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Simultaneous determination of phenolic acids and 2,4-dihydroxy-7-

Journal of Chromatography A,864(1999)315–321

https://www.sodocs.net/doc/7d13658229.html,/locate/chroma

Simultaneous determination of phenolic acids and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one in wheat (Triticum aestivum L.)by

gas chromatography–tandem mass spectrometry

a ,a a

b c

*Hanwen Wu ,Terry Haig ,Jim Pratley ,Deirdre Lemerle ,Min An

Farrer Centre for Conservation Farming ,Charles Sturt University ,P .O .Box 588,Wagga Wagga ,NSW 2678,Australia

NSW Agriculture ,Wagga Agricultural Institute ,Wagga Wagga ,NSW 2650,Australia

Environmental and Analytical Laboratories ,Charles Sturt University ,P .O .Box 588,Wagga Wagga ,NSW 2678,Australia

Received 23June 1999;received in revised form 24September 1999;accepted 27September 1999

Abstract

A procedure using gas chromatography and tandem mass spectrometry (GC–MS–MS)has been developed for the identi?cation and quanti?cation of some allelochemicals in wheat (Triticum aestivum L.).The quantities of allelochemicals in wheat shoots ranged from 2.9to 110mg per kilogram of dry shoot https://www.sodocs.net/doc/7d13658229.html,pared with gas chromatography–mass spectrometry (GC–MS),the GC–MS–MS technique signi?cantly increased instrument selectivity and sensitivity,thereby providing more reliable quantitation results in the determination of the phytotoxic compounds examined during this allelopathy research.?1999Elsevier Science B.V .All rights reserved.

Keywords :Wheat;Allelochemicals;Triticum aestivum ;Phenolic acids;Dihydroxymethoxybenzoxazinone

1.Introduction

been regarded as some of the major phenolic acids predominantly identi?ed in wheat stubbles and in the Wheat (Triticum aestivum L.)has been found to soil [1,5].In addition to the phenolic acids,one of possess allelopathic potential [1,2]and studies have the hydroxamic acids,2,4-dihydroxy-7-methoxy-1,4-been conducted to apply wheat allelopathy for benzoxazin-3-one (DIMBOA),has also been re-biological weed control [3,4].Wheat cultivars dif-ported as an active allelochemical in wheat [6].The fered in their allelopathic ability to suppress the phytotoxicity of these compounds has been clearly growth of annual ryegrass (Lolium rigidum G.)and demonstrated [7,8].

the degree of allelopathic inhibition was highly Allelopathic compounds are often found as com-associated with the total phenolic contents contained plex mixtures and their determination presents an in each wheat extract [4].p -Hydroxybenzoic,van-analytical challenge.Many traditional separation illic,p -coumaric,syringic and ferulic acids have

techniques such as paper,thin-layer,and column chromatography have been used for the separation and identi?cation of phenolic compounds,but these *Corresponding author.Tel.:161-2-6933-2749;fax:161-2-6933-methods are limited in separation power [9].High-2812.

E -mail address :11134425@https://www.sodocs.net/doc/7d13658229.html,.au (H.Wu)performance liquid chromatography (HPLC)gives

316H.Wu et al./J.Chromatogr.A864(1999)315–321

better results and has been widely used for the ence and the GC-grade diethyl ether was purchased analysis of phenolics and DIMBOA in plants from BDH.The derivatising reagent,N-methyl-N-[5,7,10].The best separation power has been(trimethylsilyl)tri?uoroacetamide(MSTFA),was ob-achieved by capillary gas chromatography(GC)in tained from Alltech Australia.

the determination of phenolic mixtures[9].Gas

chromatography coupled with mass spectrometry 2.2.Analytical procedure

(GC–MS)has been regarded as a powerful ana-

lytical tool for the characterisation of complex 2.2.1.Preparation of calibration standards organic mixtures and has successfully been em-Seven standard solutions each containing all six ployed in the identi?cation and quantitation of active target compounds,i.e.,p-hydroxybenzoic acid,van-allelopathic compounds in vulpia(Vulpia myuros L.illic acid,syringic acid,p-coumaric acid,ferulic acid Gmel)[11].Recent advances in instrumentation have and DIMBOA,were?rst prepared in methanol at further boosted the analytical power for the de-concentrations of0.05,0.1,0.5,1,5,10,20m g/ml, termination of allelopathic compounds.Ion trap respectively.In separate analyses,1ml from each of detectors have been improved to perform tandem the seven methanolic standard solutions was pipetted mass spectrometry(MS–MS),which enables the ion into a2-ml minivial and dried by nitrogen blow-trap to isolate an ion of interest and then produce down.A1-ml volume of internal standard p-chloro-characteristic progeny ions by collision-induced dis-benzoic acid at the concentration of5m g/ml in sociation(CID).Compared to single-stage MS,MS–methanol was then pipetted into the dry minivial and MS technology enhances instrumental selectivity and dried again with nitrogen prior to the derivatisation sensitivity and has become a favoured tool for the step.

