Tandem MS for Drug Analysis

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 bioanalysis, quadrupole, time-offlight (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 Ion source MS-1 Single ion MS-2 3 Fragments

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

GC/MS LC/MS/MS Immunoassay

(ELISA/FPIA etc.)

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. 5

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

Separation

MS
Mass Analyze MS1

Identification

Inlet

Ionize

Fragment

Mass Analyze
MS2

Detect

Collision Cell

MS/MS

Mass Spectrometry
+CH 3 +COH

CH3COCH3

CH3+COCH3

CH3C+OCH3

+COCH 3

Sample Inlet

Ionization & Adsorption of Excess Energy

Fragmentation (Dissociation)

Mass Analysis

Detection

Multidimensional Analyses
m/z
response m/z

m/z

chromatogram
time 8

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

10

Analytical Quadrupole

11

Pre-filter

Quadrupole Theory
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. 12

Tandem Quadrupole
MS1
Collision cell MS2

13

Components of Tandem Mass Spectrometer

Ionization Source Mass Spectrometer ESI APPI APCI MALDI Quatrupole Magnetic Sector Collision Cell Argon Xenon Mass Detector Spectrometer Quatrupole Magnetic Sector Time-of-flight

MS1

Collision cell

MS2

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

15

The Macabre History of Electrospray

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 !

F. Lemire, LCGC Europe LC-MS Supplement, December 2001, p29-35

16

The Electrospray Phenomenon

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

17

Ionization Source

18

Ionization Source
Spraying Needle Sample Cone
Orifice = 400m

Vacuum Isolation Valve

19

Ion Sources make ions from sample molecules

Electrospray ionization:
Pressure = 1 atm Inner tube diam. = 100 um Sample Inlet Nozzle (Lower Voltage) Partial vacuum

N2
+ + + + ++ ++ + + ++ ++ + + ++ + ++ + ++ + ++ + ++ + ++ + ++

MH+
+ + + + + + + +

Sample in solution N2 gas

+
+

MH2+ MH3+

High voltage applied to metal sheath (~4 kV)

Charged droplets

20

ESI Spectrum of Trypsinogen (MW 23983)

1599.8

M + 15 H+ M + 14 H+

M + 16 H+
1499.9

1714.1

M + 13 H+

1411.9

1845.9 1999.6 2181.6

m/z

Mass-to-charge ratio 21

APCI

22

APPI

23

MALDI: Matrix Assisted Laser Desorption Ionization

Sample plate hn

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) 24

The mass spectrum shows the results

MALDI TOF spectrum of IgG
40000

MH+

Relative Abundance

30000

(M+2H)2+
20000

10000

(M+3H)3+
0

50000

100000

150000

200000

Mass (m/z)

25

Components of Tandem Mass Spectrometer

Ionization Source Mass Spectrometer ESI APPI APCI MALDI Quatrupole Magnetic Sector Collision Cell Argon Xenon Mass Detector Spectrometer Quatrupole Magnetic Sector Time-of-flight

MS1

Collision cell

MS2

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.
27

Full Scan Acquisition Mode

MS1

Collision cell

MS2

MS1

Collision Cell

MS2

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]+
100
315.1
CH3 O CH3 CH3

%
316.1

0 200

220

240

260

280

300

320

340

360

380

m/z 400

29

MS1

Collision cell

MS2

Product ion scanning

Argon gas

Products Precursor

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

Argon gas
O CH3 CH3
H3C

CH3
O

CH2 CH2

CH3

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)

31

Product ion scanning

Product Ion Spectrum: Progesterone

O

100
CH3

CH3

CH3

315.1

% 0 300

Precursor ion
305
CH2

316.1

Mass Spectrum from MS1

m/z 330

310
O CH3

315

320

325

97.0

100 % 0

109.0

CH2

H3C CH3

Product ions
100 125 150 175

Product ion spectrum from MS2

m/z 200 225 250 275 300 325

32

 collision energy > fragmentation

Product ion scanning

100 % 0 100 % 0 100 % 0 100 % 0 100 % 0 20 40 60 80 100 120 140

5eV

10 eV
20 eV 30 eV 40 eV
160 180 200

33 220

m/z

Precursor ion scanning

Precursor Ion Scan

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.34

Acylcarnitines
R=0 to 18 carbon alkyl chain.

