The Basics of LC/LC MS
Transcript of The Basics of LC/LC MS
1
The Basics of LC/LC MS
Bene Technology Co., Ltd. MKT
2
Part I : HPLC
1. LC/HPLC 의 역사
2. HPLC 의 구조, Terminology
3. 컬럼 의 분리 단, 구조
4. 분리모드
5. Reversed Phase Column선택하는 법
Appendix
1. LC/HPLC 의 역사
1903 ; Mikhail Tswett Separates Plant Pigments on Chalk Columns 1970년 이전 ; few reliable chromatographic methods 1970’년대 ; open-column chromatography, paper chromatography, and thin-layer chromatography => similar compounds, Mesh,… Pressure liquid chromatography => flowthrough/purification by small particle column /Resolution. Pump Technology => constant flow rate or constant pressure 1970년대 중반 : High Pressure LC, column packing materials and on-line detectors => High Performance Liquid Chromatography I970년대 후반 Reverse Phase Chro.(Mixed Mode) => Similar/Multi Compounds/Speed
1980년대 이후 ; 대부분의 chemical compounds 분리. New techniques=> Computers and automation, typical column(15-25cm L, 3-5 mm ID) => micro/capillary columns(1-2mm ID, 3 µm-300 µm ID), affinity/chiral columns and Fast HPLC(Length 3-5mm, smaller particles size10, 5, 3um), optimal analysis ; various types of columns, a variety of detectors of the compound. 2000년대 ; GLP, UHPLC(Sub 2um), Quantificaton/Qualification(MS,…), Safety Issue, Prep, Impurity Profile,… 응용영역 : biotechnological, biomedical, biochemical research, pharmaceutical industry, cosmetics, energy, food, and environmental industries,…
4
2. HPLC/LC MS 구조
Mass Spec
UV/Vis
Detector
LP Gradient
Pump
Pump
Column ELS
Detector Manual/Auto
Injector
Fluores.
Detector
Conventional LC
HT LC : UPLC, RRLC, …
Scale ? Speed Separation
Sensitivity Reproducibility
…
5
HPLC Terminology
용매 =>펌프 =>시료주입기 =>컬럼 =>검출기 ->Data System
LC/LCMS용 수용성 용매 지용성 용매 휘/비 Buffer Filter Degassing
Isocratic HP/LP G . 유속 압력 RunTime
수용성 용매 지용성 용매 Buffer Filter 시료주입량 Inject_Start
Separation Reversed P Normal P IE, SEC,… Mixed Chiral,… Pre-heat /cool
Chromatogram RI, UV/Vis Fluo., PAD ELSD,…
Spectrum MS ; MRM/SRM DDA,…
Quan/Qual Purity
FTIR, NMR,…
Automation Validation IQ/OQ/PQ Traceable
유로(시스템/Tubing/컬럼) ; 내경, 길이, Void Volume(w/column) => Equilibrium Time, Injection Volume,… Teflon, SUS316, In-line filter, HDPE, Quarz,… => 이동상 Nut Ferrule, Hand-tight fitting,… => Easy Handling * Compound Specific Information : m/z, formula, structure, Melting/Boiling Point,…
6
3. 컬럼의 분리 단
분배계수
KA = [A]u phase/[A]l phase
KA = [A]sol phase/[A]lliq phase
CPC
small particle Column
Serial Liquid-Liquid Extraction Chromatography
7 N= 5 25 100
A 100(98/2) B 100(2/98)
A 2 98 B 98 2
A 100(98/2) B 100(2/98) 평형 후 이동 이동 후 채우기 평형 후 이동
Up Layer
Low Layer KA = [A]U phase/ [A]L phase
8
Column의 구조의 활용
Vt = Vo + Ve
Vt는
GPC/GFC의 경우 V의 약 75-80%
HPLC의 경우 V의 약 60-65%
안정화 부피 : Vt 의 10-20배 이상
시료 주입량 : Vt 의 0.5-10% 이하
시료 농도 : 시료 주입량의 0.I -10% 이하
Tubing Frit 분리관
충전물
9
Column 충전물의 구조
Porous Beads Non-Porous Beads
Totally Porous, Superficially Porous, Surface Porous
10
Column 충전물의 Surface의 특징
Silica Surface : NPC
Area m2/g Chemistry
Si-OH’s Bonding RPC
IC, SEC,…
Pore
Size 분포도
Particle
Size 분포도
Chemistry 분자량
/Hydrodynamic Volume
분리 능
• Silica는 일반적으로 pH 2-6.5 에서 사용하고 Bonding에 의해서 pH 1-11 까지 가능함
• pore size 60-120A은 4-10K 이하, 200-300A은 20-100K이하 분리함