quantitative analysis of the complex matrixes en-

countered in biological and environmental applica- 2.2.2.Preparation of wheat samples

tions[12].However,this powerful technique has not Surface-sterilised and pre-germinated wheat seeds yet been much employed in allelopathy studies.(Triticum aestivum,cv.Triller)were grown in a Although both phenolic acids and cyclic hydrox-nutrient-free agar medium under aseptic conditions amic acids have been reported in relation to wheat in a controlled growth cabinet with a daily light/dark allelopathy,no attempt has been made to simul-cycle of13h/11h and a temperature cycle at taneously determine these two distinct groups of258C/138C.The?uorescent light intensity in the

allelopathic compounds.This study was designed to cabinet was3.5660.16?10lux.Shoots of17-day-apply gas chromatography–tandem mass spec-old wheat seedlings were harvested and immediately trometry(GC–MS–MS)to selectively identify and freeze-dried(Christ Alpha1-4freeze dryer,B.Braun quantify some phenolic acids and DIMBOA in a Biotech International).An amount of0.100g of very complex matrix of wheat shoot extract.freeze-dried wheat shoots was cut into2-mm lengths,

ground into powder with a mortar and pestle after the

addition of liquid nitrogen and macerated with3ml 2.Experimental of0.001M HCl.The entire macerate was transferred

into a labelled glass scintillation vial and sonicated at 2.1.Chemicals and reagents58C for15min(Unisonics,Australia).The resulting

mixture was centrifuged at20000rpm at108C for p-Hydroxybenzoic acid(PHB),vanillic acid15min to remove the debris(Avanti J-30I Cen-(V AN),syringic acid(SYR),p-coumaric acid trifuge,Beckman,USA).The supernatant was then (COU),ferulic acid(FER)and the internal standard collected and extracted three times with10-ml (p-chlorobenzoic acid)were obtained from Sigma–portions of diethyl ether.The ether layers were Aldrich.DIMBOA was kindly provided by https://www.sodocs.net/doc/7d13658229.html,bined and evaporated on a rotary evaporator William S.Chilton of the Department of Botany,under reduced pressure at358C until the volume of North Carolina State University,Raleigh,NC,USA.residual solution was approximately2ml.The2ml HPLC-grade methanol was obtained from EM Sci-ether solution was then transferred to a2-ml minivial

H.Wu et al./J.Chromatogr.A864(1999)315–321317 and dried with nitrogen blow-down.A1-ml volume99.9999%and its linear velocity was34cm/s. of internal standard p-chlorobenzoic acid at the Injector temperature was maintained at2808C,and concentration of5m g/ml in methanol was then the injection volume was1m l with the splitless pipetted into the dry minivial and dried again with mode.

nitrogen prior to the derivatisation step.The electron impact ionization(EI)mode with

automatic gain control(AGC)was used for MS.The 2.2.3.Derivatisation electron multiplier voltage for MS–MS was1450V, The silylation of calibration standards and the AGC target was10000counts and?lament emission wheat shoot sample was accomplished by the addi-current was60m A with the axial modulation am-tion of1.00ml of MSTFA at608C for30min.The plitude at4.0V.The ion trap was held at2008C and silylated samples were analysed by GC–MS–MS or the transfer line at2508C.Manifold temperature was GC–MS.The large excess of MSTFA ensured that set at608C.Mass spectral scan time from m/z50to the derivatization was complete.Care was taken to450was 1.0s(using3microscans).Nonresonant ensure anhydrous conditions during the preparation CID was used for MS–MS.The associated parame-and derivatization process because of the high sen-ters for the MS–MS method were optimised for each sitivity of trimethylsilyl(TMS)derivatives towards individual compound(Table1).The method was moisture.divided into10acquisition segments so that different

ion preparation?les could be used to optimise the 2.2.4.GC–MS–MS instrumentation and conditions conditions for the TMS derivatives of the chemically GC–MS–MS analysis was carried out on a Varian distinct internal standard,phenolic acids and DIM-3400CX gas chromatograph coupled with a Varian BOA.Standard samples of both p-coumaric acid and Saturn2000ion trap mass spectrometer.Samples ferulic acid consisted of trans and cis isomers so that were introduced via a DB-5MSITD(Ion Trap four segments were required to characterise the p-Tested)fused-silica capillary column of30m30.25coumaric and ferulic acids.The?rst segment was a mm I.D.,with a stationary phase thickness of0.259-min solvent delay,which was necessary for the m m(J&W Scienti?c,Alltech,Australia).The gas protection of the electron multiplier from the large chromatographic conditions for the analysis of wheat solvent peak signal.

allelochemicals were slightly modi?ed from An[7].

