used for precolumn derivatization of the non-absorbing herbicides.The three analytes were separated by CE in9min with25mM borate buffer at pH9,followed by detection with a UV de-tector at260nm.We demonstrate how the detection limit can be enhanced by using acetonitrile-salt mixtures. With acetonitrile-salt mixtures,the limit of detection(LOD)was in the10-7M range.Linearity of more than two orders of magnitude was generally obtained.Precisions of migration times and peak areas were less than 0.9%and7.5%,respectively.The applicabilities of the method for the analysis of ground water and lake water were examined.Keywords:Capillary electrophoresis;Acetonitrile-salt mixture;Herbicides;Glyphosate;Glufosinate;Aminomethylphosphonic acid.INTRODUCTIONGlyphosate[N-(phosphonomethyl)glycine](GLYP) and glufosinate[DL-homoalanine-4-yl(methyl)phosphinic acid](GLUF)are non-selective herbicides for control of long grasses and broad-leaved weeds.They kill plants by interfer-ing with the formation of amino acids and other important en-dogenous chemicals.1GLYP ranks top among conventional pesticides used worldwide.The maximum contaminant level (MCL)of GLYP for safe drinking water set by the US Envi-ronmental Protection Agency(EPA)is0.7µg×mL-1.2In addi-tion,it is anticipated that the worldwide usage of GLUF will increase greatly.Therefore,there is a need to develop a rapid, simple,and sensitive analytical method for the determination of GLUF,GLYP,and aminomethylphosphonic acid(AMPA), the main metabolite of GLYP,in a variety of sample matrices.The analytical methods of GLYP,GLUF,and related compounds have been reviewed by Stalikas.3In GC analy-sis,4-8an exhaustive derivatization step is required to enhance the volatility of the analyte.High-performance liquid chro-matography(HPLC)is more practical than GC for the separa-tion of these herbicides because it is more suitable for polar and non-volatile samples.They do not have a chromophore or a fluorophore in their structure;therefore,pre-or post-column derivatization has to be used to achieve detection. p-Nitrobenzoyl chloride(PNBC)9has been reported to deri-vatize GLUF in plasma with UV detection.9-Fluorenylmeth-yl chloromate(FMOC-Cl),10-12p-toluenesulphonyl chloride13 and o-phthalaldehyde(OPA)14-18were the common deriva-tizing agents for GLYP and AMPA with fluorescence detec-tion.Post-column indirect UV detection19was also reported where the background fluorescence is provided by an Al3+-Morine(3,5,7,2¢,4¢-pentahydroxyl flavone)complex.In ad-dition,LC methods coupled with mass spectrometry20,21and condensation nucleation light scattering detection22have been reported for the determination of GLYP and AMPA.In recent years,capillary electrophoresis(CE)has become an important separation technique due to its high resolving power and speed.p-Toluenesulphonyl chloride23was used to derivatize GLYP and AMPA prior to separation by CE,fol-lowed by detection with an UV absorbance detector.Fluo-rescein isothiocyanate(FITC),5-(4,6-dichloro-s-trazin-2-yl-Journal of the Chinese Chemical Society,2005,52,785-792785Dedicated to Professor Ching-Erh Lin on the Occasion of his66th Birthday and his Retirement from National Taiwan University *Corresponding author.Tel:+886-423590248ext201;Fax:+886-423590426;E-mail:ychang@.twamino)fluorescein(DTAF)and5(6)-carboxyfluorescein N-succinimidyl ester(CFSE)were used as labeling reagents for the determination of GLYP,GLUF,and AMPA by micellar electrokinetic chromatography(MEKC)with laser-induced fluorescence(LIF)detection.24-26Ribonucleotides,272,6-pyr-idinedicarboxylic acid28and phthalate29have also been em-ployed to provide the background signals for indirect UV de-tection in CE analysis of GLYP and AMPA.In addition, fluorescein30was used as the background electrolyte for indi-rect fluorescence detection of GLUF,GLYP,and AMPA.Re-cently,a new linker-assisted