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Tandem MS for Drug Analysis

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.

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.

Mixture of ions

Single

ion

Ion source
Ion
source
MS-1
MS-1
MS-2
MS-2
What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –

Fragments

What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –
What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –
What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –
What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –
What is Tandem MS? • Uses 2 (or more) mass analyzers in a single instrument –

3

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

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.

MS vs. MS/MS

GC HPLC CE
GC
HPLC
CE
Inlet
Inlet
Ionize
Ionize
Mass Analyze
Mass
Analyze
MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass
Detect
Detect
MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass
MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass
MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass
MS
MS

Separation

MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass

Identification

MS vs. MS/MS GC HPLC CE Inlet Ionize Mass Analyze Detect MS Separation Identification Mass Mass
Mass Mass Inlet Ionize Fragment Detect Analyze Analyze Collision MS1 MS2 Cell
Mass
Mass
Inlet
Ionize
Fragment
Detect
Analyze
Analyze
Collision
MS1
MS2
Cell

MS/MS

6

Mass Spectrometry

CH 3 COCH 3
CH 3 COCH 3
+ COH + CH 3 CH 3 + COCH 3 CH 3 C + OCH 3
+ COH
+ CH 3
CH 3 + COCH 3
CH 3 C + OCH 3
+ COCH 3
Mass Spectrometry CH 3 COCH 3 + COH + CH 3 CH 3 + COCH 3
Sample Inlet
Sample
Inlet

Ionization & Adsorption of Excess Energy

Fragmentation

(Dissociation)

Mass Analysis

Detection
Detection

Multidimensional Analyses

m/z
m/z
m/z
m/z

m/z

response

chromatogram

time

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

LC-MS/MS

LC-MS/MS 10

10

Analytical Quadrupole

Analytical Quadrupole 11

Quadrupole Theory

Pre-filter Quadrupole Mass Filter Stable Trajectory
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.

Tandem Quadrupole

Collision MS2 cell MS1
Collision
MS2
cell
MS1

13

Components of Tandem Mass

Spectrometer

ESI APPI APCI MALDI
ESI
APPI
APCI
MALDI

Ionization Source

Mass

Collision

Mass

Detector

Spectrometer

Cell

Spectrometer

     

Quatrupole

 

Argon

 

Magnetic Sector

Xenon

 
 
Collision MS2 cell MS1 14
Collision
MS2
cell
MS1
14

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

The Macabre History of

Electrospray

16
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 Electrospray Phenomenon

The Electrospray Phenomenon J. Zelene, Phys. Rev ., 10 , 1-6 (1917) 17

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

17

Ionization Source 18

Ionization Source

18
18
Ionization Source 18

Ionization Source

Spraying Needle Sample Cone Orifice = 400µm Vacuum Isolation Valve
Spraying Needle
Sample Cone
Orifice = 400µm
Vacuum
Isolation Valve

19

Ion Sources make ions from sample molecules

 
 

Electrospray ionization:

Partial Sample Inlet Nozzle (Lower Voltage) vacuum MH + + + + + + + +
Partial
Sample Inlet Nozzle
(Lower Voltage)
vacuum
MH +
+
+
+
+
+
+
+
+
+
+ +
+
+ +
+
+
+ +
+
+
+
+ +
+
MH 2
+
+
+ +
+
+
+
+
+
+
+
+ +
+
+ +
+ +
+
+
+ +
+
MH 3
+
Ion Sources make ions from sample molecules Electrospray ionization: Partial Sample Inlet Nozzle (Lower Voltage) vacuum

Charged droplets

20

Pressure = 1 atm

Inner tube diam. = 100 um

N 2

Ion Sources make ions from sample molecules Electrospray ionization: Partial Sample Inlet Nozzle (Lower Voltage) vacuum

Sample in solution N 2 gas

Ion Sources make ions from sample molecules Electrospray ionization: Partial Sample Inlet Nozzle (Lower Voltage) vacuum
Ion Sources make ions from sample molecules Electrospray ionization: Partial Sample Inlet Nozzle (Lower Voltage) vacuum
Ion Sources make ions from sample molecules Electrospray ionization: Partial Sample Inlet Nozzle (Lower Voltage) vacuum

High voltage applied to metal sheath (~4 kV)

ESI Spectrum of Trypsinogen (MW 23983)

1599.8 M + 15 H + M + 16 H + M + 14 H +
1599.8
M + 15 H +
M + 16 H +
M + 14 H +
1499.9
1714.1
M + 13 H +
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
21

APCI

APPI

APPI 23

23

MALDI: Matrix Assisted Laser Desorption Ionization

Sample plate

Laser
Laser
MH +
MH +
hn
hn
MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser MH + hn Grid (0 V) 1.

