Mass Spectros

PHARMACEUTICAL ANALYSIS-III Mass Spectroscopy
PHARM-4709
Mass spectroscopy/Spectrometry (MS) is a quantitative and qualitative analytical technique by which we can
measure the molecular mass and formula of a compound and the record is known as mass spectra.
Mass spectra is useful-
 To establish the structure of a new compound

 To give the exact molecular mass
 To give the molecular formula
 To indicate the presence of functional group in a compound
The instrument is called mass spectrometer. It is an instrument which measures the masses of individual
molecules that has been converted into ions. Therefore the molecule must be in the vapour state.
Basic function:-
The mass spectrometer is designed to perform four basic functions –
 To vaporize the compound by increasing volatility.

 To generate the ions from the neutral compound in resulting vapor pressure.
 To separate the ions according to their mass to charge ratio (m/z, m/e) in a electric field.
 To collect/detect the mass and record.
Applications of MS
1. Identification: It helps in the identification of known compounds by spectral comparison.
2. Structure elucidation: It helps in the structure elucidation of compounds by
a) giving accurate mol.wt.
b) givingmol.Formula.
c) revealing the presence of some structural unit within the molecule.
3. It helps to establish the structure molecules with MS-MS technique.
4. It can analyze complex mixtures.
5. It helps in the study of drug testing and discovery, food contamination detection, pesticide residue analysis,
isotope ratio determination, proteins, peptide and DNA sequencing etc.
e.g. Mass spectrometric method used for quantitative analysis of some drugs having hypnotic, sedative and
tranquilizing properties, that is, benzodiazepine, thioxanthene, butyrophenone, methadone and
diphenylbutylpiperidine in whole blood.
Advantages of MS
1. Widespread application: Can be applied to both organic and inorganic compounds.
2. Accuracy: It is the most accurate method for determination of molecular mass & molecular formula of a
compound.
3. Sensitivity: It can analyze samples in the nanogram or even femtogram level.
Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

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PHARM-4709
 femtogram (plural femtograms) A unit of mass equal to 0.000 000 000 000 001 (10-15) grams. Symbol:
fg.
 molecular mass: The sum of the atomic masses of all atoms in a molecule.
4. Structure elucidation of complex compounds: Mass spectrometry is becoming more important in the
structure elucidation of very complex compounds due to introduction of MS-MS technique.
5. Analysis of mixtures: Some samples which remain mixtures even after exhaustive chromatographic
separations can be analyzed successfully with MS-MS technique.
6. Characterization of biomacromolecules: MS-MS is very helpful in the study of protein and peptide
sequencing, DNA sequencing, protein folding etc.
Principle/ Theory of Mass Spectroscopy
1. Molecular ion production:
Mass spectrometer is a device for the production and weighing of ions.
Molecules are subjected to bombardment by a stream of high-energy electrons, converting some of the
molecules cons. The molecular ions are usually radical cation and some may be radical anion.
[M] -e- [M] + or, [M] +e- [M]-
2. Fragmentation:
When the molecule has been bombarded by high-energy electrons to produce ions, the molecule absorbs
sufficient energy and undergo fragmentation.
B+ neutral
A+
C+ neutral
Decompose to produce new ions
D+ neutral
If fragment B and C have sufficient energy they may fragment further e.g. D+ ion
3. Separation of ions:
 The mixture of ions are separated according to the mass charge ratio in the analyzer and then recorded.

