lcms technology connects to your application · atmospheric pressure ionization (api) esi apci 22....
TRANSCRIPT
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LCMS Technology
Connects to Your Application
June, 2018 (LCMS Product Specialist)
Dr.Rittichai Charoensapyanan
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Fundamental of Liquid Chromatography (LC)
Fundamental of Mass Spectrometer (MS)
LCMS Applications
Topics
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Fundamental of Liquid Chromatography
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Liquid Chromatography (LC)
Stationary Phase
Mobile phase (continuous)1.9
4.7
6.3
Retention time Identification
Peak area Quantification
• Liquid Chromatography (LC) : Separation technique which liquid is used as mobile phase
• Separation : Between two phases (Stationary phase and Mobile phase)
• Compounds are separated from each other based on their difference in affinity for the
stationary or mobile phase.
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• Degasser : Remove air bubble in solvents
• Pump : - Mix solvents
- Control the flow rate of mobile phase and analytes
• Autosampler : Inject the sample into a running system
• Column : Separate each components
• Column Compartment : Control a column temperature
• Detector : Detect signal from analytes after separation
HPLC System
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DAD (UV, VIS) Fluorescence Reflective Index Mass Spectrometer
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HPLC System Range
Basic Automated
Highly economic & reliable
620 bar UHPLC compatible
Flow rates up to 10 mL/min
100 Hz detector range
Modular flexibility
Standard
3rd Generation Modules
620 bar UHPLC compatible
Flow rates up to 10 mL/min
Oven temp. 5 – 80 ºC
100 Hz DAD, MWD, VWD, FLD, CAD
Highest flexibility
x2 Dual LC
Two systems in one
620 bar UHPLC compatible
Flow rates up to 10 mL/min
Oven temp. 5 – 80 ºC
Automated Application Switching
Parallel and Tandem LC
Online SPE-LC
Automated method scouting
Turn key Viper kits for ease of use
RSLC
Binary and Quaternary UHPLCs
1000 bar up to 5 mL/min
800 bar up to 8 mL/min
Oven temp. 5 – 110 ºC
200 Hz DAD, MWD, VWD, FLD
Improved sub 2-µm particle column
compatibility
Ultrafast/ultra resolution system
x2 Dual RSLC
x2 Dual UHPLC System
Two systems in one
1000 bar up to 5 mL/min
800 bar up to 8 mL/min
Oven temp. 5 – 110 ºC
200 Hz DAD, MWD, VWD, FLD
Parallel and Tandem LC
Online SPE-LC
Automated method scouting
Offline 2D-UHPLC
Turn key Viper kits for ease of use
RSLCnano
UHPLC system for Nano/Cap/Micro
20 nL/min – 50 µL/min up to 800 bar
Continuous direct flow
New standard in retention time
precision
Snap-in valves
nanoViper fitting system for easy
operation
Basic Standard x2 Dual LC RSLC x2 Dual RSLC RSLCnano
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VanquishTM
Max Pressure 1517 bar
The Highest Pressure HPLC
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1.9µm
5µm
u opt
u opt
Linear Velocity (mm/s)
H E
T P
(µ
m)
5
10
15
0 61 2 3 4 5
3µm
u opt
Incr
easi
ng C
olum
n Ef
ficie
ncy
Increasing Flowrate
Advantage of Small Particle
Higher efficiency, independent of flow rate means…
Faster runs without loss of performance
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Efficiency is the key!!!
5µm
1.9µm N = 142,000 plates/m
(189% higher)
N = 75,000 plates /m
Higher resolution – narrower peaks
Higher sensitivity – taller peaks
Higher peak capacity (more peaks / unit time) – narrower peaks
( )k
kNRs
+−=
1 1
41
αα
Selectivity Efficiency Retention
Advantage of Small Particle
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Increase Speed, Maintain Resolution 200x2.1mm
0 2 4 6 8 10 12 14 16 18Time (min)
12µm
8µm
5µm
3µm
1.9µm600µl/min 655 bar
400µl/min 190 bar
250µl/min 102 bar
100µl/min 56bar
150µl/min 68 bar
Spee
d
Speeding up analysis with 1.9 µm Hypersil GOLD
Advantage of Small Particle
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The UltiMate™ 3000 HPLC Systems
Pump
Autosampler
Column Compartment
Detector
With ValvesStandard
Quaternary Dual-GradientBinary
VWD MWD/DAD Fluorescence Corona
Standard Thermostatted + FractionationBasic Automated
Isocratic
Coulochem
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HPLC Applications
• Built-in column compartment with 2-position, 6-port
switching valve
Switching Valve (2-position, 6-port)
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HPLC Applications
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Load Position
• Online SPE: Extraction and
separation at the same time !!!
