Tandem MS for Drug Analysis Lecture

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