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Analisis dengan LC-MS

Tri Joko Raharjo

Lingkup Materi
• Pengantar HPLC
• Pengantar MS
• Ion Source
• Mass Analyser
• Analisis Kimia dengan LC-TQD MS
• Analisis Kimia dengan LC-HRMS

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Pengantar HPLC

Kromatografi

• Teknik pemisahan komponen

dalam campuran
• Analit terdistribusi dalam fase
gerak (mobile phase) dan fase
diam (stationary phase)
• Untuk analisis kualitatif,
kuantitatif dan preparatif

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Kromatografi Kolom vs HPLC
Kolom
• How does the mobile phase flow?
(Gravity)
• What are the consequences?
• Slow  time consuming
• Peak broadening  low Rs
LOW PERFORMANCE
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HPLC
• High Pressure Liquid Chromatography
(HPLC) : high pressure to flow the liquid
into packed columns.
• Pressure: 500 psi (35 bar, 1 bar = 0,9868
atm)
• The early 1970’s
• HPLC up to 6,000 psi (400 bar)
• Improvement detectors
• Improvement of columns.
Performance
improvement
High Performance Liquid 6
Chromatography (HPLC).
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Kromatogram

• Plot respon detektor terhadap

waktu
• Data: tR: waktu retensi (kualitatif)
• Luas peak: peak area (kuantitatif)
• Tinggi puncak: peak height
(kuantitatif)
• Width: lebar peak

Selektivitas Vs Resolusi (Rs)

• The selectivity factor,a,
describes the separation of band a = (tR)B-tM)/(tR)A-tM)
centres,
• The resolution, Rs, of two
species, A and B, is defined as
• Baseline resolution is achieved
when Rs = 1.5

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Van Deemter plots

HETP = A + B / u + C u
u = average velocity of the
mobile phase.
A, B, and C = band broadening
factors
A - Eddy diffusion
B - Longitudinal diffusion.
C - Resistance to mass transfer.
HETP = L / N

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N menunjukkan Efisiensi pemisahan

Pemisahan kolom semakin efisien, jika:
1. N semakin tinggi (untuk HPLC minimal 2000)
2. H semakin rendah
3. Ukuran partikel semakin kecil  N semakin besar

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Fase diam di dalam kolom

Three primary HPLC separations :
• Polarity (NP Vs RP)
• Electrical Charge
• Molecular Size
• Specific interaction

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Polarity (NP Vs RP)

Normal Phase (NP) Reversed Phase (RP)

The hydrophobic compounds • The hydrophilic compounds
elute more quickly than do elute more quickly than do
hydrophilic compounds hydrophobic compounds

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Detektor
• UV - Ultraviolet
• ED - Electrical Conductivity
• RI - Refractive Index
• FD - Fluorescence
• MS - Mass Spec
• Mass to charge ratio (m/z)
• Allows specific compound ID
• Several types of ionization techniques
• ELSD - Evaporative Light Scattering Detector

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LC-MS LC-MSMS

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Pengantar MS

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Mass Spectrometry (MS)

• Analytical technique for identification,confirmation, quantitation of
compound based on the measurement of mass to charge ratio ( m/z )

• key is to be charge of the compound can be identification ms

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MS Principles: nominal vs. exact mass

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What is resolution in MS
• Resolving power
m
R m (FWHM)
m
m 10%

m 500
• Quadrupole MS R   833
m 0.6

m 500
• Orbitrap MS R   100 ,000
 m 0 .005

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What is Mass Accuracy

 Mass accuracy usually expressed as ppm or mDa error

mmeas  mtrue 6
m / z  10
mtrue
500.1  500.0 6
• Quadrupole MS m / z  10  200ppm 100 mDa error
500

• Orbitrap MS 500.10414 500.10314 6

m / z  10  2 ppm 1 mDa error
TOF MS 500.10314

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Why Do We Need Mass Accuracy: Molecular Formula

ppm error Number of hits
150 11
10052_520 #157 RT : 2.23 AV: 1 NL: 1.62E5
T : FT M S {1,1} + p ESI Full m s [100.00-1500.00]
m/z 127.07263 50 4
127.07263
C, N, O, H
10 1
100

95

10052_5 20 #118 RT : 1.67 AV: 1 NL: 1.91E6

90

85
T : FT MS {1,1} + p ESI Full ms [100.00 -15 00.00]
5 1
210
80

75
200

190 2 1
70
180

170
m/z 707.21625
65 160

150
C, N, O, H
60
Relative Abundance

140

130
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ppm error Number of hits