The column temperature was initially held at808C 2.2.5.Identi?cation and quantitation

for1min,then programmed to1608C at a rate of Two user libraries(MS and MS–MS library)were 108C/min,from160to2358C at a rate of58C/min generated with the injection of TMS-derivatised and from235to2808C at a rate of508C/min,with a authentic reference compounds by GC–MS or GC–?nal hold time of5min(total run time,29.9min).MS–MS analysis.The MS library recorded the Helium was used as the carrier gas with purity of retention times and the mass spectra of TMS deriva-Table1

Ion trap MS–MS method parameters

Acquisition segment

2345678910 Compound(silylated)p-Chlorobenzoic acid PHB V AN cis-COU SYR cis-FER trans-COU DIMBOA trans-FER Segment time(min) 4.100.40 2.60 2.000.300.50 1.10 1.808.10 Retention time(min)9.7813.3015.7416.2318.2318.6319.0620.1621.91 Chosen precursor ion(m/z)213267297293297323293340323 Excitation amplitude(V)546443.64543.6414546.541.2 Excitation storage level(m/z)608065656565657565 Quantifying product ion(m/z)169223267249253293249194293

a Mass isolation window(m/z)was set at3and excitation time(ms)at20.

b Segment one was the solvent delay.

318H.Wu et al./J.Chromatogr.A864(1999)315–321

tives of authentic standards under the chosen chro-acid,p-hydroxybenzoic acid,vanillic acid,syringic matographic conditions.Similarly,the MS–MS li-acid,p-coumaric acid,ferulic acid and DIMBOA by brary recorded the retention times and the daughter comparing retention times and the mass spectra with mass spectra derived from the speci?c precursor ions those of TMS derivatives of their authentic com-of TMS derivatives of each authentic standard after pounds under identical conditions.However,the p-CID with helium gas.The allelopathic substances hydroxybenzoic acid,cis-p-coumaric acid,trans-p-were then identi?ed by comparing retention times coumaric acid,syringic acid,cis-ferulic acid and and mass spectral data with those in either user DIMBOA were strongly coeluted with background library,depending upon the type of analysis.All substances(Fig.2),although p-chlorobenzoic acid, quantitation was performed by the method of internal vanillic acid and trans-ferulic acid were well sepa-standardisation using p-chlorobenzoic acid at a rated.Owing to the complexity of the sample matrix, concentration of5m g/ml as the internal standard.for each of the six co-eluted analytes shown in Fig. The quantitation of these compounds was based on2,there could not be found unique characteristic the peak area of the selected daughter ions listed in GC–MS ions of suf?cient intensity to serve as Table1and is reported in units of milligrams per suitable quantifying ions.In order to help illustrate kilogram of dry matter.All calibration standards and this dif?cult background matrix problem,the more the wheat samples were run in triplicate.characteristic analyte ion chromatograms and total

ion chromatograms of Fig.2have been sup-

plemented with indicative background ion chromato-3.Results and discussion grams from coelutants.Coelution with these analytes

is also further evident in the asymmetry of the more Underivatised phenolic acids and DIMBOA have characteristic analyte ion pro?les.The simple GC–relatively low volatility and are not suitable for direct MS results were therefore not considered adequate capillary GC analysis.MSTFA was chosen as the for accurate quantitation.

derivatisation reagent to convert analytes into vola-The tandem mass spectrometry technique was tile compounds.The GC–MS analysis of silylated introduced in order to?lter out unwanted chemical wheat shoot extract was repeated three times(Fig.backgrounds.After preliminary experimentation,the 1).Analysis by GC–MS was able to identify all the MS–MS conditions shown in Table1were used to target allelopathic compounds,i.e.,p-chlorobenzoic analyse the phenolic acids and DIMBOA in wheat

Fig. 1.Reconstructed total ion current chromatogram(RTIC)of derivatized wheat shoot extract obtained from GC–MS.Peaks: 15p-chlorobenzoic acid,25p-hydroxybenzoic acid,35vanillic acid,45cis-p-coumaric acid,55syringic acid,65cis-ferulic acid,75trans-p-coumaric acid,852,4,-dihydroxy-7-methoxy-1,4,-benzoxazin-3-one,95trans-ferulic acid.

