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雨果英文名言翻译导读：1、衰老是从眼睛开始的。
Aging starts from the eye.2、理想是具有冒险性的。
Ideal is a risk.3、痛苦总是守在欢乐旁边。
Pain is always keep beside the joy.4、人生是花，而爱是花蜜。
Life is a flower, and love is the honey.5、何必遗憾本不能的事情。
Why sorry this can't.6、碰到毒蛇，就得把它扼死。
Met snakes, have to strangle it.7、哪里有阴影，那里就有光。
Where there is shadow, there is light.8、谨慎比大胆要有力量得多。
Caution is much more powerful than bold.9、给予是能使人产生优越感的。
To give is to be able to make the person produces the superiority.10、人生是花，而爱便是花的蜜。
Life is a flower, and love is the honey of the flower.11、没有被听见不是沉默的理由。
Has not been heard not the silence of the reason.12、做好人容易，做正直的人却难。
Do people easily, the upright do is difficult.13、他是情场做戏，她却一片痴情。
He is fair in love and acting as she is a piece of infatuation.14、自由只有通过友爱才得以保全。
Only through love and preserve the freedom.15、虔诚的开端，带来美好的结束。
The beginning of piety, bring a good end.16、最可怕的刑罚莫过于受人嘲讽。
阿那克西曼德的分离生成观阿那克西曼德是一位在20世纪初的意大利哲学家，他提出了「分离生成观」的学术理论，对哲学和社会科学领域影响深远。
阿那克西曼德的分离生成观指的是：世界上所有的事物，在某种程度上都是由其他事物分离出来而形成的。
这个理论从“分离”和“生成”两个方面解释了物质形态的发展和演变。
阿那克西曼德对“分离”这一概念的解释，指的是从一种状态分裂出另一种独立的状态，这个过程是不可避免的。
这意味着物质实体并非一成不变，而是以不同的方式和形式不断更新和变化。
例如，一个人的身体是由无数个单独的细胞组成的，而这些细胞又是分离出来的，由无数个分子和离子组成。
这个理论还涉及到“生成”这一概念。
阿那克西曼德认为，物质形态的发展离不开不断地“生成”。
每一个事物都需要经历一定的“生成”过程，才能不断进化和演变。
阿那克西曼德认为，事物之间的相互作用和相互影响，会导致一些物质形态的生成。
这些物质形态之间的相互作用和影响又会进一步造成更多的生成，最终演化成当前的世界。
阿那克西曼德的分离生成观对哲学、物理、生物、人文历史以及人类社会等领域都有很大的影响。
对哲学而言，这个观点指出了世界和我们自身的动态和变化，同时提醒人们认识到人类自身是社会和文化环境的产物。
对物理学而言，这个理论启示人们对各种物质形态的研究和探索方式；对生物学而言，这个理论指出了生命体的分化和进化过程。
在历史和社会学领域，这个理论还阐明了社会和社会各种元素的演化过程，深入剖析了人文社会进化的基石。
总之，在现代学术领域，阿那克西曼德的分离生成观为我们认识世界提供了新的角度和思路。
我们应该以此为基础，不断探索各个领域的发展和变化的规律。
禪修基本須知及專用詞彙手冊巴利－中文－英文Ven. Dhammasiri編(請用foreign1字型)台灣法雨道場印行禪修基本須知及專用詞彙手冊巴利－中文－英文第一章：如何修行安般念到入禪第二章：如何以其它法門修定第三章：如何修行四梵住與四護衛禪第四章：如何辨識色法第五章：如何辨識名法第六章：如何透視緣起法的環結第七章：如何培育觀智以透視涅槃參考資料帕奧修程之巴~中~英文對照補充回目錄第一章：修行安般念到入禪Chapter 1: Develop Mindfulness-of-Breathing to Absorption KAMMATTHANA業處MEDITATION SUBJECTASSASA-PASSASA入出息BREATH IN AND OUTNIMITTA禪相THE SIGN OF CONCENTRATION四種能使氣平息的因1即：硬、粗、流動、支持、推動等。
2無常(anicca)、苦(dukkha)、無我(anatta)的性質。
禪相的現象APPEARANCE OF THE SIGNSSAMADHI禪定CONCENTRATIONPABCAPACCAYO平衡五根BALANCING THE FIVE CONTROLLINGBHAVANA兩種禪修法JHANA禪那ABSORPTIONSATTA BOJJAðGA平衡七覺支PABCA JHANAðGA五禪支PABCA VASI-BHAVA五自在其他OTHERS回目錄第二章：其它修定的法門Chapter 2: Develop Absorption on Other Subjects三十二身分涅槃的三門THE THREE ENTRANCES TO NIBBANAATTHIKA白骨觀THE SKELETON MEDITATIONCATU ARæPA-JHANA四無色禪回目錄第三章：四梵住與四護衛禪Chapter 3: Develop the Sublime Abidings and Proctective-MeditationsCATUBRAHMAVIHARA四梵住METTA BHAVANA慈心觀LOVINGKINDNESS不應當對兩類人修慈心觀LOVINGKIDNESS SHOULD NOT BE DEVELOPED TOWARDS TWO TYPES OF應當對四類人修慈心觀最初的時候不應當對四類人修慈心觀IN THE VERY BEGINNING YOU SHOULD NOT DEVELOPED LOVINGKIDNESS對自己培育慈愛的四種意念THE FOUR TYPES OF THOUGHTS FOR DEVELOPING LOVINGKIDNESS對敬愛的人散發慈愛SIMASAMBHEDATHE BREAKING DOWN OF BOUNDARIES廿二類遍滿THE TWENTY-TWO CATEGORIES OF PERVASION五類不限定的遍滿FIVE CATEGORIES OF UNSPECIFIED PERVASION七類限定的遍滿3你會發現對敬愛者與對親愛者的慈心變成相等，你可以將他們合為一類。
SAT写作名言警句汇总今天文都国际小编给大家带来的是SAT写作名言警句，学会运用这些句子，可以让同学们的作文在考试中锦上添花，博得高分。
成功幸福根源类Genius is one percent inspiration and ninety-nine percent perspiration。
——Thomas EdisonFew things are impossible to diligence and skill。
——Samuel Johnson Success is the sum of small efforts， repeated day in and day out。
——Robert Collier American writerI’m a great believer in luck， and I find the harder I work， the more I have of it。
——Thomas Jefferson the 3rd American president Luck is what happens when preparation meets opportunity。
Luck is not chance。
It’s toil。
Fortune’s expensive smile is earned。
——Emily Dickinson挑战权威类Dare and the world always yields。
If it beats you sometimes， dare it again and again and it will succumb。
——W.M Thackeray Imagination is more important than knowledge。
Knowledge is limited。
Imagination encircles the world。
奥格·曼狄诺名言名句
奥格·曼狄诺（OgMandino）是美国著名的励志作家、销售培训师，他的作品深受读者喜爱。
以下是一些奥格·曼狄诺的名言名句：
1.今天虽不是我全部的人生，但今天一定会影响我余下的人生。
2.生命的最后一句话是：我想起我一生中那些错过的机会，那些失败的尝试，以及那些因害怕而错失的可能性。
3.每一个早晨，当你睁开眼睛，都是新的一天，你可以选择感恩或抱怨，你的选择决定了这一天的品质。
4.生命并不是等待风暴过去，而是学会在风雨中跳舞。
5.不管多少次失败，都要继续尝试，因为下一次尝试可能就是成功的关键。
6.成功者坚持到最后，失败者则在一半放弃。
7.贵在坚持，只要坚持，就一定能够成功。
8.成功并不在于你拥有什么，而在于你成为了什么样的人。
9.你的过去不决定你的未来，你的行动才决定你的未来。
10.人生最大的成功，是在你完全做到自己最好的时候，对自己的良心还能够宁静安然。
1/ 1。
Always remember that when a man goes out of the room, he leaves everything in it behind. (Alice Munro, Canadian writer) 永远记住，当一个男人从房间里走出来时，他已经将一切回忆甩在身后——艾丽斯·芒罗(加拿大作家)Because if you let go of your grief even for a minute it would only hit you harder when you bumped into it again. (Alice Munro, Canadian writer) 只要你试图放下自己的忧伤、哪怕仅仅一分钟，就会在下一次遭遇忧伤时被打击的更狠。
——艾丽斯·芒罗(加拿大作家)You can't know the limit beforehand, but you will know when you've reached it. I believe this. (Alice Munro, Canadian writer) 你不能预知这个界限在何处，但我相信，当你达到自己所能承受的极限时，你就会知道了。
——艾丽斯·芒罗(加拿大作家)The limit you will put up with, for love, is just as the same as the limit to the amount of mess you can stand around a house. (Alice Munro, Canadian writer) 这就如同你对脏乱的房间有一个忍受限度一样。
——艾丽斯·芒罗(加拿大作家)There is a limit to the amount of misery and disarray you will put up with, for love, just as there is a limit to the amount of mess you can stand around a house. (Alice Munro, Canadian writer) 对于爱情，你所能承受的痛苦和混乱一定有一个限度。
determination翻译基本解释●determination：决心，坚定，确定●/dɪˌtɜːr.mɪˈneɪ.ʃən/●n. 决心，坚定●n. 确定，测定变化形式●n. 复数形式：determinations具体用法●●n. 决心，坚定o同义词：resolve, resolution, perseverance, tenacity, steadfastness o反义词：indecision, hesitation, doubt, uncertainty, waveringo例句：●Her determination to succeed in her career was evident toeveryone who knew her. 她在事业上取得成功的决心对所有认识她的人来说都是显而易见的。
●Despite the challenges, his determination never wavered, andhe continued to pursue his dreams. 尽管面临挑战，他的决心从未动摇，并继续追求他的梦想。
●The team's determination to win the championship wasfueled by their previous losses. 团队赢得冠军的决心是由他们之前的失败激发的。
●With determination and hard work, she managed toovercome all the obstacles in her path. 凭借决心和努力，她设法克服了道路上的所有障碍。
●His determination to learn a new language impressed histeachers and classmates alike. 他学习新语言的决心给他的老师和同学留下了深刻的印象。
密涅瓦黑格尔原句-概述说明以及解释1.引言1.1 概述密涅瓦黑格尔原句是哲学家黑格尔（Georg Wilhelm Friedrich Hegel）在其著作《逻辑学导论》中提出的重要概念。
密涅瓦黑格尔原句一词源自希腊语，意为“原初形成的命题”。
在哲学领域中，密涅瓦黑格尔原句被认为是思维的起点，是任何哲学推理的基础。
本文将探讨密涅瓦黑格尔原句的背景、含义以及对现代哲学和思维方式的影响，旨在加深对这一概念的理解，并探讨其在当今世界的意义和价值。
1.2 文章结构文章结构部分主要是对整篇文章的布局和框架进行说明，帮助读者更好地理解文章的组织结构。
在这里我们可以介绍文章的章节划分以及各个章节内容的概括。
在这篇长文中，文章结构部分的内容可以包括以下几个方面：1. 引言部分：介绍文章的主题和背景，引出密涅瓦黑格尔原句的重要性和影响。
2. 正文部分：分为密涅瓦黑格尔原句的背景、含义和影响三个小节，分别详细探讨这些内容，从不同角度展现密涅瓦黑格尔原句的意义和作用。
3. 结论部分：总结全文的主要观点，强调密涅瓦黑格尔原句的重要性，并对其未来发展进行展望和思考。
在文章结构部分，可以简要介绍每个章节的内容和重点，让读者能够明确整篇文章的框架，有助于他们更好地理解和消化文章的内容。
1.3 目的撰写本文的目的是通过深入探讨密涅瓦黑格尔原句的背景、含义和影响，来揭示这一哲学原理在哲学史上的重要性和影响。
通过对密涅瓦黑格尔原句的分析，我们可以更好地理解黑格尔哲学体系中的关键概念和思想，以及它对后世哲学和社会思潮的影响。
同时，本文也旨在引发读者对密涅瓦黑格尔原句的思考，并展望其在未来的研究和发展方向，为哲学界的进步做出贡献。
通过全面剖析和探讨密涅瓦黑格尔原句，我们可以更好地认识和理解这一重要的哲学原理，并探讨其在当今世界的意义和价值。
2.正文2.1 密涅瓦黑格尔原句的背景密涅瓦黑格尔原句，又称哲学原句，是由德国哲学家黑格尔和法国哲学家密涅瓦共同提出的概念。
Environ Fluid Mech(2009)9:549–567DOI10.1007/s10652-009-9149-0ORIGINAL ARTICLEDetermination of the Manning roughness coefficientinfluenced by vegetation in the river Aa and Biebrza riverL.De Doncker·P.Troch·R.Verhoeven·K.Bal·P.Meire·J.QuintelierReceived:11August2009/Accepted:21August2009/Published online:4September2009©Springer Science+Business Media B.V.2009Abstract The aim of this study was to investigate the variation of channel bed roughness in two rivers,as important parameter in hydraulic modelling especially with regard toflood control.The universities of Ghent(UG)and Antwerp(UA)are conducting scientific research in the river Aa in Belgium and the Biebrza river in Poland in order to better understand the phenomena involved and to come to a more accurate determination of the different parame-ters influencingflow.In this paper,the determination of the roughness coefficient‘n’from the Manning equation is used.This coefficient is not easy to determine and is varying con-stantly.It is influenced by the meandering character of the river,the bed material and the average grain size,the channel bed forms,the channel obstructions,the geometry changes between sections and the vegetation in the