Derivatization and Fragmentation

H CH COOH

Precursor ion scanning

RCOO (CH3)3N CH2 CH

Butylation
RCOO (CH3)3N CH2 CH H CH - RCOOH -(CH3)3N -C4H8

COOC4H8

CID

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

[ CH2

CH (m/z 85)

CH

COOH

]+ 35

Precursor ion scanning

Normal Acylcarnitine Profile

100

d3-free carnitine

d3-C16 carnitine

C2 carnitine
%

d3-C3 carnitine C16 carnitine d3-C8 carnitine

0 225 250 275 300 325 350 375 400 425 450 475 500

m/z

36

MS1

Collision cell

MS2

Neutral loss scanning

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.

37

Neutral and Acidic Amino Acids

Derivatization and Fragmentation (Generic)
O R OH NH2
O

HO

+
CH3

HCl

R NH2

CH3

Neutral or Acidic AA
O R NH3
+

Butanol

Amino acid butyl ester

O
NH2
+

Fragmentation R
O CH3

+
HO

CH3

Neutral or Acidic AA

Fragment

Butyl formate Neutral loss of 102Da 38

Normal Amino Acid Profile

Neutral loss scanning
Pro
100

Deuterated internal standards for quantification

d3-Leu
d5-Phe
%

d6-Tyr

d4-Ala Ser Gly d8-Val d3-Met

Glu

0 140 160 180 200 220 240 260

m /z 280

39

Multiple Reaction Monitoring

MS1

Collision cell

MS2

Argon gas

Precursor(s)

Product(s)

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. 40

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 41

HPLC-UV Analysis of Sirolimus in Whole Blood

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Wash all glassware in methanol x2 and tert-butyl methyl ether (TBME) x2. Place 50L of internal standard (in methanol) into each screw-cap glass tube. Add 200L Sirolimus calibrator (5x concentrated in methanol) or 200L methanol for patient samples. Add 1.0mL blank whole blood to calibrators or 1.0mL patient whole blood. Add 2.0mL 0.1M ammonium carbonate buffer. Mix thoroughly. Add 7.0mL TBME and extract for 15min. Transfer upper layer to clean tube and re-extract lower layer with 7.0mL TBME. Combine TBME extracts and evaporate to dryness. Redissolve residue in 5.0mL ethanol and evaporate to dryness. Redissolve residue in 1.0mL ethanol, transfer to Eppendorf tube and evaporate to dryness. Redissolve residue in 100L 85% methanol. Inject 80L (equivalent to 800L whole blood) and analyse using two 4.6mm x 250mm C18 columns connected in series (30min run time).

42

Sirolimus: HPLC - UV Example

43

Immunosuppressant Sample Preparation

LC-MS/MS Analysis
Whole Blood (10L - 40L) Add ZnSO4 Soln. Add 2 volumes MeCN with IS, Seal & Vortex Mix

Centrifuge, Inject 5 - 20L

44

Full Scan Acquisition Mode

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

45

Sirolimus:
Single ion monitoring (MS)

LC-MS (SIM) vs LC-UV

100

30g / L HPLC-MS SIR m/z 821

%

0 100

1.5 min

HPLC-UV

0.50

1.00

1.50

Time

46

Full Scan Acquisition Mode

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

47

Product ion scanning

MS1

Collision Cell

Ar (2.5 3.0e-3mBar)

MS2

Products Precursor

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

NH4+

Product ion scanning

100

821.5

Mass spectrum from MS1

822.5

%
826.5 827.5 810.5

0 790

795

800

805

810

815

820

825

830

835

840

845

m/z 850

100

768

Product ion spectrum from MS2

%
576 548 558 718 750 786 821

0 200

250

300

350

400

450

500

550

600

650

700

750

800

850

m/z 900

49

Multiple Reaction Monitoring

MS1

Collision Cell

Ar (2.5 3.0e-3mBar)

MS2

Precursor(s)

Product(s)

Static (m/z 821.5)

Static (m/z 768.5)

MS/MS : Compound-Specific Monitoring 50

Multiple Reaction Monitoring

Sirolimus
LC-MS(SIM) vs LC-MS/MS (MRM)
100 100

3g / L
% %

30g / L

SIR m/z 821

0 100

0 100

MRM m/z 821>768

0.50

1.00

1.50

Time 0

0.50

1.00

1.50

Time

51