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 The record is known as the mass spectrum. It is the record of abundance of each ion against its m/z
value.
4. Mass spectrum:
The mass spectrum is a plot of ion current intensity (ion abundance) versus m/z value.
The most abundant peak will give the tallest peak of the mass spectra. This peak is known as the base peak and
its mass arbitrarily assigned a value of 100%. The heaviest peak is the molecular ion peak (M+) and its mass
will give the mass of the molecule.
C+
Relative D+ B+
abundance
A+
m/z value

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Isotope peak:
Isotopes present in the molecule may generate additional peak termed as isotopic peaks.. Due to the occurrence
of isotopes we also observe M+1, M+2, M+3 isotopic peaks. The relative abundances of these isotopic peaks
are proportional to the abundance of the isotope in nature (e.g. the natural abundance of 13C is 1.1% of the 12C
atoms. For an ion containing 'n' number of carbon atoms, the abundance of isotope peak is n x 1.1% of the 12C
containing peak.
M+1 peaks are made by - 13C, 2H, 5N, 33S
M+2 peaks are made by - 18O, 34S, 37Cl, 81Br
Base peak
M+ peak
Relative
M+1
absorbance
peak
M+2
m/z value
M+1 and M-2 peak in benzene:
Benzene shows molecular ion peak at m/z value 78 due to C6H6. It will also show M+1 peak at m/z 79 due to
13
CC5H6+ or, C6H6D+.
M+2 peak will also show at m/z 80 due to 13CCH5D+ or 13C2C4H6+ or, C6H4D2+
The relative abundances of this isotopic can be used to determine molecular formula.

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Table: Atomic weight and natural abundance of some isotope
Isotope Atomic weight Natural abundance (%)

1H 1.0078 99.985
2H 2.014 0.015
12C 12.000 98.9
13C 13.003 1.1
16O 14.003 99.64
14N 15.001 0.36
15N 15.994 99.8
17O 16.999 0.04
18O 17.999 0.2
Ionization method:
Previously, the requirement was that the sample be able to be vaporized, but modern ionization techniques
allow the study of such non-volatile molecules as proteins and nucleotides.
Different techniques have been developed to produce ions from the compounds. In ionization method
compound are divided into 2 groups –
a) lonization of volatile materials

b) lonization of nonvolatile materials
a) lonization of volatile materials -
Two methods are commonly used to produce ions from thermally volatile compound -
1) Electron impact ionization (ET)
2) Chemical ionization (CI)
a) Electron impact ionization:
Electron impact ionization plays an important role in the routine analysis of small molecules. Samples are
introduced into the ion source of the mass spectrometer with the help of a probe. Readily volatile sample like
liquids and low molecular weight solids are volatilized by heating under low pressure outside the ion source and
then allowed to diffuse into the source. Electrons are accelerated from a hot filament to an anode.
 A direct probe tip is used near to a healed filament which provides electron and is heated in the ionization
chamber causing vapor from the sample.
(Handle) (metal sheet) (Ceramic tip with sample)

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 Electrons are accelerated from the hot filament to an anode, usually through a potential difference of about
70ev.
 A 70ev has sufficient energy not only to ionize an organic molecule but also to cause extensive
fragmentation.
 Molecules are ionized due to bombardment with high-energy electrons (70eV) by removal of an electron.
The product is cation radical.
M+ e  M+ + 2e (Here, M= analyte molecule being ioninzed, e= electron, M+ = resulting molecular ion)
Electron capture to produce an anion radical does not occur to a significant extent the bombarding electrons
have such high energies that they cannot be captured.
The molecular ion produced by EI usually have sufficient energy and undergo extensive fragmentation which
has both an advantage and a disadvantage as well. The advantage is that it gives rise to a pattern of fragment
ions which can help to characterize the compound. The disadvantage is the frequent absence of a molecular ion
peak.
2. Chemical ionization (CI):
Most modern mass spectrometers are designed so that electron impact ionization and chemical ionization can be
carried out interchangeably.
 In this technique a reagent gas (methane, isobutane or ammonia) allowed to pass into the ion chamber at
low pressure.
 The gas is ionized by using electron impact, which can then undergo ion molecule reaction.

