tandem ms for drug analysis lecture
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1
Tandem MS for Drug Analysis
2
Mass Spectrometers
• Separate and measures ions based on their mass-to-charge (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 bioanalysis, quadrupole, time-of-flight (TOF) and ion traps are most used.
3
Ion source
MS-2MS-1
Mixture of ions
Single ion
Fragments
What is Tandem MS?
• Uses 2 (or more) mass analyzers in a single instrument– One purifies the analyte ion from a mixture
using a magnetic field.– The other analyzes fragments of the analyte
ion for identification and quantification.
4
Analytical Assays used in Pharmaceutical Industry Labs for New
Chemical Entities
Method 1990 1998 2000 2006
HPLC(UV &Fluorescence)
75% 50-60% 20% 2%
GC/MS12% 3% 2% 0
LC/MS/MS3% 40-50% 60-75% 98%
Immunoassay(ELISA/FPIA etc.)
10% 10% 10% 0
5
Applications of Tandem MS
• Biotechnology & Pharmaceutical– To determine chemical structure of drugs and drug
metabolites.– Detection/quantification of impurities, drugs and their
metabolites in biological fluids and tissues.– High through-put drug screening– Analysis of liquid mixtures– Fingerprinting
• Nutraceuticals/herbal drugs/tracing source of natural products or drugs
• Clinical testing & Toxicology– inborn errors of metabolism, cancer, diabetes, various
poisons, drugs of abuse, etc.
6
InletInlet DetectDetectMass
Analyze
MassAnalyz
e
IonizeIonize
MSMS
InletInlet FragmentFragmentMass Analyz
e
Mass Analyz
e
IonizeIonizeMass
AnalyzeMass
AnalyzeDetectDetect
MS1MS1 CollisionCell
CollisionCell
MS2MS2
MS/MSMS/MS
MS vs. MS/MS
GCHPLCCE
Separation Identification
7
CH3COCH3CH3COCH3
Sample Inlet
Sample Inlet
CH3+COCH3CH3+COCH3
Ionization& Adsorption
of Excess Energy
Ionization& Adsorption
of Excess Energy
Mass AnalysisMass Analysis
CH3C+OCH3CH3C+OCH3
+COCH3+COCH3
+CH3+CH3
+COH+COH
Fragmentation(Dissociation)
Fragmentation(Dissociation)
DetectionDetection
Mass Spectrometry
8
Multidimensional Analyses
time
response
chromatogram
m/zm/z
m/z
9
Different Types of MS
• Tandem MS– Triple Quatrupole– Hybrid Instruments
• ESI-QTOF– Electrospray ionization source + quadrupole mass filter +
time-of-flight mass analyzer
• MALDI-QTOF– Matrix-assisted laser desorption ionization + quadrupole
+ time-of-flight mass analyzer
10
LC-MS/MS
11
Analytical Quadrupole
12
Quadrupole TheoryPre-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.
13
Tandem Quadrupole
Collision cellMS1
MS2
14
Components of Tandem Mass Spectrometer
CollisionCell
MassSpectrometer
MassSpectrometer
Detector
Ionization Source
ESIAPPIAPCIMALDI
ArgonXenon
QuatrupoleMagnetic Sector
QuatrupoleMagnetic SectorTime-of-flight
Collision cellMS1
MS2
15
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
– Atmospheric Pressure Ionization (API)• Electrospray• MALDI• APCI• APPI
16
The Abbé 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 !
F. Lemière, LC•GC Europe “LC-MS Supplement”, December 2001, p29-35
The Macabre History of Electrospray
17J. Zelene, Phys. Rev., 10, 1-6 (1917)
The Electrospray Phenomenon
18
Ionization Source
19
Sample ConeOrifice = 400µmSample ConeOrifice = 400µm
Spraying NeedleSpraying Needle
Vacuum Isolation Valve
Vacuum Isolation Valve
Ionization Source
20
High voltage applied to metal sheath (~4 kV)
Sample Inlet Nozzle(Lower Voltage)
Charged droplets
++
+++
+
++
+ +++
++
+ +++
+++
+++
+++
++
++
+
++
+
+
+
+++
+++
+++
MH+
MH3+
MH2+
Pressure = 1 atmInner tube diam. = 100 um
Sample in solution
N2
N2 gas
Partialvacuum
Electrospray ionization:
Ion Sources make ions from sample molecules
21
ESI Spectrum of Trypsinogen (MW 23983)
1599.8
1499.9
1714.1
1845.91411.9
1999.6
2181.6
M + 15 H+
M + 13 H+
M + 14 H+M + 16 H+
m/z Mass-to-charge ratio
22
AP
CI
23
AP
PI
24
h Laser
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.