H.Wu et al./J.Chromatogr.A864(1999)315–321319

Fig.2.GC–MS run showing the coelution of analytes with background signals(*:analytes were identi?ed by comparing retention times and mass spectral data with those in the user MS library;a:precursor ion of the analyte;b:background ion).

shoots.The GC–MS–MS analysis of the shoot compounds were much lower in GC–MS than in extract was repeated three times.A highly-resolved GC–MS–MS(Table3).The signal/noise ratios for chromatogram was successfully obtained(Fig.3).p-chlorobenzoic acid,p-hydroxybenzoic acid and The MS–MS technique successfully removed most vanillic acid in the shoot sample were51,25and29 of the unwanted chemical signals and provided a times higher under MS–MS conditions than under clear background for the quanti?cation of the chosen MS conditions.This technique was then employed to allelopathic compounds.The precursor ion of the quantify the allelochemicals in wheat(Table2). particular compound was isolated and then CID with Wheat shoots contained a higher amount of DIM-helium carrier gas gave rise to a characteristic BOA than of phenolic acids.The allelochemical daughter mass spectrum for each analyte(Table2).contents in wheat shoots were110mg/kg dry mass The preferential isolation of the particular ions by the for DIMBOA and2.9mg/kg dry mass for cis-p-MS–MS technique yielded much stronger analyte coumaric acid.The dif?culties in the separation and signals than those of single stage MS analysis,identi?cation of cis and trans mixtures have been thereby signi?cantly reducing the effect of matrix reported[13].However,the cis and trans isomers of noise.The signal/noise ratios for all the allelopathic p-coumaric and ferulic acids were successfully sepa-

320H.Wu et al./J.Chromatogr.A864(1999)315–321

Fig.3.Reconstructed total ion current chromatogram of derivatized wheat shoot extract obtained from GC–MS–MS.Peaks as in Fig.1. Table2

Important ions present in the daughter mass spectra of silylated compounds in wheat extract by GC–MS–MS and their concentrations a

Peak https://www.sodocs.net/doc/7d13658229.html,pound m/z Quantity6SD

(mg/kg dry mass)

1p-Chlorobenzoic acid213,169(100%),141,138,133,131,105,91,75Internal standard

2PHB267,251,225,223(100%),207,193,179,147,1032262.3

3V AN297,267(100%),253,237,225,224,223,181,1475664.3

4cis-p-COU293,251,249(100%),247,233,231,223,219,203 2.960.1

5SYR297,271,255,253(100%),225,223,193,179,1331160.7

6cis-FER323,294,293(100%),279,264,249,233,179,175 3.960.2

7trans-p-COU293,251,249(100%),247,233,231,223,219,2033761.5

8DIMBOA340,296,268,250,221,220,194(100%),192,14711064.8

9trans-FER323,294,293(100%),279,264,249,233,179,1758962.0

a Identi?ed as trimethylsilyl(TMS)derivative.

b The?rst ion in each compound list is the precursor ion;all other ions are greater than5%of the base peak.

rated under the present chromatographic conditions Table3

despite their similar daughter mass spectra(Table2). Signal/noise ratio of GC–MS and GC–MS–MS

Results showed that trans-ferulic and trans-p-Compound Signal/noise ratio6SD

coumaric acid were present in higher quantities than GC–MS GC–MS–MS their cis isomers in wheat shoots.

p-Chlorobenzoic acid3469.61700652.8

PHB1162.2270649.1

V AN8867.72500638.0 4.Conclusions

cis-p-COU2463.52662.1

SYR1365.76865.0

Application of state-of-the-art analytical tools can cis-FER1767.34264.1

trans-p-COU1769.3180614.1generate unambiguous results in the determination of DIMBOA 3.362.31763.0responsible allelopathic compounds in a complex trans-FER35611.0250617.2mixture of plant origin.In the present study,a

H.Wu et al./J.Chromatogr.A864(1999)315–321321 GC–MS–MS technique has been successfully used provision of GC–MS–MS instrumentation from the to obtain a highly resolved chromatogram to de-Environmental and Analytical Laboratories at termine several key phenolic compounds and DIM-Charles Sturt University.

BOA.The enhanced sensitivity and selectivity of

GC–MS–MS have provided reliable quantitative

results for the biologically active compounds in References

complex mixtures encountered in this allelopathy

research and can be employed to screen cultivars for[1]W.D.Guenzi,T.M.McCalla,Agron.J.58(1966)303.

[2]R.W.L.Kimber,Aust.J.Agric.Res.18(1967)361.

their differential production of allelopathic com-

[3]J.A.Spruell,Dissertation Abstr.Int.,B Sci.Eng.45(1984) pounds in a large number of wheat accessions.This

1102B.

technique coupled with DNA technology will facili-

[4]H.Wu,J.Pratley,D.Lemerle,T.Haig,B.Verbeek,in:Proc. tate the identi?cation of genetic markers conferring9th Aust.Agron.Conf.,Wagga Wagga,1998,p.567.

the biosynthesis of allelopathic compounds.The[5]U.Blum,T.R.Wentworth,K.Klein,A.D.Worsham,L.D.