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CH4 + e → CH4 + + 2e
CH4 + + CH4 → CH5+ + CH3
 If the sample molecules are volatilized into mixture of ions, CH3+, act as a strong acid and protonates the
sample.
M + CH5+ MH+ +CH4
The protonated sample (MH+) normally has very little internal energy and therefore it does not fragment
extensively. As a result, we obtain mass spectra with intense molecular ion peak.
Thus in positive ion CI-spectra, the observed m/z value is one unit greater than the true molecular weight.
 In CI-technique, negative ion CI-spectra may occur for molecules with electron accepting properties like
trifluoroacetates, quinines and nitro compounds. For these molecules negative ion chemical ionization mass
spectra are obtained.
M+e M-
Negative ion chemical ionization spectra can also be obtained by generating a proton abstracting ion like CH3O-
in the ion source. CH3O- acts as a base and abstract a proton from the sample molecule.
M + CH3O- (M – H)- + CH3OH
Here the observed m/z value is one unit lesser than the true molecular weight.
 Chemical ionization produces very simple mass spectra that provide molecular mass, but very limited
structural information.

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b) Ionization of non-volatile materials-
If the molecules have low molecular weight (e.g200Daltons) but have numerous polar functional groups, or
have high molecular weight (>800 Daltons) ; usually don't pass into the gas phase at high temperature and at
low pressure. For such molecules special ionization techniques have been developed.
1. Field desorption:
 Here, the probe tip is replaced by a thin wire on which sharp needles have been grown.
 The wire is supported between two posts on the probe.
 A solution of small amount of a sample is deposited on the wire
 In the mass spectrometry the wire is maintained at +8kv and can be heated and this can cause the
discharge of an electron from In this way molecules are thrown into the gas phase as a positive
molecular ion without thermal decomposition.
Figure: Field desorption technique

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3. Desorption ionization by particles or radiation:
For desorption ionization by particles or radiation, energy is given in such a way that intermolecular bonds
e.g. H- bonds are broken in preference to covalent bonds and the sample is desorbed from its environment
into the gas phase within a time of the order of 10-12 seconds. Thus thermal decomposition does not occur.
The techniques are as follows-
 Laser desorption (LD) e.g. MALDI

 Fast atom bombardment (FAB)
 Californium plasma desorption (CPD)
 Secondary ion mass spectrometry (SIMS)
 Based upon giving a large pulse of energy to the sample

 Bonds are broken and the sample is desorped from its environment into the gas phase within 10-12 sec.
 So thermal decomposition doesn't occur.
Laser desorption
 In this technique, the sample is bombarded with short duration, intense pulses of laser light.
 Efficient and controllable energy transfer to the sample requires resonant absorption of the molecule at the
laser wavelength.
 Therefore, lasers emitting either in the UV or IR are employed.
 Laser pulses are applied for 1-100ns.
One disadvantage is some thermo labile compounds may be degraded with the laser beam resulting in a
spectrum of fragment ions. To overcome this problem, a matrix is used and the technique is known as matrix
assisted laser desorption ionization technique (MALDI).
MALDI:
 The efficient and directed energy transfer during a matrix-assisted laser induced desorption event
provides high ion yields of the intact analyte, and allows for the measurement of compounds with high
accuracy and sensitivity.
 MALDI provides for the nondestructive vaporization and ionization of both large and small
biomolecules. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a
matrix compound, usuallya UV-absorbing weak organic acid, after which laser radiation of this
analyte–matrix mixture results in the vaporization of the matrix which carries the analyte with it.
 The matrix also serves as a proton donor and receptor, acting to ionize the analyte in both positive and
negative ionization modes, respectively.

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 In MALDI, a low concentration of the sample is embedded either in a liquid or a solid matrix (molar
ratio 1:100 to 1:50000) which is selected to absorb strongly the laser light. In this way, the energy is
transferred indirectly to the sample, and in a controlled manner which avoids sample decomposition.
 Used in conjunction with a suitable method for ion Analysis, MALDI can give approximate molecular
weight determinations for biomolecules, even in the range 100000-200000 Daltons.
Some common MALDI matrices:
Matrix Application
2.5-dihydroxy benzoic acid (DHB) Peptides, protein, lipids, oligosaccharides
3,5-dimethoxy-4-hydroxy cinnamic acid Peptides, protein, glycoproteins
MALDI is a two step process:
First, Desorption is triggered by a UV laserbeam. Matrix material heavily absorbs UV laser light leading to the
ablation of the upper layer of the matrix material.Hot plume gets produced during ablation.
Second, the analyte molecules are ionized in the hot plume. Ablated species may participate in the ionization of
analyte.
Advantages of MALDI
i)Mild ionization technique and nondestructive.
ii)Suitable for nonvolatile samples.
iii)High sensitivity and accuracy.
Disadvantages of MALDI
i)Compound must be soluble in matrix in case of liquid matrix.