immunoassay approach31was developed to analyze GLYP in water.Previously,we have developed a CE-indirect fluores-cence detection method for the simultaneous determination of GLUF,GLYP,and AMPA30at theµM level.However,the analysis was interfered with by the matrix of ground water. In order to enhance the sensitivity and selectivity,derivatiza-tion was required before CE analysis.FMOC-Cl has been used to derivatize GLUF,AMPA,and GLYP12in LC analysis. It hasn’t been used in CE analysis of these herbicides yet.In this study,FMOC-Cl was used for precolumn derivatization of the herbicides followed by detection with a UV detector at 260nm.Due to the short optical pathlength of the capillary, the sensitivity of CE with UV detection is not sufficient for practical application.The sensitivity can be enhanced by us-ing acetonitrile-salt mixtures.Linearity,reproducibility,and detection limits were examined.The feasibility of this method for the analysis of water samples was also investigated.EXPERIMENTALChemicalsGlufosinate ammonium was purchased from Riedel-deHaen(Milwaukee,WI,USA).Glyphosate and aminometh-ylphosphoric acid were obtained from Aldrich(Milwaukee, WI,USA).FMOC-Cl and NaCl were obtained from Merck (Darmstadt,Germany).Sodium borate was from Showa(To-kyo,Japan).All other chemicals were of reagent grade.Water purified with a Barnstead NANOpure system(Dubuque,IA, USA)was used for all solutions.The CE running buffer con-taining25mM sodium borate was prepared with deionized water.The pH of the running buffer was adjusted by addition of NaOH.CE SystemThe CE system was assembled in-house.A0-30kV power supply(GAMMA High Voltage Research,Inc.,Or-mond Beach,FL,USA)provided the separation voltage.The capillary used for separation was50µm ID´360µm OD´57cm total length(Polymicro Technologies,Phoenix,AZ, USA).The effective length of the capillary is45cm.Before use,the capillary was washed with CE buffer for5min.The capillary was then equilibrated with the buffer under an elec-tric field of368V/cm for1h.Samples were injected by rais-ing the anodic end18cm above its normal position for10-480 s.UV absorbance detection was performed at260nm with a commercial UV absorbance detector(Model number:SSI 500;Thermo Separation Products,San Jose,CA,USA) equipped with a CE flowcell module.Recording of electro-pherograms and quantitative measurements of peak area were performed with a computer connected to a SISC data ac-quisition interface(Scientific Information Service Corpora-tion,Taipei,Taiwan).FMOC-Cl Derivatization ProcedureStock solutions of GLUF,GLYP,and AMPA at a con-centration of1mM were prepared in water and diluted with water to achieve the final desired concentration.50µL of her-bicides standards were mixed with10µL of125mM borate buffer(pH9.5).140µL of FMOC-Cl(4mM in acetonitrile) was then added and thoroughly mixed.The resulting solution was shaken for30min using a Vortex shaker.RESULTS AND DISCUSSIONDerivatizationGLUF and AMPA contain primary amine while GLYP contains secondary amine.FMOC-Cl can be used to deriva-tize GLUF,GLYP,and AMPA in sodium borate solution(pH 9.5).The reaction mixture was injected into the CE system. Fig.1shows the electropherogram of30µM mixture of GLUF,GLYP,and AMPA.The three analytes were separated by the buffer solution containing25mM sodium borate at pH 9.0.At pH9.0,GLYP derivative has three negative charges while GLUF and AMPA derivatives have two negative charges.Therefore,the migration time of GLYP is longer. The migration times for GLUF,AMPA,and GLYP deriva-tives are5.8,6.2,and8.3min,respectively.The peak ap-pearing at3min is due to the excess labeling reagent.The FMOC-Cl derivatization was relatively fast.We found that the peak areas of the derivatized products increased with in-creasing time of derivatization reaction.After30min,the786J.Chin.Chem.Soc.,Vol.52,No.4,2005Chang