Injection Position
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HPLC Applications
• Parallel LC: Analysis of
two distinct methods !!!
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Fundamental of Mass Spectrometer
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“The basis in mass spectrometry (MS) is the production of ions, that aresubsequently separated or filtered according to their mass-to-charge(m/z) ratio, and detected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a function of the m/z ratio.”
Niessen et al., LC-MS: Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.
What is Mass Spectrometer?
• Measure gas-phase ions
• Operate at very low pressure (10-5 to 10-7 torr)
• Mass spectrometer work with IONS
• Determine the mass are separated according to
their mass-to-charge (m/z) ratio
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Information Rich Data
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(1023.566 x 1) - 1
= 1022.6
(512.287 x 2) - 2
= 1022.6
Mass to charge (m/z) = ( molecular weight + charge ) / charge
Mass Spectrum
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Liquid
ChromatographyIonization Mass Analysis
Mass Spectrometry: Block Diagram
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• Ion source : Converts sample molecules (neutral) into charged molecules or molecular ions.
• Type of ionization techniques
o Matrix Assisted Laser Desorption Ionization (MALDI)
o Atmospheric Pressure Ionization (API)
- Electrospray Ionization (ESI)
- Atmospheric Pressure Chemical Ionization (APCI)
Ionization
Ion Source
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No one ionization technique is applicable to all classes of
chemical species !
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Atmospheric Pressure Ionization (API)
ESI APCI
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• Ions formed by gas phase chemistry
• Good for volatile / thermally stable
• Good for non-polar analytes
• Good for small molecules (steroids)
• Ions formed by solution chemistry
• Good for thermally labile analytes
• Good for polar analytes
• Good for large molecules (protein/peptide)
Atmospheric Pressure Ionization (API)
ESI APCI
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Mass Analysis
Mass Spectrometry: Block Diagram
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Mass Analyzer
• Triple Quadrupole (QqQ)
• Orbitrap
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Mass Analyzer: Triple Quadrupoles (QqQ)
High-capacity transfer tube (HCTT)
Active collision cell (Q2)
Electrodynamic ion funnel (EDIF)
Ion beam guide with neutral blocker
Asymetric RF drive
HyperQuad quadrupole mass filter (Q1)
Dual-mode discrete-dynode detector
HyperQuad quadrupole mass filter (Q3)
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Mass Analyzer: Triple Quadrupoles (QqQ)
• Q1 and Q3 are “Mass filter” where
ions are scanned by varying the
DC/AC & RF voltages across the
quadrupole set
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Mass Analyzer: Triple Quadrupoles (QqQ)
• Q2 is “Collision Cell” where precursor
ions are fragmented and pass through
Q3 for ion sorting again
Precursor Ion
Fragmentation
(Collision gas: Ar)
Product Ions
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Scan Modes in QqQ
Purpose: Survey scan
of a chromatographic
peak
Purpose: Quantitation
on a specific m/z
range of ions
m/z 200-400
m/z 250
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Scan Modes in QqQ
Purpose: Targeted quantitation
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Fixed m/z: 400
m/z 300 m/z 500
m/z 400
m/z 400 m/z 500
Fixed m/z: 500
m/z 300 m/z 400
m/z 400 Fixed m/z: 400 Fixed m/z: 400
m/z 400
m/z 400
Scan Modes in QqQ
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SRM
m/z 300 m/z 400
m/z 400
m/z 250
Q1: Precursor Ion Q3: Product IonQ2: Fragmentation
m/z 400
m/z 250
m/z 150m/z 400
m/z 400
m/z 400
m/z 300
m/z 100
m/z 250
Fixed m/z: 250Fixed m/z: 400
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Fragment
