This document provides an overview of the application of mass spectrometry in pharmaceutical and biomedical analysis. It discusses the basic components and working of a mass spectrometer, including ionization, mass analysis, and detection. It also covers mass spectrometry techniques such as LC-MS, GC-MS, and MS-MS, and their uses in applications like structure elucidation, impurity profiling, metabolomics, and proteomics. The document concludes with a discussion of hyphenated techniques that combine chromatography with mass spectrometry for improved sensitivity, selectivity, and specificity.

1. Application of Mass Spectrometry (MS)
in Pharmaceutical and Biomedical
Analysis
Dr. M. Rudrapal
e-mail: rsmrpal@gmail.com

2. 2
Spectroscopy vs. Spectrometry

3. 3
Techniques
UV-VIS, IR
NMR
(1H & 13C)
MS
Spectroscopic Techniques in Molecular
Structure Analysis
UV-VIS:
Conjugation
IR:
Functional Groups
NMR:
Groups / C-H Skeleton
MS:
Mass & Structure

4. 4
• MS Basic Theory and Instrumentation
• MS Spectrum: Features
• Rules of Fragmentation in MS
• MS Hyphenated Techniques
• Applications
Outline

5. 5
• Instrumental analytical technique used for the
determination of the composition of a sample or
molecule and elucidation of the chemical
structure of molecules, such as peptides and
other chemical compounds including drug
substances
• Technique used for measuring the molecular
weight and determining the molecular formula
(elemental composition) of an organic compound
Mass Spectrometry (MS)

6. 6
Mass Spectrometer
Ion Spectra of Different m/z

7. Ionizer
Sample
+
_
Mass Analyzer Detector

8. 8
Mass Spectrometer - Components
• Sample Inlet: Introduction of sample into the instrument (under low pressure)
• Ion Source: Generation of sample ions in the gas phase
• Mass Analyzer: Separation of positively charged ions on the basis of differences
in m/z (mass to charge ratio)
• Detector: Detection of signals and counting ions
• Data system (acquisition and processing): Processing of signal into the spectra

9. 9

10. 10
Create ions
Separate ions Detect ions
Mass Spectrometric Analysis - Steps
1. Vaporization of sample (solution) under high vacuum
2. Bombardment of sample with high energy electron beam
– EI mode (conversion of gas phase neutral molecule to
radical cation, positively charged fragments)
3. Ions (positive) are then accelerated by an electric
(negative potential) field
4. Separation of ions (cations/fragments) based on mass
(mass-to-charge ratio) in a strong magnetic field
5. Measurement of the relative abundance of each ion as
function of their m/z

11. 11
Mass Spectrometry - Working
• In a mass spectrometer, a molecule is vaporized
under vacuum and then ionized by bombardment with
a beam of high-energy electrons causing the loss of
an electron (EI-mode)
• The energy of the electrons is ~ 1600 kcal (or 70 eV,
1eV=23Kcal/mol), only ~100 kcal of energy to cleave a
typical s bond

12. 12
• When the high energy electron beam ionizes the
neutral molecule, the species that is formed is
called a radical cation, and symbolized as M+•
• The radical cation M+• is called the molecular ion
or parent ion and the mass of M+• represents the
molecular weight of M
• Because M is unstable (excess energy), some
ions decompose to form fragments of radicals
and cations that have a lower molecular weight
than M+•

13. 13
• The ions (cations) are then accelerated through a
potential of about 10,000 volts and collected by a
detector
• Some mass spectrometers cause the ions to pass
through a magnetic field which deflects the ions
and results in a range of ion weights spread across
the detector. Only the cations are deflected by the
magnetic field
• Lighter ions are deflected more than heavier ones
• Amount of deflection depends on their m/z

14. 14
•The detector signal is proportional to the number
of ions hitting it
• By varying the magnetic field, ions of all masses
are collected and counted
• Other spectrometers are linear and measure the
time of flight of the ions (TOF)
• Heavier ions move more slowly than lighter ones

15. 15

16. 16
ESI-QTOF:
Electrospray ionization (ESI) source +
Quadrupole mass filter (Q) + Time-of-flight
(TOF) mass analyzer
MALDI-QTOF:
Matrix-assisted laser desorption ionization
(MALDI) + Quadrupole (Q) + Time-of-flight
(TOF)
LC-CID-(LTQ-Orbitrap) MS
LC-TSP-FAB (MS)
Types of Mass Spectrometer
Triple Quadrupole Analyzers (QqQ)
Soft techniques - ideal for biomedical analysis

17. 17
Orbitraps (HRMS)
Qualitative:
Tripple Quadrupole (URMS)
Quantitative:
TOF (HRMS)
Qualitative:
HRMS: High Resolution Mass Spectrometry
UMR: Unit Resolution Mass Spectrometry

18. 18
• The mass spectrometer analyzes the masses of
cations (masses are graphed or tabulated a
according to their relative abundance)
• A mass spectrum is a plot of the amount of each
cation (its relative abundance) versus its mass to
charge ratio (m/z, where m is mass, and z is
charge)
• Since z is almost always +1, m/z actually
measures the mass (m) of the individual ion
Mass Spectrum -
Structure Elucidation