Electron Configuration - Zirconium : [Kr] 5s2 4d2 , (2,8,18,10,2 : 40) - Titanium : [Ar] 3d2 4s2 , (2,8,10,2 : 22) - Silicon : [Ne] 3s2 3p2, (2,8,4 : 14)
Lewis Acid/Base Bronsted Acid/Base
Column 충전물의 Surface의 특징
Electron Configuration - Zirconium : [Kr] 5s2 4d2 , (2,8,18,10,2 : 40 ; Zr4+ ionic radius 0.720 Å ) - Titanium : [Ar] 3d2 4s2 , (2,8,10,2 : 22 ; Ti4+ ionic radius 0.605 Å) => strong electron donor-acceptor complexes of d orbitals - Silicon : [Ne] 3s2 3p2, (2,8,4 : 14 ; Si4+ ionic radius 0.400 Å)
12
4. 분리모드 1 ; A, B, C term 이해
A. Eddy Diffusion : 경로차
B. Longitudinal Diffusion : 확산
C. Mass Transfer : Pore Surface Interaction Lateral(수평) Diffusion
13
Van Deemter Plot
14
분리모드 2 ; 그림으로 보는 분리
Injection
Vo
VE
VR
Log M
Retention Time
GPC/GFC
Steric(Entropic) Interaction HPLC(RPC, NPC, IEX,..)
Adsorption(Energetic) Interaction
Adsorption
Steric Interaction
Adsorption Interaction: 컬럼 내/외 Surface
15
분리모드 3 ; Resolution
Theoretical Plate Selectivity Retention Factor
K0 , T0 Time
Response
TR1 TR2
수식으로 보는 분리 : Resolution
R > 1.5 -2.0 : Baseline Resolution
K’ : Capacity Factor /Correct Mobile Phase Strength
α : Selectivity Factor /Right Chemistry
N : Efficiency Factor /Correct Column Dimension
16
A.1 Capacity/Retention Factor
2<K<10 K’=(Ta-T0)/T0
K 2 5 7.5 10
R ∝
Mobile Phase Strength ↓ K’ ↑ => R↑ RPC, NPC, IEX, …
17
↑
Mobile/Stationary Phase (C18, C8, C4) Strength
18
∝
A.2 Selectivity
α > 1.2
α=KA’/KB’
How to Interact with compound
- Stationary/Mobile Phase - pH
- Temperature - Additives
19
Chemical Selectivity : C18 end capping process
20
TMS => - CH3
Batch Synthesis
Flow Synthesis/Microreactor : Tri step end-capping
Anthocyanin/Anthocyanidin
21
Chiral Column Selection Guide_Optical Isomer
22
-Chirality
a. Chiral/Asymmetric_center/axis/plane b. non-superposable mirror images c. Enantiomers (pure/enriched), Racemates d. Non-enantiometric : Diastereomer
- Chemical Type Classification
a. Brush type (Pirkle) b. Cellulose c. Cyclodextrins d. Crown ethers e. Chiral Ligand Exchange (CLEC) f. Macrocyclic Antibiotics g. Proteins
- Enantiomer Separation
1. chiral stationary phase 2. chiral mobile phase additive 3. diastereoisomeric derivative
- Irving Wainer Classification
a. Type I CSP : attractive interactions. hydrogen bonds/- interactions/dipole stacking.
b. Type II CSP : attractive interactions and inclusion complexes . cellulose derivatives.
c. Type III CSP : chiral cavities /inclusion complexes. cyclodextrin /poly(triphenylmethyl methacrylate).
d. Type IV CSP : diastereomeric metal complexes/Chiral Ligand Exchange Chromatography
e. Type V CSP : Proteins, hydrophobic and polar interactions
Chiral Column Selection Guide_Optical Isomer
23
Aromatic -N< -COOH Group I no no no
Group G Group H
yes yes
-N< -COOH -OH Group F no no no
Group D Group E
yes yes
yes
-COOH -OH Group C no no
yes
Group A Group B
yes yes Group J
Asymmetry center : S, P, B, N,…
∝
A.3 Efficiency/Speed
5000<N<25000
Clength ↑ N ↑ Dparticle↓ N ↑
R ↑
Minimize Extra Column Volume Mixing Performance
Optimum Velocity(Flow Rate)
24
25
Column의 분리 능 vs 선 속도 : Van Deemter Plot
Optimal Linear Velocity
26
Column Dimension Downscale : N/Res.
