This Project Aims to Describe the basics of Mass Spectrometry with a general overview on how to read a mass spectrum and a case study which used UHPLC-MS in Forensic Toxicology

1. Mass Spectrometry
Submitted by-
Bhanu Pratap Singh Yadav
MSFS Instrumental Techniques

2. EM spectrum and
Interaction of
EMR with matter
What is
spectroscopy?
What is Mass
spectroscopy?
Instrumentation
of MS.
Case study
Contents:

3. What is
Spectroscopy?
Spectroscopy is basically an experimental
subject and is concerned with the
absorption, emission or scattering of
electromagnetic radiation by atoms or
molecules.
Spectroscopic techniques are one of the
main sources of molecular geometries, that
is, bond lengths, bond angles, and torsion
angles, and can also yield, as will be seen,
significant information about molecular
symmetry, energy level distributions,
electron densities, or electric and magnetic
properties

4. Electromagnetic Radiation
Electromagnetic radiation includes, in addition to what we commonly refer to as ‘light’, radiation of longer and
shorter wavelengths. As the name implies it contains both an electric and a magnetic component which are
perpendicular to each other and to the direction of propagation of the wave.
The distance between two points of the same phase in
successive waves is called the "wavelength”, λ.
1 Å = 10−8
𝑐𝑚 = 10−1
𝑛𝑚
The frequency, ν, is the number of waves in the distance
light travels in one second
𝜈 =
𝐶
𝜆
(𝑠𝑒𝑐𝑜𝑛𝑑−1
)
The third parameter, which is most common to vibrational
spectroscopy, is the "wavenumber," 𝝂.
𝜈 =
𝜈
𝑐
=
1
𝜆
(𝑐𝑚−1
)

5. From the early work of Bohr on atomic spectra, it could be established that absorption or emission of
radiation is possible because of the quantization of atomic and molecular energy levels. If a molecule
interacts with an electromagnetic field, a transfer of energy from the field to the molecule can occur
only when Bohr's frequency condition is satisfied. Namely,
Δ𝐸 = ℎ𝜈 = ℎ
𝑐
𝜆
= ℎ𝑐𝜈
Here Δ𝐸 is the difference in energy between two quantized states, h is Planck's constant (6.626 ×
10−27
erg s) and c is the velocity of light. Thus, 𝜈 is directly proportional to the energy of transition.
Suppose that,
Δ𝐸 = 𝐸2 − 𝐸1
where 𝐸2 and 𝐸1 are the energies of the excited and ground states, respectively. Then, the molecule
"absorbs" Δ𝐸 when it is excited from 𝐸1 to 𝐸2 , and "emits" Δ𝐸 when it reverts from 𝐸2 to 𝐸1 .
Finally, we have
Δ𝐸 = 𝐸2 − 𝐸1 = ℎ𝜈 = ℎ
𝑐
𝜆
= ℎ𝑐𝜈
𝐸2 (Excited state)
𝐸1 (Ground state)
Δ𝐸
Absorption Emission

6. Interaction of Electromagnetic Radiation with
Matter:
Electromagnetic
radiation can
interact with
matter in
various ways,
including
absorption,
reflection, and
transmission.
Absorption of electromagnetic radiation increases particle energy, causing heating or chemical reactions.
Absorption amount depends on material properties like density, conductivity, and structure. Metals are
good absorbers; glass is not.
Reflection is electromagnetic radiation bouncing off a material. Reflectivity, the fraction reflected, depends
on material properties and angle. Mirrors have high reflectivity; paper reflects poorly.
Transmission is radiation passing through a material without absorption. Transmittance, the fraction
transmitted, depends on material properties and thickness. Air and glass are good transmitters; metal is
poor.
Emission of secondary radiation includes fluorescence (immediate re-emission) and phosphorescence
(delayed re-emission). Ionization occurs when radiation removes electrons, forming ions. Excitation matches
energy levels, leading to emission.
Scattering, deflection of radiation, occurs in forms like elastic, inelastic, and Raman scattering. Elastic
conserves energy; inelastic results in different energy. Raman involves energy transfer to/from material
vibrational energy.
Creation or destruction of particles, e.g., pair production, happens with high-energy radiation generating
particle-antiparticle pairs from the vacuum.

