LC MS Chemistry

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1 LC MS Chemistry

2 LC MS chemistry? HPLC RRLC Ion Source Interface (Key to Success) MS(SQ,TOF, IT) MSMS(QQQ,Q-TOF) Column 에의한 Sample matrix 의분리 Target 성분을가스모분자이온으로만든다 Gas Phase 분자이온을 m/z 차이로분리검출한다 RT 정성 1. Column Chemistry 2. Making Gas Phase Ion 3. Fragmentation (CID, CC) MS spectra 정성 2. MS/MS spectra 정성

3 LC chemistry (Separation) 원칙 1 : HPLC column 에주입된시료 Matrix 는반드시배출되어야합니다. GPC Isocratic Gradient LC

4 LC chemistry (Separation) 원칙 2 : HPLC column 에주입된시료 Matrix 는반드시분리되어야합니다. 이동상, ph, Buffer, 온도 조정 Particle size/function, RRLC Column 1. Ultra fast 2. High Resolution

5 MS chemistry : Making Gas Phase Ion

6 LC MS Interface : Ion Source Atmosphere Pressure Ionization Electro Spray API-ES Atmosphere Pressure Chemical Ionization APCI Multi Mode Ionization MMI Atmosphere Pressure Photometric Ionization APPI Matrix Assisted Laser Desorption & Ionization MALDI On-line Nano-Electro Spray Nano source

7 ESI 구조및작동원리 AgilentLC inlet Nebulizer gas inlet Charged Droplets Analyte Ions Nebulizer Solvent Ion Clusters Salts/Ion pairs Neutrals -5,000V heated N 2 Evaporation Rayleigh Limit Reached Coulombic Explosions - - Solvent Ion Cluster Analyte Ion Waste

8 Enhanced sensitivity Buffer/matrix tolerant Broader flow rate range Optimized geometry Higher signal-to-noise Reduced downtime/maintenance Orthogonal Spraying

9 Selectivity of LC/MSD Extracted ion chromatograms of morphine standards by ESI-LC/MS 6000 m/z Normorphine pg m/z 286 Morphine 50 pg m/z 462 Morphine-3-Glucuronide 100 pg Morphine-6-Glucuronide 50 pg min

10 ESI Chemistry ESI 는 LC MS 에서가장많이사용되는이온소스입니다. Target 성분을이동상분무내에서이온화하고이동상을 Drying 하는방식으로구조에따라서 Single /Multiple charge 이온을생성합니다. Nubilizer 출구부근에서가장이온화가잘일어나므로유속이낮을수록감도가좋습니다. Catechol-Amines, Sulfate Conjugates, Quaternary Amines, Menthol 등은 Single Charge 이온을 Protein, Peptide, Oligo-Nucleotides 등은 Multiple Charge 이온을생성합니다그리고 Heteroatoms, Carbamates, Benzodiazepines 등을함유하는성분분석에사용합니다이동상은 Formic Acid 같은휘발성 Buffer 를 0.1% 이하사용하는데특히 Polar 한성분에유리합니다. 시료농도가진하면 Adduct 생성이잘됩니다. 이동상의 ph 에따라서 2 이하에서는 Multiple Positive Charge, 3-9( 일반적으로 Ammonium Acetate 기준으로조정 ) 에서는 Single Positive Charge, 10 이상에서는 Single Negative Charge, 이온생성이유리합니다. Ion Pairing 시약은 Basic 시료에는 TFA, PFPA, HFnBA 등이사용이되고 Acidic 시료에는 Di-n-Propyl Ammonum Acetate, DnBAA, DnHAA 등이사용되는데감도가감소하므로 0.1% 미만으로적게사용합니다. 휘발성이좋은 ACN/MeOH 의조성 % 가높을수록감도가좋습니다. 단물은항상필요합니다. Nubulizer 출구부근에서이온형성이주로이루어지므로유속은 0.5ml/mn 미만으로낮을수록유리합니다.

