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不同离子源的适用范围以及API串联四级杆质谱特性 (7).pdf

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1、1 AB SCIEX Triple QuadQuantitative Applications Hua-fen Liu(刘华芬) Sr. Scientist Applied Biosystems, FosterCity, CA 2 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Outline Introduction of general ion source and unique features of API instruments不同离子源的适用范围以及 API串联四级

2、杆 质谱的特性 Improved fastLC support更好的超高速液相支持系统 Qtrapfunctions and MRM 3 for quantitation QTRAP功能以及 MRM 3 在定量中的应用 21CFR compliant software tools and consistent performance for validatedstudies 符合 21CFR法规的软件以及稳定仪器性能更适用于认证的 实验2 3 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE &

3、BEYOND 不同离子源的适用范围以及 API串联四级杆质 谱的特性 4 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 不同离子源的适用范围3 5 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Atmospheric Pressure Ionization* (API) Sources Three major types of API sources: El

4、ectrospray (ESI)电喷雾离子化 ionizes compounds in the liquid phase. Atmospheric Pressure Chemical Ionization (APCI)大气压化 学离子化 ionizes compounds in the gas phase using a corona discharge. Atmospheric Pressure Photo大气压光离子化 -Ionization (APPI) ionizes compounds in the gas phaseusingUV radiation and a dopantto

5、induce ionization. 6 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 电喷雾离子化 (ESI) 显示正极模式 Curtain Gas (CUR) Evaporation Coulomb Explosion Ion Spray Voltage (IS) Nebulizing Gas (GS1) LC Flow Formation of Charged Droplets Orifice (DP) Mass Analyzer Vacuum Atmospheric

6、Pressure TurboHeater (TEM) /Heated Gas (GS2) 1. 2. 3. 1. 形成带电液滴 2. 液滴蒸发,液滴里的场强增加 3. 离子从液滴中迸发出来,在接口处聚焦4 7 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Reaction with analyte molecules Corona Discharge Needle (NC) Ionization of solvent molecules Orifice (DP) Mass A

7、nalyzer X = Solvent Molecule M = Analyte Curtain Gas (CUR) Vacuum Atmospheric Pressure LC Flow Formation of clusters Heated Nebulizer (TEM) Nebulizing Gas (GS1) 大气压化学离子化 (APCI) 1. 2. 3. 1. 离子源在源内使 N2或 O2离子化 2. N2或 O2将电荷传给溶剂蒸汽 3. 蒸发的带电溶剂将电荷传给化合物 8 October, 2008 2008 Applied BiosystemsInc. and MDS Inc

8、. Joint Owners ABOVE & BEYOND 大气压光离子化 (APPI) APPI中俩个离子化机制 : 1. 电荷在非极性化合物 间传递 (上面所 示 ) 2. 极性化合物 溶剂传递 . . .5 9 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND APPI Source 加 热 雾化器 适合非离子非极性 热 稳定化合物 探针 加 热有助 于蒸发 需要 dopantpost-column和 UV光子 诱 发离子化 操作 范围 类似 APCI Turbo V TM

9、 源 10 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Comparison of optimized parameters 20 41 67 109 331 4 39 67 97 331 CXP CE DP Q3 Q1 TIS 20 43 70 109 331 4 43 70 97 331 CXP CE DP Q3 Q1 APCI 20 43 69 109 331 4 43 69 97 331 CXP CE DP Q3 Q1 APPI Voltages are the s

10、ame for all three sources!6 11 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 离子源 优缺点 ESI 优点 适合大范围的化合物 -大 小和 极性 热 不稳定性 和挥 发性不 是问题 最温和 的 分 子 碎裂方 式 可分析 大 分 子量 (多 电荷 ) 化合物 高 灵敏度 缺点 离子 抑 制 : 离子 竞争 离 开 液滴 ? 灵敏度受流动 相 组 成的 影响 APCI & APPI 优点 低 极性化合物离子化 宽动态 范围 (4 -5 个 数 量级

