LC-MSMS (En)

INTRODUCTION TO LC-MS/MS
Presented by: Hunh Khnh Duy Application Manager
TP. HCM 09/2013

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CHROMATOGRAPHY
Solvent
Column
0
t
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CHROMATOGRAPHY
Flow of mobile phase
Stationary phase
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CHROMATOGRAPHY
Weak interaction
Strong interaction
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HPLC
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HPLC
Sample
A
D
B
C
Chromatogram
B
E
C
Abundance
D
0
10
15
20
Time (minutes)
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LC-MS
LC-MS = LC (Liquid Chromatographer) coupled to MS (Mass Spectrometer).

LC: separation.
MS: detection.
) Compounds have a unique mass number according to their structure. This mass
number is measured by a mass spectrometer.
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MASS SPECTROMETER
Molecules cannot be measured directly by MS They have to be ionized.
From mass spectrometer:
Information about the molecules (molecular weight-related ions),
Information about forcibly dissociated fragment ions (Collision Induced
Dissociation; CID), and product ions generated from specific ions.
M
H+
CID
H+
M
Fragment ions
H+ removed
bound
H+
Specific
compound
CID
Product ions generated from specific ions

Monitoring specific product ions = TQ MRM
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MASS SPECTROMETER
+C
CH
H
CH3COCH3
+C
COH
3
CH3+COCH3 C
CH3C+O
OCH
C 3
+COCH
Sample
Inlet
Ionization
& Adsorption
of Excess Energy
Fragmentation
(Dissociation)
Mass Analysis
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Detection
MASS SPECTROMETER
O
H3C
C H3
N
CH
Mass
Spectrometer
N
H
Typical sample: isolated

compound (~1 nanogram)
194
Mass Spectrum
67
109
55
82
42
136
94
40
60
80
100
120
140
165
160
180
200
Mass (amu)
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10
LC-MS
Relative Intensity
Signal Intensity
Chromatogram
Time
Mass Spectrum
Base peak: 100%
intensity
Mass spectra:
- Molecular weight
- Chemical structrure
mass-to-charge ratio (m/z) of ions

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LC-MS
M/Z
M/Z
M/Z
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12
LC-MS
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LC-PDA vs LC-MS
PDA (photodiode array)

UV spectra
(2)
(4)
20 ppm
AU
(4)
nm
(2)O 580 nm
(3)O 210 nm
(3)
(5)
AU
time
nm
MS
(3)
(2)
MS spectra
(1)
init.
10 ppb
(1)TIC
(2)m/z 582
(3)m/z 193
(4)
(5)
(5)
time
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(4)
m/z
init.
(5)
m/z
14
LC vs LC-MS
Mobile phase preparation errors
An impurity coincides with the target:
Fluctuations in peak retention times
Changes in area value
Peak misidentification
Incorrect quantitation
A B
If
an
impurity
coincides with the
target component:
A B
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LC vs LC-MS
Merits
The greatest merit in using an MS instrument as an LC detector:
In addition to retention times, mass information for each peak can be obtained simply at
the same time.
Mass information is a powerful tool for reducing the risks
associated with LC analysis, such as the following:
Peak identification (i.e., qualitative) errors
Quantitative errors due to the elution of
unpredicted impurities
The peaks (including

those that cannot be
separated by time) can be
separated using mass
information.
p
This reduces the risk of
qualitative and
quantitative errors.
m/z 267
m/z 281
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m/z 264
m/z 278
16
LC vs LC-MS
m/z=100
A
B
TIC
A:100
B:100
D:150
C:150
m/z=150
C
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LC vs LC-MS
Main product
PDA
Impurity
Int.
325.1
1500e3
MS
1000e3
500e3
156.0
0e3
224.1
200
Int.
150e3
306.2
300
388.4 432.5476.0520.4 564.5608.4

400
500
600
m/z
Int.
339.1
311.1
300e3
MW = 328
MW = 330
100e3
200e3
50e3
0e3
180.2
200
306.8
299.9
300
100e3
400
- 18 -
564.0
375.1 433.1
500
600
m/z
153.0
0e3
262.1
200 2013 300

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352.1
345.2 391.7
400
477.0 521.5
500
592.8
600
695.7
m/z
Impurity Profiling of
medicine tablet
18
LC vs LC-MS
Chromatograms of plasma sample
mAU
(x10)
ALB
AL
B
PRO
PRO
VER
VER
Int.
254nm,4nm (1.00)
2.0
1.0
ANT
AN
T
ANT :Antipyrine
PIR : Piroxicam
ALB : Albedazole
PRO : Propraolol
VER : Verapamil
3.0
PIR
PIR
4.0
How can these

compouinds can be
quantified
compounds
0.0
-1.0
PDA detector
-2.0
0.0
1.0
2.0
3.0
4.0
6.0
5.0
min.
(x100,000)
Int.
PRO
PR
O
VER
VE
R
PIR
PI
R
7.5
ANT
AN
T
ANT:m/z 189.0 (6)

PIR:m/z 260.0 (4)
ALB:m/z 266.0 (60)
PRO:m/z 331.9 (6)
VER:m/z 455.0 (2)
ALB
AL
B
10.0
5.0
MS detector
2.5
0.0
0.0
1.0
2.0
3.0
3
0
4.0
4
0
5.0
6.0
min.
19
LC vs LC-MS
Detection of 0.03% impurity in product
-0.00125
Lansoprazole
UV: 254nm
-0.00130
-0.00135
0.03%
-0.00140
-0.00145
S/N = 2
AU
-0.00150
AU
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.00155
-0.00160
-0.00165
Impurity
-0.00170
-0.00175
-0.00180
4.80
5.00
5.20
5.40
Minutes
5.60
5.80
6.00
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Minutes
0.85
0.80
6
2.0x10
6
1.8x10
0.75
0.70
0.65
SIR: 298.22 m/z
Intensity
0.55
6
1.2x10
6
1.0x10
0.50
0.45
AU
S/N = 870
6
1.6x10
6
1.4x10
0.60
0.40
5
8.0x10
5
6.0x10
0.35
0.30
0.25
5
4.0x10
5
2.0x10
0.20
0.15
0.10
0.0
0.05
4.80
0.00
5.00
5.20
5.40
5.60
Minutes
5.80
6.00
-0.05
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
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LC-MS
Qualification
) Retention time of a compound is identical in same conditions (column,
tempearature, flow rate, etc.).
Unknown sample
Compound A
tA
Standard sample
(mixture of A and B)
Compound B
tB
Sample Injection
) Mass spectrum:
Confirmation of existence.
Impurity and purity check.
Identification (library).
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LC-MS
Quantification
) Amount of a compound that passes through the detector is a function of Peak

area (or peak height)
Conc.
(ppm)
Compound A tA
10
0
70
VL of sample
Area: 700
V L of 100 ppm
standard solution of A
Compound A
Peak
Area
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700
100
0
22
LC-MS
HPLC
Mass analyzer
Interface
Aqueous/organic
solvent with buffers
Remove
solvent
A high vacuum
environmental
Non-volatile analyte
molecules
Ionize analytes
Analyze ions, m/z
Direct introduction of LC elute into MS and use of EI ion source (like in GC/MS) are
not the method of choice.
Research on interfacing HPLC to MS began in the 1970s; API (atmospheric

pressure ionization) sources were commercialized after 1987.
API interfaces: electrospray ionization (ESI), atmospheric pressure

chemical ionization (APCI) and AP photo ionization (APPI).
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LC-MS
HPLC
Mass Analyzer
Interface
Ionization probe
Atmospheric
Pressure
Q-array
Octopole
Quadrupole
10-310-4 Pa
80150 Pa
TMP
Detector
TMP
Rotary Pump
Mass spectrometer
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LC-MS
Configurations
HPLC
Interface
Ion Transmission
Mass Analyzer
- ESI
-Quadrupole (Q)
-APCI
- Ion Trap (IT)
-APPI
- TOF
-Nanospray
- Tandem/Hybrid
Ion Detector
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MASS SPECTROMETER
Ionization methods
Electrospray Ionization (ESI)
Extremely soft ionization method. Suitable for the
ionization of high-polarity compounds.
Atmospheric Pressure Chemical

Ionization (APCI)
Hard ionization method than ESI. Suitable for the
ionization of medium- to low-polarity compounds.
Gas capillary
Charged droplet formation
Liquid
sample
Nitrogen gas
Heater
Ion evaporation
High voltage (r3 to 5 kV)
+ creates positive ions
- creates negative ions
Liquid
sample
Sample molecule
Nitrogen gas
Solvent molecule
Heater
Corona needle
Functional
groups
High voltage (r3 to 5 kV)