Peptide Mapping
27
Small Particle or Large Particle의 특성
HETP
선 속도
5um
2um
TR
N
5um
2um
HETP vs u TR vs N
Column Permeability Large Peak Capacity => Large Particle Size Very Fast Separation => Small Particle Size
5. Reversed Phase Column 선택 하는 법 1
Reversed Phase Column 선택 하는 법 2
30
Polar, Mid to Non polar Compounds
31
Polar Compounds
32
Basic Compounds
33
Reversed Phase Column 선택 하는 법 3 : SRM 870
34
Appendix . DAC Column/YMC Prep LC
35
Prep Process
Part II MSMS based 주요 기능 및 분석 요약
MSMS market survey용
1. History of Mass Spectrometer
2. Mass Spectrometer의 구성및 종류
3. Mass Spectrum 해석
4. MS의 주요 Data, 기능 및 Spec.
* 농약시료의 분석 특징 5. Sample Matrix Effects in Pesticide Analysis
6. MSn to MSMS, Datadase구축? 3.1 MRM mode 3.2 Product Ion Scan(SIM/SCAN) 3.3 Information/Data Dependant Analysis 3.4 MS3 의 활용 3.5 Precusor Ion Scan (SCAN/SIM), Neutral Loss (SCAN/SCAN) 3.6 Mass Defect Filtering
7. MSMS의 주요 응용 영역
1. History of Mass Spectrometer
37
Thomson J.J. Cathode/Canal Rays_ Electron/Isotope Philosophical Magazine, 1897, 44:293
Product 의 발전
1946 Time of Flight 1949 Ion Cyclotron Resonance(ICR) 1953 Quadrupole Analyzer 1956 GC MS 1966 Chemical Ionization, Tandem Mass Spectrometry 1968 Electrospray Ionization Collision Induced Dissociation 1975 APCI 1978 TQ Mass Analyzer 1985 MALDI 1989 ESI
응용의 발전
1980-1990 BIO(P/P, Olignucleotide,…) 2000-2010 Food Safety/Environmental,… Impurity Profile
Cathode Ray : Electron
Anode Ray : Isotope
+ +
2 Electrons
2 Protons 1 or 2 Neutron
2. Mass Spectrometer의 구성및 종류
Mass Spectrometer는 시료중의 Target 성분들을 Ion Source에서 가스 phase 이온(m/z)으로 만들고 가속 Focus하여 Mass Filter의 진공 안(10-5-10-7 torr)에서 수 ms-수백 ms 의 빠른 속도로 진행하는 이온분자(m/z)의 경로및 속도차이로 전개 분리하여 이온/빛 증폭 검출기에서 증폭 검출한 Mass Spectrum 을 제공하는 장비입니다
38
Liquid Chromatography
Ionization Source
Mass Analyzer Detector/ 이온분자 Collection
ESI APCI APPI ,…
•Quadrupole •TOF •Ion Trap
•Hybrids ; QQQ, Q-Trap Q-TOF, TOF-TOF LTQ-Orbitrap ,…
•Many columns •Many solvent
Vacuum •Electron Multiplier, •Photo Multiplier •,..
QqQ의 세부 구성및 작동
Ion detection
Ion filtering
Ion processing
Ion filtering
Ion transport
Ion production
39
MSMS, QqQ
RF only Collision Cell
Scanning RF/DC
Scanning RF/DC
RF only
2.1 Ion Source의 종류
AP-ESI : Atmospheric Electro Spray Ionization, AP-CI : Atmospheric Chemical Ionization AP-PI : Atmospheric Photo Ionization MMI : Multi Mode Ionization Nano ESI : Nano ElectroSpray Ionization CHIP source MALDI : Matrix Aided Laser Desorption & Ionization Fast Atom Bombardment (FAB) Electron Ionization (EI, or electron impact) Chemical Ionization (CI) ,…
40
Ion Source의 응용 별 선택
41
Ion Source 작동원리_ESI
Orthogonal design & Curtain Gas
Resistant to dirty matrix
Ruggedness / reproducibility
42
모 분자 이온, Adduct & Cluster
+ + +
+ + + + + +
+ -
- - - -
N2 Nubilized Charged Droplets
N2 Sprary+Capillary Voltage
Rayleigh
Limit
Reached
+ + +
+ +
+ +
+ +
+ - - - - -
In source CID or MS/MS ?
