Tandem MS for Drug Analysis

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Tandem MS for Drug Analysis
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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.

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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
Fragments
Ion source
MS-2
MS-1
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Analytical Assays used in Pharmaceutical
Industry Labs for New Chemical Entities
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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.

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MS vs. MS/MS
GC HPLC CE
Separation
Identification
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Mass Spectrometry
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Multidimensional Analyses
m/z
m/z
response
m/z
chromatogram
time
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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

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LC-MS/MS
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Analytical Quadrupole
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Quadrupole Theory
Pre-filter
Quadrupole Mass Filter

• 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.

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

• Ion Souce
• Transforms sample molecules to ions
• Soft ionization
• Places positive or negative charge on the analyte
  without significantly fragmenting the analyte
• M1 ion (or M-1 ion)
• No need to volatilize
• Down to fmol detection limits
• Atmospheric Pressure Ionization (API)
• Electrospray
• MALDI
• APCI
• APPI

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The Macabre History of Electrospray
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, LCGC Europe LC-MS Supplement,
December 2001, p29-35
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The Electrospray Phenomenon
J. Zelene, Phys. Rev., 10, 1-6 (1917)
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Ionization Source
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Ionization Source
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Electrospray ionization
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ESI Spectrum of Trypsinogen (MW 23983)
M 15 H
1599.8
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
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APCI
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APPI
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Sample plate
Laser
hn
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
MALDI Matrix Assisted Laser Desorption Ionization
Grid (0 V)
/- 20 kV
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The mass spectrum shows the results
MALDI TOF spectrum of IgG
MH
Relative Abundance
(M2H)2
(M3H)3
50000
100000
150000
200000
Mass (m/z)
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Components of Tandem Mass Spectrometer
Ionization Source
Mass Spectrometer
Collision Cell
Mass Spectrometer
Detector
ESI APPI APCI MALDI
Quatrupole Magnetic Sector Time-of-flight
Argon Xenon
Quatrupole Magnetic Sector
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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.

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Full Scan Acquisition Mode
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.
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Mass Spectrum Progesterone
MH
Full Scan Acquisition Mode
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Argon gas
Product ion scanning
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.
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Collision induced dissociation

• 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)

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Product Ion Spectrum Progesterone
Product ion scanning
Product ion spectrum from MS2
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? collision energy gt ? fragmentation
5eV
10 eV
Product ion scanning
20 eV
30 eV
40 eV
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Precursor Ion Scan
Argon gas
Precursor ion scanning
Product
Precursors
Static
Scanning
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.
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Acylcarnitines Derivatization and Fragmentation
R0 to 18 carbon alkyl chain.
Precursor ion scanning
All compounds of this type fragment to produce
the 85 ion.
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Normal Acylcarnitine Profile
d3-C16 carnitine
d3-free carnitine
Precursor ion scanning
C2 carnitine
d3-C3 carnitine
C16 carnitine
d3-C8 carnitine
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Argon gas
Neutral loss scanning
Products
Precursors
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.
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Neutral and Acidic Amino AcidsDerivatization and
Fragmentation (Generic)
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Normal Amino Acid Profile
Neutral loss scanning
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Argon gas
Multiple Reaction Monitoring
Product(s)
Precursor(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.
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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

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HPLC-UV Analysis of Sirolimus in Whole Blood
1. Wash all glassware in methanol x2 and
tert-butyl methyl ether (TBME) x2.2. Place 50?L
of internal standard (in methanol) into each
screw-cap glass tube.3. Add 200?L Sirolimus
calibrator (5x concentrated in methanol) or 200?L
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
100?L 85 methanol.13. Inject 80?L (equivalent
to 800?L whole blood) and analyse using two
4.6mm x 250mm C18 columns connected in series
(30min run time).
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Sirolimus HPLC - UV Example
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Immunosuppressant Sample PreparationLC-MS/MS
Analysis
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Sirolimus MS Spectrum
MNH4
Full Scan Acquisition Mode
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SirolimusLC-MS (SIM) vs LC-UV
30µg / L
SIR m/z 821
HPLC-MS
Single ion monitoring (MS)
1.5 min
HPLC-UV
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Sirolimus MS Spectrum
MNH4
Full Scan Acquisition Mode
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Ar (2.5 3.0e-3mBar)
Collision Cell
MS2
MS1
Products
Product ion scanning
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.
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NH4
Product ion scanning
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Ar (2.5 3.0e-3mBar)
Collision Cell
MS2
MS1
Product(s)
Multiple Reaction Monitoring
Precursor(s)
Static (m/z 821.5)
Static (m/z 768.5)
MS/MS Compound-Specific Monitoring
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SirolimusLC-MS(SIM) vs LC-MS/MS (MRM)
3µg / L
30µg / L
SIR m/z 821
Multiple Reaction Monitoring