+/- 20 kV

Grid (0 V)

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.

The mass spectrum shows the results

MALDI TOF spectrum of IgG

MH + 40000 30000 (M+2H) 2+ 20000 10000 (M+3H) 3+ 0 50000 100000 150000 200000 Relative
MH +
40000
30000
(M+2H) 2+
20000
10000
(M+3H) 3+
0
50000
100000
150000
200000
Relative Abundance

Mass (m/z)

Components of Tandem Mass

Spectrometer

Ionization Source

Mass Collision Mass
Mass
Collision
Mass

Spectrometer

Cell

Spectrometer

   

Quatrupole

 

Argon

Magnetic Sector

Xenon

 
ESI APPI APCI MALDI
ESI
APPI
APCI
MALDI

Detector

Quatrupole

Magnetic Sector

Time-of-flight

Collision MS2 cell MS1 26
Collision
MS2
cell
MS1
26

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.

Full Scan Acquisition Mode

Collision MS2 cell MS1 Collision MS1 Cell MS2
Collision
MS2
cell
MS1
Collision
MS1
Cell
MS2
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass
Full Scan Acquisition Mode Collision MS2 cell MS1 Collision MS1 Cell MS2 Scanning Rf only, pass

Scanning

Rf only, pass all masses

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.

28

Full Scan Acquisition Mode

Mass Spectrum: Progesterone

[M+H] + O CH 3 CH 3 315.1 100 CH 3 O % 316.1 0 m/z
[M+H] +
O
CH 3
CH 3
315.1
100
CH 3
O
%
316.1
0
m/z
200
220
240
260
280
300
320
340
360
380
400

Product ion scanning

Collision MS2 cell MS1
Collision
MS2
cell
MS1
Product ion scanning Collision MS2 cell MS1 Precursor Argon gas Products Static (m/z 315.1) Scanning The
Precursor
Precursor
Product ion scanning Collision MS2 cell MS1 Precursor Argon gas Products Static (m/z 315.1) Scanning The
Argon gas Products
Argon gas
Products
Product ion scanning Collision MS2 cell MS1 Precursor Argon gas Products Static (m/z 315.1) Scanning The
Product ion scanning Collision MS2 cell MS1 Precursor Argon gas Products Static (m/z 315.1) Scanning The

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.

30

Collision induced dissociation

O
O

Argon gas

CH 3
CH 3
CH 3 CH 3
CH 3
CH 3

O

Precursor ion

O CH CH 3 2 CH 2 O HC 3 CH 3 Product ions
O
CH
CH
3
2
CH
2
O
HC
3
CH
3
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)

Product ion scanning

Product Ion Spectrum:

Progesterone

CH 3
CH 3

O

315.1

100

%

CH 3

Product ion scanning Product Ion Spectrum: Progesterone CH 3 O 315.1 100 % CH Mass Spectrum

Mass Spectrum from

Precursor ion CH 3 O
Precursor ion
CH 3
O
310 305
310
305
315
315
315
316.1 MS1

316.1

MS1

320 325
320
325
CH 3
CH
3
O CH 2
O
CH
2
Product ion scanning Product Ion Spectrum: Progesterone CH 3 O 315.1 100 % CH Mass Spectrum

0

300

m/z

330

%

0

97.0 CH 2 109.0 O HC 3 CH 3 Product ions
97.0
CH
2
109.0
O
HC
3
CH
3
Product ions

100

Product ion spectrum from MS2

m/z

100

125

150

175

200

225

250

275

300

325

collision energy > fragmentation

100 5eV % 0 100 10 eV % 0 100 20 eV % 0 100 30
100
5eV
%
0
100
10
eV
%
0
100
20
eV
%
0
100
30
eV
%
0
100
40
eV
%
33
0
20
40
60
80
100
120
140
160
180
200
220
Product ion scanning

m/z

Precursor ion scanning

Collision MS2 cell MS1 Precursor Ion Scan
Collision
MS2
cell
MS1
Precursor Ion Scan
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Precursors
Precursors
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Argon gas
Argon gas
Product
Product
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static
Precursor ion scanning Collision MS2 cell MS1 Precursor Ion Scan Precursors Argon gas Product Scanning Static

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.

