Separation Purification Basics

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Separation_Purification Basics CPC_ HPLC_ Prep LC_ MSMS

Bene Technology Co., Ltd. (사내교육용)

사내교육용

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내용

1. Separation/Purification

- Preparation Process

2. CPC

- CPC의 장점

3. HPLC/UHPLC/Prep LC

- 분리 원리 및 C18/ODS/RP18

4. LC MS/MSMS

- MS의 주요 응용

사내교육용

1. Separation/Purification

H P L C

용매, Isocratic HP/LP Gradient Pump, 시료주입, 컬럼, Chromatogram(UV/Vis,…) Spectrum(MS,…), Validation(IQ/OQ/PQ),

Prep, Scale-up, 유로, Automation, Compound Specific Information, MRM/SRM

Isotope, Profiling, DDA/IDA,…

PREP L C

CPC

LC

MSMS

, . . .

Qualification/Quantification

What

How

Why QC

구조분석 Library구축

Metabolomics Bio Activity

,… 생산

Impurity 유해물질 분석

,…

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Preparation Process 예

Nature Related Products

Extraction

Crude Extract

Isolation

Fraction 1 Fraction 2 Fraction 3

Active ? Active ? Active ?

Pure ? Pure ?

More Isolation

Structural Elucidation

Wastes

Scale-up

Bioactive Test

Purity Test

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Separation 원리/분배계수

분배계수

KA = [A]u phase/[A]l phase

KA = [A]sol phase/[A]lliq phase

CPC

small particle Column

사내교육용

Serial Liquid-Liquid Extraction Chromatography

6 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

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2. CPC in HPLC

Partition Disc

Partition Disc Pack

Partition Channels 사내교육용

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CPC 의 내부구조 및 작동

Upper Rotary Joint

Lower Rotary Joint

Descending By-Pass

Ascending

Control Panel

CPC 240

사내교육용

CPC 의 종류 (CPC 80/240/1400/5400)

(18 Discs)

(12 Discs)

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CPC prep 응용 분야 및 분취

CPC system

Column

Volume

(ml)

Partition Cell

(개) 분리유속

분리 시

일반

Rotation Speed

시료 주입량

(이론 치 1%, ml)

CPC 80 80 712 5-10 ml/mn 1500 0.8

CPC240 240 2,136 5-20ml/mn 1500 2.4

(100mg-1g)

CPC1400 1,400 1,296 25-50ml/mn 1100 14

(100mg-20g)

CPC5400 5,400 1,040 100-200ml/mn 1000 54

주요 응용산업 분야

: Nutraceutical, Fine Chemicals, Pharmaceutical, Bio-Medical, Biotechnology, Fats and Oils, Fermentation 주요 성분

Saponins, Alkaloids, Chlorophylls, Tannins, Carotenoids, Phospholipids, Fat soluble vitamins, Mono/Oligo-saccharides, Anthocyanins, Lignans, Phenolic compounds, Synthetic Compounds, Other active compounds present in plants(e.g., herbs and spices) and protein/peptide…

사내교육용

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Scale up Strategy

Target

Amount

응용 영역

ug level Enzyme 분리 등

mg level Biological/biochemical testing

구조 및 특성 연구 : 생산공정 불순물, 대사체, 천연물

g level STDs제조

독성연구 : 고 순도 주 성분, side product

kg level 산업 생산, 상품화(Drug, 활성물질 등)

High 분리능, 압력, 비용 Low

표준

주입량

PS um

ID(mm)

Spherical Irregular

5(9만) 10(4만) 15(2만) 20(만) 50(5천) 230/70mesh

연구분야 4.6, 6.0 O O O O O O

수십 mg 10, 20 O OO O O O O

수백 mg 50 O OO O O O O

수 g 100, 150 O O O O O

수십 g 200 O O O O

수백 g 300 이상 O O O 사내교육용

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2.1 CPC의 장점 1 : High Sample Load

0

10

20

30

40

50

60

70

80

90

100

Mobile

phase

Stationary

Phase

Silica

(충전제)

Total

Volume

CPC

LC Column

% Mobile phase

Stationary Phase

Silica (충전제)