7. What is Mass Spectroscopy ?
• Mass spectroscopy is a crucial technique in analytical
chemistry, requiring minimal samples for microanalysis (few
nanomoles).
• Involves ionization, separation, and measurement of ionized
molecules and their products.
• Despite its destructive nature, it provides unique mass
spectra, serving as chemical fingerprints for sample
characterization.
• Used to determine molecular mass, quantify known
compounds, and elucidate compound structures.
• Converts samples into gaseous ions, characterizing them by
mass and charge ratios (m/z) and relative abundance.
• Applied for both pure samples and complex mixtures.
This Photo by Unknown Author is licensed under CC BY-SA-NC

8. History of Mass Spectrometry
• Mass spectrometry originated in the early 20th century during research on ionized gases.
• J.J. Thomson's 1913 work led to the development of the first mass spectrometer, utilizing a
magnetic field and a photographic plate.
• Francis Aston advanced the technology in 1918, creating a practical mass spectrometer with
expanded analytical capabilities.
• In the 1930s and 1940s, mass spectrometry found applications in studying the molecular weights
of organic substances.
• During the 1950s and 1960s, mass spectrometry gained prominence in researching proteins and
nucleic acids.
• F.W. McLafferty and D.M. Stein's work in 1955 led to the development of peptide mapping.
• In the 1970s and 1980s, mass spectrometry played a crucial role in biochemistry, including the
sequencing of the first protein, insulin, by K. Biemann in 1984.
• Today, mass spectrometry is indispensable in various fields, including proteomics, metabolomics,
and forensics, and it continues to evolve with advancements in technology.

9. Mass Spectrometry: Basics
Principle:
• Molecules bombarded with energetic electrons.
• Ionization and fragmentation occur, producing positive ions.
• Each ion has a specific mass-to-charge ratio (m/e ratio).
• For most ions, the charge is one, making the m/e ratio equal to the
molecular mass.
• Ions traverse magnetic and electric fields.
• Detected at the detector, signals recorded for mass spectra.
Theory:
• Mass spectroscopy examines characteristic fragments (ions) from the
breakdown of organic molecules.
• A mass spectrum plots ion abundance against the mass/charge ratio.
• In organic mass spectrometry, energetic electrons bombard vapor to form
positively charged molecular ions.

10. • Electron energy breaks molecular bonds, causing fragmentation into neutral or positively
charged species.
• Fragmentation may result in the formation of even electron ions and radicals.
• Positive ions formed are accelerated and deflected by magnetic or electric fields based on mass,
charge, and velocity.
• Multiple beams with the same m/z values are obtained.
• The resulting beams strike a photographic plate, creating separate lines with recorded intensity.
• Mass spectra visually present m/z values against relative abundance, with the most abundant ion
as 100% (base peak).
• Unlike IR, NMR, and UV, mass spectrometry involves no selective absorption of radiation.
• Mass spectrometry induces irreversible chemical changes, unlike the reversible physical
changes in other methods.
• Mass spectral reactions are more drastic than typical chemical reactions.

11. The Mass
Spectrometer
Parts of a mass spectrometer:
1. Inlet device
2. Ion Source
3. Analyzer system
4. Detector
5. Vacuum system

12. Types of Mass Spectrometer
Ionisation
Ion Source
Ion separation
Mass Analyzer
Ion Detection
Detector
Depending upon the methods of ionization, ion separation and detection, there are various types of mass
spectrometers as shown.
• Electron Ionisation (EI)
• Chemical Ionisation (CI)
• Fast Atom
Bombardment (FAB)
• Electrospray Ionisation
(ESI)
• Matrix-Assisted Laser
Desorption/Ionisation
(MALDI)
• Quadrupole
• Magnetic Sector Field
• Electric Sector Field
• Time of Flight (TOF)
• Ion Trap
• Electron multiplier
• Faraday Cup
• Multichannel plate
• Tandem MS

13. Inlet Systems
• Probe: The insertion probe/plate offers a
straightforward method for introducing samples into
an instrument. Initially, the sample is positioned on the
probe and subsequently inserted into the mass
spectrometer's ionization region, often via a vacuum
interlock.
• Infusion: Capillaries or columns introduce gas or
solution samples to a mass spectrometer, allowing
direct infusion without compromising vacuum
integrity. These components interface with gas
chromatography (GC) and liquid chromatography (LC),
separating and analyzing samples efficiently.

14. Ion Sources
• Electron Ionization.
• Chemical Ionization.
• Field Ionization/Desorption.
• Fast Atom Bombardment/Secondary
Ion Mass Spectrometry.
• Matrix-Assisted Laser
Desorption/Ionization.
• Electrospray Ionization.