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Fast Atom Bombarment (FAB)

In this process, the energy is provided by a beam of neutral atom (xenon atoms). In FAB, the sample is
dissolved in a matrix of low volatility (e.g. glycerol). A few micrograms of the sample are dissolved in a few
microlitres of glycerol as matrix and a beam of fast xenon atoms bombards the solution.
This fast xenon atoms are prepared by accelerating xenon ions and then neutralizing these ions by charge
exchanger at a low pressure.
Xe+  Xe+
These are then passed through stationary xenon atoms at low pressure. Electron transfer takes place and fast
xenon atoms are produced.
Xe+ + Xe → Xe + Xe+
 Due to bombardment with high energy xenon atoms, the sample is desorbed in to the gas phase often as
an ion.
 Other matrixes used in FAB are-
 Thioglycerol: Diglycerol (1:1)

 Tetragol
 Teracol
 Glycerol
 Both positive and negative ion FAB mass spectra can be obtained.
Advantages of FAB
i) High resolution and rapid ionization.
ii) Reduces sample fragmentation.
iii) Good for nonvolatile, thermally unstable compound.
Disadvantages of FAB
Not suitable for separating the samples.

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Californium plasma desorption-
 Here the sample to be analyzed is deposited on a thin metal foil (nickel).

 Spontaneous fission of the radioactive Californium nucleus (252 Cf) occurs, and each fission event gives
rise to two fragments travelling in opposite directions. A typical pair of fission fragments are and
+
of high kinetic energy.
 When such a high energy fission fragment passes through the sample foil, produce a high temperature of
10000K
 Consequently the molecules in this plasma zone are desorbed from the foil with the production of both
positive and negative ions. These ions are then accelerated out of the source into the analyzer system.
 Californium plasma desorption technique produces better molecular peak for molecules having
molecular weight between 10000-20000 Dalton.
Different technique for analyzing ions in a mass spectrometer:
a. Magnetic sector
b. Time of flight
c. Quadrupole
d. Ion cyclotron resonance
e. Ion trap
Magnetic sector analyzer/mass analyzer:
 The ions may be separated according to their mass to charge ratio (m/z) using a magnetic field.
 Here the ions of larger mass are deflected less than the ions of smaller mass according to the equation.
=
Where, r= radius of circular of path in which ion is traveling
H= magnetic field strength
V= potential difference of ion

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The equation clarifies that by varying the magnetic field strength or accelerating potential, the ions of all m/z
value can be successively allowed to pass through the detector slit & mass spectrum recorded.
Time of flight mass analyzer:
 TOF mass analyzer separates or resolves the ion beam by measuring the flight time of the ions. The
technique requires that all the ions produced in the ion source should leave at the same time.
 The ions are accelerated by a potential difference and then allowed
to pass into the filed free region.
Since all the singly charged ions will acquire the same kinetic energy, the largest mass will have the
lowest velocity and the longest time of flight over a given distance. Mathematically,
It is quicker than any other mass analyzer and applicable for all masses.
Quadrupole:
 Here four parallel rods arranged symmetrically around an ion flight path and a direct current and a radio
frequency are applied to the rods resulting an oscillating electrostatic field.
 The ions when pass through the region, will acquire an oscillation in the electrostatic field. The ions of
incorrect m/z ratio undergo an unstable oscillation and strikes one of the rods.

