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