+ creates positive ions
- creates negative ions
DUIS
Dual-ion source for both ESI
and APCI.
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MASS SPECTROMETER
Ionization methods ESI
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MASS SPECTROMETER
Ionization methods APCI
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MASS SPECTROMETER
Ionization methods ESI
ESI & APCI normally do not cause fragmentation of
molecules - Soft ionization
Main ions generated:
Protonated molecule [M+H]+ at m/z(Mr+1).
Deprotonated molecule [M-H]- at m/z(Mr-1).
Other ions often observed:
Sodium adduct ions [M+Na]+.
Solavted ions [M+H+CH3CN]+, [M+H+CH3OH]+.
Neutral lose of small molecules.
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MASS SPECTROMETER
Ionization methods
Int.
(a) GCMS, EI spectrum of riboflavin (vitamine B2)
m/z
Int.
40e3
(b) LCMS, ESI (API) spectrum of riboflavin

HO
Riboflavin C17H20N4O6
30e3
Exact Mass: 376.14

20e3
OH
OH
N O
[M+Na]+
NH
N
O
10e3
0e3
377 [M+H]+
OH
50
100
150
200
250
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300
399
350
400
m/z
30
MASS SPECTROMETER
Ionization methods
O
,QW
185
1
APCI
Positive
mode
350e3
300e3
250e3
200e3
(H3C)3C
N
100e3
197
207
50e3
66
82
105
128 142 155 167
100
150
N
H
NH2
O
Diketometribuzin (DK)
C7H12N4O2
Exact Mass: 184.10
Mol. Wt.: 184.20
150e3
0e3 57
50
227
243 257268 280 295
200
250
317
300
341
391
369
350
425
404
400
453 469 482 497

450
P]
,QW
183
500e3
APCI
Negative mode
400e3
300e3
200e3
100e3
0e350
50
71
113
89
100
141 153 168

150
193 207
200
228
243
250 265 281 297
250
300
322
356 368
350
405 416
400
435
461
450
484 499
P]
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MASS SPECTROMETER
Ionization methods
LC conditions: ODS column (75x4.6
mm), CH3CN/water = 80/20
Interface: ESI
[MH-2H2O]+
Int.
1000e3
375
Positive
433
500e3
OH
474
CH3
297
315
250e3
0e3
141 163
203
150
200
333 357
350
255
237
279
250
300
350
Int.
393
411
400
409
Negative
449
457
500
550
m /z
[M-H]-
O
H3C
75000
CH2
CH3
O
OH
490510 534553 580
450
100000
H
CH3
CH3
OH
Forskolin
C22H34O7 MW: 410
50000
25000
0
CH3
[M+H]+
750e3
106 134
150
173
349
335 368 395 426445
201 225 246 273

200
250
300
350
400
450
479 508 537

500
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550
574
m /z
ESI-Q spectra by LCMS-2010EV

32
MASS SPECTROMETER
Ionization methods
MS mode
Positive
Negative
Mass peak
(m/z)
Ion
Type of Ion
474
433
411
[M+Na+ACN]+
[M+Na]+
[M+H]+
393
375
357
333
315
297
409
[M+H-H2O]+
[M+H-2H2O]+
Protonated ion with neutral
+
[M+H-3H2O]
loss of small molecules
+
[M+H-H2O-HAc]
[M+H-2H2O-HAc]+
[M+H-3H2O-HAc]+
[M-H]Deprotonated ion
Solvated-Na-adduct ion
Na-adduct ion
Protonated ion
No simple Na-adduct ion can form in negative mode.

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MASS SPECTROMETER
Ionization methods
Sodium adduct ions:
Na+ ions attach (non-covalent interaction) to the
target molecules M to form [M+Na]+
Solvent adduct ions:
Solvent molecules attach to target molecules to form
[M+Sol+H]+ (Sol: MeOH, CH3CN, THF etc); both ESI
and APCI, more often for latter
Additive adduct ions:
Such as [M+NH3+H]+, [M+FA+H]+, [M+TFA+H]+
Dimmer ions [2M+H]+, [2M+Na]+
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MASS SPECTROMETER
Ionization methods
) In most case, adduct ions can be recognized easily: span

of adjacent mass peaks = adduct molecule:
e.g., [M+H]+ and [M+NH3+H]+: 'm/z = 17
[M+H]+ and [M+MeOH+H]+: 'm/z = 32
[M+H]+ and [M+Na]+: 'm/z = 22
[M+H]+ and [M+FA+H]+: 'm/z = 46
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MASS SPECTROMETER
Ionization methods
Natural pesticides azadirachtin as the active component
Neem extract mainly from seeds: very complex in composition
Conventional method reverse phase HPLC with UV detector (217

nm), for major components (Azadirachtin-A and -B, nimbin, salannin
etc)
Difficulty in identification of minor components due to lack of

standards, such as other azadirachtins etc.
LCMS: identification of components based on MW and neutral loss

spectrum
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MASS SPECTROMETER
Ionization methods
mAbs
25
UV, 217
nm
20
15
10
5
0
10
15
20
TIC(1.00)
27.5e6 703.00(2.00)
645.00(2.00)
25.0e6 499.00(2.00)
555.00(2.00)
541.00(2.00)
22.5e6
597.00(2.00)
30
35
40
45
50
55
min
28
22
MS-TIC
MS-MIC
30
29
26
20
21
25
24
23
14
13
15
16
17
18
19
9
10
11
5
6
7
3
4
1
2
15.0e6
20.0e6
17.5e6
25
27
12
0
Int.
12.5e6
10.0e6
7500e3
5000e3
2500e3
0e3
-2.5e6
10
15
20
25
30
35
40
45
50
55
min
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MASS SPECTROMETER
Ionization methods
Peak 22: 6-Desacetyl

nimbin 499
Int.
Int.
5000e3
Peak 28:
Salannin
597
5000e3
2500e3
2500e3
m/z
m/z
22
Peak 23: 3-Desacetyl

salannin
555
45
6
7
17.5e6
20.0e6
9
11 10
13
14
15
16
17
18
19
499.00(2.00)
25.0e6
30
TIC(1.00)
645.00(2.00)
555.00(2.00)
541.00(2.00)
22.5e6
597.00(2.00)
703.00(2.00)
27.5e6
29
650
1
2
3.0e6
650
655 677
600
Int.
Int.
600
27
623
550
28
550
500
26
500
450
23
25 24
450
619 642 660

677 694
419 437 461 479 497 519 541 565
20
21
0e3403
400
0e3401
400
544 562
578
485
12
467
449
15.0e6
2.0e6
12.5e6
10.0e6
1.0e6
0.0e6
400
419 437 455 479497

450
537
500
572
550
7500e3
600 618
634 662679
600
650
5000e3
m/z
2500e3
0e3
-2.5e6
Peak 24: OhichinolideB

625
Int.
10
15
20
25
30
35
40
45
50
55
min
Peak 27: Nimbin
1500e3
Int.
1000e3
5000e3
541
688
500e3
465
0e3
450
506 525 549 568585 607

500
550
600
647 670
650
2500e3
704 721
700
509
m/z
421 449
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0e3 404
400
450
481
527
500
559 586
550
604
621 645 665 689
600
650
m/z
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MASS SPECTROMETER
Ionization methods
Int.
Peak 12
3.0e6
m/z
703
685
585
567
743
703
2.0e6
685
567
1.0e6
585
507
743
603
555
525
500
550
Int.
600
Peak 14
750e3
766
667
625 642
0.0e6
721
650
700
784
750
m/z
Azadirachtin A, MW = 720
m/z
645
627
545
527
685
645
685
627
545
500e3
250e3
701
527
509
563 585
609
726
663
743
710
759
789
0e3
500
550
600
650
700
750
Ion
[MH-H2O]+
[MH-2H2O]+
[MH-2H2O-TgOH]+
[MH-3H2O-TgOH]+
[MNa]+
m/z
Ion
[MH-H2O]+
[MH-2H2O]+
[MH-H2O-TgOH]+
[MH-2H2O-TgOH]+
[MNa]+
Azadirachtin B, MW = 662
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MASS SPECTROMETER
Ionization methods
H2O
O
TgOH
Mr 100
CH3
H3CO
O
O
OH
CH3
CH3
CH3
O
CH3
H
OH
OH
O
H3CO
H
O
H2O
H2O
18
100
23
18
18
[MH]+ [MNa]+
[MH-H2O]+
[MH-H2O-TgOH]+
[MH-2H2O]+
m/z
+
[MH-2H2O-TgOH]
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MASS SPECTROMETER
Ionization methods
Peak No
[MH-H2O]+
(Base peak)
Azadirchtin
Type
Formula
MW
C32H42O12
618
601
645
G or H
C33H42O14
662
10
641
C34H44O14
676
12
703
C35H44O16
720
14
645
C33H42O14
662
15
687
C35H44O15
704
16
647
C32H40O14
664
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MASS SPECTROMETER
Ionization methods
Depend on structures and properties of compounds