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 Ions of correct m/z ratio undergo stable oscillation of constant amplitude and pass through analyzer to
reach the recorder.
Quadrupole analyzer is a relatively compact instrument and inexpensive.
Low-resolution mass spectrometer (LRMS)

LRMS employs a single stage analyzer. It resolves only integral masses and it can't differentiate the molecules
e.g. CO (28), CH2=CH₂ (28), N₂ (28), as all have the same integral mass 28. Since, it can't give exact masses,
molecular formula can't be determined.
High-resolution mass spectrometer (HRMS): HRMS employs multiple stage analyzer such as magnetic and
quadropole sectors linked in series. The accuracy of these types of instruments enables the distinction between
different isotopes such as 13C vs. 12C. The high-resolution data are obtained at an accuracy of 0.0001 amu
(atomic mass unit) and consequently this permits a distinction between species of the same mass unit such as -
CO (28), CH2=CH2 (28), N2 (28). Therefore data from HRMS are essential for unambiguous determination of
molecular data.
a) Double focusing mass spectrometer are used to obtain high resolution in which the beam ions are pass
through an electric field region before entering the magnetic field.
b) In a single focusing mass spectrometer, there is a lack of uniformity of ion energies that is all ions do not
have precisely same velocity. The result is peak broadening and low to moderate resolution.
Electrospray ionization (ESI):

'Electrospray' is applied to the small flow of liquid (1-10μl/min) from a capillary needle when a potential
difference of 3-6k is typically applied between the end of the capillary and a cylindrical electrode located 0.3-2
cm away.
 Under these circumstances, the liquid leaving the capillary does not leave as a drop, but rather as a
spray.
 The spray consists of highly charged liquid droplets, and these droplets may be positively or negatively
charged depending on the sign of the voltage applied to the capillary.
 If the liquid spraying from the capillary contains sample molecules, then a molecular ion of these sample
molecules can be obtained by evaporation of the solvent.
ESI is an excellent technique for the production of molecular ions from large polar molecules, and it will be
seen subsequently that, since it frequently produces multiply charged ions, it is a very powerful tool in the
analysis of biopolymers. This is especially true since the method can be conveniently used to analyze directly
the effluent from an HPLC column.

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Gas chromatography-mass spectrometry (GC/MS):
 The separation and detection of components from a mixture of organic compound is readily achievable
by gas chromatography. Furthermore, limited characterization of unknown components is often possible
from retention times appropriate to the particular column used.
 Mass spectrometry, because of its high sensitivity and fast scan speeds, is the technique most suited to
provide definite structural information from the small quantities of material eluted from a gas
chromatograph.
 The association of the two techniques provided a powerful means of structure identification for the
components of natural and synthetic organic mixtures even though the components may be present in
nanogram quantities and eluted over periods of only a few seconds.
 The interface between the GC and the MS is a jet separator.
Such a combination is useful as an aid in determining the structures and chiralities of amino acids. The
amino acids are first derivatized as follows: by treatment with trifluoroacetic anhydride in the first step
and by isopropanol / HCI in the second (to render them volatile):
NH2CH(R)COOH CF3CONHCH(R)COOH CF3CONHCH(R)COOCH(CH3)2
Since trifluoroacetyl is a good electron capture group, the mass spectra are determined in the negative
ion mode. The mixture of derivatized amino acids (frequently from 6N HCl hydrolysis) is simply
injected on to a chiral GC column, where the retention times are not only dependent on structure but also
on absolute configuration of the amino acids. Thus separation, molecular weight, and chirality can all be
determined in one experiment.
Liquid chromatography-mass spectrometer (LC/MS)
 HPLC is a powerful method for the separation of complex mixtures, especially when many of the
components may have similar polarities. In reverse-phase HPLC, the column substrate is such that
starting with an aqueous solution of a mixture of polar components; the most polar components are
eluted first. The later-eluted hydrophobic components are often encouraged to leave the column by
gradually increasing the concentration of acetonitrile (CH3CN) in the otherwise aqueous developing
solvent.
 If the mass spectrum of each component can be recorded as it eluates from the HPLC column, quick
characterization of the components is greatly facilitated.