Solvent Ion Cluster
Salts/Ion pair adducts
Neutrals adducts
Evaporation
Heated Drying N2
+ 모분자 Ion / Isotope / Frag.
Scan & SIM
43
Coulomb Explosions
+ +
+ + + + - -
+ +
+
+ + + - - +
LC EI MS w/supersonic molecular beams
44
1. Library searchable EI mass spectra 2. Enhanced molecular ion and mass spectral structural and
isomer information 3. Highly non polar samples 4. Low Ion suppression 5. Automatic sample identification : the elimination of matrix
effects and the use of deconvolution software 6. No nitrogen gas generator 7. Both GC-MS and EI-LC-MS 8. True molecular ion is provided unlike in ESI or APCI which
enables easier structural elucidation with MS-MS
2.2 MS Filter의 종류
Quadrupole Sector Time of Flight FT-ICR Ion Trap (2D, 3D) Orbitrap
RF-DC Voltage Generator
45
Mass Filter의 작동 원리(속도/경로차)
+
+
DC
Octapole
- round focusing
DC
Sector Mass
Quadrupole
Time of Flight
+
+
1-3 ppm 이하
Beam Shaper
- line focus
DC+RF
100-200 ppm
46
a. Mass Accuracy의 의미
Mass(m/z) Accuracy 와 Fragmentation Spectrum
Formula Feature/MW에 의한 Database search
=> HRMS
SIM 정량=>SQ, MSn 정성=> (Linear) Ion Trap
Fragmented library, Screening Search
=> SQ, TOF (w/LC separation) Soft fragmented library Or Amart-flag
=> Ion Trap (2D/3D, Linear IT)
b. Fragmentation Spectrum의 의미
MSn
SIM/SCAN
In Source CID/SCAN
SIM/SCAN
47
Mass Filter 의 응용 별 선택
48
2.3 증폭검출기(Multiplier)의 종류
49
GP이온
Micro-channel Plate
GP이온
Electro-Multiplier : Off-Axis Photo-Multiplier : V or W
e- hv
3. Mass Spectrum 해석_Chloride _MW35.453 g/mol
50
Cl_odd ion_EI
Cl2_odd ion_EI_70.906
34.969
69.938
Chloramphenicol_C11H12Cl2N2O5 MW323.132 g/mol
51
+ H+
C11H12Cl2N2O5+H+ _Even Ion_ESI
C11H14N2O5+H+_Even Ion_ESI
Malathion_C10H19O6PS2 MW330.358 g/mol
52
ESI TOF in-source CID_ESI
Adduct Ion _ ESI
53
Cluster Ion _ ESI Gas Phase Ions
54
Parent Ion Fragment Ion Cluster Ion?
Protein/Peptide Mass Spectrum _ De-convolution
m/z
Ab M+Z+2
M+Z+1
M+Z
M+Z+3
M+Z+4
P1 P2
1000 1500
100000
120000
20000
40000
60000
80000
706.8
5
726.2
5
746.1
5
767.5
5 7
89.9
5
839.4
5
866.4
5
895.4
5
926.1
5
959.1
5
994.8
5
1032.9
5
1074.1
5
1118.8
5
1167.5
5
1220.4
5
1278.7
5
1342.6
5
1413.2
5
1579.6
5
26700
26800
26900
27000
200000
400000
600000
800000
1000000
Deconvoluted spectrum
MW = 26828.84
Equation for
Molecular Weight Calculation
P1 = (M+Z) / Z
P2 = (M+(Z+1) / Z+1
.
.
De-convolution Software
55
MSMS Terminology _ Separation/Purification
Orthogonal In-source CID EI/CI ESI/APCI/APPI MALDI
Odd Ion Even Ion Cluster ion m/z
Separation 1 SQ TOF Trap(3D) LIT(2D)
Qual/Quan Matrix MC Isotope LC Library/DB Deconvolution Profiling s/w PCA
Separation 2 SQ TOF
Trap(3D) LIT(2D)
Orbitrap
Detection EM PM MCP Off-axis
Frag. CID Ar N2 Triwave Ion Mobility
Focus Accelation Ion Mobiility
Accurate Mass : 1-수백 ppm, SIM Chromatogram/SCAN Spectrum, Formula, Structural information, Isotope