and Weipeak areas did not increase significantly.The derivatization reaction was complete in30mins.The peak areas of deriva-tized products did not change for at least10hours at room temperature.Sample stackingUV detector is the most common detector for CE.How-ever,the sensitivity is not good enough due to the short opti-cal pathlength of the capillary.On-line concentration tech-nique was needed to improve sensitivity.Shihabi et al.have described a unique type of stacking that occurs when a sam-ple contains66%acetonitrile and salt.32-35Unlike other low ionic strength stacking methods,there is no need to remove the sample matrix before sample injection.With regard to simplicity and ease of operation,acetonitrile-salt mixtures are a suitable method for on-line concentration of the herbi-cides in CE.In our experiments,acetonitrile was used to dis-solve the labeling reagent(FMOC-Cl)and induce sample stacking in CE.In the derivatization mixture,the content of acetonitrile is about70%.Acetonitrile is a low conductivity solvent and can bring some stacking due to the high field strength.33Accelerating the analyte ions in the sample zone causes them to stack in a sharp zone when they reach the sep-aration buffer.The stacking effect will also be enhanced by the addi-tion of sodium chloride in the samples.32-35The sodium chlo-ride will migrate rapidly early on ahead of the analytes leav-ing behind an area of higher field strength.The analytes ions in the high field strength accelerate while those in the low field strength slow down leading to a concentration of the sample ions.33Different concentrations of NaCl were incor-porated into the borate buffer before derivatization.With in-creasing NaCl concentration from0to200mM in borate buffer,the peak heights of the three analytes increased.When excess sodium chloride(1M in borate buffer)was added,the split peaks were observed for GLUF and AMPA.This may be caused by that the GLUF and AMPA in the low field strength region can’t combine with those in the high field strength re-gion before they enter the separation buffer.With addition of 200mM NaCl,the peak heights of the three analytes in-creased44-59%compared to samples free from NaCl.The influence of buffer pH on the stacking effect was also studied in the pH range8-10.At pH8,the GLUF and AMPA can’t be separated.At pH9-10,the three analytes were separated.The best peak height and efficiency were obtained at pH10.Then the pH of CE buffer was changed to10.Injection volume and sensitivityFig.2compared the electropherograms of different in-jection times.The amount injected into CE can be increased to480s at18cm height without peak overlapping.Sample volume of480s injection corresponds to21%of the capillaryDetermination of Herbicides by Capillary Electrophoresis J.Chin.Chem.Soc.,Vol.52,No.4,2005787Fig.1.Electropherogram of a mixture of GLUF,AMPA,and GLYP.Peak identities:1,GLUF(3´10-5M);2,AMPA(3´10-5M);3,GLYP(3´10-5M).Conditions:fused-silica capillary,50m m´360µm OD´57cm total length(45cm to the window);applied voltage,21kV;hydrodynamic injection,10s(18cm);electrophoretic buffer,25mM sodium borate,pH9.0.volume.Fig.3A shows that the peak heights of GLYP in-creased with an increase of injection time from10-480s.For GLUF and AMPA,the peak heights increased with an in-crease of injection time from10-180s.A further increase in injection time did not increase the peak heights of GLUF and AMPA.Fig.3B shows that the peak area increased non-linearly with an increase of sample volume for the three analytes. With180s injection time,a factor of18-23in signal enhance-ment was obtained for the three analytes.However,the theo-retical plate number N dropped from92159to40824.The limits of detection(LOD)were estimated based on a sig-nal-to-noise ratio of3for different injection times(10,120 and180s).Those results are summarized in Table1.The