Total Volume

CPC 25 75 0 100

LC Column 60 5 35 100

Flow Direction

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CPC의 장점 2 : 다양한 분리용매

•Triphasic System • Beta CD • Ionic Liquid, …

Biphasic Solvent System

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CPC의 장점 3 : Biphasic Elution/No Loss

Very polar

Very Hydrophobic

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Descending vs Ascending

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CPC장점 4 : Biphasic Extraction/Injection System

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Solvent Density(g/ml)

Hexane 0.695

Ether 0.708

Toluen 0.867

Ethyl Acetate 0.897

H2O 1

Dichloromethane 1.325

Chloroform 1.492

사내교육용

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3. HPLC/UHPLC

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

…

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UPLC(sub 2um-2.5um)의 History

1. Waters Acquity UPLC ® (2004)-15,000 psi – UPLC H-Class* and Bio* (2010) 2. Jasco Extreme LC (2004) -15,000 psi 3. Thermo Accela* (2006) -15,000 psi – Accela 1250* (19,000 psi, 2010) 4. Dionex Ultimate 3000* (2008) -12,000 psi – (15,000 psi, 2010) 5. Perkin Elmer Flexar* (2009) -18,000 psi 6. Agilent 1290 Infinity (2010) – 18,000 psi – 1260* (9,000 psi, 2007) 7. Shimadzu Nexera (2010) – 19,000 psi

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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,…

사내교육용

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3.1 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

사내교육용

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Column 충전물의 구조

Porous Beads Non-Porous Beads

Totally Porous, Superficially Porous, Surface Porous 사내교육용

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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

사내교육용

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) 23 사내교육용

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3.2 분리모드 1 ; A, B, C term 이해

A. Eddy Diffusion : 경로차

B. Longitudinal Diffusion : 확산

C. Mass Transfer : Pore Surface Interaction Lateral(수평) Diffusion

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Van Deemter Plot

사내교육용

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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

사내교육용

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분리모드 3 ; Resolution

Theoretical Plate Selectivity Retention Factor

K0 , T0 Time

Response

TR1 TR2

사내교육용

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

28 사내교육용

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, …

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↑

사내교육용

Mobile/Stationary Phase (C18, C8, C4) Strength

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∝

A.2 Selectivity

α > 1.2

α=KA’/KB’

How to Interact with compound

- Stationary/Mobile Phase - pH

- Temperature - Additives

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Chemical Selectivity : C18 end capping process

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TMS => - CH3

Batch Synthesis

Flow Synthesis/Microreactor : Tri step end-capping

사내교육용

Anthocyanin/Anthocyanidin

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Chiral Column Selection Guide_Optical Isomer

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-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

사내교육용

Chiral Column Selection Guide_Optical Isomer

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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,…

사내교육용

∝

A.3 Efficiency/Speed

5000<N<25000

Clength ↑ N ↑ Dparticle↓ N ↑

R ↑

Minimize Extra Column Volume Mixing Performance

Optimum Velocity(Flow Rate)

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Column의 분리 능 vs 선 속도 : Van Deemter Plot

Optimal Linear Velocity

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Column Dimension Downscale : N/Res.

Peptide Mapping

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Small Particle(분석연구) or Large Particle(분취생산)의 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

사내교육용

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Dynamic Axial Compression Column (YMC Prep)

사내교육용

3.3 Reversed Phase Column 선택 하는 법 1

41 사내교육용

Reversed Phase Column 선택 하는 법 2

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4. LC-MS/MSMS

Purification : Prep

Column Chemistry Separation

=> NPC, RPC, IEX, GPC,…

Resolution

분석시간 : 수 분-수 십분

정성(RT)/정량

감도 Sub PPM : UV/Vis

SQ

Accurate Mass: Formula, Structural information

m/z Separation

=> gas phase 분자이온의 경로/속도차

FWHM

분석시간 : 수 ms- 수 십 ms

정성(m/z)/정량

감도 Sub PPB (LOQ)

Mass Spectrum Chromatogram

Compound specific Information

Q-TRAP

LTQ Orbitrap

43 사내교육용

LC-MS/MS 의 Data

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Abundance

1. 모분자 m/z

2. Fragment ion m/z

사내교육용

Data Mining Profile/Statistic/PCA

LC MS/MS가 필요한 이유

• HPLC가 필요한 이유

– 온도에 약한 물질

– 비 휘발성물질

– Analysis of more polar compounds without derivatization.