channel.Furthermore,due to these parameters, the roughness of the channel is not equally distributed over the channel,the banks and the floodplains.So,using literature data does not always lead to satisfactory results,due to the different situation in thefield(Werner et al.J Hydrol314:139–157,2005).Therefore,mea-surements are necessary to determine the variation of the Manning coefficient.The Manning coefficient is a function of the discharge,but will also vary over the time due to the mentioned influences.In a multidisciplinary research project on the fundamental exchange processes in river ecosystems,hydraulic measurements were performed on a regular base in the river L.De Doncker(B)·P.Troch·R.VerhoevenHydraulics Laboratory,Department of Civil Engineering,Ghent University,Sint-Pietersnieuwstraat41, Ghent9000,Belgiume-mail:liesbet.dedoncker@ugent.beK.Bal·P.MeireEcosystem Management Research Group,University of Antwerp,Universiteitsplein1,Antwerp2610, BelgiumJ.QuintelierLaboratory Soete,Department of Mechanical Construction and Production,Ghent University,Sint-Pietersnieuwstraat41,Ghent9000,BelgiumJ.QuintelierDepartment of Mechanics,University College Ghent,Schoonmeerstraat52,Ghent9000,BelgiumAa.During these measurement campaigns,velocity and discharge measurements were car-ried out in multiple cross-sections.Once a month,the discharge and the water levels were measured at the upstream and the downstream end of the test stretch.On the river Biebrza, similar intensive measurement campaigns took place along a6km stretch in the upstream part of the river.An accurate determination of the Manning coefficient according a seasonal variation is an important tool in hydraulic modelling.Keywords Ecohydraulics·Environmental engineering·Hydraulic measurements·Manning roughness coefficient·Vegetated rivers1IntroductionKnowledge of the movement of water is essential as waterflow is the driving factor for all other water related processes.Measurements of discharge and water level depict the actual situation,but hydraulic modelling allows to evaluate parameter influence and future pros-pects.Hydraulic modelling is of big concern with respect to water resources management, strategic planning and crises management.A well calibrated hydraulic model provides reliable information on different aspects of the real situation.The numerical model is used to simulate the actual behaviour of the river, but it is also an important tool to produce a forecast on possiblefloodings,peak discharges, low water levels,etc.For that,a representative model,with determination of the topography of the river and river banks and registration of the discharges and water levels,is necessary.A parameter of main importance is the roughness of the river channel and the banks[9].Here, the Manning coefficient is used as roughness parameter.The channel roughness is affected by a lot of factors which are difficult to translate into a single value.Green[13]stated that aquatic macrophytes are often the dominant factor influencingflow conditions within the channels they occupy.Furthermore,it is more dif-ficult to determine the Manning coefficient for vegetated streams than for open channel flow[4].Also Watson[25]showed that seasonal variations in the aquatic vegetation have an important influence on theflow resistance.It is possible to estimate the value,but the deviation from reality may be large.Therefore,inflood analysis,a conservative evalua-tion is advisable.The scope of formulas,experiences,tables,pictures or other techniques to estimate the friction factor is wide and therefore,a case specific analysis is interesting [12].Lowland rivers often contain extensive stands of aquatic macrophytes which can increase channel resistance and water stage,while reducing averageflow velocities[13]. Flow resistance of vegetated channels can typically be an order of magnitude greater than that of unvegetated channels[7,8].So,macrophytes can contribute to localflood-ing,and their removement is part of the water management strategy.Mowing of the grasses in the river channel and the removal of the aquatic vegetation is another impor-tant water management related topic[2].Study of the influence of different mowing patterns is done in[24],but is not seen in thefield.On the one hand,the presence of vegetation in the river leads to a wealthy ecosystem with habitats for differentfish and other species.On the other hand,theflooding risk is big,especially during the growing season.Presence of vegetation in water courses has a lot of advantages from ecological point of view,so the mowed area has to be limited[22].Next to a low per-centage of mowing,also a the avoidance of specific trajectories of the water is eco-logically important[22].This can be obtained by mowing following a‘chessboard’pattern which causes an important structural variation.As a result,erosion creates areas with high velocities next to areas with stationary water where sedimentation can take place.A classification of the mowing patterns is presented by the Flanders Hydraulics Lab[24].A mowing pattern with a low roughness(Manning coefficient)has to be cho-sen to limit the backwater effect.Out of these patterns,the most ecologically friendly one is retained.According the environment,more or less attention is paid to a good drainage(living areas)or to an ecological development(green areas)[2].Besides the mowing pattern,also the frequency of mowing,the time and the mowing method is important for the maintenance of the water course and for guaranteeing its ecological quality.In afirst phase,the importance of the roughness coefficient in two monitored river stretches is investigated,using the Manning equation and hydraulic modelling of surface water.The one-dimensional numerical model HEC-RAS[16]has been selected for the modelling.Field measurements have been carried out andflow characteristics have been collected.Based on these data,the Manning coefficient is calculated as a function of time,distance and water level.Further,this coefficient is also determined for studying the effect of vegetation on the drainage of the river.2Study area’s:the Biebrza river and the river AaTwo study areas are compared:the river Aa and the Biebrza river.Both of them are lowland rivers and highly influenced by vegetation growth in different periods over the year.They are both subject from extensive measurement campaigns from the past up till now.While the river Aa is situated in an urbanized region,the Bierbza river and its wetlands is part of an undisturbed peatland.2.1The river AaIn Belgium,focus of the study is the downstream part of the river Aa.The catchment basin of the river Aa is situated in the region of Antwerp in Belgium and is hydrographically part of the Nete basin.The most important rivers of this basin are the Kleine Nete and the Grote Nete, both influenced by the tidal bore.More than40%of the water in the Nete basin is carried by the river Aa,which is consequently the most important tributary of the Kleine Nete.The river Aaflows into the Kleine Nete near the city of Grobbendonk.The well of the river Aa is found in the northern part of the Kempic Plateau near the communities of Merksplas and Turnhout.It has a total length of36.8km and a drainage area of about23,700ha.The study area is focused on the downstream part of the Aa,near the village of Poederlee(Fig.1),on a 1.4km reach controlled by two weirs at the up-and downstream end.The river Aa is a typical lowland river with low velocities,a small fall and a sinusoidal charactar.Over the years,the river has been straightened and the section was enlarged.The water inflow originates from drainage of rain water and seepage of ground water.The water is rather acid,without chalk and a low amount of minerals,not suitable for organisms.Living conditions for them are caused by food supply in the way of organic drainage of fertilizers from thefields of agriculture along the banks of the river.The subsoil[11]is predominantly formed by coarse