Tandem MS for Drug Analysis

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 9 months ago)

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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
-
2

MS
-
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/MS

12%

3%

2%

0

LC/MS/MS

3%

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

Inlet

Detect

Mass

Analyze

Ionize

MS

Inlet

Fragment

Mass
Analyze

Ionize

Mass

Analyze

Detect

MS1

Collision

Cell

MS2

MS/MS

MS
vs.

MS/MS

GC

HPLC

CE

Separation

Identification

7

CH
3
COCH
3

Sample

Inlet

CH
3
+
COCH
3

Ionization

& Adsorption

of Excess Energy

Mass Analysis

CH
3
C
+
OCH
3

+
COCH
3

+
CH
3

+
COH

Fragmentation

(Dissociation)

Detection

Mass Spectrometry

8

Multidimensional Analyses

time

response

chromatogram

m/z

m/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 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.

13

Tandem Quadrupole

Collision
cell

MS1

MS2

14

Components of Tandem Mass
Spectrometer

Collision

Cell

Mass

Spectrometer

Mass

Spectrometer

Detector

Ionization Source

ESI

APPI

APCI

MALDI

Argon

Xenon

Quatrupole

Magnetic Sector

Quatrupole

Magnetic Sector

Time
-
of
-
flight

Collision
cell

MS1

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

17

J. Zelene,
Phys. Rev
.,
10
, 1
-
6 (1917)

The Electrospray Phenomenon

18

Ionization Source

19

Sample Cone

Orifice = 400µm

Spraying Needle

Vacuum

Isolation Valve

Ionization Source

20

High voltage applied


to metal sheath (~4 kV)


Sample Inlet Nozzle

(Lower Voltage)

Charged droplets

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

MH
+

MH
3
+

MH
2
+

Pressure = 1 atm

Inner tube diam. = 100 um

Sample in solution


N
2

N
2
gas

Partial

vacuum

Electrospray ionization:

Ion Sources make ions from sample molecules


21

ESI Spectrum of Trypsinogen (MW 23983)

1599.8

1499.9

1714.1

1845.9

1411.9

1999.6

2181.6

M + 15 H
+

M + 13 H
+

M + 14 H
+

M + 16 H
+

m/z

Mass
-
to
-
charge ratio

22

APCI

23

APPI

24

h
n


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

MALDI: Matrix Assisted Laser Desorption Ionization

25

The mass spectrum shows the results

Relative Abundance

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

Collision

Cell

Mass

Spectrometer

Mass

Spectrometer

Detector

Ionization Source

ESI

APPI

APCI

MALDI

Argon

Xenon

Quatrupole

Magnetic Sector

Quatrupole

Magnetic Sector

Time
-
of
-
flight

Collision
cell

MS1

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.

MS1

MS2

Collision

Cell

Scanning

Rf only, pass all masses

Collision
cell

MS1

MS2

Full Scan Acquisition Mode

29

Mass Spectrum: Progesterone

200
220
240
260
280
300
320
340
360
380
400
m/z
0
100
%
315.1
316.1
[
M+H]
+

O
O
C
H
3
C
H
3
C
H
3
Full Scan Acquisition Mode

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.

Argon gas

Precursor

Products

Collision
cell

MS1

MS2

Product ion scanning

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
C
H
3
C
H
3
C
H
3
Precursor ion

Product ions

O
C
H
2
C
H
2
C
H
3
O
C
H
3
C
H
3

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
330
m/z
0
100
%
315.1
316.1
Mass Spectrum from
MS1

100
125
150
175
200
225
250
275
300
325
m/z
0
100
%
109.0
97.0
Product ion spectrum from MS2

Product ion scanning

Product ions

O
C
H
2
C
H
2
C
H
3
O
C
H
3
C
H
3
O
O
C
H
3
C
H
3
C
H
3
Precursor ion

33

20
40
60
80
100
120
140
160
180
200
220
m/z
0
100
%
0
100
%
0
100
%
0
100
%
0
100
%
5eV

10 eV

30 eV

40 eV

20 eV



collision energy >


fragmentation

Product ion scanning

34

Static

Scanning

Precursor Ion Scan

The first quadrupole mass analyzer is
Scanning

a mass range while the
second quadrupole is fixed, or
S
tatic
, at the mass
-
to
-
charge ratio (
m/z
)
of a product ion known to be common to the analytes in a mixture.

Argon gas

Precursors

Product

Collision
cell

MS1

MS2

Precursor ion scanning

35

-

RCOOH

-
(CH
3
)
3
N

-
C
4
H
8

CID

Butylation

CH
2

CH

CH

RCOO

H

COOH

(
CH
3
)
3
N

CH
2

CH

CH

RCOO

H

CO
OC
4
H
8

(
CH
3
)
3
N

CH
2

CH

CH

COOH

[

]
+

(
m/z 85)

Acylcarnitines

Derivatization and Fragmentation

All compounds of this type
fragment to produce the 85
ion.