King,T.M.Gerig,S.W.Lyu,J.Chem.Ecol.17(1991)1045. possibilities for genetic manipulation of crop al-

[6]H.M.Niemeyer,Phytochemistry27(1988)3349. lelopathic potential have been reviewed[14].The

[7]M.An,Ph.D.Thesis,Charles Sturt University(1995). genetic manipulation of crop cultivars might provide

[8]F.J.Perez,Phytochemistry29(1990)773.

crop plants with strong allelopathic potential to[9]C.S.Creaser,M.R.Koupai-Abyazani,G.R.Stephenson,J. compete with weeds,thereby reducing the input of Chromatogr.478(1989)415.

[10]F.J.Perez,J.Ormeno-Nunez,J.Chem.Ecol.17(1991) herbicides into agroecosystems.

1037.

[11]M.An,J.E.Pratley,T.Haig,Rapid Commun.Mass Spec-

trom.10(1996)104.

Acknowledgements[12]C.K.Van Pelt,H.Haggarty,J.T.Brenna,Anal.Chem.70

(1998)4369.

[13]E.M.Gaspar,H.C.Neves,Allelopathy J.2(1995)79. The authors are thankful for?nancial support from

[14]H.Wu,J.Pratley,D.Lemerle,T.Haig,Weed Res.39(1999) the Australian Cooperative Research Centre for

171.

Weed Management Systems and acknowledge the

motivation, Determination and dedication

Motivation, Determination and Dedication What makes an original contributor in science is often not only ability, but also something else, something apparently intangible, and not easily detected. This extra something lies deep within the individual and needs to be nurtured and tested. Motivation is a personal trait that is primarily instilled by seniors such as teachers or parents. An important aspect in developing motivation is the setting of goals. A person probably has set long-range goals, or at this point more like dreams, such as winning the Nobel Prize. This is great as long as the individual is realistically working toward short-range also. These are the day-to-day accomplishments that really make working hard seem fun. Proficiency in anything requires a great deal of determination and self-discipline. In addition, a person’s ability to cope with frustration is also an important factor in one’s life career. Repeated failures at making experiments may be too much for many talented would-be scientists. The determination to continue, with the realization that everything worthwhile takes a great deal of patience, is an essential requirement. These factors, together with inherent dedication, will bring about the realization of one’s aspirations. Through all this it is not the triumph but he struggle that brings about the complete personal satisfaction in knowing that you as a scientist have given your all.

Measures for the Determination of High and New Tech Enterprises

国科发火〔2008〕172号关于印发《高新技术企业认定管理办法》的通知各省、自治区、直辖市、计划单列市科技厅（局）、财政厅（局）、国家税务局、地方税务局：根据《中华人民共和国企业所得税法》、《中华人民共和国企业所得税法实施条例》的有关规定，经国务院批准，现将《高新技术企业认定管理办法》及其附件《国家重点支持的高新技术领域》印发给你们，请遵照执行。附件：高新技术企业认定管理办法科技部财政部国家税务总局二ＯＯ八年四月十四日 Administrative Measures for the Determination of High and New Tech Enterprises (No. 172 [2008] of the Ministry of Science and Technology) The science and technology departments (bureaus), finance departments (bureaus), offices of the State Administration of Taxation and local taxation bureaus of all provinces, autonomous regions, municipalities directly under the Central Government and cities under separate state planning, In accordance with the Enterprise Income Tax Law of the People’s Republic of China and Regulation on the Implementation of the Enterprise Income Tax Law of the People’s Republic of China, and upon approval of the State Council, the Administrative Measures for the Determination of High and New Tech Enterprises and the annex, i.e. the High and New Tech Fields under the Key Support of the State, are hereby printed and distributed to you for your compliance. Annex: Administrative Measures for the Determination of High and New Tech Enterprises Ministry of Science and Technology, Ministry of Finance, State —1—

The POWER of determination 决心的伟大力量

The POWER of determination The little country schoolhouse was heated by an old-fashioned , pot-belled stove . A little boy had the job of coming to school early each day to start the fire and warm the room before his teacher and his classmates arrived . One morning they arrived to find the schoolhouse engulfed in flames . They dragged the unconscious little boy out of the flaming building more dead than alive . He had major burns over the lower half of his bady and was taken to the nearby county hospital . From his bed the dreadfully burned , semi-conscious little boy faintly heard the doctor told his mother that her son would surely die - which was for the best , really - for the terrible fire had devastated the lower half of his body . But the brave boy didn't want to die . He made up his mind that he would survive . Somehow , to the amazement of the physician , he did survive . When the mortal danger was past , he again heard the doctor and his mother speaking quietly . The mother was told that since the fire had destroyed so much flesh in the lower part of his body , it would almost be better if he had died , since he was doomed to be a lifetime cripple with no use at all of his lower limbs . Once more the brave boy made up his mind . He would not be a cripple . He would walk . But unfortunately , from the waist down he no motor ability . His thin legs dangled there , all but lifeless . Ultimately he was released from the hospital . Every day his mother would massage his little legs , but there was no feeling , no control , nothing . Yet his determination that he would walk was as strong as ever . When he wasn't in bed , he was confined to a wheelchair . One sunny day his mother wheeled him out into the yard to get some fresh air . This day , instead of sitting there , he therw himself from the chair . He pulled himself across the grass , dragging his legs behind him . He worked his way to the white picket fence bordering their lot . With great effort , he raised himself up on the fence . Then , stake by stake , he began dragging himself along the fence , resolved that he would walk . He started to do this every day until he beside the fence . There was nothing he wanted more than to develop life on those legs . Ultimately , through his daily massages , his iron persistence and his resolute determination , he did develop the ability to stand up , then to walk haltingly , then to walk by himself - and then to run .