Ratio,…
Mass Resolution : FWHM, HR/LR
Identification Point System
m/z Separation : gas phase 분자이온의 경로/속도차
분석시간 : 수 ms- 수 십 ms
정성(m/z)/정량
감도 Sub PPB (LOQ), LOD
진공 10-5-10-7 torr
Sample 주입
Ion Guide
Ion Source MS 1 MS 2 Cell Multiplier
Data System
GC LC CE ICP DIP
MSMS market survey용
Target 정제 Solution
분취
Extraction
시료 전처리
Column
,…
Signal Data Spectrum
PDA
MS
MSMS
,…
4. MS의 주요 Data, 기능 및 Spec.
Mass Filter 주요기능
SQ TOF Ion Trap(3D) 정성/정량
SIM chromatogram Yes No XIC
Yes 정량
MS Scan spectrum Yes Yes(Resolution) Yes(Trap) 정성
MSn No
In-source CID
No In-source CID
Post source decay Yes 정성
Mass Resolution 1Da 수 mili-Dalton 0.1 Da 정성
Mass Accuracy 수백 ppm 수 ppm 수십ppm 정성
•MSn : MRM(SIM/SIM)/Scheduled MRM, SRM, MS3, Product Ion Scan(SIM/SCAN), Precusor Ion Scan(SCAN/SIM), Neutral Loss(SCAN/SCAN),
Information/Data dependant analysis
MSMS market survey용
Enhanced MS Scan _ Deconvolution
MSMS market survey용
MS Scan sensitivity Pure Protein from Prep
LC ?
Ab M+Z+2
M+Z+1
M+Z
M+Z+3
M+Z+4
MW1 MW2
Calculation Equation
MW1 = (M+Z) / Z
MW2 = (M+(Z+1)) / Z+1
. .
M = 26.83K Da
농약시료의 분석 특징
MSMS market survey용
분석 항목 제재물 잔류물
성분 Raw materials Impurites 보조제
Raw materials Impurities 변환체(생물학적, 화학적,…)
시료의 이력 GMP의 Products 일반식품 생산/유통과정의 결과물
시료의 형태 제품 다양함
시료전처리 대부분 필요없슴 항상 필요함
재현성 중요함 ≤ 10-30%
감도 0.01-수% ppt-ppb-ppm
정성 Stds 대비 RT Compound Specific Information => Scan Spectrum(MS, NMR, IR), Parent/Product Ion Ratio,…
구조확인 Std Area 대비 0.1%이하
필요함(Toxicology)
5. Sample Matrix Effects in Pesticide Analysis
Targets in Sample Matrix : solid/liquid/gas polar/non-polar/ionic/small/large compounds
추출 Clean-up
정성 정량
용매 추출 회수율 방해요인 1. 고체 덩어리 2. 추출 용매에 녹지 않는 성분 3. 온도 변화, Chemical Reaction,…
선택적인 검출/정량 방해요인 1. Signal Addition : Ghost peak, RT shift, => Matrix Matched Calibration 2. Signal Suppression : Ion Suppression,… => Isotope Labeled Standard Calibration
MSMS market survey용
Reducing and Changing of Sample Matrix(Interference)
1. Universal Extraction : 다 성분 분석, 농약 원재료 잔류물 분석등
Sample Matrix 용매추출
Interference 석출법 Chemical
Dispersive Solid Phase
Freeze Drying 재 용해,
필터 후 분석
2. Partial Extraction : 부분 성분 분석, 농약 Metabolites 잔류물 분석등
Sample Matrix 용매추출 Interference 제거
SPE흡착용 용매로 희석 On-line SPE?
SPE 선택적 흡착 선택적 용리
필터 후 분석
3. Selective Extraction : Impurity 분취, Prep Process(Prep LC, HPCPC,…) , ImAC(Mycotoxin,…), MIP, …
MSMS market survey용
시료전처리 종류 및 분석장비
Sohxlet Precipitation Ultrafiltration
LLE (On/Off–line, Micro)
QuEChERS
GPC/RAM SPE
(On/Off–line, Micro)
ImmunoAC
MIP
LC MSMS GC MSMS
(Compound Specific Information)
LC MS GC MS
(RT, 유도체화)
LC FLD LC UV/Vis
GC ECD/NPD GC FID
Multi-Residue Selectivity
Clean-up & Concentration Matrix Effect Specificity Metabolites
추출 Clean-up
정성 정량
MSMS market survey용
6. MSn to MSMS, Datadase구축?
A. MSMS in Time : 농축(Trap/Enhanced)/Mass필터 - Ion Trap(3D) : 1/3 Cut-off - Ion Trap(3D)-TOF - Q-Trap (Hybrid Linear Ion Trap) - LTQ-Orbitrap (Hybrid Linear Ion Trap) B. MSMS in Space - QQQ - Q-TOF - TOF-TOF
MSMS market survey용
6.1 MRM mode
추가적인 정성
MSMS market survey용
Target Quan/Qual
Agilent QQQ
많은 시료, 다 성분 screening : UPLC+sMRMmode
MSMS market survey용
High Throughput ?