15. Electron Ionization (EI)
• Heated filament emits electrons towards an anode.
• Optimal wavelength for organic molecules: 70 eV kinetic
energy, 1.4 Å.
• Energy transfer during interaction with analyte molecules leads
to ionization.
• Compact source design with sample input, electron
inlet/outlet, and ion ejection gap.
• Magnetic field guides electrons for enhanced analyte
interaction.
• Vacuum required for ionization; samples heated for gas phase
transition.
• Negative ions generated through electron capture with voltage
reversal.
• Electrons are associated with a wave whose wavelength is
given by;
𝜆 =
ℎ
𝑚 ⋅ 𝑣

16. Chemical Ionization
(CI)
• Chemical ionization (CI): low-energy technique,
minimizes fragmentation, aids easy recognition
of molecular species.
• Complementary to electron ionization, CI
produces ions through collisions with primary
ions in the source.
• Ionization plasma forms through subsequent
reactions, generating positive and negative ions
of the substance.
• Produced ions, termed ions of the molecular
species or pseudomolecular ions, facilitate
molecular mass determination.
• Molecular ions in CI refer to M•+
𝑜𝑟 M•−
ions.
In the first step, the reagent gas is ionized, followed by a proton transfer to
the analyte:

17. Field Ionization/desorption (FI/FD)
Field Ionization (FI)
High positive electric potential at a pointed electrode
creates a potential gradient, causing molecular orbitals
distortion and electron tunneling, leading to the formation
of a positive ion [M]•+
The formed positive ion is repelled by the positive
electrode (emitter) and enters the mass spectrometer.
Field Desorption (FD)
FD is closely related to FI and is employed for studying
non-volatile compounds. The sample is directly coated on
the emitter via solution evaporation, and the application
of a field in high vacuum induces desorption of intact
molecular ions from regions of high field strength.
FD spectra are dominated by the [M]^+ ion for neutral
compounds, while singly charged salts provide
[𝐶𝑎𝑡𝑖𝑜𝑛]+
𝑜𝑟 [𝐴𝑛𝑖𝑜𝑛]−
as base peaks in positive and
negative ion modes, respectively.
 Both FI & FD are ‘soft’ ionization techniques, minimizing
energy imparted to the molecule, leading to reduced
fragmentation, and often producing an abundant [M]•+
peak.

18. Fast Atom Bombardment & Secondary
Ion MS (FAB/SIMS)
FAB and SIMS employ high-energy atoms for one-step sputtering and
ionization, focusing either a rare gas beam (FAB) or an ion beam (SIMS)
on liquid or solid samples.
Effective for compounds with molecular weights up to a few 10,000 Da,
especially useful for thermally labile compounds.
FAB requires analyte dissolution in a liquid matrix, while SIMS, used for
surface species and solid samples, doesn't use a matrix.
SIMS is sensitive for surface chemistry and materials analysis but can be
challenging to quantify results.
FAB: Inert gases, e.g., Ar or Xe, are ionized and accelerated to 5 keV, with
radicals neutralized at the exit, maintaining momentum as neutral
species.
SIMS: Primary ion beam: 𝐶𝑠+ ions generated by heating a cesium salt
pellet, accelerated to 30 keV, and focused by lenses onto the sample-
containing target.
The fast-moving beam of atoms/ions blasts the matrix and analyte into
the gas phase. The secondary ions that are mass analysed either are
originally charged or acquire a positive charge from protonation (or
association with another charged species such as 𝑁𝑎+
) or a negative
charge by deprotonation

19. Matrix-Assisted Laser Desorption/Ionization
(MALDI)
• MALDI efficiently produces intact gas-phase
ions from large compounds (over 300 kDa)
using a matrix for both desorption and
ionization.
• The mechanism involves dissolving analyte
with a matrix, forming crystals, and ablating
them with intense laser pulses.
• The laser energy induced heating of the solid
matrix forms a plume that experiences a
phase transition. This process results in the
desorption and ionization of the embedded
analyte, generating ions through gas-phase
ion/molecule reactions.
• Advantages include high sensitivity, minimized
sample damage, and universality in
wavelength adjustments.

20. Electrospray Ionization
(ESI)
• Electrospray ionization (ESI) creates highly
charged microdroplets in a strong electric
field.
• A charged capillary produces an electrospray,
generating charged droplets.
• Desorption occurs through thermal and
pneumatic methods.
• Solvent evaporation causes Coulomb
explosion, resulting in bare analyte ions.
• The electrospray ion source operates at
atmospheric pressure, interfacing with a
mass analyzer via skimmer cones.
• Electrochemical processes at the capillary
influence observed ions in ESI mass spectra.