SAP自动科目确定Account Determination

SAP自动科目确定Account Determination 理论概述： Account Determination：通过设定实现物料异动是自动决定会计凭证产生的科目 Automatic Account Determination涉及如下操作： Goods receipt Goods issue Transfer posting Incoming invoice Price change Debit/credit Material Account maintenance 配置介绍： 1、OBYC/OMWB Materials ManagementàValuation and Account AssignmentàAccountDeterminationàAccount Determination WithoutWizardàConfigure Automatic Postings 如下图：在物料异动时由以下涉及到的不同条件决定借贷方分别抛什么样的科目， ① Chart of Accounts: ② (valuation modification)Valuation Grouping code: 当valuation grouping code被激活，可以通过将不同的valuation area进行分组得到的grouping code座位参数来决定G/L account no. ③ Valuation Class:由物料决定 ④ Transaction: Posting transactions 在系统中是固定的，用来控制是否抛入存货，差异科目，GRIR清帐科目等等。

921_WATER DETERMINATION_usp38

á921? WATER DETERMINATION Many Pharmacopeial articles either are hydrates or contain water in adsorbed form. As a result, the determination of the water content is important in demonstrating compliance with the Pharmacopeial standards. Generally one of the methods giv-en below is called for in the individual monograph, depending upon the nature of the article. In rare cases, a choice is allowed between two methods. When the article contains water of hydration, Method I (Titrimetric), Method II (Azeotropic), or Method III (Gravimetric) is employed, as directed in the individual monograph, and the requirement is given under the heading Water.The heading Loss on Drying (see Loss on Drying á731?) is used in those cases where the loss sustained on heating may be not entirely water. METHOD I (TITRIMETRIC) Determine the water by Method Ia, unless otherwise specified in the individual monograph. Method Ia (Direct Titration) Principle—The titrimetric determination of water is based upon the quantitative reaction of water with an anhydrous solu-tion of sulfur dioxide and iodine in the presence of a buffer that reacts with hydrogen ions. In the original titrimetric solution, known as Karl Fischer Reagent, the sulfur dioxide and iodine are dissolved in pyridine and methanol. The test specimen may be titrated with the Reagent directly, or the analysis may be carried out by a residual titra-tion procedure. The stoichiometry of the reaction is not exact, and the reproducibility of a determination depends upon such factors as the relative concentrations of the Reagent ingredients, the nature of the inert solvent used to dissolve the test speci- men, and the technique used in the particular determination. Therefore, an empirically standardized technique is used in order to achieve the desired accuracy. Precision in the method is governed largely by the extent to which atmospheric moisture is excluded from the system. The titration of water is usually carried out with the use of anhydrous methanol as the solvent for the test specimen. In some cases, other suitable solvents may be used for special or unusual test specimens. In these cases, the addition of at least 20% of methanol or other primary alcohol is recommended. Apparatus—Any apparatus may be used that provides for adequate exclusion of atmospheric moisture and determination of the endpoint. In the case of a colorless solution that is titrated directly, the endpoint may be observed visually as a change in color from canary yellow to amber. The reverse is observed in the case of a test specimen that is titrated residually. More commonly, however, the endpoint is determined electrometrically with an apparatus employing a simple electrical circuit that serves to impress about 200 mV of applied potential between a pair of platinum electrodes immersed in the solution to be titrated. At the endpoint of the titration a slight excess of the reagent increases the flow of current to between 50 and 150microamperes for 30 s to 30 min, depending upon the solution being titrated. The time is shortest for substances that dissolve in the reagent. With some automatic titrators, the abrupt change in current or potential at the endpoint serves to close a sole-noid-operated valve that controls the buret delivering the titrant. Commercially available apparatus generally comprises a closed system consisting of one or two automatic burets and a tightly covered titration vessel fitted with the necessary electro-des and a magnetic stirrer. The air in the system is kept dry with a suitable desiccant, and the titration vessel may be purged by means of a stream of dry nitrogen or current of dry air. Reagent—Prepare the Karl Fischer Reagent as follows. Add 125g of iodine to a solution containing 670 mL of methanol and 170 mL of pyridine, and cool. Place 100 mL of pyridine in a 250-mL graduated cylinder, and, keeping the pyridine cold in an ice bath, pass in dry sulfur dioxide until the volume reaches 200 mL. Slowly add this solution, with shaking, to the cooled iodine mixture. Shake to dissolve the iodine, transfer the solution to the apparatus, and allow the solution to stand overnight before standardizing. One mL of this solution when freshly prepared is equivalent to approximately 5 mg of water, but it dete-riorates gradually; therefore, standardize it within 1 h before use, or daily if in continuous use. Protect from light while in use.Store any bulk stock of the reagent in a suitably sealed, glass-stoppered container, fully protected from light, and under refrig-eration. For determination of trace amounts of water (less than 1%), it is preferable to use a Reagent with a water equivalency factor of not more than 2.0, which will lead to the consumption of a more significant volume of titrant. A commercially available, stabilized solution of Karl Fischer type reagent may be used. Commercially available reagents con-taining solvents or bases other than pyridine or alcohols other than methanol may be used also. These may be single solutions or reagents formed in situ by combining the components of the reagents present in two discrete solutions. The diluted Re-agent called for in some monographs should be diluted as directed by the manufacturer. Either methanol or other suitable solvent, such as ethylene glycol monomethyl ether, may be used as the diluent. Test Preparation—Unless otherwise specified in the individual monograph, use an accurately weighed or measured amount of the specimen under test estimated to contain 2–250 mg of water. The amount of water depends on the water equivalency factor of the Reagent and on the method of endpoint determination. In most cases, the minimum amount of specimen, in mg, can be estimated using the formula: FCV / KF