11 Summary of ESI

12 APCI 구조및작동원리 Nebulizer Heater [SolvH] A --> Solv [AH] Analyte containing aerosol Heat Charge transfer to analyte Corona needle Waste heated N 2 Vapor Charged reagent gas formed Analyte ions

13 APCI Chemistry APCI 는 LC MS 에서두번째로많이사용되는이온소스입니다. 이동상의전자구름대를 Corona Needle 주위에만들고이것에의하여 Drying 된 Target 성분을이온화하는방식으로유속은 ml/mn 정도가많이사용됩니다. 주로 Single Charge 이온을생성합니다. PAHs, PCBs, Fatty Acids, Phthalates. 등중간극성을가지거나 Hydrocarbons, Alcohols, Aldehydes, Ketones, and EstersSteroid, Carotene 같은비극성성분 Heteroatoms: Ureas, Benzodiazepines, Carbamates 를포함하는성분분석에유리합니다. 비극성시료의분자량이 이상커지면감도가다소감소합니다. 이경우는 MALDI 가유용할수있습니다. 이동상은비극성용매를사용하는데 MeOH 같은 Polar 용매 (Positive/Negative) 나 Methyl Morpholine 같은 Buffer(Negative 분석용 ) 가포함되어야합니다. 이동상에 Buffer 는잘사용하지않고이동상, 알칼리 Metal 등의 Adduct 를잘형성하지않습니다. 감도는 Target 의 Proton Affinity(Positive) 와 Acidity(Negative) 가이동상보다좋아야증가합니다. Corona Needle 부근에서이온화가일어나므로 0.5-2ml/mn 유속이사용됩니다. 대표적인이동상은 H2O, ACN, MeOH, Ethanol, IPA, Butanol, Ethyl Acetate, THF 이고 ESI/APCI 양쪽에사용됩니다. HFIP, 암모니아수등은 ESI 에주로사용되고 Toluene, CS2, Hexane, Chloroform, Acetone 등은 APCI 에사용되므로첨가제추출용매에따라서선택을하면됩니다. 그리고알칼리 (Na, K ), metal, Detergent, H3PO4, H2SO4 등비휘발성 Buffer 는절대사용하지않습니다. 일반적으로 Plastic 용기는이동상및시료저장에사용하지않습니다

14 Summary of APCI Advantages Complementary to API-Electrospray for less polar analytes Good sensitivity for compounds of intermediate MW and polarity Less sensitive to solution chemistry effects than API-ES Tolerates higher flow rates without decrease in sensitivity Disadvantages Less useful for thermally labile compounds Requires some compound volatility

15 MMI : ESI APCI

16 APPI 외관및작동원리 Analyte containing aerosol Photon ionizes analyte Lamp Source instead of APCI Discharge Needle Evaporation h Analyte ions Vapor h Dopant is photoionized and acts as reagent gas

17 APCI vs APPI APCI (Atmospheric Pressure Chemical Ionization) APPI (Atmospheric Pressure Photoionization)

18 APPI 의특징 1. Vaporization of LC eluent and exposure to <11 ev photon energy /Krypton Lamp, APCI vaporization chamber - Direct ionization of analyte to radical cation - Ionization of solvent or dopant with charge exchange to analyte 2. APPI may ionize compounds that do not ionize well by ESI or APCI - PAHs - THC, Benzoic acid 3. 일반특성 APPI exhibits appropriate linear dynamic range and reproducibility for quantitative applications APPI can be used in positive and negative modes APPI can be used with pos/neg switching APPI is a complimentary API technique relative to APCI and ESI

19 APPI Mechanisms

20 GC /EI vs LC /ESI, APCI, APPI GC/MS

21 MALDI 30,000v Pulsed laser light (Proton Beam) H H H Sample and matrix on tip of solid probe Matrix: nicotinic acid, cinamic acid, Molecule ion desorbed from matrix

22 Plate Spotting

23 Matrix

1020.1 1101.5 2044.1 1193.2 1789.1 1301.4 1590.4 1431.4 Nano- SPRAY : spectral sensitivity 100 Theoretical average MW: 14305.

25 Nano- SPRAY : spectral sensitivity 100 Theoretical average MW: Curve fitted MW: For dedicated proteomics : Flow rate: 5 to 500nl m/z

26 MW calculation using multiple charge Ab MZ4 MZ3 MZ2 MZ1 P2 MZ P1 m/z Calculation Equation P1 = (MZ) / Z P2 = (M(Z1) / Z1... MW = Dalton Deconvolution Bioanalysis Software Deconvoluted spectrum MW =

27 Fragmentation : CID(MS), CC(MSMS)

28 MS, MS/MS Solution MS for LC detection MS/MS for quantification LC/MS Single Quad MS/MS for identification MS for accurate mass and profiling 6410 QQQ Profiling and MS/MS accurate mass identification LC/MS Ion Trap LC/MS TOF => Agilent 6000s MS SQ : 6110/6120/6130/6140 TOF : 6210/6220 IT : 6310/6320/6330/6340 QQQ : 6410 Q-TOF : 6510/ QTOF