sand.One million year old tertiary sand(as the Formations of Diest,Kasterlee and Berchem)is covered by10,000years old quartaire sand.Under these permeable sandy soils,a less permeable clay layer,the Boomse Klei,is present[1].Fig.1Study area of the river Aa,Poederlee,province of Antwerp,Belgium2.2The river BiebrzaThe Biebrza River and surrounding wetlands are situated in the northeastern part of Poland, in an ice-marginal valley,an area of195,000ha.This region forms the last extensive,fairly undisturbed river-marginal peatland in Europe,containing endangered plant and animal spe-cies in a large variety of ecosystems.There are three major basins[27],respectively identified as the Upper Basin reaching from the springs of Biebrza to the mouth of the Netta River;the Middle Basin covering the area from Netta to the mouth of Rudzki Channel and the Lower Basin situated in the southern part of the valley up to the alluvial cone of the recipient Narew River.The Biebrza river,as a whole,is a typical lowland river.It has mild slopes(in average about10cm/km)and a strong meandering character.It features varying cross-sections and an irregular longitudinal profile.The variability of the hydrological characteristics along the river is also typical.This is due to the fact that the valley intensively drains the surrounding plateau and the outwash plain into the river.The surface water system is quite complicated consisting of a complex drainage system,network of inundationfields and storage areas.The flow is highly influenced by the dense vegetation[23].3Methodology3.1Hydraulic measurementsDischarge measurements are carried out from a bridge and from a boat in different sections along the stretch of both the river Aa and Biebrza.The method used is the integration ofthe velocityfield over the cross section as is explained in[14].Velocities were measured at different depths on different verticals along each section.Two systems are used for measuring the velocity of the water.In case of open water(no vegetation),hydrometric propellers(Type:OTT,C31Universal Current Meter)are used.In locations where vegetation might hinder the mechanical functioning of the propeller an elec-tromagnetic instrument(Type OTT,Nautilus C2000/SENSA Z300and Valeport,Type801)is applied.The latter instrument has no moving parts which can interfere with the macrophytes. The device is wear-resistant and maintenance free.The accuracy of these electromagnetic devices is up to0.5%[21]of the velocity while for the propellers the accuracy is1%[19].Furthermore,deviations are larger for the lower range of velocities.Theflow characteristics and the precision of measuring water depths also influence the accuracy.Measurements over the year show an overall accuracy of2–5% for the determination of the discharge.Accuracy calculation of the discharge is based on the measurement error on the velocity and the wetted cross section area,as is described in[10].3.1.1The river Aa,BelgiumSeveral measurement campaigns have been organised to collect hydraulic data(water lev-els and discharges).The discharge of the river Aa was also measured monthly upstream and downstream the selected stretch since September2004.Water levels at the weirs on both sides of the stretch are registered continuously by the Hydrological Information Centre(HIC)of Flanders Hydraulics Research.Water levels are registered making use of levelled staff gauges (accuracy0.5cm).In this study,the Manning coefficient for the river Aa is calculated according to three different approaches,in the following mentioned as‘Approach1’,‘Approach2’and ‘Approach3’.‘Approach1’calculates the roughness coefficient directly using Manning’s equation.Second way(Approach2)of calculating the Manning coefficient is by using the numerical model HEC-RAS solving the complete Saint-Venant equations(in steady state conditions known as the Bresse equation).Fitting allows to determine the rough-ness value.‘Approach3’is similar to‘Approach2’but is using a simplified geome-try.3.1.2The river Biebrza,PolandOn the river Biebrza,several measurement campaigns were carried out in the period April–June from1999up to2006.Spring is the period with a wealthy plant growth,so this period is the most interesting from both,ecological and hydraulical point of view.In Fig.2the schematic plan of the Biebrza river shows the locations where measurements are carried out.A substantial contribution of the tributaries to the total discharge in the river and the importance of the groundwater inflow between the tributaries becomes clear.3.2Biomass determinationIn the river Aa,macrophytes found are various leaved water-starwort(Callitriche platycarpa), rigid hornwort(Ceratohyllm demersum)andfloatingleaf pondweed(Potamogeton natans). Sampling is carried out upstream,downstream and half way the stretch.In each of theseFig.2Study area of the Biebrzariver,Poland and schematic view,with indication of the measuringpointscross-sections,the amount of macrophytes is determined each meter,at the same position were the discharge measurements are carried out.First the fresh weight(g/m2)of the plants is defined,afterward,also the dry weight(g/m2)is postulated.For the Bierbza river,the same procedure is followed,but plants occuring are sparganium (Sparganium),floatingleaf pondweed(Potamogeton natans),reed canarygrass(Phalaris a-rindinacea)and yellow pondlily(Nuphar luteum).The sampling of the macrphytes is carried out with a sampler as described in[18].This instrument has no moving parts,its primary components are a cutting bladefixed to the base of a vertical shaft to shear off plant stems at the substrate surface,and a collection rake to allow retrieval of the freed vegetation.The sampler is well suited for the measurements in this study,because a large variety of macro-phytes over a range of conditions can be sampled,while possessing the design features of prime importance:lightweight,low cost and easily used.3.3Quality of the measurements3.3.1The river AaIt was necessary to check the sensitivity of the results to measurement errors,there small variation on the Manning coefficient has major implications on its use in hydraulic modelling [9].Therefore,a comparative study of the variation of the Manning coefficient,based on thedischarges;measured by UG,calculated out of the calibrationformula and calculated by HICmeasurements of Flanders Hydraulics Research,Hydrological Information Centre(HIC)and the measurements of UG is carried out.Furthermore,the discharge measurements of UG are compared to the values of HIC and to the values calculated out of the calibration formula of the downstream weir.Therefore,the water height over theflap of the weir is measured. Results for that are in the same range of the measured and registered values.For the com-parison,river characteristics(as the wetted cross section and the river width)are used as measured by UG.The Hydrological Information Centre(HIC)of Flanders Hydraulics Research has regis-tered the water levels and the discharge in the studied stretch during2004and2005.The Manning coefficient was calculated using the HEC-RAS model as this numerical approach gave the best results.For each of the monthly measured values,the upstream and downstream discharge is determined.There is only seen a small difference between both values(Fig.3), due to groundwater inflow/outflow,to little tributary inflow between the sections and to inac-curacy of the measurements.Besides,also the discharge based on the calibration formula of the downstream weir,is calculated.In general,both values are in good agreement.The small deviations that sometimes occur can be explained by vegetation patches disturbing the free flow over the weir.It should be mentioned that the values of HIC are estimated starting from the measure-ments in two neighbouring stations and taking into account the surface of the corresponding catchment areas.Due to this approximative determination of the discharges,differences can be remarked.Three different