9 High proton affinity: C-O, C-N double and triple bonds,
basic compound tend to form positive ions.
9 Low proton affinity: COOH, -F, -Cl, -HSO3, phenols,
aniline and sugars tend to form negative ions due to
stronger tendency to donate proton.
9 Many compounds form both positive & negative ions
due to multi function groups.
Acquire positive & negative mass spectra and compare
sensitivity and spectrum quality.
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MASS SPECTROMETER
Ionization methods
Functional group
Positive ion
R- H2
R-NH
protonation
R- H-R
R-NH
HR- HR-SH
R-OH
R-O--R
R-O
R R-C=O
R
[benzene ring]
g]-OH
RR-COOH
R- O3HR--PO
R-SO
O3H
Negative
egative ion
deprotonation
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MASS SPECTROMETER
Ionization methods
ESI: CH3CN/H2O and MeOH/H2O

APCI: MeOH/H2O recommended; CH3CN/H2O may
cause coking on the corona needle in negative mode.
THF can be used for LCMS ionization.
The ratio is not so critical, but higher water content may
cause lower ionization efficiency.
Gradient elution with changing aqueous phase between
0 and 100% can be used.
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MASS SPECTROMETER
Ionization methods
Non-volatile buffer like phosphate is not recommended

Using volatile buffer to replace phosphate buffer:
NH4Ac/HAc, NH3/TFA, NH3/NH4Ac etc
pH control:
pH 1.8 ~ 2.5
: TFA, conc. < 0.1%
pH 2.5 ~ 4
: FA, conc. ~ 0.1%
pH 4 ~ 5
: HAc, conc. 0.1~5%
pH 7
: NH4Ac
pH > 7
: NH3 aqueous solution
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MASS SPECTROMETER
Other ionization methods
APPI : Atmospheric Pressure Photo ionisation [Non Polar]
DART : Direct Analysis in Real Time [Solid samples]
DESI : Desorption Electro Spray Ionisation [Min. sample prep
for bio fluids, Tablets, creams etc. [Thermo]
ASAP : Atmospheric Solids Analysis Probe [Solid Sample
probe] Waters
DUIS : Combined ESI + APCI [Shimadzu]
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MASS SPECTROMETER
Other ionization methods - DUIS
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MASS SPECTROMETER
Other ionization methods - DART
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48
IONIZATION METHODS
Analysis target: Areas differ according to ionization method

GC/MS
(Also if derivatization is
required.)
Molecular weight
10,000
x Non-polar
LC/MS
ESI
(High polarity)
10,000
1,000
LC/MS
APCI
GC/MS (Low/medium
polarity)
Contents:
polyvalent ions
x Volatile
x Thermally stable
LC/MS(MS)
(Simple, requiring almost
no pretreatment.)
x High polarity
x Non-volatile
Nonpolar
Medium
polarity
High
polarity
x Large mass numbers

x Thermally unstable
HKDUY 2013
49
MASS SPECTROMETER
Type of Mass filter
Quadrupole
Time-of-flight (TOF)
Ion-trap
Magnetic-sector
Fourier-transform
Tandem MS (TQ, IT-TOF, )
9Qualification of unknown samples
Quadrupole < Ion trap < TOF
9Sensitivity, quantitation
Quadrupole (SQ/SIM) < TOF (Scan) < Quadrupole (TQ/MRM)
HKDUY 2013
50
MASS SPECTROMETER
Type of Mass filter
Quadrupole MS: Good for quantification. Ions selected by four poles.
Good for quantification of unknown substances.

General-purpose, most widely used type. Low cost and easy
Features:
maintenance. Acquires SCAN and SIM data.
Advantages: (1) Compact and lightweight, (2) Easy to operate, (3) High sensitivity
excellent for quantification.
Disadvantages: Poor resolution. Little qualitative information (nominal mass only).
HKDUY 2013
51
MASS SPECTROMETER
Type of Mass filter
Advantages:
Better detector than UV/PDA.
More sensitive.
Useful for screening and Quantitation.
Disadvantages:
No Fragmentation.
Not very sensitive.
Not useful for Identification and confirmation of unknown
compounds.
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52
MASS SPECTROMETER
Type of Mass filter
R&D Pharma / API
Reaction Monitoring.
Basic Impurity Identification.
Chemical Synthesis
Reaction monitoring.
High Thro put screening.
Food testing
Analysis of permitted colours.
Mycotoxins.
Academia
53
HKDUY 2013
MASS SPECTROMETER
Type of Mass filter
Ultra-fast detection of catechins in Pos/Neg modes by LCMS-2020
(x100,000)
Analysis of catechins in tea
m/z 307
m/z 291
m/z 459
1
10
m/z 473
m/z 471
HO
negative
m/z 441
10
OH
m/z 289
m/z 457
OH
m/z 305
Positive
m/z 443
1: (-)-gallocatechin,
2: (-)-epigallocatechin,
3: (+)-catechin,
4: (-)-epicatechin,
5: (-)-epigallocatechin gallate,
6: (-)-gallocatechin gallate,
7: (-)-epicatechin gallate,
8: (-)-catechin gallate
9, 10: methylated catechins
min
HKDUY 2013
OH
OH
(-)-epicatechin
54
MASS SPECTROMETER
Type of Mass filter
Triple Quadrupole MS
Full Scan MS
Q1
Selection of Ions in
selected m/z range
Q2 Collision cell
Only RF to move
ions
Q3
Only RF to move
ions to detector
55
HKDUY 2013
MASS SPECTROMETER
Type of Mass filter
Full Scan MSMS
Q1
Selection of
Precursor Ion
Q2 Collision cell
Fragmentation of
Precursor Ion
HKDUY 2013
Q3
Scanning of all
Product Ions
56
MASS SPECTROMETER
Type of Mass filter
MRM
Q1
Selection of
Precursor Ion
Q3
Selection of
Product Ion
Q2 Collision cell
Fragmentation of
Precursor Ion
57
HKDUY 2013
MASS SPECTROMETER
Type of Mass filter
SIM
(x10,000,000)
TIC
1.25 405.80 (10.00)
417.80 (10.00)
483.70 (10.00)
485.70 (10.00)
1.00 495.70 (10.00)
497.70 (10.00)
561.60 (10.00)
563.60 (10.00)
573.60 (10.00)
0.75 575.60 (10.00)
TIC
799.50 (10.00)
811.50 (10.00)
959.40 (10.00)
971.40 (10.00)
0.50
0.25
5.0
10.0
15.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
MRM
(x100,000)
2:405.80>245.90 (1.00)
7.0 3:417.80>257.90 (1.00)
5:485.70>325.70 (1.00)
6:497.70>337.70 (1.00)
6.0 7:563.60>403.70 (1.00)
8:575.60>415.70 (1.00)
9:643.60>483.70 (1.00)
(1.00)
5.0 10:655.60>495.70
11:721.40>561.60 (1.00)
12:733.40>573.60 (1.00)
13:959.40>799.30 (1.00)
4.0 14:971.40>811.30 (1.00)
3.0
2.0
1.0
5.0
20.0
HKDUY
2013 25.0
30.0
35.0
40.0
58
MASS SPECTROMETER
Type of Mass filter
Hexa-BDE (BDE-153)
Tri-BDE (BDE-028)
SIM
(x100,000)
405.80
245.90
(x10,000)
405.80>245.90
5.0 405.80>247.90
3.0
MRM
SIM
(x10,000)
483.70
2.0 643.60
4.0
4.0
1.5
MRM
(x10,000)
643.60>483.70
643.60>485.70
3.0
3.0
2.0
1.0
2.0
0.5
1.0
2.0
1.0
1.0
11.5
12.0
12.5
11.5
13.0
12.0
12.5
23.0
13.0
23.5
24.0
(x100,000)
1.25 563.60
403.80
1.00
SIM
24.5
23.0
23.5
24.0
24.5
Hepta-BDE (BDE-183)
Penta-BDE (BDE-100)
MRM
(x100,000)
563.60>403.70
2.5 563.60>405.70
SIM
(x1,000)
6.0 561.60
721.50
5.0
(x10,000)
5.0 721.40>561.60
721.40>563.60
MRM
4.0
2.0
4.0
0.75
3.0
1.5
0.50
3.0
1.0
0.25
0.5
19.0
19.5
20.0
20.5
2.0
2.0
1.0
1.0
18.5
19.0
19.5
20.0
26.0
26.5
27.0
(x1,000)
483.70
643.60
7.5
SIM
MRM
(x10,000)
643.60>483.70
3.0 643.60>485.70
(x100,000)
799.50
1.25 959.40
2.5
1.00
26.0
26.5
27.0
27.5
SIM
(x100,000)
959.40>799.30
7.5 959.40>801.30
MRM
5.0
2.0
5.0
27.5
Deca-BDE (BDE-209)
Hexa-BDE (BDE-154)
0.75
1.5
0.50
1.0
2.5
22.0
22.5
23.0
23.5
2.5
0.25
0.5
22.0
22.5
23.0
23.5
HKDUY
2013
37.0
37.5
38.0
38.5
37.0
37.5
38.0
38.559
MASS SPECTROMETER
Type of Mass filter
Advantages:
Very fast and highly sensitive.
Useful for screening and Quantitation of trace level impurities.
So far, the best tool for trace level quantitation.
Disadvantages:
Low Resolution, Mass accuracy is less.

