Mass spectrometry is a powerful analytical technique that measures the mass-to-charge ratio of ions to identify molecules. It involves ionizing samples, separating the ions by their mass-to-charge ratio using electric or magnetic fields, and detecting the ions. Common applications include clinical analysis of metabolites and proteins. Key components include the ion source, which ionizes samples using techniques like electrospray ionization; the mass analyzer, such as quadrupoles, that separate ions; and the detector. Mass spectrometry provides qualitative and quantitative analysis of a wide range of clinically relevant compounds.

1. MASS SPECTROMETRY
Dipesh Tamrakar
M.Sc. Clinical Biochemistry
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2. Mass spectrometry
• powerful qualitative & quantitative analytical technique
• Use to measure wide range of clinically relevant analytes
• Universal detector & detects everything (theoretically)
• Coupled with either gas or liquid chromatography results in
expanded analytical capabilities with widespread clinical
applications
• Key tool in emerging field of proteomics
A mass spectrometer is a device that measures the mass-to-
charge (m/z) ratio of ions.
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3. The concept of mass spectrometry was put forth by Sir J.J
Thomson, English physicist who discovered the electron in
1887; NP-1906
Thomson II Experiment
The beam of electron get deflected in magnetic field
The degree of deflection depends on mass and charge
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Video 1

4. PRINCIPLE
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• IONIZATION
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• MASS
ANALYZER
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• DETECTION
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Video 2

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7. 1. Inlet
• Solid samples with lower vapor pressure: Directly inserted into
ionization chamber & volatilization is controlled by heating the probe
• Liquids : are handled by hypodermic needle injection through a silicon
rubber dam
• Gaseous samples : are leaked into the ionization chamber directly by
the help of mercury manometer
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8. Ionization
• In 2002, the Nobel Prize was shared by John Fenn & Koichi Tanaka for
their development of electrospray and laser desorption ionization
respectively
• If low energy or soft ionization technique are used, the mass of the
target molecule can be determined
• Advances in soft ionization techniques have extended the use of MS
to the direct measurement of peptide & protein mass
• Ionization at higher energy results in more extensive fragmentation of
target molecules
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9. Ionization
• Loss of electrons from a molecule leads to radical cation
• The ion sources is the part of MS that ionizes the material under
analysis (the analyte)
• The ions are produced:
By removing or adding the electron
 By removing or adding the proton (H+)
By addition of entities s/a NH4+ or CH5+
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10. 2. Ion source
Electron ionization and chemical ionization are ionization techniques used
when gas phase molecules can be introduced directly into the analyzer from
a gas chromatography
• In HPLC-MS, following ionization
sources are used
• Electrospray ionization
• Sonic spray ionization
• Atmospheric pressure chemical
ionization
• Atmospheric pressure
photoionization
• Other ionization technique
• Inductively Coupled Plasma
• Matrix Assisted Laser
Desorption Ionization (MALDI)
• Atmospheric pressure-MALDI
• Fast Atom Bombardment
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11. a. Electron Ionization (EI)
• Gas phase molecules are bombarded by electrons emitted from a
heated filament & attracted to a collector electrode
• Occurs in vacuum to prevent filament oxidation
• A p.d. of 70eV is enough to bring ionization
• Positive ions are repelled or drawn out of ionization chamber by an
electric field
• The cations are then electrostatically focused and introduced into the
mass analyzer
2. Ion source
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12. a. Electron Ionization (EI)
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13. b. Chemical ionization
• Soft ionization technique (proton transfer)
• Typical reagent gases are methane, ammonia, isobutane & water
• An electron beam produces reactive sps. Such as CH5+ for methane
• Collision between the relative reagent gas and the analyte cause proton
and energy transfer
• Because the protonated molecule Is not highly excited in this process,
relatively little fragmentation occurs
• This is major advantage for analyte molecular mass determination and for
its quantification
• Negative ion electron capture CI has become popular for quantification of
drugs such as benzodiazepines
• Used in Quadrupole Ion Trap in lower gas pressure
2. Ion source
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15. c. Electrospray Ionization (ESI)
• A technique in which a sample is ionized at atm. Pressure before
introducing into mass analyzer
• A sample is passed through a narrow metal or fused silica capillary to