curves,used for the calculation of the Manning coefficient,are presented in Fig.4.First,the Manning coefficient calculated out of the measured values upstream of UG(UG measured),second,based on the downstream measured values of UG,third,the Manning coefficient based on the HIC-values registered at the same moment of the mea-surements of UG(HIC measured).All these values are calculated by the numerical model Hec-Ras.The Manning coefficient is increasing from May until full summer,reaches the highest values in July and is decreasing from October–November on.This trend accompanies the vegetation growth in spring and summer.Maximum values of the Manning coefficient in 2005are somewhat larger than0.4(UG)and0.5(HIC).As can be seen,the deviations of the Manning coefficient based on the datasets of UG are rather small,result of good registration and ing the discharges estimated byManning coefficients;calculations based on themeasurements of UG and theregistrations of HICHIC,the differences become bigger.The biggest deviations can be remarked from July2005 to October2005,probably due to the mentioned reason:difference in the discharges based on the estimations of HIC.This comparison illustrates the importance of accurate information on hydraulic data with regard to the determination of the Manning coefficient as a key-value in numerical modelling.It is clearly shown that presence of vegetation adds an extra roughness to the channel and influences the water levels.As there is a big variation in the amount of vegetation over the year,the roughness coefficient will change substantionaly.3.3.2The river BiebrzaSeveral intensivefield-measuring campaigns have been developed in different seasons.In all these locations(3.1.2)discharges were ually,the measurements were con-ducted twice in order to increase the measurement accuracy.The local water surface has been recorded in multiple,discrete cross-sections along the river leading to a fairly accurate recognition of the longitudinal water surface profile.3.4Determination of the Manning coefficientThe measured discharges and water levels are used for the calculation of the roughness coefficient of the stretch,making use of the Bresse equation and the Manning equation.In general,hydraulic models for open channelflow are based on the Saint-Venant equations [5].These equations(continuity equation and momentum equation)are the one dimensional simplification of the Navier Stokes equations,which describefluidflow in three dimensions. By calculation of the discharge and the water levels,the Saint-Venant equations allow for the calibration of the roughness of the channel(expressed by the roughness coefficient or fric-tion factor)by comparing withfield data.Here,this roughness is represented by the Manning coefficient n and is calculated from the energy slope.For steadyflow,the momentum equation is simplified and is known as the Bresse equation (Eq.1).dh ds =S0−S f(1−S20)−Q2Bg A3(1)with Q=discharge(m3/s),A=wetted cross section(m2),B=channel width(m),g=gravity (m/s2),S f=energy slope,S0=bottom slope(−),h(m)=water depth,s(m)=distance along the channel.The Manning coefficient n(m−1/3s)is easily linked to the Bresse equation(Eq.1)and the expression for the energy slope S f(Eq.2)by the roughness coefficient of Darcy-Weisbach f [−](Eq.3):S f=f P Q28g A3(2)f[−]=8gn2R1/3(3)with P=wetted perimeter(m),R=hydraulic radius(m)and n=Manning coefficient(m−1/3s).In steady state conditions and assuming uniformflow,the energy slope is equal to the bottom slope(S0=S f)and discharge,water levels and Manning coefficient are linked directly by Manning’s equation(Eq.4)[5].The roughness coefficient is determined out of the measurements of discharge and water levels by(Eq.4).Further,channelflow is connected with the hydraulic and geometric characteristics of the channel.Substituting Eq.3in Eq.2yields:n[m−1/3s]=S1/2f A5/3Q P(4)i.e.the Manning equation.Hydraulic data as water levels and discharges are necessary,but also topographical data of the river bed and banks has to be collected.While carrying out velocity measurements in the river,the water depth and consequently,the bottom profile is registered.However,this is not sufficient to collect a useful topographical data set.Therefore,the study area of the river Aa is monitored in more detail.The stretch covers1.4km and every50m,a section is surveyed.So a set of30sections is available containing detailed information on the different cross sections and the bottom slope of the river.The cross sections are irregular due to the meandering aspect of the river.The average bottom slope(from upstream to downstream)is 0.0002m/m.In the Biebrza river,measurements have been carried out each year in spring,to measure the influence of the present vegetation.The campaigns started in1999and are still going on. Specific measurement points on the river are selected as is indicated on Fig.2.Furthermore, in a specific stretch,the variation of the Manning coefficient over the distance is measured.3.5Roughness coefficient according different methodsTheoretical calculation of the Manning coefficient is difficult.The Manning formula is most used for expressing resistance[4].It might be determined by empirical formula,e.g.[6] who splitted channel resistance into several parts,including the bed material,presence of vegetation in the river,meandering,etc.n=(n0+n1+n2+n3+n4)m(5) wheren0=basic value,for a straight,uniform channel,n1=irregularities of the bottom,n2=variations in the geometry of the channel,n3=obstacles,n4=vegetation,m=correction factor for meanderingImportant is to take into account not to double conditions that are already included in another parameter.The equation requires an estimate of separate n factors for different chan-nel conditions.However,the large variability of the coefficients involved leaves too many degrees of freedom to allow an accurate determination of the roughness coefficient.Another method-ology is to use a set of pictures from literature which represent a comparable situation or to make use of graphs and tables[12].One canfind photographs and descriptive data of typical rivers for which the Manning coefficient is determined in[3],for United States riv-ers,and[15],for New Zealand rivers.Their main advantage derives from a wider range of bank vegetation types and density and multiple roughness values for each river calcu-lated at different ing the books asks for a lot of experience in thefield and looking for comparable rivers in the Flanders region as in the United States or New Zea-land.As this research started from scratch,measurements over the year were carried out to have an idea of the roughness coefficient of the river.Next to these mentioned meth-ods,also formulas to determine the roughness coefficient based on the bed material are published.[17]related the Manning coefficient to hydraulic radius and particle size on the basis of samples from11stream channels having bed material ranging from small gravel to medium-sized boulders.Analogous research,based on the characteristic bed material, is carried out by[20].Using Cowan’s formula for the river Aa leads to values from0.04 m−1/3s for winter situations(low amount of vegetation)to0.15m−1/3s for summer situa-tions(high amount of vegetation).Using the tables with Manning values mentioned in[4], leads to the same range.The calculated Manning coefficient,based on measurements of water level and velocity confirms these values for autumn and spring situations,but dur-ing summer,Manning coefficients are much higher and can reach values up0.4and even 0.5m−1/3s.Therefore,the Manning coefficient is considered as the result of the Bresse equation.Channel roughness is influenced by grain size of the bed material,the surface irregularities of the channel,the channel bed forms(such as ripples and dunes),erosion and deposition char-acteristics,meandering tendencies,channel obstructions(downed trees,exposed root wads, debris,etc.),geometry changes between channel sections,vegetation along the bankline and in the channel,etc.