Fragmentation is only up to MS2.
Not useful for Identification and confirmation of unknown
compounds
HKDUY 2013
60
MASS SPECTROMETER
Type of Mass filter
R & D Pharma / API

Quantitation of trace level impurities.
BA/BE studies.
PK/PD studies.
Impurity profiling [limited]
Chemical Synthesis
High Through put screening.
Environmental Food safety
Screening of wide range of impurities in water and food.
Analysis of Banned colours.
Mycotoxins.
Trace level impurities like pesticides.
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61
MASS SPECTROMETER
Type of Mass filter
Clinical
New born screening.
Immunosuppressant.
Vitamin D.
TDM.
Clinical trials phase I to III.
Forensic and Toxicology
Screening of banned substances in various matrices.
Quantitation and confirmation of banned substances.
Academia
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62
MASS SPECTROMETER
Type of Mass filter
Ion-trap: Good for structure analysis. Ion-trap + MSn combination possible..
Good for structure analysis.

Features:
Comparatively small and
cheap. Easy operation and maintenance.
Permits SCAN and SIM.
Advantages: (1) High sensitivity by
detecting all trapped ions.
(2) Permits MSn.

Disadvantages: Unsuited to quantitation.
Poor resolution. Inferior dynamic range to
quadrupole MS due to limit on trapped
quantity.
63
HKDUY 2013
MASS SPECTROMETER
Type of Mass filter
Ion-trap: Good for structure analysis. Ion-trap + MSn combination possible..
Ar
Ar
1. Selection of precursor ion
2. Fragmentation by
acceleration and collision with
Argon
0V
+V
0V
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-V
0V
64
MASS SPECTROMETER
Type of Mass filter
TOFMS: Good for qualification. Measures ion time of flight.
Good for qualification of unknown substances.

Features:
Has become commonly used in recent years. Comparatively large
and expensive. Acquires SCAN data.
Advantages: (1) High resolution, (2) High mass measurement accuracy, (3) High
spectral sensitivity.
Disadvantages: Unsuited to quantitation. Requires high vacuum. Difficult to handle.
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MASS SPECTROMETER
Type of Mass filter
Resolution (R):
R = m/'m
'm = mass difference of two adjacent resolved peaks (typically m = mass

of first peak or average).
Example: R = 500 (low resolution)
resolves m/z=50 and 50.1, and m/z=500 and 501
Example: R = 150,000 (high resolution)
resolves m/z=50 and 50.0003, and m/z=500 and 500.0033
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66
MASS SPECTROMETER
Type of Mass filter
785.8419
R=5901
100
786.3435
R=5900
RP 7500
0.2 s
80
786.8447
R=5900
60
40
787.3463
R=6000
785.5934
R=6200
20
0
785.8421
R=23801
Relative Abundance
100
787.8453
R=5800
786.3434
R=23900
RP 30000
0.5 s
80
786.8446
R=24000
60
40
787.3457
R=24100
785.5992
R=24300
20
787.8471
R=15600
785.8419
R=48101
100
786.3435
R=47700
80
786.8446
R=48200
60
40
787.3458
R=47500
785.5994
R=47100
20
RP 60000
0.9 s
787.8477
R=42000
785.8413
R=94801
100
786.3428
R=95200
80
786.8442
R=93600
60
40
787.3458
R=98000
785.5989
R=95800
20
RP 100000
1.6 s
787.8477
R=89200
0
785.0
785.2
785.4
785.6
785.8
786.0
786.2
786.4
786.6 786.8
m/z
787.0
787.2
787.4
787.6
787.8
788.0
788.2
67
HKDUY 2013
MASS SPECTROMETER
Type of Mass filter
455.3
RUnregistered
= 1,000
100
90
80
70
455.29
60
50
Unregistered
R=
3,000
90
40
80
30
70
20
60
10
50
455
456
Quadrupole
& ion trap
457
40
458
30
455.2910
459
460
RUnregistered
= 12,000
461
90
20
80
10
70
0
455
456
457
60
458
50
459
460
461
40
30
TOF
20
10
0
HKDUY 2013
455
456
457
458
459
460
46168
MASS SPECTROMETER
Type of Mass filter
Inten.(x1,000,000)
Inten.
2.0
H3C
1.9
455.2897
1.8
CH3
CN
H3CO
CH3
N
OCH3
1.7
1.6
H3CO
1.5
FWHM: 0.08 Da
OCH3
C27H38N2O4
Exact Mass: 454.2832, Mol. Wt.: 454.6017
1.4
1.3
1.2
Resolution: 12,000
1.1
Mass accuracy = -2.9 ppm
1.0
< 5 ppm
0.9
R > 10,000
0.8
0.7
0.6
0.5
0.4
1 Da
0.3
0.2
0.1
0.0
Bovine Insulin spectrum [M+6H]6+
453.0
454.0
455.0
456.0
457.0
458.0
459.0
460.0
461.0
462.0
463.0
464.0
m/z
Verapamil spectrum [M+H]+
High resolution means high mass accuracy - we can determine very

accurately the mass of ion to 3rd or 4th decimal point.
HKDUY 2013
69
MASS SPECTROMETER
Type of Mass filter
Mass Accuracy :
For example
C9H12NO3
Theoretical mass = 182.0811
Observed mass = 182.0819

Mass Accuracy = 4.01 ppm
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MASS SPECTROMETER
Type of Mass filter
9 Identification and confirmation.

9 Non- Targeted Screening approach.
9 High level confidence in Research.
9 Analysis of compounds in Highly complex matrix.
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71
MASS SPECTROMETER
Type of Mass filter
Advantages:
High Resolution and Accurate mass.
High speed of analysis.
Disadvantages:
No Trapping of ions, No fragmentation.
Just TOF is not enough for Identification and Confirmation.
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72
MASS SPECTROMETER
Type of Mass filter - QTOF
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73
MASS SPECTROMETER
Type of Mass filter - QTOF
Advantages:
High Mass Range [20,000 or more].
MS2 fragmentation helps in better confirmation.
Works well for both Small Molecules and Big Molecules.
Disadvantages:
No MSn.
Quantitation is not so good.
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74
MASS SPECTROMETER
Type of Mass filter IT-TOF
Stage 1: The ions enter the octopole during a definite time
Lens 1
(+60 V)
Skimmer (+8 V) Open

V
Skimmer (-30 V) Closed

Lens 1
Skimmer
Stage 2: The entry of the octopole is closed

75
HKDUY 2013
MASS SPECTROMETER
Stage 3: The ions are transferred to the trap
Lens 1
(-80 V)
Skimmer (-30 V) Closed

Lens 1
Skimmer
HKDUY 2013
76
MASS SPECTROMETER
Octopole
lens 1
0V
-V
During entry of the ions, no RF is

applied to the ring electrode
0V
Ion trap
77
HKDUY 2013
MASS SPECTROMETER
When ions are in the trap, the RF is applied

and focuses them in the center
Ar
Ar
Octopole
0V
RF
0V
Ion trap
HKDUY 2013
78
MASS SPECTROMETER
Detector
TOF
0V
+V0V
RF
-V0V
Ion trap
Reflectron
Simultaneous ejection (BIE: Ballistic Ion Extraction) of

all the ions into the TOF to obtain a TOF mass
spectrum
79
HKDUY 2013
MASS SPECTROMETER
Ion Trap for ion
selection, CID
and MSn
ESI
Ion inlet CDL
Octopole
DQ Array
MCP detector
3D Ion trap
p
HKDUY 2013
TOF of high
resolution spectrum
of MS and MSn
Time of Flight
g
80
MASS SPECTROMETER
Multi stage CID
in the ion trap
MS duty cycle
Ion trapping
Ion cooling
Ejection to TOF
MS/MS duty cycle
octopole
Lens 1
0V
0V
Ion trapping
Ion cooling
Ion selection
Fragmentation by CID (Ar)
Ion cooling
Ejection to TOF
MS/MS/MS
Ion trap
MS/MS/MS/MS (MSn)
HKDUY 2013
81
MASS SPECTROMETER
Advantages:
MS10 fragmentation helps in better confirmation.
Works well for small molecule applications [with less complex
matrix].
Disadvantages:
Resolution is not enough if the matrix is complex.
Specifications are lower compared to any HRAM instrument.
Not suitable for proteomics applications.
HKDUY 2013
82
MASS SPECTROMETER
Reserpine
Reserpine Degradation A
CH3
CH3
N
H H
CH3
CH3
Side Reaction
CH3
H
O
CH3
CH3
195.0650
397.2104
236.1265
365.1851
336.1566
NL212577.2504
Intact structure could be
observed as either common

product ion or neutral loss on
MS/MS spectra of reserpine and
its degradation products.
NL212
MS/MS
Analysis
Inten. (x1,000,000)
O
O
CH3
MS/MS
Analysis
448.1967
CH3
NL212
CH3
N
H H
CH3
Inten. (x1,000,000)
381.1801
NL212
227.1168 349.1538
363.1688
595.2684
m/z
Reserpine
MS/MS
spectrum
m/z
Degradation A
MS/MS
spectrum
83
HKDUY 2013
MASS SPECTROMETER
Reserpine Degradation Products
Reserpine, Theoretical: 609.2807
Column :
Mobile phase A :
Mobile phase B :
Gradient program :
Flow rate :
Injection volume :
Column temp. :
ODS column 2.0mmI.D.x50mmL