Precursor ion scanning

R=0 to 18 carbon alkyl chain.

36

225

250

275

300

325

350

375

400

425

450

475

500

m/z

0

100

%

d
3
-
free carnitin
e

C2 carnitine

C16 carnitine

d
3
-
C3 carnitine

d
3
-
C8 carnitine

d
3
-
C16


carnitine

Normal Acylcarnitine Profile

Precursor ion scanning

37

S
canning (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
cell

MS1

MS2

Neutral loss scanning

38

Neutral and Acidic Amino Acids

Derivatization and Fragmentation
(Generic)

+
Butanol

C
H
3
O
H
O
O
H
C
H
3
Butyl formate
Neutral loss

of
102Da

+
O
N
H
2
O
H
R
Neutral or Acidic AA

HCl

Amino acid butyl ester

O
N
H
2
O
R
C
H
3
Neutral or Acidic AA

O
N
H
3
+
O
R
C
H
3
Fragmentation

Fragment

N
H
2
+
R
39

140
160
180
200
220
240
260
280
m/z
0
100
%
d
3
-
Leu

d
4
-
Ala

d
3
-
Met

d
5
-
Phe

d
6
-
Tyr

d
8
-
Val

Gly

Ser

Pro

Glu

Deuterated internal standards for quantification

Normal Amino Acid Profile

Neutral loss scanning

40

Both the first and second quadrupole mass analyzers are held
S
tatic

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 gas

Precursor(s)

Product(s)

Collision
cell

MS1

MS2

Multiple Reaction Monitoring

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 50

L 潦oi湴敲湡 獴慮摡牤s⡩渠浥m桡h潬o i湴漠敡捨獣牥s
-
捡瀠杬慳猠

瑵扥t

3
.

Add 200

L 卩S潬o浵猠捡汩扲慴潲b⠵砠捯湣敮瑲慴敤ai渠整桡湯l⤠潲o㈰2



浥m桡h潬o景爠灡瑩敮琠獡浰汥献

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 100

L 㠵8 浥m桡h潬o


.

Inject 80

L ⡥煵楶慬敮琠瑯t
㠰8

L wh潬攠扬潯b
⤠慮a
慮慬a獥s畳楮朠uwo



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 ZnSO
4

Soln.

Whole Blood

(10

L
-

40µL)

Add 2 volumes MeCN

with IS, Seal & Vortex Mix

Centrifuge,

Inject 5
-

20

L

Immunosuppressant Sample
Preparation

LC
-
MS/MS Analysis

45

Sirolimus: MS Spectrum

790
795
800
805
810
815
820
825
830
835
840
845
850
m/z
0
100
%
821.5
810.5
822.5
826.5
827.5
[
M+H
]
+

[
M+NH
4
]
+

[
M+Li
]
+

[
M+Na
]
+

[
M+K
]
+

Full Scan Acquisition Mode

46

Sirolimus:

LC
-
MS (SIM)
vs

LC
-
UV

0.50
1.00
1.50
Time
0
100
%
0
100
%
SIR
m/z
821

30µg / L

1.5 min

HPLC
-
UV

HPLC
-
MS

Single ion monitoring (MS)

47

Sirolimus: MS Spectrum

790
795
800
805
810
815
820
825
830
835
840
845
850
m/z
0
100
%
821.5
810.5
822.5
826.5
827.5
[
M+H
]
+

[
M+NH
4
]
+

[
M+Li
]
+

[
M+Na
]
+

[
M+K
]
+

Full Scan Acquisition Mode

48

MS1

MS2

Collision

Cell

Static (m/z 821.5)

Scanning

The first quadrupole mass analyzer is fixed, or
S
tatic
, 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
-
3
mBar)

Precursor

Products

Product ion scanning

49

790
795
800
805
810
815
820
825
830
835
840
845
850
m/z
0
100
%
821.5
810.5
822.5
826.5
827.5
Mass spectrum from MS1

200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
m/z
0
100
%
768
576
558
548
718
750
786
821
Product ion spectrum from MS2

Product ion scanning

NH
4
+

50

MS1

MS2

Collision

Cell

Static (m/z 821.5)

Static (m/z 768.5)



Ar (2.5


3.0e
-
3
mBar)

Precursor(s)

Product(s)

MS/MS : Compound
-
Specific Monitoring

Multiple Reaction Monitoring

51

Sirolimus

LC
-
MS(SIM)
vs

LC
-
MS/MS (MRM)

SIR
m/z
821

0.50
1.00
1.50
Time
0
100
%
0
100
%
0.50
1.00
1.50
Time
0
100
%
0
100
%
MRM
m/z
821>768

3µg / L

30µg / L

Multiple Reaction Monitoring