1. LC MSMS based 주요 기능 및 분석 요약
MSMS market survey
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2. 내용 MS의 주요 Data, 기능 및 Spec. 2. 시료 전처리 및 분취의 필요성 3. MS, MSMS, MSn 의 응용
* MSMS Terminology
MS의 주요 Data, 기능 및 Spec.
* 농약시료의 분석특징
2. 시료 전처리 및 분취의 필요성
3. MS, MSMS, MSn 의 응용
* Conventional LC vs UPLC
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
4. MSMS의 주요 응용 영역
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3. MSMS Terminology _ Separation/Purification
Sample 주입
Ion
Source
Ion
Guide
Data
System
MS 1
Cell
MS 2
Multiplier
Orthogonal
In-source CID
EI/CI
ESI/APCI
APPI
MALDI
Odd Ion
Even Ion
m/z
Frag.
CID Ar N2
Triwave
Ion Mobility
Separation 2 SQ
TOF
Trap(3D)
LIT(2D)
Orbitrap
Separation 1 SQ
TOF
Trap(3D)
LIT(2D)
Detection EM PM
MCP
Off-axis
Qual/Quan
Matrix MC
Isotope RC
Library/DB
Deconvolution
Profiling s/w
PCA GC LC CE
ICP
DIP
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 torr
Target 정제
Solution 분취
Extraction
시료 전처리
Column ,…
Signal Data
Spectrum
PDA MS
MSMS
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4. 1. MS의 주요 Data, 기능 및 Spec. Mass Filter 주요기능 SQ TOF Ion Trap(3D) 정성/정량
SIM chromatogram
Yes No
XIC 정량
MS Scan spectrum
Yes(Resolution)
Yes(Trap) 정성
MSn
In-source CID
Post source decay
Mass Resolution
1Da
수 mili-Dalton
0.1 Da
Mass Accuracy
수백 ppm
수 ppm
수십ppm
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

5. Enhanced MS Scan _ DeconvolutionAb
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
MS Scan sensitivity
Pure Protein from Prep LC ?
MSMS market survey용

6. 농약시료의 분석 특징 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)
MSMS market survey용

7. 2. 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
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8. 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
Selective Extraction : Impurity 분취, Prep Process(Prep LC, HPCPC,…) , ImAC(Mycotoxin,…), MIP, …
MSMS market survey용

9. 시료전처리 종류 및 분석장비 Sohxlet LC MSMS Precipitation GC MSMS Ultrafiltration추출
Clean-up 정성 정량
Sohxlet
Precipitation
Ultrafiltration
LLE
(On/Off–line, Micro)
QuEChERS
GPC/RAM
SPE
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
MSMS market survey용

10. 3. 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

11. Conventional LC vs UPLC : Column 충전물
Porous Beads
Non-Porous Beads
Totally Porous, Superficially Porous, Surface Porous

12. Conventional LC vs UPLC : Van Deemter Plot
Optimal Linear Velocity
Column
Downscale

13. Target Quan/Qual
3.1 MRM mode
Agilent QQQ
추가적인 정성
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14. 많은 시료, 다 성분 screening : UPLC+sMRMmode
High Throughput ?
AB Sciex
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15. 3.2 Product Ion Scan (SIM/SCAN)
Metabolic route에 따른 Mass Shift
Phase I Metabolism
Phase II Metabolism
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 N2
-H2
P80
SO3
Sulfation
N14
-CH2
dealkylation
P42
CH2CO
Acethylation
N28
-C2H4
N30
-O2+H2
MSMS Scan Sensitivity
Database ?
-H2O
-C4H4
Double Bond
Oxidation
Ring Opening
MSMS market survey용

16. 3.3 Information/Data Dependant Analysis
Thermo Finnigan LTQ
AB Sciex Q-Trap
MSMS 주요기능 Q1 Q2 Q3
ePI Scan
SIM
Fragment
Trap, Scan
MS3
Trap, Isolate, Fragment, Scan
SRM
Scan
MSMS market survey용

17. Information/Data Dependant Analysis
MS SCAN and MSMS SCAN switching
Inclusion/Exclusion list
764
MS-MS SCAN
Survey SCAN
Database 비교?
764
MSMS market survey용

18. Information/Data Dependant Analysis
MS and MSn switching, Inclusion/Exclusion list
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
MSMS market survey용

19. Information/Data Dependant Analysis
SRM and MSMS SCAN switching
Quantitation and Identification/Confirmation
SRM
262(Buferalol) and 278(M1, M2, M3,…)
SRM Triggered
Enhanced PI MSMS
MSMS market survey용

20. Precusor Ion Scan/ ePI Scan(remikiren-OH)
precursor ion scan trace of m/z 254
enhanced product ion trace
(A) Precursor ion spectrum of the peak at 3.2 min
(B) Enhanced product ion spectrum of the precursor ion at m/z 647
JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2004; 39: 845–855
Published online inWiley InterScience ( DOI: /jms.659
MSMS market survey용

21. 3.4 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

22. 3.5 Precusor Ion Scan (SCAN/SIM)
MS CID MS2
Tramadol m/z => m/z 58
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23. Neutral Loss (SCAN/SCAN)
MS CID MS2
Scan TIC
Neutral Loss
Neutral Loss
176 Da Glucuronic acid
80 Da Sulfate conjugate
132 Da ,…
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24. Metabolic route에 따른 Mass Shift
3.6 Mass Defect Filtering
Waters Q-TOF
Mass Resolution/Accuracy?
Metabolic route에 따른 Mass Shift
Phase I Metabolism
Phase II Metabolism
Mass Shift
Group
Mass Defect Shift
P32 O2
P305
Glutathion
0.0682
P16 O
P192
GluA
0.027
P14
O-H2
P176
0.0321 N2
-H2
P80
SO3
N14
-CH2
P42
CH2CO
0.0106
N28
-C2H4
N30
-O2+H2
0.0055
0.04 Da 이하
0.07 Da 이하
MSMS market survey용

25. Mass Defect Filtering PCA, Profiling,… TIC Unfiltered Spectrum XIC
Signal
Purification
MDF spectrum
Mass Defect
PCA, Profiling,…
MSn with mass defect filtering for in vitro and in vivo metabolite identification
MSMS market survey용

26. 4. MSMS의 주요 응용 영역 Protein/Peptide Metabolite Natural Product Profiling
Unknown Analysis : Impurity 분석
Target Analysis : 유해 Regulation성분
etc, …
MSMS market survey용