5mmol/L ammonium formate - water
acetonitrile
20%B (0min) 80%B (15min)
0.2 mL/min
1 uL
40 deg. C
HKDUY 2013
Ionization mode :
Nebulizing gas :
Drying gas press. :
Probe voltage :
CDL temp. :
BH temp. :
ESI (+)
1.5 L/min
100 kPa
+4.5 kV
200 deg. C
200 deg. C
84
MASS SPECTROMETER
Inten. (x1,000,000)
MS2
Precursor ion: m/z609.2802
448.1967
NL 161.0835
195.0650
NL 414.2152
236.1265
NL 373.1537
397.2104
NL 212.0698
365.1851
NL 244.0951
436.1949
336.1566
NL 178.0853
CH3
N
H H
C22H30NO8+
Theoretical:436.1966
C23H30NO8+
Theoretical:448.1966CH
CH3
O
CH3
C13H18NO3
H
Theoretical:236.1281O
C10H11O4
C22H25N2O3+ CH3
CH3
C23H29N2O4+
HKDUY 2013
577.2504
Found
PI
m/z
Theoretical
195.0650
195.0652
Error
-0.0002
NL
414.2152
414.2155
-0.0003
PI
236.1265
236.1281
-0.0016
NL
373.1537
373.1526
0.0011
PI
365.1851
365.1860
-0.0009
NL
244.0951
244.0947
0.0004
PI
397.2104
397.2122
-0.0018
NL
212.0698
212.0685
0.0013
PI
436.1949
436.1966
-0.0017
NL
173.0853
85
173.0841
0.0012
PI
448.1967
448.1966
0.0001
NL
161.0835
161.0841
-0.0006
85
MASS SPECTROMETER
Inten. (x10,000,000)
MS1
Mass difference from Reserpine= -194.0567Da

C10H10O4 (Theoritical: 194.0579)
415.2235
Common neutral loss with Reserpine161.08
m/z
Peak #1 is supposed to have -194 Da difference

(C10H10O4) on the m/z 448 in the substructure of
reserpine.
Inten. (x1,000,000)
MS2
254.1381
The product ion which yields NLS 161 was m/z 448 in
reserpine
NL161
CH3
174.0905
383.1990
m/z
CH3
Putative Structure of Peak #1O

CH3
N
H H
C23H30N2O5
M+H]+: 415.2227
N
H
N
H H
CH3
O
CH3
H
H
O
O
O
CH3
CH3
m/z 448
H
OH
O
O
CH3
CH3
HKDUY 2013
86
MASS SPECTROMETER
Inten. (x10,000,000)
Difference from reserpine = -16.0304Da

593.2496 CH4 (Theoritical: 16.0313)
MS1
Common NLS with reserpine:212.07
436.1966
The product ion which yields NLS 212 was

m/z 397 in reserpine
m/z
Peak #3 is supposed to have -16 Da

difference (CH4) on the m/z 397 in the
substructure of reserpine.
Inten. (x10,000,000)
MS2
381.1801
NL212
227.1168 349.1538
CH3
m/z
CH3
m/z397 of
reserpine
N
H H
CH3
O
CH3
H
H
O
O
O
O
CH3
CH3
87
HKDUY 2013
MASS SPECTROMETER
MS3 measurement for the PI which yields NLS 212
Inten. (x1,000,000)
Reserpine
m/z609397174.0911
236.1266
192.1008
159.0668
365.1845
O
CH3
396.1950
265.1341
305.1609350.1629
CH3
C32H36N2O9
M+H+:
593.2494
CH3
Putative structure of Peak #3
CH3
N
H H
O
CH3
It is supposed that peak #3

has -16 Da (CH4) difference
from reserpine except the
structure of m/z305.
= demethylation
H
O
O
CH3
Common product ion on

reserpine and Peak #3 was
m/z305.16.
305.1628
O
CH3
CH3
CH3
m/z
Peak
m/z593381
CH3
N
H H
O
CH3
HKDUY 2013
88
MASS SPECTROMETER
Ion Detection
89
HKDUY 2013
MASS SPECTROMETER
Ion Detection
TOF detector
6.5KV
650 V
Microchannel
Plate (MCP)
e
e
e
e
6 KV
Scintillator
h
Optical lens
700V
Overall gain ~ 2x10
Photomultiplier
tube (PMT)
Ground
HKDUY 2013
90
MASS SPECTROMETERS
Triple quadrupoles
93
HKDUY 2013
LC-MS/MS
Configuration
Controller
Reservoir tray
Note) The LC detector can be removed, if not required.
Solvent delivery module

(gradient)
Degasser
LCMS-8040
Mixer
Mobile phase
Column
LC detector
Mobile phase
Autosampler rinsing solution
Autosampler
Column oven
Rotary
pump
Nitrogen gas
generator
Ionization probe
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94
LC-MS/MS
Ionization probe
Ionization probe
Inject mixed sample
Collision cell
Detector
(q2)
Mass separation unit
Mass separation unit
(Q3)
(Q1)
Atmospheric
pressure region
Vacuum region
Separation in column
Detector
Components enter MS sequentially

from components with weak retention
Mobile phase
Protons (hydrogen ions)
Q3
q2
Ionization probe
Positive ions
(protonated
molecules)
Q1
Remove
protons
Protonation
Negative ions
(deprotonated
molecules)
Ionization probe
95
HKDUY 2013
LC-MS/MS
SIM Mode
Pass through
Pass
through
Q1
Collision cell
SIM
Q3
Detector
Ions pass through MS1 and collision cell, but only specified ions pass through
MS2.
As the detection time for the specified ions exceeds the scan time, sensitivity
increases 10 to 100 times.
Analysis method for quantitation of target compounds.
Cannot analyze unknown compounds as the mass spectra are not saved.
o Effective for simultaneous full scan and SIM.
Optimal parameters must be confirmed in advance. Same as LC/MS (SQ) SCAN
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96
LC-MS/MS
MRM Mode
SIM
SIM
Fragmentation
Q1
Q3
Collision cell
Detector
Specific ions selected by MS1 undergo collision-induced dissociation (CID) in the

collision cell and specific ions can also be selected by MS2.
Precursor ion-derived product ions are selected.
Despite reduced ion quantity, significantly reduced chemical noise enhances S/N.
High selectivity is ideal for highly sensitive quantitative analysis.
Analysis method for target compounds.
97
HKDUY 2013
LC-MS/MS
MRM Mode
50000
10 ppb
Collision cell
Detector
Fragment
g
Quadrupole
p
Q1
Scan/SIM
Ion source
SIM
7500
High sensitivity but

high background
40000
30000
20000
Quadrupole
p
Q3
Scan/SIM
10 ppb
Detector
MRM
Eliminates background for
trace-level quantitation with
high S/N
5000
2500
10000
0
1.0
1.5
2.0
2.5
min
1.0
1.5
2.0
2.5
min
Ionization probe
HKDUY 2013
98
LC-MS/MS
Progesteron
100
CH3
CH3
315.1
Mass Spectrum from

MS1
CH3
316.1
Precursor ion
0
300
305
310
O
CH2
97.0
100
109.0
315
320
325
m/z
330
CH3
CH2
H3C
CH3
Product ions
Product ion spectrum from MS2
m/z
100
125
150
175
200
225
250
275
300
325
99
HKDUY 2013
LC-MS/MS
UV
MS-TIC
Tuning condition
MS-TIC
CID condition
HKDUY 2013
100
LC-MS/MS
Peak 2, RT = 7.31
Int.
232
Normal
10.0e6
5.0e6
119
0.0e6
100
171 200
246270
200
Int.
332354 384 416