19. 19
Masses of the positively charged fragments
(cations) and their relative abundance reveal
information about the structure of the molecule

20. 20

21. 21
• Bar plot between relative ion abundance (intensity) vs.
mass of ions
• Line spectrum of positive ions
• Characterized by sharp and narrow peaks
• High resolution (HRMS)
• X-axis position indicates the m/z ratio of a given ion (for
singly charged ions, this corresponds to the mass of the
ion)
• Height of peak indicates the relative abundance (conc.) of a
given ion (not reliable for quantitation)
Typical MS Spectrum: Features

22. 22
Typical Mass Spectrum
Aspirin
MW: 180
Relative
Abundance
180 m/z for singly charged ion (M+) is the mass (MW of Aspirin)

23. 23
• Molecular ion (M+) peak: Peak of highest mass is
called the molecular ion peak (except isotope
peaks)
• The tallest peak in the mass spectrum is called
the base peak (most intense peak)
• The base peak may also be the M peak, although
this may not always be the case

24. 24
• Isotopes peaks:
• Though most C atoms have an atomic mass of
12, 1.1% have a mass of 13
• Thus, 13CH4 is responsible for the peak at m/z =
87 in hexane. This is called the M + 1 peak
• Some isotopes show M + 2 peaks (18O, 34S, 37Cl
81 Br etc.)

25. 25
Relative Abundance of Isotopes

26. 26
M + 2 Peak:
• Most elements have one major isotope
• Chlorine has two common isotopes: 35Cl and 37Cl, which occur naturally
in a 3:1 ratio (M+2 is one third of M)
Thus, there are two peaks in a 3:1 ratio for the molecular ion of an
alkyl chloride
The larger peak, the M peak, corresponds to the compound
containing the 35Cl. The smaller peak, (M + 2 peak), corresponds to
the compound containing 37Cl
Thus, when the molecular ion consists of two peaks (M and M + 2) in
a 3:1 ratio, a Cl atom is present
35Cl (100 %) : 37Cl (32.5%): 3:1
• Br has two isotopes: 79Br and 81Br, in a ratio of ~1:1. Thus, when the
molecular ion consists of two peaks (M and M + 2) in a 1:1 ratio, a Br
atom is present (M+2 is equal to M)
79Br (100 %) : 81Br (98%): 1:1

27. 27
M + 2 Peak

28. 28
• Fragment ion peak: Peak of fragment ion in mass spectrum is
called fragment ion peak
• Fragmentation pattern is characteristic for a particular organic
compound
• It can be used to confirm the compound by comparing with the
library of fragmentation pattern of reference compounds
• Molecular ion can loose only
one fragment, but any
number of neutral fragments
• Once radical has been lost,
only neutral fragments can
be lost thereafter

29. 29
• Metastable ion peak: This ion is formed from the disintegration
of fragment ion in the analyzer tube of mass spectrometer.
• It is generated due to loss of kinetic energy of ion during
acceleration from ionization chamber to analyzer tube.
• This ion appears in the spectrum at m/z ratio, which depends
upon the mass of original ion from which it is formed (m1+) and
fragment ion mass (m2+)
• Metastable ion peaks usually appear at non-integral values of
m/z ratio and are often seen as broad peaks.
• This is used for the confirmation of proposed fragmentation
pattern of a molecule
m* = (m2+)2/( m1+)

30. 30
Recognition of M+ : Approaches
The Nitrogen Rule
• Hydrocarbons like methane (CH4) and hexane
(C6H14), as well as compounds that contain only C,
H, and O atoms, always have a molecular ion with
an even mass
• An odd molecular ion indicates that a compound
has an odd number of nitrogen atoms
Aspirin: C9H8O4 (180) Acetamide: C2H5NO (59)
M+ = 180 M+ = 59

31. 31
• The effect of N atoms on the mass of the molecular
ion in a mass spectrum is called the nitrogen rule:
• A compound that contains an odd number of N
atoms gives an odd molecular ion
• A compound that contains an even number of N
atoms (including zero) gives an even molecular ion
• This rule holds for all compounds that contain C, H,
O, N, S etc.
M+ W Even: Even / No N M+W Odd: Odd N

32. 32
Identifying M+ Peak from
Fragmentation Pattern
• Fragmentation at a single bond gives an odd-numbered
ion fragment from an even-numbered molecular ion,
and an even-numbered ion fragment from an odd-
numbered molecular ion
• Ion fragment must contain all of the nitrogen (if any) of
the molecular ion

33. 33

34. 34
• The intensity of the molecular ion peak depends on the
stability of the molecular ion
• The most stable molecular ions are those of purely
aromatic systems
• If substituents that have favorable modes of cleavage
are present, the molecular ion peak will be less intense,
and the fragment peaks relatively more intense

35. 35
Aromatic Compounds > Conjugated Alkenes >
Cyclic > Org. Sulfides > Normal Alkanes
Ketones >Amines > Esters> Ethers >
Acids > Aldehydes > Amides > Halides
Prominent M+ (in decreasing order)
M+ remains quite undetectable in
Aliphatic alcohols, Nitriles,
Nitro and branched compounds

