环境水中生物检测方案(固相萃取仪)

On-line versus off-line solid phase extraction for the determination of Microcystins inWater by LC-MS/MS OVERVIEW .>Microcystins (MC) are d by Cyanob ers SPE extraction Method Performance >Its toxicity has been demonstrated in both animals and human beings.The ca ensive verdamage and promote liver tumor formation in subacute exposures.>1The World Health Organization (WHO) suggested a maximum daily intake cadults. The European Water Framework Directive (2000/60/EC) has established MC in drinking water at the same level suggested by 1ug/lthe WHO， 1pg/LY CAS Nr.NominalMass101043-37-2 994 Cartridge： HySphere C18-HD cartridgem ID-7um， Spark)Conditioning solvents：1 mL MeOH(5mL/min)2 mL Water (5mL/min)Sample loading：with WaterWashing：1 mL MeOH：Water (20：80)Focusing (Elution)：HPD syringe： MeOH 200 pL 100 pL/minGradient pump： Mobile phase (ng/L) LOQ(ng/L) Run-to-run(%RSD) Dayispay Linearity MC-RR 1.2 10 2.9 3.6 0.995 MC-YR 0.4 6 1.8 2.3 0.997 MC-LR 0.8 8 2.2 2.3 0.998 Compound MC-LR Leucin Arginin 101043-37-2 994 MC-YR Tryptopharan Arginin 1010-64-48-6 1044MC-RR Arginin Arginin 111755-37-4 1037 >Satisfactory LODs were obtained in the low ng/L level. WASTE > Excellent linearity's were achieved for each compound. (A：B；5：95) 400pL/min INTRODUCTION The present work describes the first fully automated method， based on on-line Solid Phase Extraction (SPE) - ultrafast liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS) for the determination of microcystins (MC-LR，-YR， -RR) in surface waters. Combination of both on-line SPE and UHPLC has been achieved by using specialfused coreTM chromatographic columns which allowed maintaining the high efficiencies obtained with UHPLCreducing the system backpressure. The analysis of MC was then performed in a total analysis time of 15 minutes.High recoveries， ranging from 99% to 115% were obtained and the focusing mode was used during SPE elution inorder to focus analytes in the column head and to avoid peak broadening and high efficiencies were then achieved(tN=34000-80000).Thhan those obtaine quality parameted with the off-ers of the methodline SPE ranging were established； from 6 to 10 ng limits of quanti/L while lower rfication (LOQ) weun-to-run and dare similary-to-day precisionswere obtained in the range of1.8-2.9% and 2.3-3.6% respectively. MS： AB/Sciex API 3200 Qtrap MS/MS RT (min) Q1DP(V) Q3 CE(V) Q3 CE(V) MC-RR 3.46 995 116 135 87 105 127 MC-YR 3.85 1045 146 135 99 105 129 MC-LR 3.97 1038 131 135 103 127 117 Off-line SPE VS On-line SPE Off-line On-line Sample volume 100 mL 10 mL SPE Solvent volun 12mL 1.2mL Evaporation N2 Reconstitution volume 6mL Total Analysis time 2 hours 15 minutes Nodularin (I.S.) 3.68 825 96 103 127 Objectives Devel -line SPE-LC method coupled to tanden ectrometry for the determination of MC in 100 wvaate >Mainta 1efits achieved with exissttiinng off-line SPE UPLC-MS/MS lution Off-line On-line PL C- MS /MS c ou thod EXPERIMENTAL CONDITIONS ONLINE SPE-LC (SYMBIOSISTM PICO， SPARK HOLLAND) 15a2 AUTOSAMPLEFR IniInjection volume：们0mLWVater (+5%MethanolpH=7) SamplNeedllee yash： 00 lpyiL.40% ACN， 0.2% FA LClumCccolu As kpress C18 (Suippel Column over 50(Mobile phase A) ACN. 0.1% FAAEB) Water， 0.1% FA Gradienti umeie flow Off-line SPE VS On-line SPE SPE-(Off-line) UPLC-MS/MS SPE-(On-line) LC-MS/MS Rt((min) Rs N Asymm. FRt ((min) Rs N Asym. MC-RR 1.83 35130 0.8 3.46 33965 1.1 MC-YR 2.35 .0 21114 1.0 3.85 1.8 79985 1.3 MC-LF.R 2.46 ).6 23260 1. 3.97 0..8 52755 1.1 ooo o DIscUSSION and CONCLUSION >The benefits of both on-line SPE system and UHPLC technologies (coupled to tandem MS) have been suUccessfully combined for the analysis of microcystin n wate1> The combination of both techniques was performed by using a fused core particle column which allowedto obtain high efficiencies and reduced backpressures. > Th Witthhooiut lution.