300
482
400
536
611
500
667
600
232
m/z
CID
144 169
50e3
187
201
215 246269
101 129
0e3
100
398
343
200
300
442 479
400
500
600
m/z
m/z187
m/z215
H3C
CH2 C
N
H3C
m/z201
m/z155
m/z187
H2
C
H2
C
C
H
m/z169
m/z215
C
H2
H3C
NH 2
CH2 C
H3C
H2
C
C
H
C
H2
C
H2
NH 2
m/z144
101
HKDUY 2013
LC-MS/MS
Peak 13, RT = 24.81 (Main-3)

Int.
343
10.0e6
Normal
5.0e6
0.0e6 106 132155
100
214
298 326
255
200
365
300
460482 517 553
407
400
500
597 623
671 700
600
m/z
600
m/z
343
Int.
100e3
CID
298
255
50e3
112
0e3
100
155 187
223
365
328 367391
286
200
300
434
482
400
500
m/z187
m/z298
m/z155
H3C
N
H3C
CH2 C
S
H2
C
H2
C
CH3
C
N
C
H
C
H
C
S
CH2 N
CH3
m/z255
HKDUY 2013
102
MS vs MS/MS
Tetra-Dioxins
Tetra-Furans
90
NL:
2.25E5
m/z=
321.50322.50
MS
pcb124820psi-05
Relative Abundance
80
70
60
50
40
30
90
80
60
50
40
30
20
10
10
2,3,7,8
TCDD-IS
90
80
70
60
NL:
4.70E4
m/z=
333.50334.50
MS
pcb124820psi-05
NL:
2.66E5
m/z=
305.50306.50
MS
pcb124820psi-05
70
20
0
100
2,3,7,8
TCDF
RT: 16.93 - 20.35 SM: 5G

100
Relative Abundance
2,3,7,8
TCDD
RT: 16.93 - 20.35 SM: 3G

100
0
100
80
70
60
50
50
40
40
30
30
20
20
10
10
NL:
5.20E4
m/z=
317.50318.50
MS
pcb124820psi-05
2,3,7,8
TCDF-IS
90
0
17.0
17.2
17.4
17.6
17.8
18.0
18.2
18.4
18.6 18.8
Time (min)
19.0
19.2
19.4
19.6
19.8
20.0
17.0
20.2
17.2
17.4
HKDUY 2013
17.6
17.8
18.0
18.2
18.4
18.6 18.8
Time (min)
19.0
19.2
19.4
19.6
19.8
20.0
20.2
103
MS vs MS/MS
HKDUY 2013
104
LCMS-8040
UF Sweeper II
UF Lens
8030
UF Sweeper II
UF LensHKDUY 2013
106
LC-MS/MS
Cross Talk
Q1
Cross Talk: The phenomenon where ions

loses momentum by collisions with the
collision gas remain in the collision cell and
are detected by the subsequent analysis.
MRM
Transition 1
MRM
Transition 2
MRM
Transition 3
9 Ghost peaks may appear or performance in

quantitation may be deteriorated. HKDUY 2013
Q3 Detector
Collision cell
q2
Ionization
probe
Compound A
m/z 402Q1)>167Q3)
Compound B
m/z 382Q1)>167Q3)
Compound C
m/z 215Q1)>167Q3)
107
LC-MS/MS
Ultra-fast Analysis
Environment
Economy
Economy
Production Capacity
Cost
Environment
Solvent.
Energy.
# Time.
# Solvent.
HKDUY 2013
112
LC-MS/MS
Ultra-fast Analysis
Mobile phase
Mobile phase changing re-built of analysis method.
Flow Pressure.
Stationary phase
Stationary phase changing Column, Analysis methods.
Column length Separation.
# Efficiency ?
# Amount of used solvent ?
HKDUY 2013
113
LC-MS/MS
Ultra-fast LC
Column
# Packing particles < 3 m
9 Higher resolution.
System
9 Shorter analysis time.
) Pressure 1000 bar
9 Higher sensitivity.
Detector
9 Lower solvent consumption.
# High response (> 50 Hz)

# Flow-cell 2.5 L, 5 mm (traditional detector)
Use as HPLC
Easy to migrate from HPLC UHPLC
HKDUY 2013
LC-MS/MS
Ultra-fast LC
Passing through 100 m tunnel at 50 km/h
Analysis time
(minutes)
150
I.D.
6.0 mm
4.6 mm
3.0 mm
2.0 mm
Note) Relative time, taking the time

of analysis with 4.6 x 150
mm column as 100 minutes
(at constant pumping rate).
Column size example:

Reducing column size
to 1/5 reduces analysis
time to approx. 1/5.
100
Speed 50 km/h
Mobile phase
example:
Doubling pumping
rate reduces
analysis time to
approx. 1/2.
50
Column length (mm)
250
150
50 km
75
50
Speed 50 km/h
Speed 50 km/h
30
Time to pass through tunnel

reduced to 1/10
Resolution
Poor
But the resolution becomes poorer.
100
Time to pass through tunnel

reduced to 1/2
Both measures reduce

analysis time to 1/10.
Maintaining the resolution }

Particle size
5.0 Pm
3.0 Pm
2.2 Pm
Particle size:
Reducing particle size
improves resolution
(at constant pumping
rate).
Pumping rate:
Reducing particle
size maintains high
resolution, even if
pumping rate is
increased.
Select a column with

small particle size.
Speed 100 km/h
Good
Small
HKDUY 2013
Large
LC-MS/MS
Ultra-fast LC
(Low )
<<A>> Vortex diffusion

Diffusion occurring between packing particles
H = A u dp + B / X + C u dp2 u X
xH
: Height equivalent to a theoretical
plate
x dp
: Particle size of packing
x X
: Linear flowrate
x A u dp
: Vortex diffusion
xB/X
: Molecular diffusion
x C u dp 2 u X : Resistance to mass transfer
Number of
theoretical
plates
1. Size of gaps between particles
Particle size
(large)
(High)
A
B
(Slow )
(Fast )
Flowrate
<<B>> Molecular diffusion
Particle size
(small)
Natural diffusion along mobile phase flow.
<<C>> Resistance to mass transfer
2. Variability in particle size (particle size distribution)
Diffusion occurring within packing particles

Particle size
distribution
(large)
Particle size
(large)
Particle size
(small)
Particle size
distribution
(small)
HKDUY 2013
LC-MS/MS
Ultra-fast LC
) The fastest Autosampler: 10 sec/injection.
) Overlapping injection.
) Minimized carryover.
Start
0 min 0 sec
Other UHPLC
30sec for injection
Simadzu UFLC
10sec for injection
Finish
3 min 0 sec
Injection
Injection
Analysis
Analysis
Injection
Injection
Analysis
Analysis
Injection
Injection
Analysis
Analysis
33% Faster
Finish
2 min 0 sec
Nexera with
overlapping
injection
Injection
Analysis
Injection
Injection
Analysis
44% Faster
Analysis
Finish
1 min 40 sec
HKDUY 2013
LC-MS/MS
Ultra-fast LC
Low Carryover
y
9 Low carryover,
even with LC/MS/MS
Automatic
ti sample
l pretreatment
t t
t ffunctions:
ti
A
Auto injection of internal standard
(measurement by LC/MS)
9 Supports simple addition of an internal standard for correction of
suppression
(1) Take in internal standard
(3) Take in sample
(4) Start analysis
(2) Rinse
Sample
HKDUY 2013
To column
LC-MS/MS
Ultra-fast LC
mAU
125
5.4 MPa
TPN at peak 5 = 11352
Rs (4,5 = 10.849)
100
75
50
25
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
min
25 times faster
1.
2.
3.
4.
5.
Acetophenone
Propiophenone
Butyrophenone
Valerophenone
Hexanophenone
MPa
mAU
105 MPa
52.0
TPN at peak 5 = 663451.0
50.0
Rs (4,5 = 8.40)
1175
1150
1125
49.0
1100
48.0
1075
47.0
1050
46.0
45.0
0.0
C18
(4.6mm ID, 150mm,
5m)
1.0 mL/min
Water/acetonitrile = 45/55
40
245nm
0.1
0.2
0.3
0.4
0.5
0.6
HKDUY 2013
0.7
min
C18
(2.1mm ID, 50mm, 1.8 m)
1.8 mL/min
Water/acetonitrilen = 45/55
40
245nm
LC-MS/MS
Ultra-fast LC
1
2
4.7 MPa
3
4
5
40
0.0
2.5
5.0
7.5
C18
(4.6mm ID, 150mm, 5 m)
1.0 mL/min
10.0
245nm
1.
2.
3.
4.
5.
6.
7.
min
12.5
5 times higher separation efficiency