– Analysis of significantly higher masses

– Reduction of lengthy clean-up

• MS/MS가 필요한 이유

– Compound Specific Information

– Structural elucidation

– Further reduction of clean-up (?)

– Specificity ; Reducing Matrix Effect

– MS & MSMS modes : SCAN, SIM, Fragmentation

– Data Mining

45 사내교육용

4.1 History of Mass Spectrometer

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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 Photons And 1 or 2 Neutron

사내교육용

4.2 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

47 사내교육용

4.3 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

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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 •,..

사내교육용

QqQ의 세부 구성및 작동

Ion detection

Ion filtering

Ion processing

Ion filtering

Ion transport

Ion production

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MSMS, QqQ

RF only Collision Cell

Scanning RF/DC

Scanning RF/DC

RF only

사내교육용

4.4 MSn to MSMS, Datadase구축?

A. MSMS in Time : 농축(Ion 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

사내교육용 50

4.4.1 MRM mode

추가적인 정성

사내교육용

Target Quan/Qual

Agilent QQQ

51

많은 시료, 다 성분 screening : UPLC+sMRMmode

사내교육용

High Throughput ?

AB Sciex

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여러 가지의 MSMS Mode

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Q1 collision cell Q2

Q2/MS2 Scan Focus-Focus-SCAN

Q1/MS1 SIM SIM-Focus-Focus

Product Ion Scan SIM-Frag-SCAN

Precusor Ion Scan

SCAN-Frag-SIM

Neutral Loss/Gain

SCAN-Frag-SCAN

MRM/SRM SIM-Frag-SIM

모 분자 이온 조합 ; Isotope, Adduct Ion 여러 모 분자 동시

특정 모 분자 이온

조각 이온 조합 ; 특정 모 분자 선택 구조 정보

특정 조각 이온 ; 여러 모 분자 구조정보

조각 이온 Scan : +/-특정 Neutral ; 여러 모 분자 추적 특정 조각 이온 ; 여러 모 분자 추적 Parent /Daughter Ion

검출 MSMS Mode

사내교육용

4.4.2 Product Ion Scan (SIM/SCAN)

사내교육용

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 ?

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Flonicamid and metabolite of 파프리카

55 사내교육용

4.4.3 Information/Data Dependant Analysis

사내교육용

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

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Information/Data Dependant Analysis

MS SCAN and MSMS SCAN switching

Inclusion/Exclusion list

사내교육용

764

764

Survey SCAN

MS-MS SCAN

Database 비교?

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Information/Data Dependant Analysis

MS and MSn switching, Inclusion/Exclusion list

사내교육용

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

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Information/Data Dependant Analysis

SRM and MSMS SCAN switching

Quantitation and Identification/Confirmation

사내교육용

SRM 262(Buferalol) and

278(M1, M2, M3,…)

SRM Triggered Enhanced PI MSMS

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4.4.4 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.

사내교육용 60

4.4.5 Precusor Ion Scan (SCAN/SIM)

사내교육용

MS1 CID MS2

Tramadol m/z 264 => m/z 58

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Neutral Loss (SCAN/SCAN)

사내교육용

MS1 CID MS2

Neutral Loss

176 Da Glucuronic acid

80 Da Sulfate conjugate

132 Da ,…

Scan TIC

Neutral Loss

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4.4.6 Mass Defect Filtering

Mass Resolution/Accuracy?

사내교육용

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

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Mass Defect Filtering

사내교육용

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,…

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4.5 MSMS의 주요 응용 영역

1. Protein/Peptide

2. Metabolite

3. Natural Product Profiling

4. Unknown Analysis : Impurity 분석

5. Target Analysis : 유해 Regulation성분

6. etc, …

사내교육용 65