LODs of120and180s injection times were similar and which were0.8,0.9,0.8µM for GLUF,AMPA and GLYP, pared with10s injection time,the LODs of 120s injection were10times pared with the pre-viously described CE-indirect detection methods,29,30the sensitivity of the present study is better.The LODs of180s injection time were similar to those obtained with120s injec-tion time.However,the injection volume was more.This is likely due to the sample loading capacity of the capillary be-ing exceeded at180s under the stacking conditions.There-fore,the optimal injection time is120s.A series of solution mixtures containing a known amount of GLUF,AMPA,and GLYP were prepared using D.I.water as the solvent.These standard solutions were used for the construction of calibration curves.The reaction mixture was injected by hydrodynamic injection at18cm for120s.The results are summarized in Table2.Within the concentration range studies,a good linear correlation(r>0.999)between peak areas and concentrations was obtained for each analyte.788J.Chin.Chem.Soc.,Vol.52,No.4,2005Chang andWeiFig.2.Electropherograms of30m M herbicides for different injection times.(A)120s;(B)180s;(C)360s;(D)480s.Elec-trophoretic buffer,25mM sodium borate,pH10.0;peak identities and other conditions as in Fig.1.The limits of detection (LODs)were calculated based on an S/N ratio of 3,which are 0.8,0.9and 0.8µM for GLUF,AMPA,and GLYP,respectively.These values correspond to 45,50,and 44fmol for GLUF,AMPA,and GLYP in an injec-tion volume of 58nL.The repeatability of migration times and peak areas are evaluated by seven replicate injections of the three analytes.Results expressed in RSD range from 0.6to 0.9%and from 5.8to 7.5%for migration times and peak ar-eas.ApplicationIn order to assess the applicability of the developed method to environmental samples,ground water and lake wa-ter samples were used.The samples were first filtered through a 0.45m m membrane filter,followed by derivatization with FMOC-Cl.The reaction mixtures were injected into the CE system for 120s.No detectable amount of GLUF and GLYP were found.The samples were then spiked with 12m M of GLUF,AMPA,and GLYP.The electropherograms of both unspiked and spiked ground water samples are shown in Figs.4A and 4B,respectively.Figs.5A and 5B show the electro-pherograms of unspiked and spiked lake water samples.The electropherograms of unspiked ground water and lake water samples show that there is no interference from water matrix.The recoveries of GLUF,AMPA,and GLYP were determined by spiking 480µL of ground water and lake water samples with 20m L of 3*10-4M GLUF,AMPA,and GLYP standard solutions.Final concentrations of 12m M for GLUF,AMPA,and GLYP were achieved.After filtration and derivatization were performed,the calibration curve method was used to de-termine the concentrations of GLUF,AMPA,and GLYP.The recoveries were calculated by comparing the quantitation re-sults with the theoretical concentrations of the water samples.Based on triplicate measurements,the mean recoveries were found to be in the range of 92.7-103.1%(Table 3).CONCLUSIONSCE-UV absorption detection coupled with acetoni-trile-salt mixtures has been demonstrated to be an efficientDetermination of Herbicides by Capillary Electrophoresis J.Chin.Chem.Soc.,Vol.52,No.4,2005789Fig.3.Effect of injection time on (A)peak height;(B)peak area;(C)theoretical plate number.Mix-tures of GLUF (3´10-5M),AMPA (3´10-5M),and GLYP (3´10-5M)were injected at dif-ferent periods of time.Table 1.LODs of GLUF,AMPA,and GLYP for differentinjection timesInjection time10s 120s 180sSpecies LOD (m M)GLUF 7.90.80.8AMPA 8.60.90.9GLYP7.20.80.8and sensitive method for the analysis of herbicides.The LODs for GLUF,GLYP,and AMPA were in the 10-7M level.The developed method can be applied to the simultaneous de-termination of GLUF,AMPA,and GLYP in ground water