mAU
125
115 MPa
100
Acetophenone
Propiophenone
Butyrophenone
Valerophenone
Hexanophenone
Heptanophenone
Octanophenone
C18
(2.1mm ID, 300mm, 1.8 m)
0.5 mL/min
75
50
25
40
0
0.0
1.0
2.0
3.0
4.0
5.0
min
245nm
HKDUY 2013
LC-MS/MS
Ultra-fast LC
mAU
125
5.4 MPa
Rs (4,5 = 10.849)
100
C18
(4.6mm ID, 150mm, 5m)
1.0 mL/min
40
245nm
75
50
25
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
min
1.
2.
3.
4.
4
5.
14 times faster
1.5 times higher separation effciency
mAU
175
100 MPa
TPN at peak 5 = 16510 C18
Rs (4,5 = 12.21)
(2.1mm ID, 100mm, 1.8 m)
150
125
100
1.4mL/min
60
245nm
75
50
25
0
0.00
Acetophenone
Propiophenone
y p
Butyrophenone
Valerophenone
V
Hexanophenone
0.25
0.50
0.75
1.00
1.25
HKDUY 2013
1.50
1.75
min
LC-MS/MS
Ultra-fast LC
Peak capacity : 244 in 8 minutes

mAU
17.5
Column
: C18
Mobile phase
: A : 0.03 % TFA in water
Gradient
: B 5% 40 % (8 min)
2.1mm, 100mm, 1.8 m
15.0
12.5
B : 0.03 % TFA in acetonitrile
10.0
Mixer
: 180 L
7.5
Flow rate
: 0.9 mL/min
Column temp.
: 40
5.0
Detection
: 214 nm
Sample
: Trypsin digested BSA
2.5
(total of 1 pmol / uL)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
min
Bovin serum albumin triyptic digest
HKDUY 2013
LC-MS/MS
Ultra-fast LC
) Water as mobile phase Green LC
Green LC
Green LC
1
1 : Theophylline
2 : Caffeine
150
Water only
Column
: Shodex ET-RP1 (3.0mm, 150mm)
Mobile phase : water

Flow rate
: 0.5 mL/min
Column temp. : 150 oC
LC
40
30% methanol
LC
Column
: C18 (4.6mm, 150mm, 5 m)
Mobile phase
Flow rate
: 1.0 mL/min
Column temp. : 40 oC
HKDUY 2013
: methanol / water = 3/7
LC-MS/MS
Ultra-fast LC
Column: 150 mm L x 4.6 mm I.D., 5 m

Flow rate: 1 mL/min
Injection volume: 10 L
Analytical time: 60 min
Consumption volume: 60 mL/sample
0 min
60 min
Column: 75 mm L x 4.6 mm I.D., 2.2 m
Flow rate: 1 mL/min (or 2 mL/min)
Analytical time: 30 min (or 15 min)
0 min
30 min
(15 min)
HKDUY 2013
LC-MS/MS
Ultra-fast LC
Column: 150 mmL x 4.6 mmI.ID., 5 m

Flow rate: 1 mL/min
0 min
60 min
Column: 150 mmL x 2.0 mmI.ID., 5 m
Flow rate: 0.2 mL/min
0 min
60 min
HKDUY 2013
LC-MS/MS
Ultra-fast LC
) Acetonitirile consumption reduced up to > 80% while remaining the
separation efficiency
mAU
250
HPLC
Shim-pack VP-ODS (4.6 mm u 250 mm , 5m)
200
150

100
Acetonitrile: 12.8mL

50

0
0.0
5.0
10.0
mAU
250
15.0
20.0
25.0
min
UHPLC
Shim-pack XR-ODS (3.0mm u 100 mm , 2.2m)
200
150

100

50
Acetonitrile: 1.7mL

0
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
min
Mobile phase : A: 0.1% formic acid-water, B: acetonitrile, Gradient elution

Temperature : 40
Flow rate
: 1.0mL/min
Peaks
: 1:cefadroxil, 2:cephaprin, 3:cefaclor, 4:cefalexin, 5:cephradine, 6:cefotaxime. 7:cefazolin, 8 :cefuroxime, 9
:cefoperazone, 10 :cefloxitin, 11 :cefamandole A, 12 :cephalothin, 13: cefamandole B
HKDUY 2013
LC-MS/MS
Ultra-fast LC
Maximized Throughput
25 times faster analysis cycle, 10 sec injection speed , 2300 samples/day.
Solvent consumption reduced to 1/40.
Cost saving and environmentally-friendly UHPLC
Maximized Performance
5 times higher separation efficiency.
Carryover reduced to one/third.
Maximized Expandability
Expanded configurations by more than 100 optional units.
HKDUY 2013
LC-MS/MS
Ultra-fast LC
Solvent Saving
Flow Rate : 3/5 reduction
Analysis time : 1/25 reduction
= Mobile phase consumption/analysis is reduced to 1/40

Cost for power
Analysis Time : 1/25 reduction

= Power consumption is reduced to 1/25
Cost for performance
Initial cost + 5 years maintenance cost ) up to x 1.3 only
Sample / System ) up to x 5
= Cost / performance is reduced to 1/15
Cost saving for method migration
Method migration conventional LC UHPLC) tool available
HKDUY 2013
LC-MS/MS
Ultra-fast LC
The Worlds highest pressure 130MPa
S4Plunger Super Smooth Surface Sapphire
Minimized frictional heat on plunger surface
Optimized material for UHPLC
Plunger, Plunger seal, Needle seal, High-pressure valve, etc.
New high pressure valve design
S4-Plunger surface(x50)
S4-Plunger
High
h-strength
h
-st
strength
seal material
High-pressure valve
HKDUY 2013
LC-MS/MS
MS responding to performance of UFLC
What makes an MS instrument suitable for UFLC?

) The ability to acquire data at high speed without sacrificing data quality is
required!
Three things that enable ultrafast analysis:
Ability to perform scan measurement at high speed
UFscanning
Ability to switch between positive and negative ion measurement at
high speed
UFswitching
High sensitivity in high-speed measurement
UFsensitivity
HKDUY 2013
LC-MS/MS
UFLC Data
Influence of Data Sampling Points

on Peak Form
20 points
10 points
1.0
min.
)If the number of data points decreases,
the sensitivity also decreases.
This adversely affects the reproducibility.
HKDUY 2013
4 to 5 points
LC-MS/MS
Scan: Data is acquired in the desired m/z range.
m/z
Scan speed
(scan cycle)
1,100
Increase the scan speed.

100
t(s)
With conventional instruments,

the sensitivity decreases.
Decrease the scan speed.
It is difficult to handle high-speed
analysis.
m/z
Scan speed
(scan cycle)
1,100
100
t(s)
Since LCMS-2020, it has become possible to

maintain sensitivity when the scan speed is
increased.
HKDUY 2013
APPLICATIONS
Pesticides
HKDUY 2013
APPLICATIONS
Pesticides
HPLC : Nexera UHPLC system
Column
: Shim-pack XR-ODSII (75 mm x 2 mmI.D., 2.2 um)
Mobile phase
: A ; 2 mM ammonium formate containing 0.1 % formic acid water

B ; Methanol
: 5% B (0-2.5 min.)55% B (2.51-6 min.)80% B (6.01-12 min.)
100% (12-15 min.)5% (15.01-20 min.)
Gradient program
Flow rate
: 0.2 mL / min.
Column temperature
: 40 C
MS : LCMS-8040 Triple quadrupole mass spectrometer

Ionization
Ion spray voltage
: ESI (Positive / Negative)

: +4.5 kV / -3.5 kV
DL temperature
: 250 C
HB temperature
Nebulizing Gas
: 400 C
: 3 L / min.
Drying Gas
MRM
: 15 L / min.
: 276 MRM transitions (2 MRMs / compound)
Dwell time 5 msec. / Pause time 1 msec.
HKDUY 2013
APPLICATIONS
Pesticides
Number of compounds: 138