which 3 – 5 kV charge is applied
• The partial charge separation in between liquid and capillary results
instability causing expulsion of a charged droplets series from a Taylor cone
• A coaxial nebulizing gas helps direct the charged droplets towards a
counter electrode
• Labile compounds are analyzed using “cold electrospray”
• Unique feature: production of multiple charge ions from peptides/proteins
2. Ion source
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17. d. Sonic spray ionization (SSI)
• Coaxial nitrogen gas travelling at the speed of sound can be used to create
the spray and cause ionization
• As the sonic velocity gas flows over the surface of mobile phase exiting the
capillary, 2 effects:
1. Droplet fission occurs as a result of shear stress created by sonic gas
flow
2. Ionization efficiency optimized by minimum droplet size at sonic
velocity
2. Ion source
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18. e. Atmospheric Pressure Chemical Ionization (APCI)
• Similar to ESI taking place in atm. Pressure but only differ in mode of
ionization
• In APCI, no voltage is applied to inlet capillary instead a separate corona
discharge needle is used to emit a cloud of electrons that ionize
compounds
• Because eluent molecules like water, methanol are present in excess
relative to the analytes in the sample, they are predominantly ionized and
then act as a reagent gas that reacts secondarily to ionize analyte
molecules
• Relatively little fragmentation and mainly used in Tandem MS
2. Ion source
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20. f. Atmospheric Pressure Photoionization (APPI)
• ESI and APCI less effectively ionize nonpolar compounds
• APPI is similar to ESI and APCI but differ in photon flux used instead of
corona discharge needle
• Better quantitative and a potential higher dynamic range is obtained by use
of photon source
• Krypton discharge lamp with magnesium fluoride window is used
2. Ion source
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22. g. Inductively Coupled Plasma (ICP)
• It is atm. Pressure ionization method which can bring complete ionization
• Particularly useful for trace metal and heavy metal analysis in tissue or
body fluids
• ICP is extremely sensitive (e.g parts per trillion) and capable of extremely
high dynamic range
• ICP-MS is comparatively free from most interferences
2. Ion source
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23. h. Matrix-Assisted Laser Desorption Ionization (MALDI)
• Originally described in 1987 consisting purely of laser
desorption/ionization
• Due to limitation on size and stability of analyte, addition of matrix to
assist the process under vacuum
• Currently , analyte is dissolved in solution of matrix (small mol. Wt.
UV-absorbing compound)
• Generally matrix to analyte ratio is 1000:1
• As volatile solvents evaporate, the matrix compound crystallizes and
incorporates analyte molecules
• MALDI is mostly coupled with Time of Flight-MS
2. Ion source
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25. i. Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization
(AP-MALDI)
• Works as MALDI at atmospheric pressure
• Major advantage: ability to switch sources easily while coupling the
inherent speed & multiple sample wells of traditional MALDI with MS
• Major drawback as compared with ESI is poor fragmentation of the slightly
charged ions produced in MALDI
2. Ion source
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26. J. surface- Enhanced Laser Desorption/Ionization (SELDI)
• Combines affinity purification & MALDI on the target
• The most common setup involves producing a MALDI target surface
modified with some type of affinity capture property (hydrophobic, ionic,
immobilized metal affinity chromatography (IMAC), DNA, antibody, etc)
• Sample of interest is exposed to one or more of these affinity surfaces
where certain analyte will bind preferentially, then a matrix is added to
enhance desorption/ionization & analyzed by TOF
• Major advantage is low sample loss as purification & analysis occur on the
same surface
2. Ion source
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28. k. Fast Atom Bombardment (FAB)
• FAB produces ions when a high-velocity beam of atoms impacts the surface
of liquid containing analytes
• Potentization is thought to occur when analytes on the surface of
vaporized droplets are transferred to the gas state
• Since the bombardment occurs in high vacuum, the liquid used must have
a high boiling point
• Used to ionize proteins and small molecules
• Used in conjunction with Tandem MS for diagnosis of short chain fatty acid
acylcarnitine deficiencies from newborn blood spots
2. Ion source
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30. Vacuum system
• To prevent collision of ions in magnetic or electrical field
• Vacuum of 10-3torr to 10-9torr is applied depending on MA type
• Mass analyzer is maintained at elevated temp (150 – 250 C) to avoid
absorbing of molecules inside the vacuum chamber
• Efficient high vacuum pumps generally don’t operate well near atm
pressure so it should have mechanical vacuum pump to evacuate
system pressure
• Diffusion pump, Turbomolecular Pump, Cryopump can be used.