MH+
+/- 20 kV Grid (0 V)
Sample plate
MA
LD
I: M
atri
x A
ssis
ted
Las
er D
eso
rpti
on
Io
niz
atio
n
25
The mass spectrum shows the results
Re
lativ
e A
bun
dan
ce
Mass (m/z)
0
10000
20000
30000
40000
50000 100000 150000 200000
MH+
(M+2H)2+
(M+3H)3+
MALDI TOF spectrum of IgG
26
Components of Tandem Mass Spectrometer
CollisionCell
MassSpectrometer
MassSpectrometer
Detector
Ionization Source
ESIAPPIAPCIMALDI
ArgonXenon
QuatrupoleMagnetic Sector
QuatrupoleMagnetic SectorTime-of-flight
Collision cellMS1
MS2
27
Operation Modes
• Product Ion Scanning– Analyzes all products of a single precursor
• Precursor Ion Scanning – Analyzes all precursors of a single charged product
• Neutral Loss Scanning – Analyzes all precursors of a single uncharged product
• Multiple Reaction Monitoring– Analyzes for specific precursors producing specific
products.
28
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.
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.
MS1 MS2Collision
Cell
Scanning Rf only, pass all masses
Collision cellMS1
MS2F
ull S
can
Acq
uisi
tion
Mod
e
29
Mass Spectrum: Progesterone
200 220 240 260 280 300 320 340 360 380 400m/z0
100
%
315.1
316.1
[M+H]+
O
O
CH3
CH3
CH3
Ful
l Sca
n A
cqui
sitio
n M
ode
30
Static (m/z 315.1) 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.
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.
Argon gasArgon gas
PrecursorPrecursorProductsProducts
Collision cellMS1
MS2P
rodu
ct io
n sc
anni
ng
31
Collision induced dissociation
• 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)
Argon gas
O
O
CH3
CH3
CH3
Precursor ion
Product ions
OCH
2
CH2
CH3
O
CH3
CH3
• In the collision cell, the TRANSLATIONAL ENERGY of the ions is converted to INTERNAL ENERGY.
32
Product Ion Spectrum: Progesterone
300 305 310 315 320 325 330m/z0
100
%
315.1
316.1
Mass Spectrum from MS1
100 125 150 175 200 225 250 275 300 325m/z0
100
%
109.097.0
Product ion spectrum from MS2
Pro
duct
ion
scan
ning
Product ions
OCH
2
CH2
CH3
O
CH3
CH3
O
O
CH3
CH3
CH3
Precursor ion
3320 40 60 80 100 120 140 160 180 200 220m/z0
100
%
0
100
%
0
100
%
0
100
%
0
100
% 5eV
10 eV
30 eV
40 eV
20 eV
collision energy > fragmentationP
rodu
ct io
n sc
anni
ng
34
StaticScanning
Precursor Ion Scan
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.
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.
Argon gasArgon gas
PrecursorsPrecursorsProductProduct
Collision cellMS1
MS2P
recu
rsor
ion
scan
ning
35
- RCOOH-(CH3)3N
-C4H8
- RCOOH-(CH3)3N
-C4H8
CIDCID
ButylationButylation
CH2CH2 CHCH CHCH
RCOORCOO HH
COOHCOOH(CH3)3N(CH3)3N
CH2CH2 CHCH CHCH
RCOORCOO HH
COOC4H8COOC4H8(CH3)3N(CH3)3N
CH2CH2 CHCH CHCH COOHCOOH[[ ]+]+
(m/z 85)(m/z 85)
Acylcarnitines Derivatization and Fragmentation
All compounds of this type fragment to produce the 85 ion.
Pre
curs
or io
n sc
anni
ngR=0 to 18 carbon alkyl chain.
36225 250 275 300 325 350 375 400 425 450 475 500
m/z0
100
%
d3-free carnitined3-free carnitine
C2 carnitineC2 carnitine
C16 carnitineC16 carnitine
d3-C3 carnitined3-C3 carnitine
d3-C8 carnitined3-C8 carnitine
d3-C16 carnitined3-C16 carnitine
Normal Acylcarnitine ProfileP
recu
rsor
ion
scan
ning
37
Scanning (M-102)Scanning (M)
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.
