Yy

1. APPLICATIONS OF MASS
SPECTROMETRY
BY:
K.Bhagyasri,
Pharmaceutical
Analysis.

2. MASS SPECTROMETRY:
 An analytical technique for measuring the mass-to-charge
ratio (m/z) of ions, most commonly positive ions.
 Mass spectrum: a plot of the relative abundance of each ion
versus mass-to-charge ratio
 Base peak: the most abundant peak; assigned an arbitrary
intensity of 100
 The relative abundance of all other ions is reported as a %
of abundance of the base peak

3. Relative abundance
Parent ion peak
Base peak
TYPICAL MASS SPECTRUM

4. APPLICATIONS OF MASS SPECTROMETRY
 Molecular weight determination.
 Determination of molecular formula.
 Structure determination.
 Relative abundance of isotopes.
 Detection of impurities.
 Qunatitative analysis.
 Determination of bond dissosciation energies.
 Identification of drug metabolites.
 Distinction between cis and trans isomers.

5. Molecular weight determination:
 The mass spectrum is a line spectrum, each line
corresponding to a positive ion of specific mass.
 In the case of a compound which has simply lost one
electron, the parent peak correspond to the exact molecular
weight of the compound.
 By a process known as peak matching, the molecular weight
of an ion can be determined to six decimal places on a
double focussing mass spectrometer. The peak
corresponding to the unknown mass, is matched with a
known flourocarbon peak in that mass region.

6. Accuracy of this order permits the absolute identification of
ions of identical rough mass.
 For example, the ions CO,+ N2
+, CH2N+, C2H4
+ each
correspond to a mass of 28.
 Mass measurement to six decimal places establishes the
identity and elemental composition of each ion.
CO - 27.994914
CH2N - 28.018723
N2 - 28.006154
C2H4 – 28.031299

7. Determination of molecular formula:
 Mass spectrum is a plot representing the m/e values of
various ions against their corresponding relative abundances.
 Consider a compound that forms peaks at m/e values of 100,
85, 71, 57, 43 etc. Clearly it is a straight chain alkane
because fragment peaks are formed 14 units apart. In case of
straight chain hydrocarbon , a peak due to C3H7
+ is most
abundant i.e., base peak. Thus a molecular formula of a
compound can be obtained.

8.  In case an organic compound gives fragment as well as
parent peaks in pairs which are two units apart, then
 If the pair of peaks are in the intensity ratio of 1:3, then it
must be a chloro compound.
 If the pair of peaks appear in the intense ratio of 1:1, then it
must be a bromo compound.

10. Structure determination:
 Each compound has a characteristic fragmentation pattern that
can be used to identify the compound.
 Since no 2 compounds can be ionised exactly in the same
manner, the mass spectrum brings a finger print for each
compound and is unique.
 From the parent peak and other major peaks, one may extract the
information regarding the nature of the fragment and the
structure of the original molecule.
 Molecular weight is one of the most useful parameter used in the
identification of a compound.

11. Relative abundance of isotopes:
 Another method of determining the element make-up of an
ion is by a consideration of isotope peaks.
 In a compound possessing only one carbon atom both 12C
and 13C occur in the ratio 98.982 to 1.108.
 Compounds with more than one carbon atom have a
correspondingly greater chance of possessing a 13 C
nucleus.

12.  A molecular ion having a single carbon atom affords a mass
peak(M) and (M+1) peak in the ratio approximately 99:1
 Important contributions to an M+1 peak are made by 13C,
2H, 15N and 33S, and to an M+2 peak by 18O, 34S, 37Cl and
81Br.
 The ratios of intensities of M+1 and M+2 peaks to the
parent peak correspond to specific elemental composition of
the molecular ion.

13. Detection of impurities:
 The mass spectrometer is able to detect as little as few ppm
of an impurity in a compound, particularly if the structure of
impurity is quite different from that of the main component.
 Ex: Detection of trace amounts of xylene in acetylacetone.
In the mass spectrum of the acetylacetone sample(M+=100)
extra peaks were observed at m/e 106, 105 and 91
suggesting the presence of higher mol.wt. impurity
(M+=106).

14.  Fragmentation of the acetylacetone molecular ion cannot
give rise to the 91 peak since this would involve the loss of 9
hydrogen atoms.
 The anomolous peaks may be explained by the presence of
xylene as a contaminant, the ion of which , through a loss of
proton, may rearrange to a tropylium ion or by loss of a
methyl fragment yields the 91 peak (M+ -15). The presence
of xylene was traced.

15. The presence of impurities in a sample may also be
established by lowering the intensity of the ionising
electron beam. If all peak heights do not decrease
proportionally then a contaminant is present.

16. Quantitative analysis:
 The method of determining quantitatively the components in
a mixture of similar compounds is known as the isotope
dilution method.
 For this the compounds to be assayed must be available in
isotopically labelled form ex: with 13 C, 15N, 131I. A known
amount of labelled compound of known purity is added to
the mixture to be estimated and a small quantity of sample
isolated; the isotope ratio of compound isolated is
determined by MS.

17.  From the ratio of labelled to non-labelled , taking in to
account and the purity of the labelled standard , the amount
of compound in the unknown mixture may be calculated..
 The technique has been widely applied in aminoacid studies.
 Drugs like diphenylhydantoin, caffiene and several
barbiturates and their metabolites have been measured in
picogram and nanogram range with the aid of stable isotope
labelled drugs.

18. Determination of bond dissosciation energies:
Bond dissosciation energies of molecules can be
determined by the appearance potential of a fragment ion.
The value of ionization potential can be determined
experimentally from ionisation-efficiency curves
AB + e- AB++ 2e-
AB+ A++B.
AB+ A.+B+

19.  The appearance potential of A+ for the step 2 can be
measured from the ionisation efficiency curve of the ion A+.
As A+ is only formed after the ionisation of AB, the
appearance potential is equal to the sum of the energies
required to dissosciate AB and to ionise the radical A.. Then
A(A+) = I (A.)+D (A-B)
where A(A+) = Appearance potential of A+
I(A.) = Ionisation potential of the radical A
D(A-B) = Bond dissosciation energy of AB

20. Identification of drug metabolites:
 Drug metabolites usually arise from relatively minor
structural modifications of the parent molecule.
 The most obvious method of identifying the metabolites is to
compare directly spectra of the pure drug with those of its
biotransformation products.
 Direct insertion of metabolites , after isolation by
preparative TLC, HPLC, GC in to the ion source of a
instrument, followed by accurate mass measurement of each
molecular ion , affords their molecular formula.

21.  Differences in molecular formula indicate the
transformation involved, and the fragmentation pattern of
a metabolite will often yield information concerning the
position of gain or loss of a molecular unit.
Ex: identification of metabolites of diazepam.
 Combined GC-MS is now a common technique for the
identification of organic compounds in admixture and in
high dilution

22. REFERENCES:
 PRACTICAL PHARMACEUTICAL CHEMISTRY ( 4TH
EDITION) BY A.H. BECKETT AND J.B.STENLAKE.
 ELEMENTARY ORGANIC SPECTROSCOPY BY
Y.R.SHARMA.
 INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS
BY GURDEEP. R. CHATWAAL