21. Mass Analysers
Single Focussing
Sector Analysers
Magnetic Sector Analyser
Electrostatic Sector Analyser
Double Focussing
Sector Analysers
Quadrupole Analyser
Time-of-Flight (TOF)
system
Fourier Transform Ion
Cyclotron Resonance

22. Magnetic
Sector Analyser
In these, ions leaving the ion
source are accelerated to a high
velocity. The ions then pass
through a magnetic sector in
which the magnetic field is
applied in a direction
perpendicular to the direction
of ion motion. Therefore, these
sector(s) follow an arc; the
radius and angle of the arc vary
with different instrument
designs.
Mass to Charge ratio under the influence of a
magnetic field:
𝑚
𝑧
=
𝐵2𝑟2
2𝑉
Momentum analyzer: The magnetic sector does not directly separate ions by
mass. Rather it effects ion separation by their momentum and this feature can
can be used as a measure of mass provided all ions possess equal kinetic

23. Electrostatic Sector
Analyser
• An electrostatic sector analyzer
comprises two curved plates with
equal and opposite potential.
• As ions move through the electric
field, they experience deflection.
• The force on the ion in the electric
field equals the centripetal force,
focusing ions with the same kinetic
energy.
• Ions with different kinetic energies are
dispersed in this process.
Energy filter: The ESA affects energy dispersion. Thus, the kinetic
energy distribution of an ion beam can be reduced. The ESA does
not allow for mass separation among monoenergetic ions.

24. Double Focussing
Sector Analyzers
These employ both Magnetic and Electrostatic Sectors, usually one
each but nowadays alternative multiple sector arrangements are being
used like BEB/EBE/EBEB types.
• EB analyser
• BE analyser or Reverse EB analyser

25. Quadrupole
• These consist of two pairs of metal rods spaced
equidistantly and biased with equal and opposite
potentials.
• These twin potentials contain a fixed DC and an
alternating RF component, with adjustable RF
strength.
• Ion trajectories are deflected proportionally to their
m/z values upon entering the quadrupole.
• At specific RF values, only ions with a particular m/z
value resonate and reach the end for detection.
• Ions with different m/z values collide with the
quadrupoles, lose charge, and go undetected.

26. Time-of-Flight (TOF) Analyzer
• Time-of-flight (TOF) mass analyzers utilize a flight tube to separate ions based on
their travel time.
• They feature a straightforward design with fixed voltages, eliminating the need
for a magnetic field.
• During operation, ions are swiftly generated by a rapid ionization pulse and then
accelerated into the flight tube through an electric field.
• Ions traverse a field-free region within the flight tube, with the time taken to
reach the detector determined by factors such as drift region length, mass-to-
charge ratio, and acceleration voltage.
• Mass spectrum acquisition involves the detection of low m/z ions reaching the
detector first, enabling the measurement of the detector signal over time for
each ion pulse.
• TOF instruments exhibit high transmission efficiency, leading to an improved
signal-to-noise ratio and absence of an upper m/z limit, low detection limits, and
rapid scan rates.

27. Linear TOF:
The analyte on a sample holder is pulsed with a laser, and
ions desorbed during this pulse are continuously extracted
and accelerated into the gas phase using an applied
acceleration voltage (U) between the target and a grounded
counter electrode.Upon leaving the acceleration region (𝑠0),
ions possess equal kinetic energies.
Ions drift down a field-free flight path (s) of about 1–2 meters
before hitting the detector.
Reflector TOF:
Reflectron TOF focuses ions with varying kinetic energies,
often using multistage designs.
Allows linear mode operation with a detector behind
when the reflector voltage is off.
Basic design includes ring-shaped electrodes creating a
retarding electric field, with 𝑈𝑟 set slightly higher than U.
Ions enter, reach zero kinetic energy, and are expelled,
improving resolving power and focusing ions.