Argon gas
Precursors
Products
Collision cellMS1
MS2N
eutr
al lo
ss s
cann
ing
38
Neutral and Acidic Amino AcidsDerivatization and Fragmentation
(Generic)
+
Butanol
CH3
OH
O
OHCH3
Butyl formate Neutral loss of
102Da
+
O
NH2
OHR
Neutral or Acidic AA
HCl
Amino acid butyl ester
O
NH2
OR
CH3
Neutral or Acidic AA
O
NH3+
OR
CH3
Fragmentation
Fragment
NH2+
R
39140 160 180 200 220 240 260 280
m/z0
100
%
d3-Leu
d4-Ala
d3-Met
d5-Phed6-Tyr
d8-Val
Gly
Ser
Pro
Glu
Deuterated internal standards for quantification
Normal Amino Acid ProfileN
eutr
al lo
ss s
cann
ing
40
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 intense product ion, respectively.
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 intense product ion, respectively.
Static (m/z 315.1) Static (m/z 109.0)
Argon gasArgon gas
Precursor(s)
Precursor(s)
Product(s)Product(s)
Collision cellMS1
MS2M
ultip
le R
eact
ion
Mon
itorin
g
41
Specificity of Detection for LC
• UV – chromophore– all compounds with a chromophore responding at the
selected wavelength will interfere
• MS – molecular mass – interference from isobaric compounds – chemical noise
• MS/MS – molecular mass and structural information– interference from structural isomers only
42
1. Wash all glassware in methanol x2 and tert-butyl methyl ether (TBME) x2.2. Place 50L of internal standard (in methanol) into each screw-cap glass tube.3. Add 200L Sirolimus calibrator (5x concentrated in methanol) or 200L methanol for patient samples.4. Add 1.0mL blank whole blood to calibrators or 1.0mL patient whole blood.5. Add 2.0mL 0.1M ammonium carbonate buffer.6. Mix thoroughly.7. Add 7.0mL TBME and extract for 15min.8. Transfer upper layer to clean tube and re-extract lower layer with 7.0mL TBME.9. Combine TBME extracts and evaporate to dryness.10. Redissolve residue in 5.0mL ethanol and evaporate to dryness.11. Redissolve residue in 1.0mL ethanol, transfer to Eppendorf tube and evaporate to dryness.12. Redissolve residue in 100L 85% methanol.13. Inject 80L (equivalent to 800L whole blood) and analyse using two 4.6mm x 250mm C18 columns connected in series (30min run time).
HPLC-UV Analysis of Sirolimus in Whole Blood
43
Sirolimus: HPLC - UV Example
44
Add ZnSO4 Soln.
Whole Blood(10L - 40µL)
Add 2 volumes MeCNwith IS, Seal & Vortex Mix
Centrifuge,Inject 5 - 20L
Immunosuppressant Sample Preparation
LC-MS/MS Analysis
45
Sirolimus: MS Spectrum
790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5
827.5[M+H]+
[M+NH4]+
[M+Li]+
[M+Na]+
[M+K]+
Ful
l Sca
n A
cqui
sitio
n M
ode
46
Sirolimus:LC-MS (SIM) vs LC-UV
0.50 1.00 1.50Time0
100
%
0
100
%SIR m/z 821
30µg / L
1.5 min
HPLC-UV
HPLC-MS
Sin
gle
ion
mon
itorin
g (M
S)
47
Sirolimus: MS Spectrum
790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5
827.5[M+H]+
[M+NH4]+
[M+Li]+
[M+Na]+
[M+K]+
Ful
l Sca
n A
cqui
sitio
n M
ode
48
MS1 MS2Collision
Cell
Static (m/z 821.5) 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.
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.
Ar (2.5 – 3.0e-3mBar)Ar (2.5 – 3.0e-3mBar)
PrecursorPrecursorProductsProducts
Pro
duct
ion
scan
ning
49
790 795 800 805 810 815 820 825 830 835 840 845 850m/z0
100
%
821.5
810.5
822.5
826.5827.5
Mass spectrum from MS1Mass spectrum from MS1
200 250 300 350 400 450 500 550 600 650 700 750 800 850 900m/z0
100
%
768
576
558548718 750
786
821
Product ion spectrum from MS2Product ion spectrum from MS2
Pro
duct
ion
scan
ning
NH4+
50
MS1 MS2Collision
Cell
Static (m/z 821.5) Static (m/z 768.5)
Ar (2.5 – 3.0e-3mBar)Ar (2.5 – 3.0e-3mBar)
Precursor(s)Precursor(s)Product(s)Product(s)
MS/MS : Compound-Specific Monitoring
Mul
tiple
Rea
ctio
n M
onito
ring
51
SirolimusLC-MS(SIM) vs LC-MS/MS (MRM)
SIR m/z 821
0.50 1.00 1.50Time0
100
%
0
100
%
0.50 1.00 1.50Time0
100
%
0
100
%
MRM m/z 821>768
3µg / L 30µg / L
Mul
tiple
Rea
ctio
n M
onito
ring
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