11、 ) 质量 敏感(依赖流 速 ) 可耐受 高 缓冲 液 浓度 缺点 化合物 热 稳定 (最 高 130- 150C) 挥 发性 需要掺杂剂 (APPI ) 12 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Turbo V离子源 (针对 API 3200, API 4000, & API 5000系统 ) 垂直和水平方向可精细调节 内置 的气 路和配线方便使 用 直角设计保 证 了 更 有效 的 去 溶剂并提 高 了耐 用性 对称 加 热装置 , 最 高 温度可 达 750

12、C 含 2个离子源 : ESI & APCI7 13 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 加 上 的 挡板减少 了 再循环 流 速范围 5-2000 L/min 与 以 往 的离子源相 比 有 5 至 20X S/N TIS 增强 Recirculation Entrainment 对称 的 高瓦特密度 加 热装置设计 以 维 持 集 中喷 射 , 被 称 为夹 带 Turbo V离子源 14 October, 2008 2008 Applied Biosystem

13、sInc. and MDS Inc. Joint Owners ABOVE & BEYOND Pressures due to Differential Pumping Q1 region 10 Torr Q0, Entrance Potential (EP) 6-8 mTorr Atmospheric Pressure Curtain plate (1100 V, fixed) Orifice Hole Curtain gas Skimmer (Grounded ) Orifaceplate, Declustering Potential (DP) 1-2 Torr Interface of

14、 API 4000 System 8 15 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Turbo V IonSpray 系统加 热 喷雾器 区域 加 热 技术 LC 流 速 被 雾化 产生 细 液滴喷 雾 在液滴 和 陶瓷壁之 间 形成蒸 汽 屏障 (Leidenfrost效 应 ) 最 佳 热传 导 形成液滴 解吸附 热 梯 度 减少 化合物的 降解 Angstrom磨 光的 陶瓷 是 不 粘 的 并 且具 有 自我清洁 能 力 得到 的 峰 更 尖锐 ,显 著 提 高

15、 S/N Laminar flow Convective Heating Above Leidenfrost Temp Turbulent Mixing Vapor Film Boiling Droplet Rebound and Breakup Hotter-Cooler Vaporization Nebulizer Gas Nebulizer Gas LC Flow Below Leidenfrost Temp Laminar flow Convective Heating Above Leidenfrost Temp Turbulent Mixing Vapor Film Boiling

16、 Droplet Rebound and Breakup Hotter-Cooler Vaporization Nebulizer Gas Nebulizer Gas LC Flow Below Leidenfrost Temp 16 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Center Line Field 5.75 V Center Line Field 4.25V +1.5V Field Gradient B A A B B A A B Collision Cel

17、l Entrance Collision Cell Exit 4V: A 6V: B LINAC 碰撞池 电场 梯 度 (LINAC = 线 性加速 ) 因为 电场 梯 度 ,离子 快 速 通过碰撞池 Q2 LINAC 碰撞池减少交叉污染 , 并 允许 更 快 的 MS/MS 扫描而 不 损 伤 灵敏度9 17 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Cross-talk象交通堵塞 Highway Highway 离子, 象交通 中的 车 子, 如果被 分开 很容易 分

18、 辨。交通堵塞时 , 很难 分 辨一 个 车 子 尾 和 车 子 头。没 有 LINAC,质谱 很难 分 辨一 个质量 数 的 结束 和 另一 个质量 数 的 开 始。 Q1 Q3 Q2 traffic No traffic No LINAC LINAC 18 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND LINAC ELMINATES CROSS TALK without LINAC Collision Cell with LINAC Collision Cell Inje

19、ction of selegiline(IS) to determine the level of cross-talk in the amphetamine (none present) MRM transition in non- LinacQ2 8.66e3 cps XIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg 2500pg w LINAC 0.5 1.0 1.5 2.0 Time, min 2000 4000 6000 8000 Intensity, cps 3.76e6 cps XIC of +MRM (4 pairs)