72: LC/MS/MS
66: GC/MS/MS
LOQs of 138 pesticides in the EURL method by LCMS-8040

Technique
LOQs < 10 ppb
LOQs > 10 ppb
Not Ionization
by LC/MS/MS
72 (100 %)
0 (0 %)
0 (0 %)
by GC/MS/MS
47 (71 %)
6 (9 %)
13 (20 %)
0.089.9 ppb
80 % of compounds which was refered to GC-QqQ
were possible to be measured by LC-QqQ
HKDUY 2013
APPLICATIONS
Pesticides
HKDUY 2013
APPLICATIONS
Pesticides
HKDUY 2013
APPLICATIONS
Veterinary Drugs
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Salbutamol
5-Propylsulfonyl-1H-benzimidazole-2-amine
Lincomycin
Trimethoprim
Thiabendazole
Sulfacetamide
Ormetoprim
Ractopamine
Sulfadiazine
Xylazine
Clenbuterol
Sulfathiazole
Sulfapyridine
Sulfamerazine
Carbadox
Pyrimethamine
Thiamphenicol
Sulfadimidine
Sulfamonomethoxine
Trichlorfon (DEP)
Sulfamethoxypyridiazine
Sulfachlorpyridazine
Erythromycin
Sulfadoxine
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Sulfamethoxazole
Tiamulin
Florfenicol
Chloramphenicol
Clorsulon
Ethopabate
Sulfaquinoxaline
Sulfadimethoxine
Prednisolone
Hydrocortisone
Dexamethasone
Penicillin-G
Sulfanitran
Emamectin B1a
beta-Trenbolone
alpha-Trenbolone
Zeranol
Oxacillin
Famphur
Fenobucarb (BPMC)
Phenylbutazone
Melengestrol Acetate
Temephos (Abate)
Allethrin
HKDUY 2013
APPLICATIONS
Veterinary Drugs
HKDUY 2013
APPLICATIONS
Veterinary Drugs
HKDUY 2013
APPLICATIONS
Veterinary Drugs
Calibration point (conc.)
5, 10, 25, 50, 100, 250, 500 ng / g (=1, 2.5, 5, 10, 25, 50, 100 ng / mL)
Veterinary drug standards were spiked into the pork matrix.
Compounds
Salbutamol
Lincomycin
Trimethoprim
Thiabendazole
Sulfacetamide
Ormetoprim
Ractopamine
Sulfadiazine
Xylazine
Clenbuterol
Sulfathiazole
Sulfapyridine
Sulfamerazine
Carbadox
Pyrimethamine
Thiamphenicol
Sulfadimidine
Sulfamonomethoxine
Trichlorfon (DEP)
Erythromycin
Sulfadoxine
Calibration curve
Range (ng / g)
r2
5-500
5-500
10-1000
5-500
5-500
25-500
5-500
5-500
10-500
5-500
5-500
5-500
5-500
10-1000
10-500
5-500
50-500
10-1000
5-500
25-500
5-500
25-500
5-500
5-500
0.999
0.997
0.999
0.996
0.998
0.999
0.998
0.999
0.998
0.999
0.997
0.996
0.998
0.998
0.999
0.997
0.989
0.995
0.995
0.999
0.997
0.996
0.999
0.993
LOQ (ng / g)
1.1
2.5
1.6
3.2
2.5
21.3
1.9
2.0
5.8
1.6
0.8
1.5
2.5
1.9
4.3
1.1
16.7
1.2
0.8
10.3
2.7
21.5
0.1
1.1
HKDUY 2013
Compounds
Sulfamethoxazole
Tiamulin
Florfenicol
Chloramphenicol
Clorsulon
Ethopabate
Sulfaquinoxaline
Sulfadimethoxine
Prednisolone
Hydrocortisone
Dexamethasone
Penicillin-G
Sulfanitran
Emamectin B1a
beta-Trenbolone
alpha-Trenbolone
Zeranol
Oxacillin
Famphur
Fenobucarb (BPMC)
Phenylbutazone
Temephos (Abate)
Allethrin
Calibration curve
Range (ng / g)
r2
LOQ (ng / g)
5-500
5-500
50-500
25-500
50-500
5-500
5-500
5-500
10-500
10-500
20-1000
5-500
5-500
5-500
10-500
10-500
25-500
5-500
10-500
5-500
5-500
5-500
10-500
5-500
0.997
0.999
0.990
0.999
0.997
0.997
0.998
0.998
0.999
0.996
0.999
0.999
0.989
0.999
0.997
0.998
0.995
0.999
0.999
0.999
0.996
0.997
0.999
0.999
4.5
5.0
16.3
12.4
50.0
5.3
1.3
1.0
6.2
5.2
15.3
1.4
2.0
5.0
5.8
5.4
18.9
0.3
7.9
1.1
6.4
3.1
3.4
3.9
APPLICATIONS
Veterinary Drugs
HKDUY 2013
APPLICATIONS
Veterinary Drugs
HKDUY 2013
APPLICATIONS
Veterinary Drugs
HKDUY 2013
APPLICATIONS
Veterinary Drugs
Recovery (%)
Compounds
Salbutamol
Lincomycin
Trimethoprim
Thiabendazole
Sulfacetamide
Ormetoprim
Ractopamine
Sulfadiazine
Xylazine
Clenbuterol
Sulfathiazole
Sulfapyridine
Sulfamerazine
Carbadox
Pyrimethamine
Thiamphenicol
Sulfadimidine
Sulfamonomethoxine
Trichlorfon (DEP)
Erythromycin
Sulfadoxine
Spike level
(ng / g
5
5
5
5
5
50
5
5
50
5
5
5
50
50
50
5
50
5
50
50
5
50
5
5
Recovery (%)
Pork
Chicken
Salmon
75
108
120
92
71
104
97
61
52
90
90
84
88
86
76
101
95
104
90
99
110
112
144
84
74
99
129
114
77
91
144
133
55
91
76
62
76
74
64
86
90
84
78
123
99
122
113
86
89
101
124
116
103
157
150
107
71
82
76
74
85
83
91
97
92
83
82
151
109
154
104
84
Compounds
Sulfamethoxazole
Tiamulin
Florfenicol
Chloramphenicol
Clorsulon
Ethopabate
Sulfaquinoxaline
Sulfadimethoxine
Prednisolone
Hydrocortisone
Dexamethasone
Penicillin-G
Sulfanitran
Emamectin B1a
beta-Trenbolone
alpha-Trenbolone
Zeranol
Oxacillin
Famphur
Fenobucarb (BPMC)
Phenylbutazone
Temephos (Abate)
Allethrin
Spike level
(ng / g
5
5
50
50
50
5
5
5
50
50
50
50
5
5
50
50
50
5
50
5
50
5
50
5
Pork
Chicken
Salmon
76
104
122
121
108
85
79
81
114
139
105
116
58
101
110
108
116
123
107
98
84
99
90
94
100
118
134
156
108
116
91
93
122
114
126
116
107
129
109
109
111
133
112
109
107
87
84
89
99
121
113
119
93
112
108
91
155
127
149
116
76
130
103
110
122
116
128
116
52
76
70
63
Recovery
Sample
70 % -120 %
70 % >
120 %<
Pork
41 (85 %)
2 (4 %)
5 (10 %)
Chicken
Salmon
35 (73 %)
34 (71 %)
3 (6 %)
2 (4 %)
10 (21 %)
12 (25 %)
HKDUY 2013

LC-MSMS (En)

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INTRODUCTION TO LC-MS/MS

Presented by: Hunh Khnh Duy Application Manager

TP. HCM 09/2013

Flow of mobile phase

LC-MS = LC (Liquid Chromatographer) coupled to MS (Mass Spectrometer).

Product ions generated from specific ions

Typical sample: isolated

mass-to-charge ratio (m/z) of ions

PDA (photodiode array)

Mobile phase preparation errors

An impurity coincides with the target:

Fluctuations in peak retention times

Changes in area value

The peaks (including

388.4 432.5476.0520.4 564.5608.4

200 2013 300

Chromatograms of plasma sample

How can these

ANT:m/z 189.0 (6)

SIR: 298.22 m/z

) Amount of a compound that passes through the detector is a function of Peak

Analyze ions, m/z

Research on interfacing HPLC to MS began in the 1970s; API (atmospheric

API interfaces: electrospray ionization (ESI), atmospheric pressure

- Ion Trap (IT)

Atmospheric Pressure Chemical

High voltage (r3 to 5 kV)

Protonated molecule [M+H]+ at m/z(Mr+1).

Deprotonated molecule [M-H]- at m/z(Mr-1).

Other ions often observed:

Sodium adduct ions [M+Na]+.

Solavted ions [M+H+CH3CN]+, [M+H+CH3OH]+.

Neutral lose of small molecules.

(a) GCMS, EI spectrum of riboflavin (vitamine B2)

(b) LCMS, ESI (API) spectrum of riboflavin

Exact Mass: 376.14

128 142 155 167

243 257268 280 295

453 469 482 497

141 153 168

490510 534553 580

201 225 246 273

479 508 537

ESI-Q spectra by LCMS-2010EV

No simple Na-adduct ion can form in negative mode.

) In most case, adduct ions can be recognized easily: span

Natural pesticides azadirachtin as the active component

Neem extract mainly from seeds: very complex in composition

Conventional method reverse phase HPLC with UV detector (217

Difficulty in identification of minor components due to lack of

LCMS: identification of components based on MW and neutral loss

Peak 22: 6-Desacetyl

Peak 23: 3-Desacetyl

619 642 660

419 437 461 479 497 519 541 565

419 437 455 479497

Peak 24: OhichinolideB

Peak 27: Nimbin

506 525 549 568585 607

Depend on structures and properties of compounds

ESI: CH3CN/H2O and MeOH/H2O

Non-volatile buffer like phosphate is not recommended

: TFA, conc. < 0.1%