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Scan Modes in QqQ
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RT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + c NSI Full ms [ 400.00-1800.00] MS data14
NL: 7.97E7Base Peak m/z= 1030.90-1031.90 F: + c NSI Full ms [ 400.00-1800.00] MS data14
SIM
Full Scan
Full Scan VS SIM
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18 19 20 21 22 23 24 25 26 27Time (min)
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
20
40
60
80
100
Rel
ativ
e A
bund
ance
0
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 2.67E4m/z= 271.50-272.50 F: + c SIM ms [236.50-237.50, 271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIM ms [236.50-237.50, 271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EI SRM ms2 237.000 [207.999-208.001] MS Genesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EI SRM ms2 272.000 [236.999-237.001] MS Genesis Probe20F
Superior Selectivity
Free from sample matrix
SRM
SIM
SIM VS SRM
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Mass Analyzer
• Orbitrap
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Mass Analyzer: Orbitrap
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Orbitrap Mass Analyzer: Principle of Operation
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
(r,z)
(r,φ)
Image Current
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qmk
z /=ω
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Many Ions Generate a Complex “Transient”
Fourier Transform
Frequency DomainImage Current
Mass Spectrum
qmk
z /=ω
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Q ExactiveTM
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Mass Analyzer
• Orbitrap
High Resolution Accurate Mass (HRAM)
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Mass Resolution
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794792790788786
100908070605040302010
794792790788786
100908070605040302010
Δm= 0.786
• Ability of a mass spectrometer to distinguish between ions of nearly equal m/z ratios (isobars).
m - measured mass
Δm - peak width measured at 50% peak intensity (Full Width Half Maximum)
Low Resolution
R = 786.6 = 1,000
High Resolution
R = 786.6 = 100,000
0.786
0.007Δm= 0.007
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Commercial High Resolution MS Technology Race
Time progression (year)
Mas
s re
solu
tion
(FW
HM
)
Bendix Tof0
50000
100000
150000
200000
250000
300000
350000
400000
450000
500000
1955 1965 1975 1985 1995 2005 2015
Orbitrap Tof / QTof
Ion Trap-Orbitrap
Quad Orbitrap
Tribrid Orbitrap
ORBITRAP’s spectacular climb in performance in a decade!
First Q-Tof
Q-Orbitrap*
New Q-Orbitrap
New Tribrid Orbitrap
Entry Q-Orbitrap
LIT-Orbitrap ETD
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• The precision of which the mass is measuredby the mass spectrometer.
• Typical way of reporting mass error in ppm(relative measure) or mDa (absolute measure)
Mass error = Measured – Exact Mass x 106
Mass Accuracy
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( )Exact Mass
C = 12.0000
H = 1.0078
N = 14.0031
O = 15.9949
S = 31.9721
Good
786.6003 (-1.19 ppm)
794792790788786
100908070605040302010
786.60124
787.60463
788.60773
789.61068
786.70 (+124 ppm)
error = 786.6003 – 786.60124 x 106
786.60124
( )error = 786.7000 – 786.60124 x 106
786.60124
( )
Not so Good
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• Typical mass accuracy capability for various MS types
Source: Metabolomics Fiehn’s lab
Mass Accuracy
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Mass Accuracy
47
• Increases confidence in identification
CxNxOxHx
C12H22O11
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Measured Mass Mass Error (Da) Possible Formula Exact Mass
32.0 ± 0.2
O2 31.9898
CH3OH 32.0261
N2H4 32.0374
S 31.9721
32.02 ± 0.02CH3OH 32.0261
N2H4 32.0374
32.0257 ± 0.002 CH3OH 32.0261CH3OH
• Main advantage: the possibility to determine the elemental composition of individual
molecular or fragment ions, a powerful tool for the structural elucidation or confirmation.