20、: for 188.2 / 91.1 amu from Seleg 2500pg w LINAC 0.5 1.0 1.5 2.0 Time, min 1e6 2e6 3e6 Intensity, cps 25 ng Selegiline Cross-talk for amphetamine 3.67e4 cps XIC of +MRM (4 pairs): for 136.1 / 91.1 amu from Seleg2500pg No-LINAC Dwell100msec 0.5 1.0 1.5 2.0 Time, min 20 40 60 80 % Intensity 1.04e6 cps

21、 XIC of +MRM (4 pairs): for 188.2 / 91.1 amu from Seleg2500pg No-LINAC 0.5 1.0 1.5 2.0 Time, min 20 40 60 80 % Intensity 25 ngSelegiline Cross-talk for amphetamine 100 10010 19 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Sensitivity of API 4000 source over exte

22、nded flow range 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 Time, min 0.0 5000.0 1.0e4 1.5e4 2.0e4 2.5e4 3.0e4 3.5e4 4.0e4 4.5e4 5.0e4 5.5e4 6.0e4 6.5e4 7.0e4 7.5e4 8.0e4 Intensity, cps Minoxidil 830 fgon column no split was used 1mm-60uL/min 2mm-200uL/min 4.6mm 1000u

23、L/min 20 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND LLOQ of Acetylcholine in Solvent 5500 Blank 0.0002pg/L 0.002pg/L Lloq0.001pg on column 11 21 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 更好的超高速液相支持 系统 22 October, 2008 2

24、008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Advantages of Fast LC Increased response Increased throughput Better resolution Conventional LC 1.8 mm Columns 600 mL/min Instrument Requirements Faster cycle times! Maintain response at high LC flow rates12 23 October, 2008 2008 Ap

25、plied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 2.9 3.0 3.1 3.2 3.3 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 9.1e4 3.1 cycle time 2.9 3.0 3.1 3.2 3.3 Time, min 0.0 2.0e4 4.0e4 6.0e4 8.0e4 9.1e4 3.1 cycle time Need for faster MRM scanning with Fast LC 2.9 3.0 3.1 3.2 3.3 Time, min 0.00 2.0

26、0e4 4.00e4 6.00e4 8.00e4 1.00e5 1.11e5 3.1 cycle time 2.9 3.0 3.1 3.2 3.3 Time, min 0.00 2.00e4 4.00e4 6.00e4 8.00e4 1.00e5 1.11e5 3.1 cycle time Longer dwell times-poor precision Short dwell times-better precision 24 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND

27、 Scheduled MRMAlgorithm Improving MRM Method Efficiency by Maximizing AnalyteUtilization Each MRM monitored only across its expected elution time concurrent MRMs Maintain cycle time and dwell time effective duty cycle for every peptide Maintain analytical precision13 25 October, 2008 2008 Applied Bi

28、osystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Scheduled MRM -(sMRM) -Setup 26 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND QTRAP 5500 Systemvs. 4000 QTRAPSystem Performance comparison using fast chromatography (2 min separation drugs, inhibitors, metabol

29、ites) XIC of +MRM (22 pairs): 152.0/110.0 amu Expected RT: 0.4 ID: Acetaminophem from Sample 11 (Sample001 - sMRM - 1) of Quant 550. Max. 1.9e5 cps. 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time, min 0.0 1.0e5 2.0e5 3.0e5 4.0e5 5.0e5 6.0e5 7.0e5 8.0e5 9.0e5 1.0e6 1.1e6 1.2e6 1.3e6 1.4e6 1.5e6 1.6e6 1

30、.7e6 1.8e6 1.9e6 2.0e6 2.1e6 2.2e6 2.3e6 2.4e6 0.42 All experiments had at least 12 points per peak14 27 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND CVs 4000 QTRAPSystem vs. 5500 QTRAPSystem and sMRM5500 QTRAP CMPD Q1 Q3 1 Sulfadiazine 251.1 156.0 2 Sulfamethox