and lake water.This simple,sensitive,and selective method is suitable for the monitoring of environmental water sam-ples.The sensitiviy of the developed method can be enhanced by the use of a fluorescence detector or a laser-induced fluo-rescence detector.790J.Chin.Chem.Soc.,Vol.52,No.4,2005Chang and WeiTable 2.Calibration data and relative standard deviations of migration times and peak areasfor GLUF,AMPA,and GLYP RSD%Species Linear range (m M)Slope Intercept r Migration timepeak area GLUF 1.5-300 4.9*10936230.99980.6 5.8AMPA 1.5-300 5.7*10937130.99970.6 4.0GLYP1.5-3008.0*109-11030.99970.97.5Fig.4.Electropherograms of GLUF,AMPA,and GLYP in ground water.(A)Ground water,(B)ground water spiked withGLUF (1.2´10-5M),AMPA (1.2´10-5M),and GLYP (1.2´10-5M).Electrophoretic buffer,25mM borate,pH 10.0.Peak identities and other conditions as in Fig.1.Table 3.Recoveries of GLUF,AMPA,and GLYP in groundwater and lake water (n =3)Ground water Lake water Species Recovery (%)RSD (%)Recovery (%)RSD (%)GLUF 97.1 1.2100.1 1.5AMPA 94.80.292.7 1.6GLYP101.5 2.8103.10.7ACKNOWLEDGEMENTSFinancial support from the National Science Council of Taiwan is gratefully acknowledged.Received February21,2005.REFERENCES1.Tomlin,C.D.S.The Pesticide Manual;British Crop Protec-tion Council,11Ed.,1997;pp.643-649.2.United States Environmental Protection Agency,ground wa-ter and drinking water,/safewater/dwh/ C-soc/glyphosa.html.3.Stalikas,C.D.;Konidari,C.N.J.Chromatogr.A2001,907,1.4.Kataoka,H.;Ryu,S.;Sakiyama,N.;Makita,M.J.Chro-matogr.A1996,726,253.5.Börjesson,E.;Torstensson,L.J Chromatogr.A2000,886,207.6.Roy,D.N.;Konar,S.K.J.Agric.Food Chem.1989,37,441.7.Mogadayi,P.S.;Louis,J.B.;Rosen,J.D.J.AOAC Int.1996,79,157.8.Alferness,P.L.;Iwata,Y.J.Agric.Food Chem.1994,42,2751.9.Hori,Y.;Fujisawa,M.;Shimada,K.;Sato,M.;Kikuchi,M.;Honda,M.;Hirose,Y.J.Chromatogr.B2002,767,255. 10.Hernandez,F.;Hidalgo,C.;Sancho,J.V.J.AOAC Int.2000,83,728.11.Hogendoorn,E.A.;Ossendrijver,F.M.;Dijkman,E.;Baumann,R.A.J.Chromatogr.A1999,833,67.12.Sancho,J.V.;Hernández,F.;López,F.J.;Hogendoorn,E.A.;Dijkman,E.;van Zoonen,P.J.Chromatogr.A1996,737,75.13.Kawai,S.;Uno,B.;Tomita,M.J.Chromatogr.1991,540,411.14.Sundaram,K.M.S.;Curry,J.J.Liq.Chrom.&Rel.Technol.1997,20,511.15.Wigfield,Y.Y.;Lanouette,M.Anal.Chim.Acta1990,233,311.Determination of Herbicides by Capillary Electrophoresis J.Chin.Chem.Soc.,Vol.52,No.4,2005791Fig.5.Electropherograms of GLUF,AMPA,and GLYP in lake water.(A)Lake water,(B)lake water spiked with GLUF(1.2´10-5M),AMPA(1.2´10-5M)and GLYP(1.2´10-5M).Electrophoretic buffer,25mM borate,pH10.0.Peak iden-tities and other conditions as in Fig.1.16.Mallat,E.;Barcel,D.J.Chromatogr.A1998,823,129.17.Patsias,J.;Papadopoulou,A.;Papadopoulou-Mourkidou,E.J.Chromatogr.A2001,932,83.18.Abdullah,M.P.;Daud,J.;Hong,K.S.;Yew,C.H.J.Chromatogr.A1995,697,363.19.Lovdahl,M.J.;Pietrzyk,D.J.J.Chromatogr.1992,602,197.20.Bauer,K.;Knepper,T.P.;Maes,A.;Schatz,V.;Voihsel,M.J.Chromatogr.A1999,837,117.21.Vreeken,R.J.;Speksnijder,P.;Bobeldijk-Pastorova,I.;Noij,Th.H.M.J.Chromatogr.A1998,794,187.22.You,J.;Koropahak,J.A.J.Chromatogr.A2003,989,231.23.Tomita,M.;Okuyama,T.;Nigo,Y.;Uno,B.;Kawai,S.J.Chromatogr.1991,571,324.24.Molina,M.;Silva,M.Electrophoresis2002,23,2333.25.Molina,M.;Silva,M.Electrophoresis2001,22,1175.26.Molina,M.;Silva,M.Electrophoresis2002,23,1096.27.Shamsi,S.A.;Danielson,N.D.Anal.Chem.1995,67,1845.28.Soga,T.;Imaizumi,M.Electrophoresis2001,22,3418.29.Cikalo,M.G.;Goodal,D.M.;Matthews,W.J.Chromatogr.A1996,745,189.30.Chang,S.Y.;Liao,C.-H.J.Chromatogr.A2002,959,309.31.Lee,E.A.;Zimmerman,L.R.;Bhullar,B.S.;Thurman,E.M.Anal.Chem.2002,74,4937.32.Shihabi,Z.K.J.Chromatogr.A1996,744,231.33.Shihabi,Z.K.J.Chromatogr.A2000,902,107.34.Friedberg,M.A.;Shihabi,Z.K.J.Chromatogr.B1997,695,193.35.Friedberg,M.A.;Hinsdale,M.;Shihabi,Z.K.J.Chro-matogr.A1997,781,35.792J.Chin.Chem.Soc.,Vol.52,No.4,2005Chang and Wei。