> Focusing mode during the SPE elution step avoided peak broadening and the high efficiencies achievedwivtith the UHPLCV re maintained > is the total analysis time wa reduced to 15 minutes yielding to a reduction in bottout >- High recoveries (99-115%) and low LOQs (6-10 ng/L) were obtained for all the compounds Off-line SPE routine：For the off-line SPE method， a Waters Acquity ultra-performance liquid chromatograph (UPLCQ) systemlityy tulJlttra mequipped with a quaternary pump system (Milford， MA， USA) using an Acquity BEH C18 column (5.0 cmx2.mm i.d.， 1.7 pm particle size) (Waters Corp.， Milford， MA，USA) was used.Waters Oasis@ HLB Cartridges were rinsed with 10 mL of methanol and 10 mL of Milli-Qwater， samples werepercolaa1ted througgth the cartridge at a flow rate of 10 mL/min. The SPE idge was washed with 8 mL of a 20imoatthmethanol aqueousus soSlution and dried with nitrogen gas fs foorr 101 minutes. Analytes were eluted from the cartridgeusing 6 mL of methanol and the extracts were stored at -20 °C. Prior to injection， the extract was evaporated todryness at 40 °C under a stream of nitrogen in a TurboVap LV evaporator from Zymark (Zymark， Hopkinton， MAeSaarmples were reconstituted in 200o pulL.c of an aqueous solution with a 5% of methanol. The final extracts werefiltered through 0.2 pm before injection Spark HollandSince 1982 Spark has provided the HPLC ancnd LC/MS markets with state-of-the-art autosamplers， column ovensand sample preparation solutions. Solid Phase Extraction with on-liAll-line elution into HPLC and LC/MS systemspioneered by Spark and introduced in the early 90's. Spark， ISO 9001 certified， does basic research， productdevelopment， production， sales and marketing in-house， guaranteeing quality from start to finish. With 25% of thenremployees working in research and development Spark continues to invest in the future， making sure we candeliver the solutions you need to improve your business results. Innovation and quality are keywords when talkingbout our development efforts Phone： +31 591631700 Fax： +31 491645900 F-m E-mail：： applications@S；Sparkholland.coWeb：www.sparkholland.com AgBar， Barcelona，spain the Agbar Group is at the head of a big hoholding made up of more than 150 companies and 140 years of historywhich operates in all the fields related to community services： complete water cycle and health， among others.AahnGIC in tthe__：. d taThe Agbar Grroup is a world leader both in the fields which constitute the core of its activities related to the watercycle and in the business lines demanded by the new society， particularly in the services of healthlts international expansion，with presence in United Kingdom， Chile， China， Algeria， Colombia， Cuba andMexico consolidates its leadership and its p il of growth as a group rendering service to rmillion inhabitants all over the world.With a workforce over 13，000 employees， operating revenues of €2，861 million and consolidated incomeattributed to the parent company of 352.5 million in 2007， the Agbar Group has established continuous rarates ofgrowth and of value creation which allow it to advance day by day， adapting to the needs of a constantly evolving world， and also to occupy a leading positon in most of the areas in which it develops its activitie  日趋严重的水体富营养化使水华(Water bloom)发生成为全球性的环境问题。水体富营养化的直接后果是导致水中藻类大量繁殖，主要为有毒的蓝藻，其产生的微囊藻素（Microcystins，简称 MC)具有明显的肝毒性。被其污染的水源若为人或牲畜所饮用，将引起中毒，甚至死亡。  微囊藻毒素是由 7 种氨基酸组成的小分子环状多肽，为单环结构。最常见的且毒性较高的三种为MC-LR、MC-YR和MC-RR型。微囊藻素的毒性表现为肝脏毒性和强致癌性，自1959年首次被分离出后，世界多个国家都相继报道了其湖泊水中微囊藻素的存在。在我国湖泊水中微囊藻素的检出率在60%以上，浓度在130ng／ml - 2ug／ml不等，而世界卫生组织对水中微囊藻素含量的规定是不超过1 ug/L。  目前对于水中微囊藻素的分析主要采用生物测试，化学分析，免疫检测等方法。而化学分析法主要以液质联用为主，并需先将被测水样进行SPE前处理。传统的离线SPE需要耗费大量的前处理时间，使用大量溶剂。与此相比，在线SPE-液质联用，以其分析周期短，准确性高，低检测限等具有明显优势。