C = 12.0000
H = 1.0078
N = 14.0031
O = 15.9949
S = 31.9721
Mass Resolution and Accuracy
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100
892.60 892.65 892.70 892.75 892.80 892.85 892.90 892.95 893.00m/z
892.7862
892.8323
892.7952
892.7377
892.8226
892.7903100
892.7378
892.8246
100892.7973
Mass Resolution and Accuracy
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• Isobaric compounds separation
R = 100,000292.02 292.04 292.06 292.08
m/z
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
292.04031C 10 H 15 O5 N P S
292.02656C8 H11 O 3 N 5 Cl S
0
R = 50,000
R = 35,000
R = 15,000
Thiamethoxam
[M+H]+ = 292.02656
Parathion
[M+H]+ = 292.04031
∆m0.0138 Da
Mass Resolution and Accuracy
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• Isobaric compounds separation
Mass Resolution and Accuracy
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• Removing interferences
High resolution is very important for samples with complex matrix (e.g. biological,
food), since they will contain a significant number of background ions
Mass Resolution and Accuracy
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LCMS Applications
53
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LCMS Applications
54
• Pharmaceutical analysis
– Bioavailability studies
– Drug metabolism studies, pharmacokinetics
– Characterization of potential drugs
– Drug degradation product analysis
– Screening of drug candidates
– Identifying drug targets
• Etc.
• Biomolecule characterization
– Proteomics
– Oligonucleotides
• Environmental analysis
– Pesticides on foods
– Soil and groundwater contamination
• Forensic and clinical analysis
• Toxicology
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Application in Food
Melamine
SRM Transitions
(Q1) 127 -> 68 (Q3)
(Q1) 127 -> 85 (Q3)
Identification and Quantitation of Melamine in Infant Milk
Varelis et al. Thermo AN62732. 2008
Sample Prep
(SPE)
LC-MS/MS
(Targeted SRM)
LC: AccelaTM
Column: BioBasic AX (Ion Exchange)
Column Temperature: 30ºC
Injection Volume: 1 µL
Mobile Phase: A) 85% ACN + 10% IPA + 5%
Ammonium acetate; B) 90% water and 10%ACN
Flow Rate: 400 µL/min Run Time: 5 min
MS: TSQ Quantum UltraTM
Ionization: Positive ESI
Modes: Targeted SRM
Instrument conditions
55
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Identification and Quantitation of Melamine in Infant Milk
• Limit of Detection (LOD): <10 ppb (below the FDA requirement at 1 ppm)
Varelis et al. Thermo AN62732. 2008
Application in Food
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Application in Halal Food
Determination of Meat Authenticity
Orduna et al. Thermo AN64677. 2016
LC & HRAM MS
Conditions
57
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Orduna et al. Thermo AN64677. 2016
Application in Halal Food
Determination of Meat Authenticity
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Application in Pharmaceutical Products
Li et al. Thermo AN64427. 2015
Discovery and Characterization of Natural Components
LC: Ultimate 3000TM
Column: Hypersil Gold C18
Column Temperature: 35ºC
Injection Volume: 1 µL
Mobile Phase: ACN + 0.1% FA (linear gradient)
Flow Rate: 300 µL/min Run Time: 50 min
MS: Q ExactiveTM
Ionization: Positive and Negative ESI
Modes: dd-MS2
Instrument conditions
59
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Application in Pharmaceutical Products
Discovery and Characterization of Natural Components
Extracted Ion Chromatogram and Isotopic Pattern
MS and MS2 Spectrum
MS2 Spectrum Interpretation
60 Li et al. Thermo AN64427. 2015
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Application in Pharmaceutical Products
Discovery and Characterization of Natural Components
• 21 Cantharidins were detected
in the blister beetle extract.
• 16 new Cantharidins were
discovered and identified with
mass accuracy <1 ppm by
fragment ion search (FISh)
function, using cantharidinimide
as the parent structure.
Li et al. Thermo AN64427. 201561
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• Peptide Labeling (multiple studies with a single experiment)
62
Application in Proteomics
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63
Application in Proteomics
• Protein Identification
• Expression Level of Protein
• Protein Modification
• Protein Localization
• Peptide Mapping
• Pathways
• Etc.
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http://planetorbitrap.com/
LCMS Technology and Applications
64
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Thank You for Your Attention
65