Determination-of-phenylethanolamine-A

Journal of Chromatography B,900 (2012) 94–99 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B j o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o m b Short communication Determination of phenylethanolamine A in animal hair,tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS Ming-Xia Zhang a ,Cun Li a ,b ,Yin-Liang Wu c ,? a Hebei University of Engineering,Handan 056038,PR China b Tianjin Agricultural University,Tianjin 300380,PR China c Ningbo Academy of Agricultural Sciences,Ningbo 315040,PR China a r t i c l e i n f o Article history: Received 6March 2012Accepted 24May 2012 Available online 2 June 2012 Keywords: Phenylethanolamine A QuEChERS Hair Animal tissues Animal feeds LC–MS/MS a b s t r a c t A simple,sensitive and reliable analytical method was developed for the determination of a new beta-agonist phenylethanolamine A in animal hair,tissues and animal feeds by ultra high performance liquid chromatography–positive electrospray ionization tandem mass spectrometry (UHPLC–ESI-MS/MS)with QuEChERS.Samples were extracted with acetonitrile/water (80:20,v/v).The extract was puri?ed through QuEChERS method,then was dried with nitrogen and residues were redissolved in mobile phase for hair sample or directly diluted with 0.1%formic acid in water for other samples,and analyzed by LC–MS/MS on a Waters Acquity BEH C 18column with 0.1%formic acid in water/methanol as mobile phase with gradient elution.The samples were quanti?ed using phenylethanolamine A-D 3as internal standards.The pro-posed method was validated according to the European Commission Decision 2002/657/EC determining speci?city,decision limit (CC ?),detection capability (CC ?),recovery,precision,linearity,robustness and stability.The CC ?values ranged from 0.10to 0.26?g/kg.The CC ?values ranged from 0.20to 0.37?g/kg.The mean recoveries of 95.4–108.9%with intra-day CVs of 2.2–5.6%and inter-day CVs of 3.1–6.2%were obtained.The method is demonstrated to be suitable for the determination of phenylethanolamine A in animal hair,tissues and animal feeds.The total time required for the analysis of one sample except animal hair sample,including sample preparation,was about 25min. ? 2012 Elsevier B.V. All rights reserved. 1.Introduction Beta-agonists are originally used in the therapeutic treatment of asthma and preterm labor in humans [1].However,these com-pounds are also misused as growth promoters in livestock by diverting nutrients from fat deposition to the production of muscle tissues in animals [2].This misuse had caused some severe acci-dental poisonings in humans [3,4].Therefore,all beta-agonists are banned as feed additives for growth promotion in animals in China and EU [5,6].With the crackdown of banned beta-agonists,such as clenbuterol,salbutamol and ractopamine,a new beta-agonist named phenylethanolamine A has been illegally used in livestock in China (Fig.1)[7].In order to control the use of phenylethanolamine A,speci?c and sensitive methods for identifying and quantifying of phenylethanolamine A in animal hair,tissues and animal feeds are required. Although gas chromatography–mass spectrometry (GC–MS)[8–11]and liquid chromatography–mass spectrometry (LC–MS) ?Corresponding author.Tel.:+8657487928060;fax:+8657487928062.E-mail address:wupaddy?eld@https://www.sodocs.net/doc/7d13658229.html, (Y.-L.Wu). [12–17]methods have been developed to identify beta-agonists in biological samples,there are little literatures on identi?cation of phenylethanolamine A in animal tissues and feeds [7,18].A LC–MS/MS method had been recently developed by Sun and Yan in swine muscle [7].However,the pre-treatment procedure of the method was very complicated and time-consuming because the combination of enzymatic hydrolysis,liquid–liquid extraction and solid phase extraction (SPE)were used to cleanup.Therefore,rapid,speci?c and sensitive methods for the identi?cation and quanti?-cation of phenylethanolamine A in hair,animal tissues and feeds are required. The QuEChERS method was previously used by Anastassiades et al.for the determination of pesticides in fruit and vegetable samples with primary and secondary amine (PSA)as the base sor-bent [19].The method has already received worldwide acceptance because of the simplicity and high throughput enabled a labora-tory to process signi?cantly a large number of samples in a given time.Moreover,the method had recently been used for the deter-mination of veterinary drug residue and mycotoxins [20,21].Due to high ef?ciency of QuEChERS,it is necessary to develop QuECh-ERS method for phenylethanolamine A in animal hair,tissues and feeds. 1570-0232/$–see front matter ? 2012 Elsevier B.V. All rights reserved.https://www.sodocs.net/doc/7d13658229.html,/10.1016/j.jchromb.2012.05.030