Relative Abundance

120

50 110 707.21625
C 30 H 31 O 11 N 10
100
45

150 38
90

40 80
723.19019
70
C 30 H 29 O 13 N 9
35
60

30 50

40
50 12
25 30

20

15
20

10 649.21008
C 27 H 33 O 13 N 6
695.20386
C 27 H 33 O 15 N 7
785.2348 6
C 30 H 45 O 15 N 10 10 3
0
650 700 750 800
10

5
m/z

128.07596
5 3
127.10697 127.41898 127.54161
0
127.0 127.2 127.4
m /z
127.6 127.8 128.0 2 2

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Element Exact Mass

H 1.007825
C 12.000000
N 14.003074
O 15.994915

288.0441 C9H21O2P1S3 Terbufos

288.0949 C13H21O3P1S1 Iprobenfos
0.1033 amu 288.1142 C15H17N4Cl1 Myclobutanil
288.1256 C11H20N4O3S1 Epronaz
288.1351 C11H21N4O3P1 Pirimethaphos
288.1474 C16H20N2O3 Imazamethabenz

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R = 100,000
R = 20,000
289.1390 100 289.1424
100
289.1022 289.1329 289.1547
90
90 289.0514

80 80
289.1215
70
70 289.1424
289.1021 289.1329
60 289.0514 60
Relative Abundance

Relative Abundance

50 289.1215 50

40 40

30 30

20 20

10 10

0 0
289.00 289.05 289.10 289.15 289.20 289.00 289.05 289.10 289.15 289.20
m/z m/z

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Importance of Mass Accuracy and Resolving

power

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Type Resolving Power

(FWHM)
FT-ICR-MS 1,000,000
FT-Orbitrap 100,000
High-Res-TOF 60,000
TOF 10,000
Quadrupole / 10,000
IonTrap in
UltraZoom mode
Quadrupole / 1,000
Iontrap

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Mass Spectrometer Intrumentation

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Ion Source

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Hard vs Soft Ionisation

Hard Soft

• GC-MS • LC-MS
• Electron impact
• Ion molecular
• Fragmentation
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Soft Ionization
• Several common modes differing by method of ion formation:
– Electrospray (ESI)
– Atmospheric Pressure Chemical Ionization (APCI)
– Atmospheric Pressure Photo-Ionization (APPI)
– New dual sources (ESI/APCI) or (APCI/APPI)
- Matrix Assisted Laser Adsorption (MALDI)

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Electrospray (ESI)
Counter electrode

Capillary
FLOW

High Voltage Power Supply

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More Negative Ions

than Positive Ions
More Negative Ions
than Positive Ions

Liquid
Flow

Positively
Charged
Taylor Cone Droplets

Electrospray Probe Tip

at high voltage

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• Droplets produced from the spray have a surface charge

• Surface charged droplets undergo solvent evaporation and droplet
fission to produce smaller droplets
• Like charge repulsion becomes greater than droplet surface tension
and fission occurs to produce smaller charged droplets

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Ion Desorption Mechanism

Charged Residue Mechanism

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Atmospheric Pressure Chemical Ionisation (APCI)

• Liquid flow is forced through a narrow capillary to give it a high linear velocity
• The APCI Probe heater combined with nebulizer gas then vaporises the liquid
flow
• The solvent and analyte vapour passes though the corona discharge region to
produce gas phase ions

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• Electrospray can operate in either positive or negative mode.

• Positive mode:
• Best suited to basic compounds that form a stable HCl salt.
• [M+H]+ is the primary ion formed
• [M+nH]n+ and [M+Na+]+ can also be formed.
• Negative mode
• Best suited to acidic compounds that form stable Na salts.
• [M-H]-, [M-nH]n-and [M+I-]-

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Mechanisms of Ion Formation

Positive Electrospray Ions

Negative ElectroSpray Ions

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Mass analyser

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• Low Resolution
• Quadrupole (Mass Filter, Ion
Trap)
• Quadrupole Tandem
Type Resolving Power
• High Resolution (FWHM)
• Time-of-Flight (Linier TOF, FT-ICR-MS 1,000,000
Reflectron)
FT-Orbitrap 100,000
• Double Focusing Magnetic
High-Res-TOF 60,000
Sector
TOF 10,000
• Orbitrap
Quadrupole / 10,000
• Ultra high resolution IonTrap in
• ICR ion cyclotron resonance UltraZoom mode
Quadrupole / 1,000
Iontrap

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Quadrupole
• Suitable for Quantitation and known compound confirmation Good
linearity
• Structural information

• Single Quad
• Mass confirmation
• Get molecular ion
• NO fragmentation

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When to use Single Quads

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Tandem Quad (MS/MS)

• Mass confirmation
• Fragmentation- for some structural information
• Structural confirmation
• More selectivity than Single Quad