[12].One single value of the roughness coefficient has to include all these parameters(cf.Bresse equation,where the roughness is presented as f).Furthermore,as vegetation is strongly depen-dent on the season,the roughness coefficient can be fairly different for summer and winter conditions.As different approaches show great variability of n,they do not guarantee accurate values for the roughness coefficient and a determination of the roughness starting from measure-ments is recommended[26].Therefore,some assumptions are made:first,the bottom slope and the cross sections are supposed to be stable,so sediment transport and transformation of the channel bed are not included.Further,the friction factor or roughness coefficient (expressed by the Manning coefficient n)is set as a constant value in the channel and at the banks.Theflow is regarded in a1-dimensional way,with uniform velocity over the cross section.As mentioned in Sect.3.4,the Manning coefficient n has been calculated in two ways:firstly,when uniformflow is supposed,the energy slope is equal to the bottom slope and n is obtained from the Manning equation.Secondly,when the Bresse equation is used,steady stateflow is assumed and the roughness coefficient n is calculated from the energy slope.Due to the wide range of factors influencing the Manning roughness coefficient,accurate measurement is difficult and very extensive,therefore,a well established model allows to compute Manning’s coefficient for any discharge on any location.The data from several measurement campaigns allow to determine the variation of the roughness coefficient[friction factor or Manning coefficient n(m−1/3s)]as a function of time,type and density of vegetation,hydraulic parameters,etc.The importance of the use of a correct Manning coefficient for the river is clearly illustrated by this example,representing the variation of aflood wave along a25km long trapezoidal shaped river stretch,increasing the roughness coefficient by a factor2,causes a large reduc-tion of the discharge.While a Manning coefficient of0.02m−1/3s reduces the peak discharge of the inflow hydrogram after12km with17%,a Manning coefficient of0.04m−1/3s reduces the value with23%.So,accurate knowledge of the Manning coefficient is necessary to guar-antee reliable predictions.4Results and discussion4.1Manning coefficient as a function of timeThe variation of the Manning coefficient as a function of time has been investigated in the river Aa.The three calculation methods(cf.Sect.3.1.1)for the Manning coefficient are applied and calculations are carried out separately for both the discharges measured up-and downstream.For each of the monthly measurements,the Manning coefficient is plotted in Fig.5.The different calculation methods are indicated with a different ing the same approach, the Manning values based on both the up and downstream discharge values are very similar.For the measurements of November2005,January and February2006,differences in the Manning coefficient are negligible.The scatter is larger for the measurements of September, May and June,which is probably due to the larger amount of vegetation.During winter,the presence of vegetation remains rather small and constant,while during spring and summer, the Manning coefficient increases rapidly with the amount of vegetation.Fig.5Manning coefficient n forthe upstream(Qu)anddownstream(Qd)value of Q:comparison of the valuescalculated using threeapproaches:Manning’s equationgiven by Eq.2(1),fitting basedon the Saint-Venant(Bresse)equations(HEC-RAS)(2),andfitting using an averagesection(3)。
Pursuing dreams is a journey filled with passion,determination,and an unwavering belief in oneself.It is about setting a course towards the horizon and not letting anything stand in the way.Here is an essay that captures the essence of chasing dreams with abandon and the freedom of spirit that comes with it.The Unbridled Pursuit of DreamsIn the vast expanse of life,there lies a path less traveled,a path that leads to the heart of our aspirations.This is the path of the dreamer,the one who dares to chase their dreams with an unbridled spirit.It is a journey that requires courage,resilience,and an unyielding faith in the power of ones own potential.The Spark of AmbitionThe pursuit begins with a spark,a moment of clarity where the heart whispers its deepest desires.It is the first step on a winding road,paved with the fragments of our imagination and the raw materials of our will.This spark is the catalyst that ignites the fire of ambition,compelling us to reach for the stars.The Wings of PassionPassion is the wind beneath our wings,propelling us forward with a force that is both exhilarating and terrifying.It is the fuel that keeps the engine of our dreams running, even when the path is steep and the skies are dark.Passion is the unwavering commitment to our goals,the relentless drive that pushes us to soar higher and reach further.The Flight of DeterminationDetermination is the compass that guides us through the storms of doubt and the valleys of despair.It is the steadfast resolve that keeps us on course when the winds of adversity threaten to knock us off our flight path.With determination as our guide,we navigate the treacherous terrain of life,confident in our ability to overcome any obstacle.The Soaring SpiritAs we chase our dreams,our spirit soars.It transcends the limitations of the physical world,reaching heights that were once unimaginable.The spirit that dares to dream is aforce to be reckoned with,a beacon of hope that shines brightly in the darkest of times.It is the essence of our being,the very core of our existence.The Landing of FulfillmentThe journey of a dreamer is not without its challenges,but the rewards are immeasurable. To land on the shores of fulfillment is to experience a sense of joy and accomplishment that is unparalleled.It is the culmination of a journey that has tested our limits and revealed our strengths.It is the moment when the dreamer can look back and see the path they have traveled,a path marked by the footprints of perseverance and the fingerprints of destiny.The Legacy of the DreamerThe legacy of the dreamer is not one of material wealth or fame,but of inspiration and influence.It is the story of a life lived with purpose,a life that has touched the hearts of others and ignited the flames of their own dreams.The dreamer leaves behind a trail of inspiration,a testament to the power of the human spirit and the boundless potential that lies within each of us.In conclusion,the pursuit of dreams is a flight of the soul,a journey that takes us to the very edges of our capabilities.It is a dance with the unknown,a leap of faith that requires us to let go of our fears and embrace the possibilities that await us.As we chase our dreams with an unbridled spirit,we discover not only the heights we can reach but also the depths of our own strength and resilience.。