04 价格决定(Price Determination)

第4单元价格决定（Price Determination）

经济学BA330 4.1 定义（Definition）市场上所有商品以当时价格完全售出后就会出现均衡价格。卖者以均衡价格买入市场的商品正由买者购买时出现均衡数量。 1.50 1.20 Pe 1.00 Q e 均衡价格和均衡数量说明：P e, Q e = 市场商品全部售出时的均衡量 D < S = 剩余 D1 > S1 = 短缺 4.2 对均衡价格和数量的影响（Effect on the Equilibrium Price and Quantity） 4.2.1 需求条件变动（但供应量不变）（When Conditions of Demand Changed (With Supply Unchanged) 如果需求条件发生变动使需求曲线左移，将出现新的均衡价格和数量，如下图(i) 所示。

BA330 经济学 Q Q (i) 需求移动 (ii) 供应移动 4.2.2 供应条件变动时（需求条件不变）When Conditions of Supply Changed (With Demand Unchanged) 如果需求条件发生变动使曲线左移，会引起新的均衡价格和数量，如上图(ii) 所示。 4.2.3需求和供应两种条件都发生变动时（When Both Conditions of Demand and Supply Changed ）如果需求和供应两种条件都发生变动，则只有当我们知道以下条件时才能决定均衡价格和数量： - 移动方向（左或者右） - 移动幅度（距离） - 需求和供应曲线的坡度 4.2.4国家征收间接税时（When Government Imposes an Indirect Tax ）征收间接税会减少一个企业准备以每种不同价格进行供给的数量，下表示出这一影响。 D 1 P P e P P e

英语达人必备经典文章---Determination

In 1883, a creative engineer named John Roebling was inspired to build a spectacular[壮观的] bridge connecting New York with Long Island. However bridge-building experts throughout the world thought that this was an impossible feat and told Roebling to forget the idea. Roebling could not ignore the vision he had in his mind of this bridge. After much discussion, he managed to convince his son Washington, an up-and-coming[初崭头角的] engineer, that the bridge in fact could be built. Working together for the first time, the father and son developed concepts of how it could be accomplished and how the obstacles[障碍] could be overcome. They hired their crew and began to build their dream bridge. The project started well, but when it was only a few months underway a tragic accident on the site took the life of John Roebling. Washington was injured, unable to walk or talk or even move. Everyone had negative[负面的] comments and felt that the project should be scrapped[抛弃] since the Roeblings were the only ones who knew how the bridge could be built. In spite of his handicap[身体的不便] Washington was never discouraged and still had a burning desire to complete the bridge and his mind was still as sharp as ever. He tried to inspire and pass on his enthusiasm[激情] to some of his friends, but they were too daunted[气馁的] by the task. As he lay on his bed in his hospital room, with the sunlight streaming through the windows, a gentle breeze blew the flimsy[薄薄的] white curtains apart and he was able to see the sky and the tops of the trees outside for just a moment. It seemed that there was a message for him not to give up. Suddenly an idea hit him. All he could do was move one finger and he decided to make the best use of it. By moving this, he slowly developed a code of communication with his wife. He touched his wife's arm with that finger, indicating to her that he wanted her to call the engineers again. Then he used the same method of tapping her arm to tell the engineers what to do. For 13 years Washington tapped out his instructions with his finger on his wife's arm, until the bridge was finally completed. Today the spectacular Brooklyn Bridge stands in all its glory as a tribute to the triumph of one man's indomitable[不屈不挠的] spirit and his determination[坚定] not to be defeated by circumstances.