29 LC MS Solutions : Choices HPLC Ion Source MS MS/MS 1200 RRLC 1200 HPLC Cap LC Nano LC ESI APCI MMI APPI Nano LC PDF-MALDI 6130/40 SQ 6220 TOF 6330/40 Ion Trap 6410B QQQ 6520 Q-TOF

30 LC MS System Basics

1. Agilent s MS, MS/MS Portfolio in LC/MS MS for LC detection MS/MS for quantification LC/MS Single Quad MS/MS for identification LC/MS Ion Trap MS for accurate mass and profiling LC/MS TOF =>

31 1. Agilent s MS, MS/MS Portfolio in LC/MS MS for LC detection MS/MS for quantification LC/MS Single Quad MS/MS for identification LC/MS Ion Trap MS for accurate mass and profiling LC/MS TOF => Agilent 6000s MS SQ : 6110/6120/6130/6140 TOF : 6210/6220/6230 IT : 6310/6320/6330/6340 QQQ : 6410/6460 Q-TOF : 6510/6520/ QQQ Profiling and MS/MS accurate mass identification 6510 QTOF

2. Agilent s Ion Source Portfolio of LC/MS MM-ESI Indole 1434.31912.0 1147.7 Max: 103701 Insulin 118.1 2867.9 0 118.

32 2. Agilent s Ion Source Portfolio of LC/MS MM-ESI Indole Max: Insulin m/z Max: MM-APCI Indole m/z Max: MM-Mixed Indole Insulin m/z MMI

33 Ion Source(Interface) 작동원리및 Noise 제거의필요성 N2 Nubilized Charged Droplets Evaporation 모분자 Ion / Isotope / Frag. Scan & SIM Rayleigh Limit Reached Coulomb Explosions - - Solvent Ion Cluster Salts/Ion pair adducts Neutrals adducts CID MS or MS/MS?

3. LC-MS 분리 : LC-MS 의구성및목적 Chromatogram Mass Spectrum Compound specific Information Ion Trap SQ Purification => Target Compound Prep Column Chemistry Separation =>

34 3. LC-MS 분리 : LC-MS 의구성및목적 Chromatogram Mass Spectrum Compound specific Information Ion Trap SQ Purification => Target Compound Prep Column Chemistry Separation => NPC, RPC, IEX, GPC 정성 (RT)/ 정량감도 Sub PPM TOF Molecular weight => Formula, Structural information m/z Separation => gas phase 분자이온의경로 / 속도차정성 (m/z)/ 정량감도 Sub PPB (LOQ)

35 Accurate Mass Ion (m/z) 의 Separation 원리 ( 속도 / 경로차 ) DC DC Beam Shaper - line focus Time of Flight 1-3 ppm 이하 DCRF Octapole - round focusing Sector Mass Quadrupole ppm

36 Mass(m/z) Accuracy 와 Fragmentation Spectrum a. Mass Accuracy Formula Feature/MW 에의한 Database search?=> SQ Formula Feature/MW 에의한 Database search OK : Chem ACX, Merck Index, Synthetic DB => TOF b. Fragmentation Spectrum SIM/SCAN SIM/SCAN Fragmented library, Screening Search CID/SCAN MS n => SQ, TOF (LC separation) Soft fragmented library Or Amart-flag => Ion Trap (3D separation)

37 Agilent 6100s SQ 의내부구조및작동원리 AgilentLC inlet Nebulizer gas inlet Capillary Skimmers Octopole Lenses Quadrupole HED detector Nebulizer Neutral Molecules Analyte Ions Clusters Salts -5,000V heated N 2 Waste Fragmentation zone (CID) SQ

38 Agilent 6200s TOF 의내부구조및작동원리 TOF

39 Agilent 6300s Ion Trap 의내부구조및작동원리

40 MS type 별비교 : MS Spectrum 의 Performance Quadrupole Ion Trap Time Of Flight MS Type MS (SIM, SCAN) MS n (SIM, SCAN, MS n) MS (Scan ) Molecular Weight (M/Z) 100 vs ppm vs ppm vs ppm Target 분자량 Small Molecule 1,350-3,000 Small Molecule 4,000 Small Molecule Large Molecule(20,000) Target Sample Known Known Unknown Known Unknown Sensitivity High(SIM) High Low(SCAN) Reproducibility Best Not Good Good