AB Sciex
6.2 Product Ion Scan (SIM/SCAN)
MSMS market survey용
Metabolic route에 따른 Mass Shift
Phase I Metabolism Phase II Metabolism
Mass Shift Group Reaction Mass Shift Group Reaction
P32 O2 Oxidation at C, N, S
P305 Glutathion Conjugation
P16 O P192 GluA Glucuronidation
P14 O-H2 Double bond
P176 GluA Glucuronidation
N2 -H2 P80 SO3 Sulfation
N14 -CH2 dealkylation
P42 CH2CO Acethylation
N28 -C2H4
N30 -O2+H2
-H2O -C4H4
Double Bond Oxidation
Ring Opening
MSMS Scan Sensitivity Database ?
Flonicamid and metabolite of 파프리카
6.3 Information/Data Dependant Analysis
MSMS market survey용
Thermo Finnigan LTQ
AB Sciex Q-Trap
MSMS 주요기능 Q1 Q2 Q3
ePI Scan SIM Fragment Trap, Scan
MS3 SIM Fragment Trap, Isolate, Fragment, Scan
SRM SIM Fragment Scan
Information/Data Dependant Analysis
MS SCAN and MSMS SCAN switching Inclusion/Exclusion list
MSMS market survey용
764
764
Survey SCAN
MS-MS SCAN
Database 비교?
Information/Data Dependant Analysis
MS and MSn switching, Inclusion/Exclusion list
MSMS market survey용
MS Scan(Baseline noise) Exclusion : 402.3, 418.4
MS Scan(Target+Baselin) Inclusion : 262.1, 358.9,
376.2
New metabolite MW 376 in Buspirone And Target ID
Information/Data Dependant Analysis
SRM and MSMS SCAN switching Quantitation and Identification/Confirmation
MSMS market survey용
SRM 262(Buferalol) and 278(M1, M2, M3,…)
SRM Triggered Enhanced PI MSMS
Precusor Ion Scan/ ePI Scan(remikiren-OH)
MSMS market survey용
(A) Precursor ion spectrum of the peak at 3.2 min (B) Enhanced product ion spectrum of the precursor ion
at m/z 647
(A) precursor ion scan trace of m/z 254 (B) enhanced product ion trace
JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2004; 39: 845–855 Published online inWiley InterScience (www.interscience.wiley.com). DOI:
10.1002/jms.659
6.4 MS3 의 활용
The ion in the MS3 spectrum at m/z 274.1549
suggests GSH addition to the 2-chloro-4-piperazin-1-
yl-phenol moiety.
MSMS market survey용
6.5 Precusor Ion Scan (SCAN/SIM)
MSMS market survey용
MS1 CID MS2
Tramadol m/z 264 => m/z 58
Neutral Loss (SCAN/SCAN)
MSMS market survey용
MS1 CID MS2
Neutral Loss 176 Da Glucuronic acid 80 Da Sulfate conjugate
132 Da ,…
Scan TIC
Neutral Loss
6.6 Mass Defect Filtering
Mass Resolution/Accuracy?
MSMS market survey용
Waters Q-TOF
Metabolic route에 따른 Mass Shift
Phase I Metabolism Phase II Metabolism
Mass Shift Group Mass Defect Shift Mass Shift Group Mass Defect Shift
P32 O2 -0.0102 P305 Glutathion 0.0682
P16 O -0.0051 P192 GluA 0.027
P14 O-H2 -0.0106 P176 GluA 0.0321
N2 -H2 -0.0157 P80 SO3 -0.0432
N14 -CH2 -0.0157 P42 CH2CO 0.0106
N28 -C2H4 -0.0313
N30 -O2+H2 0.0055
0.04 Da 이하 0.07 Da 이하
Mass Defect Filtering
MSMS market survey용
MSn with mass defect filtering for in vitro and in vivo metabolite identification
Signal Purification
TIC
XIC
Mass Defect
Unfiltered Spectrum
MDF spectrum
PCA, Profiling,…
7. MSMS의 주요 응용 영역
a. Protein/Peptide
b. Metabolite
c. Natural Product Profiling
d. Unknown Analysis : Impurity 분석
e. Target Analysis : 유해 Regulation성분
f. etc, …
MSMS market survey용