The Basics of Mass Spectrometry/Spectroscopy.pptx

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. 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 = The frequency, ν , is the number of waves in the distance light travels in one second The third parameter, which is most common to vibrational spectroscopy, is the "wavenumber," .  

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, Here is the difference in energy between two quantized states, h is Planck's constant ( erg s) and c is the velocity of light. Thus, is directly proportional to the energy of transition. Suppose that, where and are the energies of the excited and ground states, respectively. Then, the molecule "absorbs" when it is excited from to , and "emits" when it reverts from to . Finally, we have   (Excited state)   (Ground state)     Absorption Emission

6. Interaction of Electromagnetic Radiation with Matter:

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: Inlet device Ion Source Analyzer system Detector 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;  

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 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 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 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 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: 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 ) 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

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:   Momentum analyzer: The magnetic sector does not directly separate ions by mass. Rather it effects ion separation by their momentum and this feature can be used as a measure of mass provided all ions possess equal kinetic energy.

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 ( ), 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 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 from , with numerous plates achieving up to .  

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 . 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 ( 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.   This Photo by Unknown Author is licensed under CC BY

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.

39. 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.

40. 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).

41. 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

42. Types of peaks in Mass Spectrum

43. Isotopic Peaks

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