Beruflich Dokumente
Kultur Dokumente
Navnath Jaybhaye
Manager- Product development & Application
Agenda
Introduction Overview of MS Ionization Techniques Mass Analyzers Quadrupole Operations Applications Of LCMS
Analytical Assays used in Pharmaceutical Industry Labs for New Chemical Entities
Method
HPLC
(UV &Fluorescence)
1990
75% 12% 3% 10%
1998
50-60% 3% 40-50% 10%
2000
20% 2% 60-75% 10%
2006
2% 0 98% 0
3
Mass Spectrometers
Separate and measures ions based on their mass-tocharge (m/z) ratio. Operate under high vacuum (keeps ions from bumping into gas molecules) Key specifications are resolution, mass measurement accuracy, and sensitivity. Several kinds exist: for analysis, quadrupole, time-offlight (TOF) and ion traps are most used.
MS vs. MS/MS
GC HPLC CE Inlet Ionize Mass Analyze Detect
Separation
MS
Mass Analyze MS1
Identification
Inlet
Ionize
Detect
MS/MS
Mass Spectrometry
+CH 3 +COH
CH3COCH3
CH3+COCH3
CH3C+OCH3
+COCH 3
Sample Inlet
Mass Analysis
Detection
MS1
Collision cell
MS2
Sample introduction
Ion Souce
Transforms sample molecules to ions Soft ionization
Places positive or negative charge on the analyte without significantly fragmenting the analyte M+1 ion (or M-1 ion) No need to volatilize Down to fmol detection limits
The Abb Nollet experimented with electrified liquids in the 18th century ! He observed that when a person was connected to a high-voltage generator he/she would not bleed normally after cutting ...blood sprayed from the wound !
Heater
The sample is held on the surface of a capillary tube, a fine wire, or a small cup.
10
11
Ionization Source
12
Ionization Source
Spraying Needle Sample Cone
Orifice = 400m
13
Ionization Methods.
Gas Phase Ionization Electron Impact (EI) Chemical Ionization (CI) Spray Ionization / Atmospheric Pressure Ionization (API)
Electrospray (ESI) / API Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure photo Ionization (APPI)
Desorption Ionization
14
Electrospray Ionization(ESI)
16
N2
+ + + + ++ ++ + + ++ ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++
MH+
+ + + + + + + + + +
MH2+ MH3+
Charged droplets 17
TURBOIONSPRAY (TIS)
18
TM
19
M + 15 H+ M + 14 H+
M + 16 H+
1499.9 1714.1
M + 13 H+
1411.9
m/z
Mass-to-charge ratio
20
APCI
21
APPI
22
Sample plate h
Laser
MH+
1. Sample is mixed with matrix (X) and dried on plate. 2. Laser flash ionizes matrix molecules. 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X.
+/- 20 kV
Grid (0 V)
23
MH+
30000
(M+2H)2+
20000
10000
(M+3H)3+
0
50000
100000
150000
200000
Mass (m/z)
24
Differential vacuum in MS
OR IQ1
DP-FP
RNG
Collisional Focussing
QO
Q1 Analyzer
IQ2
Q3 Analyzer
DET
ST
RO1
RO3
DF
1x10-5 torr
1-5 x 10 torr
-4
Turbo
Roughing pump
25
Significance of vacuum
A vacuum is necessary to permit ions to reach the detector. It reduces or eliminates the chances of ion collisions with mass analyzer. The major reason for maintaining high vacuum is to increase the mean-free path of ions. Increases sensitivity and resolution of Mass Spectrum.
26
Types Of Analyzers
Quadrupole Ion Trap Time-of-flight
27
Analytical Quadrupole
28
29
Quadrupole Theory
Pre-filter Quadrupole Mass Filter
Stable Trajectory
Unstable Trajectories
Only ions with the correct m/z values have stable trajectories within an RF/DC Quadrupole field. Ions with unstable trajectories collide with the rods, or the walls of the vacuum chamber, and are neutralised.
30
1. Quadrupole
31
Tandem Quadrupole
MS1 Collision cell MS2
33
MS1
Collision cell
MS2
34
Operation Modes
Product Ion Scanning
Analyzes all products of a single precursor
MS1
Collision cell
MS2
MS1
Collision Cell
MS2
Scanning
SCANNING MODE: The first quadrupole mass analyzer is Scanning over a mass range. The collision cell and the second quadrupole mass analyzer allow all ions to pass to the detector.
36
%
316.1
0 200
220
240
260
280
300
320
340
360
380
m/z 400
37
MS1
Collision cell
MS2
Argon gas
Products Precursor
Scanning
The first quadrupole mass analyzer is fixed at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range.
38
100
CH3
CH3
CH3
315.1
% 0 300
97.0
Precursor ion
305
CH2
316.1
310
O CH3
315
320
325
100 % 0
109.0
CH2
H3C CH3
Product ions
100 125 150 175
MS1
Collision cell
MS2
Argon gas
Product Precursors
Scanning Static The first quadrupole mass analyzer is Scanning a mass range while the second quadrupole is fixed, or Static, at the mass-to-charge ratio (m/z) of a product ion known to be common to the analytes in a mixture. 40
Acylcarnitines
R=0 to 18 carbon alkyl chain.