Precursor ion scanning

R=0 to 18 carbon alkyl chain.

Acylcarnitines

Derivatization and Fragmentation

RCOO H (CH ) 3 N CH CH CH COOH 3 2 Butylation
RCOO
H
(CH
) 3 N
CH
CH
CH
COOH
3
2
Butylation
RCOO H (CH 3 ) 3 N CH 2 CH CH
RCOO
H
(CH 3 ) 3 N
CH 2
CH
CH
COOC 4 H 8
COOC 4 H 8
CID
CID
- RCOOH -(CH 3 ) 3 N -C 4 H 8
- RCOOH
-(CH
3 )
3 N
-C 4
H 8

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

[ ] + CH 2 CH CH COOH (m/z 85)
[
] +
CH 2
CH
CH
COOH
(m/z 85)

35

Precursor ion scanning

Normal Acylcarnitine Profile

d 3 -C16 carnitine d 3 -free carnitine 100 C2 carnitine % d 3 -C3 carnitine
d 3 -C16 carnitine
d 3 -free carnitine
100
C2 carnitine
%
d 3 -C3 carnitine
C16 carnitine
d 3 -C8 carnitine
0
225
250
275
300
325
350
375
400
425
450
475
500

m/

Neutral loss scanning

Collision MS2 cell MS1
Collision
MS2
cell
MS1
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In

Precursors

Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In

Argon gas

Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In

Products

Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In
Neutral loss scanning Collision MS2 cell MS1 Precursors Argon gas Products Scanning (M) Scanning (M-102) In

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.

37

Neutral and Acidic Amino Acids

Derivatization and Fragmentation

 

O

R

R OH
R OH

OH

NH 2

(Generic)

HO +
HO
+

HCl

Neutral and Acidic Amino Acids Derivatization and Fragmentation O R OH NH (Generic) HO + HCl

CH 3

 

O

R

R O
R O

O

NH 2

Neutral and Acidic Amino Acids Derivatization and Fragmentation O R OH NH (Generic) HO + HCl

CH 3

Neutral or Acidic AA

Butanol

Amino acid butyl ester

R

O CH 3
O
CH 3
O + NH 3
O
+
NH
3

Fragmentation

Neutral and Acidic Amino Acids Derivatization and Fragmentation O R OH NH (Generic) HO + HCl

Neutral or Acidic AA

R + + NH 2 Fragment
R
+
+
NH 2
Fragment

O

HO

Neutral and Acidic Amino Acids Derivatization and Fragmentation O R OH NH (Generic) HO + HCl

CH 3

Butyl formate Neutral loss of

102Da

38

Neutral loss scanning

Normal Amino Acid Profile

Pro

100

%

0

Deuterated internal standards for quantification d 3 -Leu d 5 -Phe d 6 -Tyr d 4
Deuterated internal standards for quantification
d
3 -Leu
d 5 -Phe
d 6 -Tyr
d
4 -Ala
Ser
d
8 -Val
d
3 -Met
Glu
Gly
m/z
140
160
180
200
220
240
260
280

Multiple Reaction Monitoring

Collision MS2 cell MS1
Collision
MS2
cell
MS1
Multiple Reaction Monitoring Collision MS2 cell MS1 Precursor(s) Argon gas Product(s) Static (m/z 315.1) Static (m/z
Precursor(s)
Precursor(s)
Multiple Reaction Monitoring Collision MS2 cell MS1 Precursor(s) Argon gas Product(s) Static (m/z 315.1) Static (m/z
Argon gas Product(s)
Argon gas
Product(s)
Multiple Reaction Monitoring Collision MS2 cell MS1 Precursor(s) Argon gas Product(s) Static (m/z 315.1) Static (m/z
Multiple Reaction Monitoring Collision MS2 cell MS1 Precursor(s) Argon gas Product(s) Static (m/z 315.1) Static (m/z
Multiple Reaction Monitoring Collision MS2 cell MS1 Precursor(s) Argon gas Product(s) Static (m/z 315.1) Static (m/z

Static (m/z 315.1)

Static (m/z 109.0)

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.

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

HPLC-UV Analysis of Sirolimus in Whole Blood

  • 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).