36. 36
Peak Loss of Group
M-15 CH3
M-18 H2O
M-31 OCH3
M-2 H2
M-3 alcohol

37. 37
Mass Spectra & Fragmentation Pattern
(α-Homolytic Cleavage)

38. 38
Drug molecules in which homolytic α-cleavage
dominates the spectrum
Since many drugs contain hetero atoms the fragmentation of
drug molecules is often directed by α-homolytic cleavage
adjacent to these atoms.
Figure 9.16 shows the mass spectrum of bupivacine, where
homolytic α-cleavage is directed by the nitrogen atom in the
heterocyclic ring resulting in an ion at m/z 140, which
dominates the spectrum

39. 39

40. 40

41. 41

42. 42
McLafferty Rearrangement:
• This fragmentation pattern is typically seen in
carbonyl compounds that have a γ hydrogen
C H
OCH2
CH2
CH2
H
C H
OCH2
CH2
CH2
H
+
++
CH3CH2CH2-CHO CH3CH2CH2 + H - C = O
++
Carbonyl Cleavage:

43. 43

44. 44
High Resolution Mass Spectrometers
• Low resolution mass spectrometers report m/z
values to the nearest whole number. Thus, the
mass of a given molecular ion can correspond to
many different molecular formulas
• High resolution mass spectrometers measure
m/z ratios to four (or more) decimal places
• The better the resolution or resolving power, the
better the instrument and the better the mass
accuracy

45. 45
Precise Mass Determination:
Sum of exact formula masses of most abundant isotopes ->
Precise molecular ion

46. 46
This is valuable because except for 12C whose
mass is defined as 12.0000, the masses of all
other nuclei are very close—but not exactly—
whole numbers
Table 13.1 lists the exact mass values for a few
common nuclei. Using these values it is
possible to determine the single molecular
formula that gives rise to a molecular ion

47. 47
• Consider a compound having a molecular ion at m/z = 60
using a low-resolution mass spectrometer. The molecule
could have any one of the following molecular formulas
In HRMS, the molecular formula is obtained
from the precise mass

48. 48
Periodic Table
Different elements can be uniquely
identified by their respective mass values

49. 49
Molecular Mass - Molecular Weight (MW)
Elemental Composition - Molecular
Formula (MF)
Structure Elucidation – Organic
Compounds
Structural Determination - Drugs, Peptides
etc.
Mass Spectrometric Determination

50. 50
Broad Areas of Applications
 Phytochemical/ Herbal Drug Analysis
 Pharmaceutical Analysis and Research
 Purity Analysis
 Impurity Profiling
 Structure Elucidation
 Drug Discovery/ Molecular Biology/ Biotechnology
 Biomolecule Characterization (Target Validation)
 Protein, Peptides, Oligonucleotides etc.
 Forensic Science
 Bioanalytical - Clinical (PK) Study & Toxicology
 Drug Metabolism Studies (Metabolomics)
 Analysis of Drug Metabolites
 Petrochemical Analysis

51. 51
 Peptide and Protein Sequence/ Structure Analysis
 Forensic/ Food/ Cosmetic/Agriculture
 Nanobiotechnology
 Biomaterials
 Computational biology

52. 52
Mass
Spectrometry
Chemical
Pharmaceutical
Cosmetic
Food/ Herbal/
Agricultural/
Forensic

53. 53

54. 54
Traditional Mass Spectrometer

55. 55
Hyphenated Instruments
(Tandem Analytical Techniques)
• Reasons behind combining techniques together, in a
tandem arrangement often chromatographic and
spectroscopic techniques
• Sensitivity of detection
• Selectivity of separation
• Specificity of separation
• Improved reliability of identification

56. 56
Separation and Detection in a
Hyphenated Technique

57. 57
Hyphenated Techniques
LC-MS
GC-MS
MS-MS

58. 58
GC-MS Analysis
Analysis of tetrahydrocannabinol
(THC) in urine by GC-MS
(Metabolomics)
M+ peak = 314

59. 59
Hyphenated MS: Tandem LC-Tandem MS
LC/LC-MS/MS: Excellent Accuracy in Measurement

60. Tandem Mass Spectrometry
• Different MS-MS configurations (depending upon analyzers)
• Quadrupole (Q)-quadrupole (Q) [low energy]
• Magnetic sector (MS)-quadrupole (Q) [high energy]
• Quadrupole (Q)-time-of-flight (TOF) [low energy]
• Time-of-flight (TOF)-time-of-flight (TOF) [low energy]
• GC-MS-MS
• LC-MS-MS
• LC-ICP-MS
• HPTLC-MS
Advanced Hyphenated
Techniques

61. Positive or Negative Ion Mode?
• If the sample has functional groups that readily
accept H+ (such as amide and amino groups
found in peptides and proteins) then positive
ion detection is used - PROTEINS
• If a sample has functional groups that readily
lose a H+ (such as carboxylic acids and
hydroxyls as found in nucleic acids and sugars)
then negative ion detection is used - DNA

62. 62
MS-MS in Proteomics
Peptide Mass Fingerprinting
Protein Identification

63. 63