Journal of Chromatography B,938 (2013) 8–13Contents lists available at ScienceDirectJournal of ChromatographyBj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c h r o mbDetermination of amantadine and rimantadine in chicken muscle by QuEChERS pretreatment method and UHPLC coupled with LTQ Orbitrap mass spectrometryHua Yan a ,Xin Liu a ,Fengyun Cui a ,Huan Yun a ,Jianhui Li a ,Shuangyang Ding b ,Dajin Yang c ,Zhaohui Zhang a ,∗aFood Laboratory of Beijing Inspection and Quarantine Testing Center,Beijing Entry-Exit Inspection and Quarantine Bureau,Beijing 100026,China bCollege of Veterinary Medicine,Chinese Agricultural University,Beijing 100094,China cChina National Center for Food Safety Risk Assessment,Beijing 100021,Chinaa r t i c l ei n f oArticle history:Received 17May 2013Accepted 13August 2013Available online 27 August 2013Keywords:Amantadine RimantadineQuEChERS method UHPLCLTQ Orbitrap MSChicken muscle tissuesa b s t r a c tA novel sample pretreatment method was developed for the quantitative determination of amantadine and rimantadine in chicken muscle tissues by ultra high performance liquid chromatography coupled with high resolution LTQ Orbitrap mass spectrometry (UHPLC-LTQ Orbitrap MS).The samples were pre-treated by modified QuEChERS (Quick,Easy,Cheap,Effective,Rugged and Safe)method,using acetonitrile (1%acetic acid,v/v)as extraction solution and C 18sorbent for clean-up.The separation was carried out on a Waters ACQUITY UPLC HSS T3column (150mm ×2.1mm,1.8m particle),using a mobile phase of acetonitrile and 0.1%aqueous formic acid solution.LTQ Orbitrap MS with resolving power of 60000was applied for analysis of the samples.Amantadine and rimantadine were identified from their accu-rate mass (within 5ppm)and retention times from the acquired full-scan chromatogram and quantified by their peak areas.The linear range for the determination of the analytes was 1–100g/kg.Limits of detection (LODs)for amantadine and rimantadine were 1.02g/kg and 0.67g/kg,respectively.The intra-and inter-day accuracy ranged from 87.5%to 102.4%,and 82.5%to 105.8%for amantadine,and 95.3%to 97.4%,and 89.4%to 93.2%for rimantadine,respectively.The precision of intra-and inter-day was between 3.9–6.3%and 5.95–13.9%for amantadine,6.0–7.45%and 7.8–12.4%for rimantadine,respec-tively.Finally,the method was applied for the determination of these antiviral agents in routine samples,and amantadine residue was detected in some cases.© 2013 Elsevier B.V. All rights reserved.1.IntroductionAmantadine and rimantadine are antiviral agents used for the treatments of influenza A virus infections [1,2].Due to the potential resistance to these compounds for human beings [3],amantadine and rimantadine have been banned as antiviral agents during poul-try farming in many countries including USA [4]and China since 2005.Nevertheless,these antiviral drugs are still illegally used for the treatment of avian influenza in poultry farming (especially in chicken farming)in China occasionally [5].Therefore,it is necessary to establish a sensitive method for the analysis of these antiviral agents in chicken muscle tissues.Up to now,different methods have been published for the determination of amantadine and rimantadine in biological flu-ids samples such as plasma and urine [6–15],including gas∗Corresponding author.Tel.:+861058619227.E-mail address:zhangzhh@ (Z.Zhang).chromatography with electron-capture detection (GC-ECD)[6],gas chromatography with mass spectrometry (GC–MS)[7],high-performance liquid chromatography with fluorescence detection (HPLC-Flu)[8–12],HPLC with UV detection (HPLC-UV)[13],cap-illary zone electrophoresis [14]and HPLC coupled with mass spectrometry (HPLC-MS)[15].As these two compounds have no prominent UV absorption and fluorescence properties,it is usu-ally required derivatization to increase its sensitivity by HPLC-UV and pared with HPLC-UV and HPLC-Flu,HPLC-MS has the advantage of simple sample treatment without derivati-zation and high sensitivity and selectivity.HPLC coupled to triple quadrupole mass spectrometry (QQQ)is currently widely used in residue analysis.A number of compounds can be analyzed by QQQ under selected reaction monitoring (SRM)mode [16–19].When applying SRM mode,at least two transitions are required because monitoring only one transition might result in false positive identi-fications [20].The application of SRM is limited for some cases when analyte ions with low molecular weight and with only one transi-tion.High resolution mass spectrometry (HRMS)such as TOF MS1570-0232/$–see front matter © 2013 Elsevier B.V. All rights reserved./10.1016/j.jchromb.2013.08.020H.Yan et al./J.Chromatogr.B938 (2013) 8–139and LTQ Orbitrap MS provides higher resolution,higher sensitivity, and higher scan speed than QQQ.For LTQ Orbitrap,excellent selec-tivity could be achieved with resolution up to100000,by which the target analytes could be easily distinguished from interfer-ences of matrix backgrounds[21].More recently,TOF MS and LTQ Orbitrap MS have been increasingly utilized in food analysis[20]. However,there were few reports about determination amantadine and rimantadine in chicken muscle using HRMS.The samples were biologicalfluids in normal such as plasma and urine owing to the most studies was focused on the pharma-cokinetic of amantadine and rimantadine.Liquid–liquid extraction and solid-phase extraction were commonly used for sample pre-treatment in plasma and urine.However,these pretreatment methods are not suitable for chicken muscle tissues.The QuECh-ERS method(acronym for Quick,Easy,Cheap,Effective,Rugged and Safe),is a promising sample preparation method for the deter-mination of residues in different food matrices[22,23].QuEChERS method is based on a single extraction with acetonitrile followed by partitioning with salt and purifying the extract using dispersive solid-phase extraction(d-SPE).d-SPE does not require SPE appara-tus,cartridges,vacuum,preconditioning of column,and many steps in experiment.It is quicker,cheaper,and provides better interaction with the sorbent for cleanup than traditional SPE.The QuEChERS method was originally applied for the analysis of pesticide residues in plant tissues[22,24],and now it has been widely used for the extraction of compounds with a wide polarity range,including both pesticide residues in plant origin food and veterinary drugs residues in animal origin matrices[25],such as bovine milk,liver [26],shrimps[27],fish[17]and chicken muscle tissue[18].It was reported that dozens of animal drug residues including quinolones,fluoroquinolones,sulfonamides,macrolides,anthelmintics,aver-mectins,nitroimidazoles,ionophores and dinitrocarbanilide can be extracted using QuEChERS method.However,as for as we know, this approach has not been applied for the extraction of amantadine and rimantadine in chicken muscle tissues.The purpose of this work is the development of a simple and sensitive method for the determination of amantadine and riman-tadine in chicken muscle tissues.The developed method involves an optimize QuEChERS method in the sample pre-treatment step followed by determination by UHPLC LTQ-Orbitrap MS.2.Experimental2.1.Reagents and materialsAcetonitrile and methanol(HPLC grade)were from Merck (Darmstadt,Germany).Formic acid,acetic acid and ammonium acetate(purity>99%)were from TEDIA(Fairfield,OH,USA). Sodium chloride and anhydrous magnesium sulfate(analytical grade)were supplied by Sinopharm Chemical Reagent Co.Ltd. (Shanghai,China).Sorbents including Cleanert C18(40–60m), Cleanert graphitized carbon black(GCB,120–140mesh),Clean-ert NH2(40–60m),Cleanert Alumina-N(150mesh),Florisil (60–100mesh),and primary-secondary-amine(PSA,40–60m) were obtained from Bonna-Agela Technologies(Tianjin,China). Certified analytical standards of amantadine and rimantadine were obtained from Dr.Ehrenstorfer GmbH(Augsburg,Germany). Individual stock standard solutions(1000g/mL)of amantadine and rimantadine were prepared by dissolving the appropriate amount of each analyte in methanol and were stored in the dark at−20◦C.A1g/mL working standard solution contain-ing both compounds was prepared by appropriate dilution of the stock standard solutions with methanol.Standard solutions for UHPLC LTQ Orbitrap-MS analysis(1–100g/L)were prepared daily from dilution of the1g/mL working standard solution in acetonitrile/0.1%formic acid aqueous solution(3:7,v/v).Ultrapure water was obtained from a Milli-Q Elix system(Millipore,Beaford, MA,USA).2.2.Instrument conditionsThe analysis was performed on an Accela liquid chromato-graphic system(Thermo Scientific,San José,USA)coupled to LTQ-Orbitrap XL MS(Thermo Scientific Bremen,Germany).The data was collected and analyzed by the Thermo Fisher Xcalibur software package(version2.1.0).The UHPLC separations were operated on a Waters ACQUITY UPLC HSS T3column(150mm×2.1mm, 1.8m particle size, Waters,Wexford,Ireland)under gradient conditions with mobile phase of0.1%formic acid aqueous solution(eluent A)and