41 LC-MSMS 분리 : LC-MSMS 의구성및목적 CSI Clean MS Spectrum

42 MSMS ( Q-TOF and QQQ ) 내부구성 Q-TOF QQQ Q-TOF Quad Mass Filter (Q1) Quad Mass Filter (Q3) Lens 1 and 2 a Collision Cell HED Detector QQQ Rough Pump Turbo Pump Turbo Pump Turbo Pump

43 MSMS ( Q-TOF & QQQ ) 의결과 279 Targeted MSMS Ion Source Q1 Collision Cell TOF MR M Ion Source Q1 Collision Cell Q2

44 MS/MS (6410 QQQ / TQ / Tandem MS) 의내부구조원리 추가적인정성

45 MS Data : MS Library vs MRM List vs Molecular Feature Database MS Library MFD MRM List Data SQ Q-TOF, TOF QQQ Acquisiton / GC SQ (EI, CI? ) Sector MS, FT MS /Tandem MS (ESI, APCI) Identification Point ( 정성점수 ) 2-3 points Fragmentation Ions 2 이상 Exact Mass (1-2ppm) points Precusor-Product Ions Sensitivity ( 최적감도 ) No Information No Information Fragmentation Voltage(CID) Collision Energy(CE) Optimized Sensitivity Target Pesticides/MRL All Pesticides/ MRL Antibiotics/MRPL Data Source Open : Wiley, NIST 약 200, ,000 Open : Chem ACX Merck Index MS/MS Vender 약 400-1,000

46 4. LC 분리와 MS 분리필요성 : Meat 중의잒류항균성분분석 (HPLC), 선택적검출? Matrix effect 1. 시료의 Impurity/matrix Peak => 선택적인검출 : 정성의중요성 2. 일반 UV/Vis 은시료 matrix effect 영향심함 Compound Spcific Information? Mass Spectrometer : SQ, TOF, IT?

47 LC-MS 분리와 MS/MS 분리필요성 : 잒류 Sulfonamide 계분석 ( DAD vs MS ), 감도개선, 구조정보? 1. 시료의 Impurity/matrix Peak 분리 => m/z 에의한선택성검출 2. 감도는 DAD 에비해 10 배이상개선됨 구조정보? MS/MS 의필요성 SIM mode DAD

48 LC-MS 분리와 MS/MS 분리필요성 : SQ 벌꿀, 새우중의 Chloramphenicol 분석 I

49 LC-MS 분리와 MS/MS 분리필요성 : QQQ 벌꿀, 새우중의 Chloramphenicol 분석 II Quant,Qual,Ratio

50 Ion Trap : Smart Frag

TOF : Resolution (FWHM) c. Resolution 1.30e4 1.20e4 1.10e4 1.00e4 9000.00 8000.00 7000.00 6000.00 5000.00 4000.00 3000.00 2000.00 1000.00 0.00 Intensity, counts 922.0092 0.

51 TOF : Resolution (FWHM) c. Resolution 1.30e4 1.20e4 1.10e4 1.00e Intensity, counts Resolution = M/ΔM 922/0.087 = >10, m/z, amu Delta M/Z Resolution and Dynamic Range for comparison between samples for Metabolite etc

52 Ion drift motion & ion mobility

IMS Ion drift motion in gas & homogeneous electric field: vd = K(P,T) E - K0(P0,T0) is a characteristic parameter for certain molecules/elements given in [cm²/vs] - Measurement conditions are given

53 IMS Ion drift motion in gas & homogeneous electric field: vd = K(P,T) E - K0(P0,T0) is a characteristic parameter for certain molecules/elements given in [cm²/vs] - Measurement conditions are given by the ratio: E/n [Td=1017 V cm2] td = s/(k E) - characteristic time spectra - very fast method (1/E) Vd : Ion velocity K : Ion mobility S : Ion path td: Drift time K0 : Reduced ion mobility E : Electric field strength n: Number Density of Buffer Gas Atoms

54 IMS Data

55 IM-MS

56 IM-MS Data

57 Traveling-wave ion mobility spectrometry (TWIMS)

58 Traveling-wave ion mobility spectrometry (TWIMS)

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$(Microsoft PowerPoint - src.ppt [\300\320\261\342 \300\374\277\353] [\310\243\310\257 \270\360\265\345])$ LC/MS, LC/MS/MS 기초개론 LC LC/MS(Liquid Chromatography/Mass Spectrometry) 액상의분석물질을기화및이온화하여고진공관에서원하는질량 (m/z) 이나질량범위만검출하여정량 정성분석하는시스템 High vacuum PC/Software Ion source Mass Analyzer Detector ESI APCI APPI