Butylation
RCOO (CH3)3N CH2 CH H CH - RCOOH -(CH3)3N -C4H8 COOC4H8
CID
[ CH2
CH (m/z 85)
CH
COOH
]+
41
d3-free carnitine
d3-C16 carnitine
C2 carnitine
%
0 225 250 275 300 325 350 375 400 425 450 475 500
42
m/z
MS1
Collision cell
MS2
Argon gas
Products Precursors
Scanning (M)
Scanning (M-102)
In a neutral loss scan the two quadrupole mass filters are Scanning synchronously at a user-defined offset. The neutral loss is known to be common to the analytes in a mixture. 43
(Generic)
O R NH2 O CH3
HO
+
CH3
HCl
Neutral or Acidic AA
O R NH3
+
Butanol
Fragmentation R
O CH3
NH2
+
+
HO
CH3
Neutral or Acidic AA
Fragment
d6-Tyr
Glu
m /z 280 45
MS1
Collision cell
MS2
Argon gas
Product(s)
Precursor(s)
Both the first and second quadrupole mass analyzers are held Static at the mass-to-charge ratios (m/z) of the precursor ion and the most 46 intense product ion, respectively.
CH3
O
CH3
Precursor ion
Product ions
In the collision cell, the TRANSLATIONAL ENERGY of the ions is converted to INTERNAL ENERGY.
Collision conditions (FRAGMENTATION) is controlled by altering: The collision energy (speed of the ions as they enter the cell) Number of collisions undertaken (collision gas pressure)
47
48
N2 CAD Gas
Exit lens
Q0
Q1
Q2
Q3
ion selection
Fragmentation
49
Ion Trap ID ~ 10 mm
50
51
3. Time-of-Flight (TOF)
Time of flight mass spectrometer measures the massdependent time It takes ions of different masses to move from the ion source to the detector. Ions are either formed by a pulsed ionization method (usually MALDI), or various kinds of rapid electric field switching are used as a 'gate' to release the ions from the ion source in a very short time.
52
53
DETECTORS
Mass analysis is the separation of bunches or streams of ions according to their individual mass-to-charge (m/z) ratio The mass analyzer sorts the ions according to m/z and the detector records the abundance of each m/z.
54
Once the ion passes through the mass analyzer it is then detected by the ion detector, The final element of the mass spectrometer.
55
56
Secondary Electrons
58
Contd
CHARECTERISTICS
Certain of these
characteristics are common, like :
61
62
1. Multiple-dynode type EM
Incident ions
Cathode
Anode
Incoming ion reaches the first dynode Ejects several other electrons by secondary emission. This process repeated at each succeeding dynode having
a higher potential than the preceding dynode.
Contains a glass pipe with a coating on its inner surface The electron flow moves along the pipe, reflecting from
the inner wall and progressively gaining electrons
Disadvantage:
Short lifetime Requires good vacuum to operate
66
IONS
The primary incoming ions passes through the inlet and strikes the
surface of the CEM The collision energy eject an electron from CEM wall Ejected electrons accelerated into interior of CEM Trigger secondary emission and the process continues to produce an output electron
67
RECORDER
Analogue signal is produced by the detector. Analogue Digital Converter sends the output to the computer.
Detector
ADC
Recorder
68
IDENTIFICATION
CHARACTERIZATION
Single MS Specificity
Precursor Ion Neutral Loss Enh.Multi Charged
QUANTITATION
SIM or MRM
TurboIonspray Heated Nebulizer (APCI) Nanospray Integrated Syringe pump Built-in divert valve
AUTOMATION
Info. Dep. Acquisition
MetID
69
Application Example 1
LC-MS/MS
Selectivity and Sensitivity comparisons Application Area: Environmental
72
Q1 fixed, =
CAD Collision
Q3 scanning
MH+
Simazine (202-132)
MH+
Atrazine (216-174)
74
75
Application Example 2
LC-MS/MS
Impurity profiling
76
77
For known impurities, targeted MRM analysis can be used. At the same time an EMS survey scan can search for unknown impurities for unknown impurities, IDA triggered MS/MS and MS3 information can be collected to aid in identification.
Dependent Scan (s) Dependent Scan (s) Dependent Scan (s) Dependent Scan (s) Add to Exclusion List
Second Dependent Second Dependent Second Dependent Scan (s) Scan (s) Scan (s)
78
OH
H N
HO
H N OH
H 3C O
79
H N O
245
80
software can help with data analysis through sample and control comparison for degradation products
2 .8 6
M a x . 8 .3 e 6 c p s .
XICs of peaks present in Sample, but not Control. MS/MS information collected through IDA
81
MS molecular mass
interference from isobaric compounds chemical noise
84
Sirolimus: MS Spectrum
[M+NH4]+
100
821.5
822.5
[M+Na]+
[M+Li]+ [M+H]+
810.5
826.5 827.5
[M+K]+
0 790
795
800
805
810
815
820
825
830
835
840
845
m/z 850
86
Sirolimus:
Single ion monitoring (MS)
1.5 min
HPLC-UV
87
Sirolimus: MS Spectrum
[M+NH4]+
100
821.5
822.5
[M+Na]+
[M+Li]+ [M+H]+
810.5
826.5 827.5
[M+K]+
0 790
795
800
805
810
815
820
825
830
835
840
845
m/z 850
88
MS1
Collision Cell
Products Precursor
Scanning
The first quadrupole mass analyzer is fixed, or Static, at the mass-to-charge ratio (m/z) of the precursor ion to be interrogated while the second quadrupole is Scanning over a user-defined mass range.
89
NH4+
100
821.5
%
826.5 827.5 810.5
0 790
795
800
805
810
815
820
825
830
835
840
845
m/z 850
100
768
0 200
250
300
350
400
450
500
550
600
650
700
750
800
850
m/z 900
90
MS1
Collision Cell
Precursor(s)
Product(s)
3g / L
% %
30g / L
0 100
0 100
0.50
1.00
1.50
Time 0
0.50
1.00
1.50
Time
92
Questions please?...
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