acetoni-trile(eluent B)at aflow rate of0.25mL min−1at30◦C.The initial mobile phase composition was80%of eluent A and20%of eluent B.The content of eluent B was increased by a linear gradient from 20%to50%in6min and increased from50%to90%in0.1min and held for2.5min.The analytical column was then equilibrated at the initial conditions for4min.The total run time for analysis was 12min.The injection volume was10L.LTQ Orbitrap MS equipped with an ESI source was operated in positive ionization mode using the following operating param-eters:electrospray voltage4.5kV,sheath gasflow rate30abu, auxiliary gasflow rate8abu,capillary temperature350◦C,cap-illary voltage47V,tube lens offset130V.Instrument calibration was performed externally prior to each sequence with a calibra-tion solution.Accurate mass spectra of[M+H]+ions were recorded from100to500m/z,the mass resolution power of the mass ana-lyzer was set to60000(m/ m).Nitrogen(99.95%)was used as sheath gas,aux gas and served as the collision gas in the HCD cell and the bath gas in the C-trap.2.3.Samples and sample extraction proceduresChicken breast samples were obtained from local supermarket (Beijing,China)and the samples were homogenized(1000rpm, 2×15s)by a homogenizer(Grindomix GM200,Retsch GmbH)and then stored at−20◦C until analysis.Blank samples were confirmed to be free of targeted analyte residues by UHPLC-LTQ Orbitrap MS after sample preparation by the procedures investigated.Extraction and cleanup were based on the QuEChERS method and modified as follows:3.0g homogenized chicken muscle sam-ples were weighted into50mL polypropylene centrifuge tubes, followed by addition of6mL of H2O,and then vortexed by IKA MS 3basic(IKA,Staufen,Germany)at3000rpm for30s.Subsequently, 10.0mL of1%HAc in acetonitrile was added to each tube and the tube was vortexed for1min.After that,3g MgSO4and1g NaCl was added to each tube and the tube was shaken vigorously for 1min.The sample was centrifuged at7000rpm for5min at15◦C. A6.0mL of the upper acetonitrile layer was transferred into15mL tube which containing150mg sorbent for cleanup.The tube was vortexed for30s and centrifuged at1500rpm for10min at15◦C. An aliquot of4.0mL of the upper layer was transferred into10mL centrifuge tube and evaporated to dryness by nitrogen-evaporator (N-EVAP TM116,Organomation Association Incorporated,South Berlin,USA)at45◦C water bath.Samples were then redissolved with0.8mL of ACN/H2O(0.1%,FA)(7:3,v/v),filtered through 0.22m cellulose acetatefilters and transferred into autosampler vials for analysis.2.4.Method validationsCalibration curves were conducted using mixed working standard solutions by plotting the peak area at concentrations of1,10H.Yan et al./J.Chromatogr.B 938 (2013) 8–132 46 8 1012Time (min)020*********R e l a t i v e A b u n d a n c e3.2215215420406080100R e l a t i v e A b u n d a n c e152.14342153.14662246810Time (min)020*********R e l a t i v e A b u n d a n c e5.3718018220406080100R e l a t i v e A b u n d a n c e180.17464181.17790ABCDNH 2NH 2H 3C m/zm/zFig.1.Extraction of exact ion chromatograms of (A)amantadine and (B)rimantadine and positive ion mass spectra of (C)amantadine ([M+H]+,m /z 152.14336),and (D)rimantadine ([M+H]+,m /z 180.17471).2.5,5,10,25,50,and 100g/L for each compound.Matrix-matched external standard calibration was adopted for quantification.Five spiked standard concentration levels of 1.0,2.5,5.0,10.0,25.0,50.0,and 100g/kg were made for matrix-matched calibration and linearities were evaluated.LODs and LOQs were defined as the signal-to-noise ratio (S /N )of 3and 10,respectively.Recoveries of amantadine and rimantadine were measured in spiked chicken muscle at three concentration levels (5.0,10.0,and 20.0g/kg)based on six replicates.The spiked samples were set-ting for 30min for sufficiently stability.The spiked samples were analyzed,and the recoveries were calculated by comparing the measured concentration to the nominal concentrations.The accu-racy and the precision of the method were described by means of absolute recovery and repeatability (intra-day and inter-day RSDs).The intra-day and inter-day RSDs were measured on the same fortified blank chicken muscle samples (n =6replicates per concentration level)and analyzed within three days.3.Results and discussion3.1.Optimization of UHPLC-LTQ Orbitrap MS conditionsDue to its high resolution power and high sensitivity,LTQ Orbit-rap MS has become increasingly popular in the field of food analysis.It is realized that for the analysis of residues in complex matrix such as animal tissues,the resolving power of a mass spectrome-try is a key parameter affecting the correct assignment of masses for analytes.At lower resolving power settings,the error in the mass assignment increased due to co-elution of analytes with interferences of similar mass.For analysis of residues in animal tis-sues,consistent and reliable mass assignment (<2ppm)and a high resolving power (>50000)was found to be required [21].LTQ Orbit-rap MS provides the special advantage of high resolution power (up to 100000)and it is possible to estimate the accurate mass of the target compounds within ±1millidalton (mDa).In this work,at the optimized conditions of LTQ Orbitrap MS,the protonated molecu-lar ions [M+H]+of amantadine and rimantadine detected were atm /z 152.14342and 180.17464(Fig.1).The accuracy of the system was within 1mDa for the target compounds (Table 1).For better separation of the target compounds,acetonitrile-0.1%aqueous formic acid solution system and methanol-0.1%aqueous formic acid solution system was compared.The gradient elution program described in the “Instruments”section was carried out.Both amantadine and rimantadine were eluted using acetonitrile-0.1%aqueous formic acid solution system and methanol-0.1%aqueous formic acid solution system within 10min.The retention time of amantadine and rimantadine under the mobile phase of acetonitrile-0.1%aqueous formic acid solution system were about 3.2and 5.3min,respectively.As a comparison,the retention time of amantadine and rimantadine under the mobile phase of methanol-0.1%aqueous formic acid solution system were about 7.3and 7.6min,respectively.The mobile phase of acetonitrile-0.1%aque-ous formic acid solution system was selected because the two compounds eluted more efficiently in time,and separated solitarily.3.2.Optimization of sample extraction procedureTypically,the QuEChERS method was developed for the extrac-tion of pesticides from plant matrix.The principle of QuEChERS method briefly involves an initial extraction with organic solvent (the usual solvents were acetone,ethyl acetate and acetonitrile for pesticide residues),liquid–liquid-partitioning after addition of a mixture of MgSO 4and NaCl,followed by dispersive-solid-phase extraction (d-SPE)clean-up step [22].The critical factors including extraction solvent and type of acid were evaluated in this study.First,organic reagents including methanol,acetonitrile,and ethyl acetate were compared.Methanol is discarded because it can-not be salted out after addition of a mixture of MgSO 4and NaCl,and the extracts were very dirty and difficult to be dried by N 2stream.Then the recoveries (spiked level at 15g/kg)of amanta-dine and rimantadine extracted by ethyl acetate and acetonitrile were compared.It was found that the recoveries were very poor (only 8.57%and 12.3%for amantadine and rimantadine,respec-tively)when ethyl acetate was used.The recoveries were more thanH.Yan et al./J.Chromatogr.B 938 (2013) 8–1311Table 1Name,formula,retention time and exact mass of the target compound.Compound nameFormulaTheoretic [M+H]+Accuracy [M+H]+Mass accuracy ( m )Retention time (min)ACNRetention time (min)MeOHAmantadineC 10H 17N152.14338152.143420.00004(0.26ppm)3.227.29Rimantadine C 12H 21N 180.17468180.174640.00004(0.22ppm)5.377.5960%when acetonitrile was used,and consequently,it was used for further experiments.In addition,the type of acid added to the acetonitrile solution was studied.Acetonitrile in 