• Higher pump capacities are associated with lower detection limits
because noise arising from the gas background is reduced
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31. 3. Mass analyzer
I. Beam type instruments
A. Quadrupole
B. Magnetic sectors
C. Time of flight (TOF)
II. Trapping mass spectrometers
A. Quadrupole ion trap
B. Linear ion trap
C. Ion cyclotron resonance
General classes of MS
I. In beam type instruments , the ions make one pass through the
instrument and then strike the detector
II. In trapping type analyzer, ions are held in a spatially confined region
of space by a combination of magnetic or electrostatic or radio
frequency electrical field
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32. 1. Beam Type Designs
A. Quadrupole
• Currently most widely used MS
• Easy to use, flexibility, adequate performance for most applications,
relatively low cost, non critical site requirements & highly developed
software systems
• It consists of four parallel electrically conductive rods arranged in a square
array forming long channel through which ion beam pass
• Ion beam entering the quadrupole have various m/z values but with only
narrow range will transport
• Ions outside the narrow range are ejected radially
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34. • Quadrupole MS rely on superposition of Radio Frequency & DC potential
applied
• Both RF & DC are fixed in SIM mode
• In scanning mode of operation, the RF &/or DC voltages are continuously
varied to scan a range of m/z values
• The effective force with pseudopotential points inward towards the
quadrupole axis & is proportional to the distance from axis
• Therefore it acts as confining force preventing ions from being ejected
radially from quadrupole assembly
1. Beam Type Designs
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35. B. Magnetic source
• Nowadays rarely used in MS
• It is versatile, reliable, highly sensitive & in their double focusing variation
are capable of very high m/z resolution & mass accuracy
• Demerits: typically expensive, large & heavy, difficult to use
1. Beam Type Designs
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36. C. Time of Flight (TOF)
• Non-scanning technique
• Advantages: unlimited m/z range, high acquisition, high sensitivity &
reasonable cost
• Significant advantage of modern TOF-MS produce exact mass
measurements, typically with low ppm accuracy
• Practical use in routine chromatography & clinical analysis
• Simply based on the fact that a lighter ion travels faster than a heavier ion,
provided they both have same kinetic energy
1. Beam Type Designs
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37. • The flight time for an ion of mass m & kinetic energy E to travel a distance L
in a region free of electric fields is given by:
T = L(m/2E)1/2
• TOF is coupled readily to pulse ionization method, MALDI (most commonly)
• TOF-MS is extremely fast, 5000 spectra/s or greater
1. Beam Type Designs
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38. 2. Trapping Mass Spectrometers
A. Quadrupole Ion Trap (QIT):
• Primarily used as GC or HPLC detectors
• Relatively compact, inexpensive & versatile instrument
• Similar physical principle as Quadrupole MS however RF field of an ion trap
is designed to trap ions in 3D
• Known for high sensitivity
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39. B. Linear Ion Trap (LIT):
• Based on modified linear Quadruple Mass Filter
• Electrostatic fields are applied to the ends to prevent ions from exiting out
of the ends of device
• An advantage is the trapping field can be turned off at will & the device
operated as a normal QMF
2. Trapping Mass Spectrometers
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40. C. Ion Cyclotron Resonance (ICR):
• ICR is a trapping technique with high sensitivity
• Based on principle that ion immersed in a magnetic field undergo circular
motion (cyclotron motion)
• A typical ICR-MS uses a high field superconducting magnet
• Disadvantages:
1. High instrument cost
2. Very demanding site requirements: space & access restriction
3. Uses high field superconducting magnet: erase of credit cards and
magnetically encoded strips
4. Cost of operation, care & maintenance is high
5. A highly skilled operator level
2. Trapping Mass Spectrometers
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Video 3

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42. D. Tandem Mass Spectrometers
• MS/MS mainly used for quantitative analysis of routine samples
• Excellent for structural characterization & compound identification
• The most important feature: very high selectivity together with good
sensitivity
• Very low interference when coupled with HPLC, low consumable cost, high
sample throughput rates
• Physical principle: 2 MS are arranged sequentially with a collision cell
placed between 2 instruments
2. Trapping Mass Spectrometers
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43. D. Tandem Mass Spectrometers
• 1st : used to select ions of particular m/z called either parent ion or
precursor ion
• 2nd: directed into collision cells, precursor ions collide with background gas
molecules & broken to form product ion
• Possible scan function involving 1st MS to select a given m/z and full
scanning through mass spectrum of product ion (structural
characterization)
• In constant neutral loss scan, 2MS are scanned synchronously with m/z