31、azole 254.2 156.0 3 Sulfathiazole 256.1 156.0 4 Sulfamerazine 265.1 156.0 5 Sulfamethizole 271.1 156.0 6 Sulfamethazine 279.2 186.0 7 Sulfachloropyridazine 285.2 156.0 8 Sulfadimethoxine 311.2 156.1 9 Phenacetin 180.2 138.1 10 Diclofenac 296.1 214.0 11 S-mephenytoin 219.3 134.1 12 Bufuralol 262.3 18

32、8.1 13 Midazolam 326.2 291.1 14 Testosterone 289.3 97.1 15 Nifedipine 347.2 315.1 16 Acetaminophem 152.2 110.1 17 4-hydroxydiclofenac 312.2 230.1 18 4-hydroxymephenytoin 235.2 150.1 19 1-hydroxybufuralol 278.2 186.1 20 1-hydroxymidazolam 342.1 203.0 21 6-hydroxytestosterone 305.3 269.2 22 oxidized n

33、ifedipine 345.2 284.1 New Collision Cell and sMRMimproves CVs 28 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND XIC of +MRM (78 pairs): 195.5/13. Max. 2.3e4 cps. 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 1.0e4 2.0e4 Intensity, cps 6.9 XIC of +MRM (78 pairs): E

34、xp 1, 1. Max. 1.9e4 cps. 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 1.0e4 1.9e4 Intensity, cps 6.9 XIC of +MRM (78 pairs): 235.2/86. Max. 1.1e6 cps. 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.00 5.00e5 1.00e6 Intensity, cps 6.5 XIC of +MRM (78 pairs): Exp 1, 2. Max. 1.0e6 cps. 4.5 5.0 5.5 6.

35、0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 5.0e5 1.0e6 Intensity, cps 6.5 XIC of +MRM (78 pairs): 213.1/72. Max. 9.1e4 cps. 9.0 9.5 10.0 10.5 11.0 11.5 12.012.5 Time, min 0.0 5.0e4 9.1e4 Intensity, cps 10.7 XIC of +MRM (78 pairs): Exp 1, 2. Max. 9.1e4 cps. 9.0 9.5 10.0 10.5 11.0 11.512.0 12.5 Time, min 0.0

36、 5.0e4 9.1e4 Intensity, cps 10.7 XIC of +MRM (78 pairs): 195.5/13. Max. 2.3e4 cps. 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 1.0e4 2.0e4 Intensity, cps 6.9 XIC of +MRM (78 pairs): Exp 1, 1. Max. 1.9e4 cps. 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 1.0e4 1.9e4 Intensity, cps 6.9 XIC of +MRM (

37、78 pairs): 235.2/86. Max. 1.1e6 cps. 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.00 5.00e5 1.00e6 Intensity, cps 6.5 XIC of +MRM (78 pairs): Exp 1, 2. Max. 1.0e6 cps. 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 Time, min 0.0 5.0e5 1.0e6 Intensity, cps 6.5 XIC of +MRM (78 pairs): 213.1/72. Max. 9.1e4 cps

38、. 9.0 9.5 10.0 10.5 11.0 11.5 12.012.5 Time, min 0.0 5.0e4 9.1e4 Intensity, cps 10.7 XIC of +MRM (78 pairs): Exp 1, 2. Max. 9.1e4 cps. 9.0 9.5 10.0 10.5 11.0 11.512.0 12.5 Time, min 0.0 5.0e4 9.1e4 Intensity, cps 10.7 (+) Caffeine (+/-)Caffeine S/N = 24.7 S/N = 20.0 (+) Lidocaine (+/-) Lidocaine S/N