1%FA,1%HAc and 1%NH 3·H 2O were evaluated,the experimental results are shown in Fig.2.It was found that recoveries of amantadine was higher than 70%when 1%HAc-ACN and 1%FA-ACN was used,and the recoveries of rimantadine was higher than 70%when 1%HAc-ACN and 1%NH 3·H 2O-ACN was used.Overall,the highest extract recoveries for both amantadine and rimantadine were obtained when 1%HAc-ACN was used,and therefore,it was used for further experiments.3.3.Choices of sorbents in clean-upSince animal muscle contains large amounts of various sub-stances that are soluble in organic solvents,further clean-up processes are commonly used to minimize chromatographic inter-ference and ion suppression.The QuEChERS method uses d-SPE for cleaning up.The suitable sorbent for d-SPE should easily remove the matrix components but let the analytes remained.In order to evaluate the performance of the sorbent for clean-up,blank matrix solution (1%HAc-acetonitrile extraction solution)was made fol-lowing “2.3samples and sample preparation procedure”and spiked with standard solution to make matrix spiked solution.150mg of C 18,NH 2,Alumina-N,PSA,GCB,and Florisil were weighted into each d-SPE tube (3replicates).5mL spiked matrix solution was added into each tube,the tube was vortexed for 30s and centrifuged.A 4mL aliquot of the upper layer was transferred into 10mL cen-trifuge tube and dried by N 2stream at 45◦C water bath.The recoveries of each group were compared.It was found there were no recoveries when Florisil was used,indicated Florisil absorbed the target analytes and thus it was discarded.For other sorbents (Fig.3),the average recoveries were in the following sequence:C 18>NH 2>PSA >GCB >Alumina-N.Lowest recoveries for amanta-dine and rimandatine were found when PSA and GCB were used,respectively.When PSA was used,the recovery of amantadine was lower than 50%,and when GCB was used,the recovery ofR e c o v e r y (%)Fig.2.Recoveries of amantadine and rimantadine extracted by different solvent sys-tems from chicken muscle samples spiked at 15g/kg.1–5represent ethyl acetate,acetonitrile,1%FA-acetonitrile,1%HAc-acetonitrile,and 1%NH 3·H 2O-acetonitrile,respectively.rimantadine was lower than 30%.C 18sorbent gave the highest recoveries (>65%)of both compounds,so it was selected for clean up.3.4.Method performance characteristics3.4.1.SpecificitySpecificity was assessed by comparing the chromatograms of blank chicken muscle tissues with the corresponding spiked mus-cle.Fig.4A shows the typical chromatograms of blank chicken muscle sample,a blank chicken muscle sample spiked with aman-tadine and rimantadine at 5g/kg was shown in Fig.4B and no significant interference was observed.3.4.2.Matrix effectThe matrix effect by using ESI source is a main problem dur-ing LC–MS analysis.Due to the interactions of co-eluting matrix components with target compounds in the ionization step,the signal suppression or enhancement of the analytes were always observed.To evaluate matrix effect,the slopes obtained in the matrix-matched calibrations were compared with those obtained with solvent standards (Table 2).Then,matrix/solvent slope ratios for each compound were obtained considering a signal enhance-ment or suppression effect as acceptable if the slope ratio ranged from 0.8to 1.2.Slope ratios higher values than 1.2or lower than 0.8indicate a strong matrix effect.The results show that a significant matrix suppression effect was observed for amantadine,whereas a tolerable matrix enhancement effect was observed for rimantadine.Matrix-matched curved was used to accurately quantify amanta-dine and rimantadine.3.4.3.LinearityLinearity was evaluated by matrix-matched calibrations at the range of 1–100g/kg.Calibration curves were obtained by least-squares linear regression analysis of the peak area versus concentration.The calibration curves showed good linearity with determination coefficients (r 2)higher than 0.990in all thecases.R e c o v e r y (%)18NH 2PSAAlumina-Nparison of recoveries for amantadine and rimantadine with different sorbent spiked at 10g/kg.12H.Yan et al./J.Chromatogr.B 938 (2013) 8–13510Time (min)20406080100020406080100R e l a t i v e A b u n d a n c e (%)3.305.39NL: 2.82E3 m/z=152.14262-152.14414 F: FTMS + p ESI Full ms [100.00-1000.00] MS JZ-5PPBNL: 1.70E4 m/z=180.17378-180.17558 F: FTMS + p ESI Full ms [100.00-1000.00] MS JZ-5PPB510Time (min)020 40 60 80 100 020 40 60 80 100 NL: 1.69E3m/z=152.14262-152.14414 F: FTMS + p ESI Full ms[100.00-1000.00] MS BLKNL: 0 m/z=180.17378-180.17558 F: FTMS + p ESI Full ms[100.00 -10 00.00] MS BLKR e l a t i v e A b u n d a n c e (%)ABFig.4.Typical chromatograms of amantadine and rimantadine obtained by extraction of (A)blank chicken muscle tissue,(B)muscle spiked with amantadine and rimantadine (5g/kg).Table 2Evaluation of matrix effects by comparing the calibration curves using matrix-matched calibration and solvent-based standards.AnalyteSolvent-based curve slopeCorrelation coefficient (r 2)Matrix-matched curve slopeCorrelation coefficient (r 2)Matrix effect (%)Amantadine 92820.998462850.9957−32.3Rimantadine217330.9992243120.998311.83.4.4.Accuracy and precisionThe accuracy and the precision of the method were described by means of absolute recovery and repeatability (intra-day and inter-day RSDs).Table 3gives an overview of the values for the intra-day and inter-day accuracy and precision at three spiking levels of 5,10,and 20g/kg.The intra-day and inter-day accu-racy of amantadine ranged from 87.5%to 102.4%(RSDs ranged from 3.9%to 6.3%)and 82.5%to 105.8%(RSDs ranged from 5.95%to 13.9%),respectively.The intra-day and inter-day accuracy of ramantadine ranged from 95.3%to 97.4%(RSDs ranged from 6.0%to 7.45%)and 89.4%to 93.2%(RSDs ranged from 7.8%to 12.4%),respec-tively.The results indicated that the method was accurate and precise.3.4.5.LODs and LOQsThe limit of detection (LOD)was considered to be the concentration that produced a signal-to-noise (S /N )ratio of 3.The limit of quantification (LOQ)was set at the lowest spiking level.The LODs were 1.02and 0.67g/kg the LOQs were 3.40and 2.21g/kg for amantadine and rimantadine,respectively.Table 3Intra-and inter-day accuracy and precision of amantadine and rimantadine assay in chicken muscle tissue.AnalyteSpiked level (g/kg)Intra-day (n =6)Inter-day (n =3)Accuracy (%)RSD (%)Accuracy (%)RSD (%)Amantadine587.5 3.9082.513.91095.7 4.05105.8 5.9520102.46.31103.97.51Rimantadine596.47.4593.212.41095.3 6.0389.412.02097.46.7191.27.833.5.Application of the methodThe developed method was applied to determination amanta-dine and rimantadine in 100chicken muscle samples.The matrix matched calibration curves were used to calculate the concentra-tions of amantadine and rimantadine.In order to assure the quality of the results,an internal quality control was applied for every batch of samples.Among the assayed samples,amantadine was detected in 3chicken muscle samples with concentrations of 4.25,37.3and 86.0g/kg.For the rest of the samples,no amantadine and rimantadine were detected by using the proposed method.4.ConclusionIn this paper,a new sample pretreatment method based on QuEChERS method coupled with UHPLC-LTQ Orbitrap MS was applied for the determination of amantadine and rimantadine in chicken muscle tissues.The method provides good repeatability,high recovery,excellent intra-and inter-day accuracy and preci-sion.And it was successfully applied for the routine analysis of amantadine and rimantadine residues in chicken muscle samples.AcknowledgementsThis work was supported by National High-Tech R&D Program of China (863Program)(2012AA101603),Science and technology planning project of General Administration of Quality Supervision,Inspection and Quarantine (AQSIQ)of PR China (2012IK145)and AQSIQ industrial public service scientific research project of the Ministry of Science and Technology of PR China (201210029).References[1]R.L.Tominack,F.G.Hayden,Infect.Dis.Clin.North 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