28. Detectors
• Photographic Plates
• Electron multiplier:
• Discrete Dynode
• Continuous Dynode
• Faraday Cup
• Photomultiplier
• Charge Coupled Device

29. Photographic Plate
• The first mass spectrometers used photographic
plates located behind the analyser as detectors.
• Ions sharing the same m/z ratio all reach the plate
at the same place and the position of the spots
allows the determination of their m/z values after
calibration.
• The darkness of the spots gives an approximate
value of their relative abundance.
• This detector, which allows simultaneous detection
over a large m/z range, has been used for many
years but is obsolete today

30. Electron
Multiplier
Detectors
Discrete Dynode EMD:
A type of electron multiplier with
12 to 20 dynodes, each with good
secondary emission properties.
Held at decreasing negative
potentials, secondary electrons
cascade through the dynodes,
producing amplified electric
current. The first dynode is at a
high negative potential (-1 to -5
kV), while the output remains at
ground potential.

31. Continuous Electron
Multiplier Detector:
Channeltron:
An electron multiplier replacing discrete dynodes
with a lead-doped glass tube. The tube has uniform
electric resistance, creating a continuous
accelerating field. Secondary particles collide with
the tube's inner wall, generating secondary
electrons that cascade, and a metal anode collects
the stream at the detector exit.
Microchannel plate (MCP)
This has Parallelly running cylindrical channels. The
electrons are multiplied by a semiconductor
material that coats each channel. The plate input
side is at a negative potential of around 1 kV.
Amplification ranges from102
𝑡𝑜 105
, with
numerous plates achieving up to 108
.

32. Faraday Cup
• A Faraday cup is made of a metal cup or cylinder with a
small orifice. It is connected to the ground through a
resistor.
• Ions reach the inside of the cylinder and are neutralized
by either accepting or donating electrons as they strike
the walls. This leads to a current through the resistor.
• The discharge current is then amplified and detected.
• It provides a measure of ion abundance. Because the
charge associated with an electron leaving the wall of the
detector is identical to the arrival of a positive ion at this
detector.
• Secondary electrons emitted by ions can cause errors in
detectors. To improve accuracy, devices like coating
carbon on inner walls capture ions efficiently and
minimize secondary electron losses.

33. Photomultiplier Detector or Daly
Counter
• In a photomultiplier (or scintillation counter) the
ions initially strike a dynode which results in
electron emission.
• These electrons then strike a phosphorous screen
which in turn releases a burst of photons.
• A photomultiplier detects these photons,
converting them into an electric current, which is
then amplified.
• - The detector's phosphorescent screen has a thin
aluminum conductor layer to prevent charge
buildup.
• Amplification ranges from 104
𝑡𝑜 105
.
• Photomultipliers are now probably the most
common detectors in modern mass spectrometers.

34. Charge Coupled Detector (CCD)
• CCD imagers have a photoactive region (usually an epitaxial layer of
silicon) and a transmission region (shift register).
• When a particle hits the sensor, it generates a charge proportional to
its energy, charging a capacitor within the pixel.
• In a CCD chip, this charge is transported row-by-row along the
column to a shift register row at the bottom of the pixel matrix.
• The charge is read out pixel-by-pixel from the shift register row, and
the last pixel transfers the charge to an amplifier and a charge-to-
voltage converter.
• Analog buffering may be included, and the information is sent off the
chip as an analog signal.
• Sampling and digitization are performed off-chip.
• Sequential readout results in relatively long readout times, scaling
with the pixel array's area.
• CCD readout pixels can be small (e.g., 10 µm × 10 µm) but achieve full
area coverage.
To Amplifier

35. Vacuum System
Mass spectrometers operate at high vacuum
(10−2
𝑡𝑜 10−5
Pa) to minimize ion collisions.
Ion collisions can lead to undesirable reactions,
neutralization, scattering, or fragmentation,
affecting mass spectra.
A two-stage pumping system is employed for high
vacuum: a mechanical pump for rough vacuum
(down to 0.1 Pa) and diffusion or turbomolecular
pumps for high vacuum.
In some cases, like ICR instruments, a third
pumping stage with a cryogenic pump is used for
even higher vacuum requirements.

36. Application of Mass Spectrometry
• Toxicological Analysis: Mass spectrometry is crucial in detecting toxic substances
in bodily samples, offering insights into the cause of death, timing, dosage, and
potential habitual substance use by the victim.
• Trace Evidence Analysis: Essential for examining trace evidence like carpet fibers
or glass splinters, mass spectrometry precisely identifies material composition.
This aids investigators in linking evidence to manufacturers, narrowing down
origins, and building cases against suspects.
• Arson Investigations: Invaluable in arson cases, mass spectrometry breaks down
residue from burn patterns, providing a precise molecular makeup report. This
helps identify unique compounds, potentially linking similar mixes at multiple
crime scenes and aiding in the identification of a serial arsonist.
• Explosive Residue Analysis: Crucial for examining explosive residue, mass
spectrometry identifies unique chemical makeups specific to each explosive
manufacturer. Even with homemade explosives, this analysis reveals materials
used, guiding investigators to identify the source.