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Tandem mass spectrometry (MS/MS):

Tandem mass spectrometer uses two mass spectrometers is tandem.
 It has a great potential value in the structure elucidation of organic compounds.

 In this technique, a compound to be analyzed is subjected to fonization and fragmentation. The mixtures
of ions are then separated in the first mass analyzer.
 From the mixture of ions, a specified ion is selected for the second mass analyzer. The magnetic field is
set to pass only the selected ion through a slit into a collision chamber. This chamber contains a high
energy reagent gas like helium (He) or argon (Ar) with which the ions collide. As a result, mixtures of
ions are produced. The process is known as collision activated decomposition (CAD). The ions are then
analyzed in the second mass analyzer.
Example -
Penazitidine A is a heterocyclic compound with a long chain. It has a methyl group on the side chain but the
position was not established by various technique (NMR & even by 2D NMR), But MS-MS can determine the
position of methyl group on the structure.
The ion at m/z 296 was selected. It was allowed to pass into the collision chamber where it is subjected to CAD.
The mixtures of ions produced are analyzed in the 2nd mass analyzer. The intense peak at m/z 182, m/z 210
indicates the position of methyl group at C12 of the side chain.
Fragmentation patterns:
For most classes of compounds, the mode of fragmentation is somewhat characteristic. The most common mode
of fragmentation involves the cleavage of one bond. In this process the odd-electron molecular ion yields an
odd-electron neutral fragment (a radical) and an even electron fragment ion (carbonium type).
Solving a mass spectrum

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Exercise 01

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PHARMACEUTICAL ANALYSIS-III Mass Spectroscopy

Mass spectra is useful-

 To establish the structure of a new compound

The mass spectrometer is designed to perform four basic functions –

 To vaporize the compound by increasing volatility.

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Principle/ Theory of Mass Spectroscopy

1. Molecular ion production:

Mass spectrometer is a device for the production and weighing of ions.

[M] -e- [M] + or, [M] +e- [M]-

Decompose to produce new ions

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

M+1 peaks are made by - 13C, 2H, 5N, 33S

M+2 peaks are made by - 18O, 34S, 37Cl, 81Br

M+1 and M-2 peak in benzene:

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Isotope Atomic weight Natural abundance (%)

a) lonization of volatile materials

a) Electron impact ionization:

(Handle) (metal sheet) (Ceramic tip with sample)

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

M+ e  M+ + 2e (Here, M= analyte molecule being ioninzed, e= electron, M+ = resulting molecular ion)

2. Chemical ionization (CI):

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

CH4 + + CH4 → CH5+ + CH3

M + CH5+ MH+ +CH4

M + CH3O- (M – H)- + CH3OH

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

b) Ionization of non-volatile materials-

 The wire is supported between two posts on the probe.

 A solution of small amount of a sample is deposited on the wire

Figure: Field desorption technique

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

3. Desorption ionization by particles or radiation:

The techniques are as follows-

 Laser desorption (LD) e.g. MALDI

 Based upon giving a large pulse of energy to the sample

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Some common MALDI matrices:

MALDI is a two step process:

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Fast Atom Bombarment (FAB)

 Thioglycerol: Diglycerol (1:1)

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

 Here the sample to be analyzed is deposited on a thin metal foil (nickel).

Different technique for analyzing ions in a mass spectrometer:

Magnetic sector analyzer/mass analyzer:

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Time of flight mass analyzer:

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Low-resolution mass spectrometer (LRMS)

Electrospray ionization (ESI):

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Gas chromatography-mass spectrometry (GC/MS):

NH2CH(R)COOH CF3CONHCH(R)COOH CF3CONHCH(R)COOCH(CH3)2

Liquid chromatography-mass spectrometer (LC/MS)

Compiled by: Md Masud Morshed ; Department of Pharmacy, IIUC

Tandem mass spectrometry (MS/MS):