offset between parent and product ion
• Another scan function is multiple reaction monitoring (MRM)
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44. D. Tandem Mass Spectrometers
• MRM primarily used for quantitative analysis of few selected target
compounds & is a closer analogue of SIM monitoring used in GC-MS
• Classification: as with single stage MS
1. Beam type instrument: Tandem in space (Triple Quadrupole)
2. Trapping instrument: Tandem in time
• Q1 as MS1, Q3 as MS3 and Q2 as function cell in triple quadrupole
• 2 magnetic sector instruments have been operated in Tandem with
collision cell placed between 2 instruments: permit high resolution, rarely
used, expensive, cumbersome to operate
• Double focusing MS: linked scanning technique; a product ion scan by
linked scanning involves low resolution for MS1 & high resolution for MS2
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45. D. Tandem Mass Spectrometers
• 2 TOF-MS gives excellent sensitivity & throughput
• Hybrid MS: combination of 2 different types of MS more popular
quadrupole-MS1 , TOF-MS2; used in proteomics & clinical lab applications
• Trapping mass spectrometers: ions are held in one region of space
• QIT & ICR can be used in tandem MS and are capable of multiple stage of
MS
• Extremely versatile but unable to perform true precursor ion scans or
constant neutral loss scans
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46. 4. Detectors
• All MS use detectors for electron multiplication except ICR-MS(ion
cyclotron resonance)
• 3 classes of electron Multipliers: (similar principle)
1. Discrete Dynode Multiplier
2. Continuous Dynode Electron Multipliers
3. Micro channel Plate Electron Multipliers
1. Discrete Dynode Multipliers (DDM):
• Cascade process that electron amplifies on striking dynode
• One electron can produce pulse of 104 – 108 electrons
• Duration of pulse is as low as 10 Nano seconds
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48. 2. Continuous dynode electron multiplier (CDEM)
• Same as DDM, only differ in physical construction: set of dynode is
replaced by single continuous surface
• The surface of the tube contain an electrically resistive layer
• The resistive layer also serves as the secondary electron emitter
• CDEM is generally fabricated from a specialized glass
3. Microchannel Plate Election Multipliers
• Microchannel Plate in a disk of glass that contains pores extending from
the upper surface to the lower surface
• Channels are 3 -30 um diameter, length 200-1000 um
4. Detectors
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50. Faraday Cup:
• The Faraday Cup is a simple electrode that intercepts the ion beam directly
• This current is then amplified using electronic amplifier
• Provides absolute measure of ion current
• Some instruments use both electron multiplier & Faraday Cup to provide
extended dynamic range of detection : useful for elemental analysis of
trace metals in samples
4. Detectors
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51. 5. Computer & Software
• MS instruments generate enormous amounts of raw data
• In toxicology lab one important function of the data system is library
searching to assist in compound identification
• Several commercial libraries with quality & quantity of available spectra are
available
• Data systems exist that aid in characterization of spectral data to identify
proteins
• Fragmentation information can also be compared with peptide databases
to identify structural mutations that may be present
• Software programs are also available to locate & identify components in
complex chromatographic separations
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Video 4

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54. Applications of MS:
GC-MS
• Gas chromatography- mass spectrometry (GC-MS) is a method that
combines the features of gas liquid chromatography and mass
spectrometry to identify different substances within a test sample
• Application of GC_MS
Drug detection
Fire investigation
Environmental investigation
Explosives investigation , and
Identification of unknown samples.
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55. Applications of Tandem MS
1. Biotechnology and Pharmaceutical
To determine chemical structure of drugs and drug metabolites
Detection/quantification of impurities, drugs and their metabolites in
biological fluids and tissues
Analysis of liquid mixtures
Fingerprinting
Nutraceuticals/ herbal drugs/ tracing source of natural products or
drugs
2. Clinical testing and Toxicology
Inborn errors of metabolism, cancer, diabetes, various poisons, drugs of
abuse, etc.
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56. • Pharmaceutical analysis
• Bioavailability studies
• Drug metabolism studies, pharmacokinetics
• Characterization of potential drugs
• Drug degradation product analysis
• Screening of drug candidates
• Identifying drug targets
• Biomolecule characterization
• Protein and peptides
• Oligonucleotides
• Environmental analysis
• Pesticides on foods
• Soil and groundwater contamination
• Forensic analysis
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57. Advantages of MS/MS as a screening tool in NBS
 Sensitive
 Specific
 Accurate Quantitation
• Internal standards: gold standard for accuracy
 High impact
• Multiple Metabolite, Multiple Disease Screening
• cost effective
 High throughput
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