37. Case Study: Application of high-resolution mass spectrometry to
determination of baclofen in a case of fatal intoxication
• The woman consumed an undetermined quantity of drugs of unknown origin, likely causing her
death.
• The woman had a history of alcohol abuse, arguments, and causing various problems.
• After arguments with her boyfriend, the woman had a history of taking various drugs and
consuming alcohol, leading to subsequent vomiting.
• She wrote to her boyfriend claiming to have taken 100 tablets of baclofen.
• Containers found include one empty Baclofen 25 mg container for 50 tablets, one full container
with 51 tablets, and one container with 12 and 1/2 tablets, which amounts to a suggested total
consumption of 87 and 1/2 tablets (maximal potential dose 2187.5 mg).
• External examination and autopsy did not reveal any pathological changes.
• No ethyl alcohol was present in her blood during autopsy.
• No acute alcohol intoxication, mechanical trauma, or underlying diseases were identified as
causes of death.
• Contradictory information exists between the claimed dose (100 tablets) and the tablets found,
adding complexity to the investigation.

38. Introduction
Baclofen is a drug that affects the central nervous system (CNS) and is derived from γ-
aminobutyric acid (GABA). Baclofen is commonly used to treat spinal cord diseases,
cerebral stroke, cerebrospinal meningitis, and severe chronic spasticity in multiple
sclerosis patients. Additionally, it is known to alleviate symptoms of alcohol craving.
The recommended oral therapeutic dose for adults is individually tailored and ranges
from 15–80 mg/day. However, significant complications and life-threatening cases
have been reported even with doses as low as 300 mg.
In the presented case, high-resolution mass spectrometry (HRMS) was employed to
determine baclofen levels in postmortem blood. The technique utilized liquid
chromatography–hybrid quadrupole time-of-flight-mass spectrometry (LC–QTOF-MS)
for comprehensive targeted forensic screening. Detection was achieved using a
quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray
ionization (ESI) source.

40. High-resolution product ion mass spectra
of baclofen obtained at different CEs.
• The accurate mass of the precursor protonated
baclofen was 214.0629.
• The application of a QTOF detector enabled
obtaining high-resolution mass spectra
(MS/MS). The MS/MS analysis allowed for the
investigation of the mechanism of product ion
formation from baclofen.
• Depending on the CE, baclofen underwent
product ion formation to three major ions at
m/z 197.03638 (CE 5 V), 151.0309 (CE 20 V)
and 116.06205 (CE 35 V), which can be used as
confirmation ions.
• In its structure, baclofen contains one atom of
chlorine, which causes the formation of a
characteristic mass spectrum evidencing the
presence of isotopic ions of this element
• The mass difference between chlorine
isotopes 35Cl and 37Cl was 1.9970; therefore,
ion fragments with 35Cl should be
accompanied by the ion containing 37Cl,
differing by the value mentioned above.

41. • The use of HRMS enabled to define the precise mass of product ions of interest and errors in their
determination as well as to analyze their structure in detail. At a 5-V CE, the most intense fragment was at
m/z 197.03638. The fragment occurred due to dissociation of the ammonium group from the baclofen
molecule. Next Slide also contains the 196.05237 ion, which results from dissociation of a water molecule.
• Examinations of the MS/MS spectra revealed that several very intense fragments were formed as a result
of baclofen fragmentation, depending on the CEs. General formulae were proposed for six of them (Table
3).

42. Conclusion
• The application of HRMS enables reliable
identification of baclofen in autopsy blood. The
method designed is characterized by high-
efficiency extraction.
• The study findings demonstrated that the
specific, simple and quick procedure described
for determination of baclofen in autopsy blood
can be successfully used for routine toxicology
testing in the cases of suspected baclofen
intoxication.
• Unambiguous identification of baclofen is
derived from the available product ion mass
spectrum.
• In such a spectrum, we observed three product
ions of baclofen (m/z 197.03638, 151.0309 and
116.06205) of high intensity.
• These product ions may be successfully
employed as confirmative ions in QTOF-MS or
triplequadrupole-MS analysis
Probable product-
ion formation
pathways from
the protonated
baclofen
precursor ion

55. Types of
peaks in
Mass
Spectrum

58. Isotopic Peaks

60. Thank You