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MS/MS

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Quadrupole
Advantages Disadvantages
• Inexpensive • Low Resolution (<4000)
• Easily Interfaced to Many • Low Accuracy (>100ppm)
Ionization Methods • MS/MS requires multiple
analyzers
• Low Mass Range (<4000)
• Slow Scanning

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Orbitrap (High resolution)

• orbitrap Orbitrap 2

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Analisis Kimia dengan LC-TQD MS

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Aplikasi MS
Qualitative
• Identification of unknown : HRMS or TOF
Quadruple : MS Scan (limited only m/z information)
• Confirmation of targeted compound:
Quadrupole : MS Scan, SIR, MS/MS, MRM
Quantitation
• Quadrupole SIR (LCMS)
• Quadrupole SIR, MRM (LC-MSMS)
• HRMS : (SIM, PRM)

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Confirmation of targeted compound

• Identification point (IP) : 4 IP • Ion molecular: 1 IP
• Each Transition (daughter ion) of
MS/MS : 1.5 IP

LC-MS/MS
Confirmed as a
compound if m/z of ion
molecular and 2 m/z of
daughter ion confirmed

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Mode Analysis TQD

• MS Scan
• SIR (Selected Ion Recording)
• MRM (Multiple Reaction Monitoring)
• Daughter Scan

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Scanning (Operating like a single quad)

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Setting parameters for MS Scan Mode

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Contoh Chromatogram MS Scan Mode

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Contoh Spectrum MS Scan Mode

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S to N pada MS Scan Mode

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SIR (Selected Ion Recording) Single Quad

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Setting parameters for SIR Mode

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Contoh Chromatogram SIR Mode

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Contoh Spectrum SIR Mode

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S to N pada SIR Mode

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MRM (Multiple Reaction Monitoring)

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Contoh kondisi MRM

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Setting parameters for MRM Mode

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Contoh Chromatogram MRM Mode

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Contoh Spectrum pada MRM Mode

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S to N pada MRM Mode

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Sensitivity of SIR vs MRM Carbofuran

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Product (Daughter) Ion Analysis

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Setting parameters for Daughter Mode

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Contoh Chromatogram Daughter Ion Mode

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Contoh Spectrum Daughter Ion Mode

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S to N pada Daughter Ion Mode

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Analisis Kimia dengan LC-HRMS

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Scan Mode
• Full scan (R=70,000, Max: 140,000)
Metabolomics / Lipidomics / Small molecule screening / Intact protein

• Full scan (R=70,000) + data dependent MS2 (R=17,500) (DDA)

Proteomics

• Targeted MS2 (R=17,000/35,000) (PRM)

Targeted quantitation / Quanformation

• Targeted SIM (Max: 140,000)

Targeted quantitation

• Data independent acquisition (R=17,500/35,000) (DIA)

High throughput targeted quanformation

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Workflow For Unknown Screening

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Workflow of Proteomics

LC-MS Data Data

Treatment Sample Pre Processing
Analysis Mining

Subcellular Separation; Digestion; Labeling; Peptide Fractionation; Desalting

DDA; MRM; DIA; Fragmentation: ETD/HCD/CID

Data Search: Sequest/MASCOT etc.

Functional Annotation/Pathways/Overrepresentation

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DDA Method
MS1 Top N peaks from MS1 Selected

1st MS2
2nd MS2
3rd MS2
4th MS2
...

[ Acquire MS2 data depends on the MS1 intensity

For discovery/non-targeted experiment ]

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ZhouLei_eGC1_5ul_FullMSddMS2 #19591 RT: 79.21 AV: 1 NL: 1.13E7

T: FTMS + p NSI Full lock ms [350.00-2000.00]
805.92627
z=2
100

90

80

70

elativeAbundance
60

50
644.30328
z=3
40 554.29718
R

z=1
843.43579
30 455.33417 z=?
z=1 690.34387
z=? 983.57703
20 z=1

10 1136.45715
z=2 1322.60962 1502.32751 1685.00903 1803.28552
z=? z=? z=? z=?
0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
ZhouLei_eGC1_5ul_FullMSddMS2 #19592 RT: 79.21 AV: 1 NL: 1.13E5
T: FTMS + p NSI d Full ms2 538.96@hcd30.00 [100.00-1670.00]
159.11290
z=1
100