39、 = 705 S/N = 922 (+) Chlorotoluron (+/-) Chlorotoluron S/N = 52.6 S/N = 48.8 No Loss in S/N when using Polarity Switching15 29 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND QTRAP功能 以及 MRM3在定量中的 应用 30 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Ow

40、ners ABOVE & BEYOND An interference peak from solvent for R-baclofen Solvent Blank (pos only method) Plasma Blank 1 ng/mLin Plasma with MRM_EPI Interference peak Interference peak Interference peak16 31 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND Infusion Tunin

41、g and MRM-EPI for Pos 214/116 50 ng/mL Infusion Tune 1 ng/mLMRM-EPI with 10L injection 1 ng/mLMRM-EPI with 10L injection Interference peak 32 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND MRM 3 Quantitation using the QTRAP 5500 system Many assays suffer from inte

42、rferences What if MRM is not selective enough? MRM 3 gives an extra level of selectivity With the added LIT sensitivity of the QTRAP 5500 system, quantitation using MRM 3 is a perfect solution for many problem assays17 33 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BE

43、YOND Fast and Selective MS/MS/MS Quantitation with MRM 3 Selection of 1 st precursor ion in Q1 Fragmentation in Q2 Product ions trapped in Q3 (Linear Ion Trap LIT) Isolation of 2 nd precursor ion in LIT Fragmentation in LIT 2 nd generation of product ions scanned out 34 October, 2008 2008 Applied Bi

44、osystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND 10 g/kg in Matrix (MRM 3 : %CV5, accuracy 90-110% ) S/N = 922 S/N = 8 S/N = 497 S/N = 147 XIC of +MRM (4 pairs): 330.9/126. Max. 1.0e5 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.00 2.00e4 4.00e4 6.00e4 8.00e4 1.00e5 Intensity, cps 2.1 X

45、IC of +MS3 (330.90),(127.10): E. Max. 2.8e7 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e6 1.0e7 1.5e7 2.0e7 2.5e7 2.8e7 Intensity, cps 2.1 XIC of +MRM (4 pairs): 330.9/98 Max. 4.5e5 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e4 1.0e5 1.5e5 2.0e5 2.5e5 3.0e5 3.5e5 4.0e5

46、4.5e5 Intensity, cps 2.1 XIC of +MS3 (330.90),(99.00): Ex. Max. 2.9e7 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e6 1.0e7 1.5e7 2.0e7 2.5e7 2.8e7 Intensity, cps 2.1 XIC of +MRM (4 pairs): 330.9/98 Max. 4.5e5 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e4 1.0e5 1.5e5 2.0e

47、5 2.5e5 3.0e5 3.5e5 4.0e5 4.5e5 Intensity, cps 2.1 XIC of +MS3 (330.90),(99.00): Ex. Max. 2.9e7 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e6 1.0e7 1.5e7 2.0e7 2.5e7 2.9e7 Intensity, cps 2.1 XIC of +MRM (4 pairs): 330.9/126. Max. 1.0e5 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, mi

48、n 0.00 2.00e4 4.00e4 6.00e4 8.00e4 1.00e5 Intensity, cps 2.1 XIC of +MS3 (330.90),(127.10): E. Max. 2.8e7 cps. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 0.0 5.0e6 1.0e7 1.5e7 2.0e7 2.5e7 2.8e7 Intensity, cps 2.1 XIC of +MRM (4 pairs): 330.9/98 Max. 4.5e5 cps. 0.0 0.5 1.0 1.5 331/99/71 matrix int

49、erference MRM 331/127 MRM 331/99 MRM 3 331/127/99 331/99/71 matrix interference MRM 318 35 October, 2008 2008 Applied BiosystemsInc. and MDS Inc. Joint Owners ABOVE & BEYOND XIC of +MRM (1 pair): 423.4/387.3 amu from Sample 1 (Ser200ppt 1V+2V acn) of Ju30-frSer200pptx30uL-70t100i9LO-2Mo500.wiff (T. Max. 9148.8 cps. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Time, m

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