Sirolimus: HPLC - UV Example

Sirolimus: HPLC - UV Example 43

43

Immunosuppressant Sample

Preparation

LC-MS/MS Analysis

Whole Blood (10L - 40µL)

Add ZnSO 4 Soln.

Immunosuppressant Sample Preparation LC-MS/MS Analysis Whole Blood (10  L - 40µL) Add ZnSO Soln. Add
Immunosuppressant Sample Preparation LC-MS/MS Analysis Whole Blood (10  L - 40µL) Add ZnSO Soln. Add

Add 2 volumes MeCN with IS, Seal & Vortex Mix

Centrifuge, Inject 5 - 20L

Immunosuppressant Sample Preparation LC-MS/MS Analysis Whole Blood (10  L - 40µL) Add ZnSO Soln. Add

Full Scan Acquisition Mode

Sirolimus: MS Spectrum

[M+NH 4 ] + 821.5 100 822.5 [M+Na] + % 826.5 [M+Li] + [M+H] + [M+K]
[M+NH 4 ] +
821.5
100
822.5
[M+Na] +
%
826.5
[M+Li] +
[M+H] +
[M+K] +
827.5
810.5
0
m/z
790
795
800
805
810
815
820
825
830
835
840
845
850

45

Single ion monitoring (MS)

Sirolimus:

LC-MS (SIM) vs LC-UV

HPLC-MS

HPLC-UV

SIR m/z 821

Single ion monitoring (MS) Sirolimus: LC-MS (SIM) vs LC-UV HPLC-MS HPLC-UV SIR m/z 821 100 %

100

%

0

Single ion monitoring (MS) Sirolimus: LC-MS (SIM) vs LC-UV HPLC-MS HPLC-UV SIR m/z 821 100 %

30µg / L

Single ion monitoring (MS) Sirolimus: LC-MS (SIM) vs LC-UV HPLC-MS HPLC-UV SIR m/z 821 100 %
Single ion monitoring (MS) Sirolimus: LC-MS (SIM) vs LC-UV HPLC-MS HPLC-UV SIR m/z 821 100 %
1.5 min 46
1.5 min
46

Full Scan Acquisition Mode

Sirolimus: MS Spectrum

[M+NH 4 ] +

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

Product ion scanning

MS1

Product ion scanning MS1 Precursor Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Products Static
Precursor
Precursor
Product ion scanning MS1 Precursor Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Products Static
Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Products
Ar (2.5 – 3.0e -3 mBar)
Collision
Cell
MS2
Products

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.

48

Product ion scanning

NH 4 + 821.5 822.5 795 800 805 810 815 820 825 830 835 840 845
NH 4 +
821.5
822.5
795
800
805
810
815
820
825
830
835
840
845

100

%

0

826.5 827.5 810.5
826.5
827.5
810.5

790

850

Product ion scanning NH 4 + 821.5 822.5 795 800 805 810 815 820 825 830
768
768

100

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

900

Mass spectrum from MS1

m/z

m/z

Multiple Reaction Monitoring

MS1

Multiple Reaction Monitoring MS1 Precursor(s) Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Product(s) Static
Precursor(s)
Precursor(s)
Multiple Reaction Monitoring MS1 Precursor(s) Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Product(s) Static
Ar (2.5 – 3.0e -3 mBar) Collision Cell MS2 Product(s)
Ar (2.5 – 3.0e -3 mBar)
Collision
Cell
MS2
Product(s)

Static (m/z 821.5)

Static (m/z 768.5)

MS/MS : Compound-Specific Monitoring

Multiple Reaction Monitoring

Sirolimus

LC-MS(SIM) vs LC-MS/MS (MRM)

100 3µg / L % SIR m/z 821 0
100
3µg / L
%
SIR m/z 821
0
100 30µg / L % 0
100
30µg / L
%
0
 

100

MRM m/z 821>768

%

0

Multiple Reaction Monitoring Sirolimus LC-MS(SIM) vs LC-MS/MS (MRM) 100 3µg / L % SIR m/z 821

0.50

1.00

1.50

100

%

Time

0

Multiple Reaction Monitoring Sirolimus LC-MS(SIM) vs LC-MS/MS (MRM) 100 3µg / L % SIR m/z 821
Multiple Reaction Monitoring Sirolimus LC-MS(SIM) vs LC-MS/MS (MRM) 100 3µg / L % SIR m/z 821

0.50

1.00

1.50

Time

51