90

80

70 ZhouLei_eGC1_5ul_FullMSddMS2 #19593 RT: 79.22 AV: 1 NL: 2.36E4

T: FTMS + p NSI d Full ms2 1082.53@hcd30.00 [100.00-2230.00]
RelativeAbundance

392.21429 701.40942
60 z=1
z=1
100
50 515.33044
200.13957
90 z=1
z=1
40
80 343.16104
z=?
30
70 801.37189
301.12952 ZhouLei_eGC1_5ul_FullMSddMS2z=1 #19594 RT: 79.22 AV: 1 NL: 2.13E4
20 401.28763
RelativeAbundance

z=1 T: FTMS 1129.59924

+ p NSI d Full ms2 782.88@hcd30.00 [100.00-1615.00]
60 z=1 885.53070 z=1
244.09296
512.19788
615.27332
z=? z=1
10 z=1
z=1
100
50
183.11311 130.08641597.28772
0 z=1 z=? z=1
90
200 40 400 600 800 1000 1200 1400 1600
1080.53528
m/z z=1
80 1308.64636
30 z=1
739.32703
70 ZhouLei_eGC1_5ul_FullMSddMS2 #19595 RT: 79.22 AV: 1 NL: 3.97E3
1521.75378
20 z=1
T: FTMS + p NSI d Full ms2 723.33@hcd30.00 [100.00-1495.00]
z=1
RelativeAbundance

60 909.46985
10 1835.21204 2093.78516
z=?
100 z=? z=?
50
0
200 400 90
600 800 1000 1200 1400 1600 1800 2000 2200
40 175.11888
294.18137 m/z
z=1 z=?
80 764.40491
30 494.25977 z=1
z=?

20 70
809.45410
RelativeAbundance

z=?
607.34601 1094.54468
10 60 z=? 776.39569
z=?
z=?

0 50
200 400 600 800 1000 1200 1400 1600
m/z
40 1257.58215
z=?
934.51245
276.13403 z=?
30
z=? 604.29236
444.83582 z=?
20 z=?

93
10

0
200 400 600 800 1000 1200 1400
m/z

How does the result look like

E:\Data\Test\20121031_RatLiver_150min 31/10/2012 8:54:34 AM

RT: 0.00 - 150.00

123.13 NL:
100 1.67E9
90 Base Peak
123.48 MS
80 20121031_
RatLiver_1
70
Relative Abundance

50mi n
60 14.38
43.05
50
59.37 65.99
40 15.67
29.59 59.04
41.23 49.66 83.51
30 69.88 75.88 142.88
13.68 16.54 54.91 61.54 144.16
20.98 132.72
20 90.30 98.53
27.56 37.73 107.84 112.14 142.15
13.02 126.13
10 119.25
0.22 10.45
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (min)

20121031_RatLiver_150min #14952 RT: 43.32 AV: 1 NL: 4.37E8

T : FT MS + p NSI Full ms [400.00-2000.00]
649.82361
z=2
100

90
80
Relative Abundance

70
60
50

40
30
20
448.23917 580.79761 716.35876 816.42767 895.47113
10 z=2 1075.56592 1160.58887 1298.63977 1660.04175 1784.01672 1906.63831
z=2 z=2
z=1 z=1 z=1 z=1 z=1 z=? z=? z=?
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z

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Peptide sequence

Extracted from: E:\Data\Test\20121031_RatLiver_150min.raw #14254 RT: 41.58

FTMS, HCD@27.00, z=+2, Mono m/z=738.84808 Da, MH+=1476.68889 Da, Match Tol.=100 mmu

350
y₅⁺
300 543.31464
Intensity [counts] (10^3)

250
[M+2H]²⁺+H
200 b₆²⁺-H₂O, b₃⁺ 739.39655
284.12430
y₁₂²⁺
150
645.79779 y₇⁺ y₁₀⁺
y₁⁺ y₂⁺ y₈⁺ y₁₁⁺
b₄⁺ 763.39838 1078.50464
100
147.11324 260.19751 892.44025 1193.53577
399.15060 y₅⁺-H₂O y₉⁺ y₁₂⁺
50 525.30170 1021.48047 1290.59949

0
200 400 600 800 1000 1200 1400
m/z

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Targeted SIM

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304.2773

m/z

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• By collecting a narrower range of ions, more target ions can be

collected within same time
100
195.0876
N=248402.81 NL: 1.94E8
[150.00-2000.00]
Gain in sensitivity (7x)
6000
80 Lowest detected
60
Full MS signal/scan 5000
40
S/N = 745 250330
4000

S/N (spectrum)
20

0
195.0877
3000
Caffeine
N=20741.58 NL: 1.12E8
100
[190.10-200.10]
80 2000
Relative Abundance

60 Lowest detected
40
SIM (10amu) signal/scan 1000

20 S/N = 5400 28240 0

0 195.082 195.084 195.086 195.088 195.09 195.092 195.094

S/N (FMS) S/N (SIM10)

Sensitivity gain 5 – 10 x with SIM mode

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PRM

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TERIMA KASIH

102

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