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This is to certify that the

thesis entitled

Use of Tandem Mass Spectrometry for Rationalizing

the Mass Spectrum of Estrone Methyl Ether
presented by

Linda Lee Bramble

has been accepted towards fulfillment
of the requirements for

M. S . degree in Ch emi 5 try

 

‘ a
Major

51427415 0&3ng

Date October 15. 1993

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES retum on or before date due.

DATE DUE DATE DUE DATE DUE |

it
MSU Is An Affirmative Action/Equal Opportunity Institution
cMMmS-nt

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

USE OF TANDEM MASS SPECTROMETRY FOR
RATIONALIZING THE MASS SPECTRUM OF ESTRONE
METHYL ETHER

By

Linda Lee Bramble

A Thesis

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Chemistry
1 993

 

ABSTRACT

USE OF TANDEM MASS SPECTROMETRY FOR
RATIONALIZING THE MASS SPECTRUM OF ESTRONE
METHYL ETHER

By
Linda Lee Bramble

The mass spectrum of estrone methyl ether was studied by tandem
mass spectrometry (MS/MS) and by analogy with model compounds.
The ions at m/z 134, 199, and 227 were selected for study. Spectral
data were also collected for ions at m/z 147, 171, 212, 214, and 242.
The methyl ether fimctionality on estrone methyl ether was deuterated
and product spectra of the deuterated compound were collected for
m/z 171, and 147, at m/z 174, and 150.

From rationalization of these spectra, and use of a model
compound, structures were proposed for several ions in the mass
spectrum.

The use of MS/MS, together with deuteration and the use of
model compounds, has been shown to provide greater insight for
understanding the fragmentation patterns in estrone methyl ether.
This should also apply to other complex molecules that can be studied
by EI mass spectrometry.

ACKNOWLEDGIVIENTS

I would like to thank first of all my advisor's, Dr. W. Reusch and
Dr. C. Enke, without their combined effort and mentoring this thesis
could not have been accomplished. Their individual contributions are
so numerous that it is difficult to state them in an acknowledgment.
However, I would like to say that they served not only as academic
advisors but as personal mentors for my professional and personal
growth. Working closely with men that I hold in such high esteem as
true scholars and gentlemen will affect my professional and personal
development for the rest of my life.

The members of both groups also deserve recognition. The
members of both groups helped to train me, both in use of the Triple
Quadrupole Mass Spectrometer in Dr. Enke's group and in organic
synthesis in Dr. Reusch's group. Both groups provided a place to
grow, a place where I could question as well as be questioned. They
helped me to keep things in perspective, not to take myself to seriously
and that I_could contribute to others growth.

I would also like to thank Dr. Albert DeVoogd. Without him I
never would have began this adventure. His love and support has
been and always will be a lighthouse for me in life's ocean. He will

always live in my heart.

iii

My husband, Michael has been a steady source of support and
encouragement. Only he and I can known how much he has
contributed to the completion of this thesis.

My children, Michael, Lori, Ricky, Shannon, and Danny have
cheered me on all the way. There were many personal sacrifices for
each of them, missed trips, dinners, sport events, concerts and times

when they just wanted Mom around.

iv

 

Table of Contents

........................................................................................................ Page
List of Tables .........................................................................................
List of Figures ........................................................................................
Chapter I ................................................................................................

Introduction ................................................................................ 1
Methodology .............................................................................. 3
Previous Studies of Estrone Methyl Ether ............................... 14
References ................................................................................ 30
Chapter H ...............................................................................................
6-Methoxy-1-Tetralone and 6-Methoxytetralin as Model Compounds
for Mass Spectrometric Behavior .............................................. 33
Conclusion ............................................................................... 47
Areas For Further Work ........................................................... 48
References ................................................................................ 52
Chapter IH ..............................................................................................
Studies of selected m/z values of estrone methyl ether ............ 53
Conclusion ............................................................................... 88
Areas For Further work ............................................................ 89
References ................................................................................ 91
Chapter IV ..............................................................................................
Experimental Methods ............................................................. 92
References ................................................................................ 98

List of Tables

Table ............................................................................................... Page
1 Precursor scans from defocused metastable ions ...................... 15
2 HRMS data on 6-methoxy estrone methyl ether ....................... 16
3 HRMS data on 6-methyl-1-tetralone ......................................... 38
4 Comparison of literature and experimental precursor data ....... 54

vi

List of Figures

Figure .............................................................................................. Page
1. Mass spectrum and Structure of Estrone Methyl Ether .................. 2
2. Magnetic analyzer ........................................................................... 6
3. Double focusing mass spectrometer ................................................ 8
4. Triple-quadrupole mass spectrometer ........................................... 11
5. Estrone .......................................................................................... 18
6. 18-norestrone methyl ether ........................................................... 18
7. Estradiol methyl ether ................................................................... 19
8. 18-nor-13-propylestrone ................................................................ 19
9. Proposed mechanism for formation of ion at m/z 199 ................... 21
10. Proposed mechanism for formation of ion at m/z 186 ................. 22
11. An alternative mechanism for formation of ion at m/z 186 .......... 23
12. Proposed structure for ion at m/z 256 from loss of ethylene ........ 23
13. Proposed structure for ion at m/z 256 from loss of

carbon monoxide ......................................................................... 23
14. Proposed mechanism for formation of ion at m/z 228 ................. 24
15. Proposed pathways for formation of ion at m/z 227 ..................... 26
16. Proposed mechanism for formation of ion at m/z 160 ................. 27
17. l-methyl estrone methyl ether ...................................................... 27
18. Proposed mechanism for formation of ion at m/z 173 ................. 28
19. Proposed mechanism for formation of ion at m/z 174 ................. 29
20. Structure 6—methoxy-1-tetralone .................................................. 34
21. Structure of 6-methoxy tetralin .................................................... 34
22. Mass spectrum of 6-methoxy-1-tetralone ..................................... 35

vii

23. Product spectrum of ion at m/z 148 in 6-methoxy—tetralone ........ 36
24. Sequential loss of 28 mass units from 6-methoxy-tetralone ......... 37
25. Product spectrum of 6-methoxy-tetralone, m/z 120 ..................... 39
26. Formation of ion at m/z 105 from m/z 120 in 6-methoxy-tetralone40
27. Product mass spectrum of ion atm/z 161 in 6-methoxy—tetralone 4O
28. Possible formation of an ion at m/z 161 from 6-methoxy-tetralone4l
29. Alternative formation of ion at m/z 161, and possible

fragmentation to ions at m/z 133 and 105. .................................. 42
30. Mass spectrum of 6-methoxytetralin ............................................ 43
31. RDA fragmentation for formation of ion at m/z 104 from

tetralln .......................................... 44
32. Two proposed processes for fragmentation of tetralin ................. 45
33. Structures from literature for m/z 104 .......................................... 45
34. Product spectrum of 6-methoxytetralin for ion at m/z 134 .......... 46
35- Mass Spectmm 0f 6-meth0xytetralin-d3 ....................................... 47
36. Reduction of ketone group in 6-methoxy-tetralone ...................... 49
37. Tricyclic intermediate structure ................................................... 49
38. Possible formation of tricyclic structure ...................................... 50
39. Mass spectrum of Michael adduct from 6-methoxy-tetralone ...... 51
40. Precursor spectrum of ion at m/z 199 .......................................... 55
41. Product spectrum for ion at m/z 199 ............................................ 56
42. Rationalization for formation of fragment ion at m/z 171 from

m/z 199 ........................................................................................ 57
43. Rationalization for formation of ion at 158 from m/z 199 ........... 58

44. Rationalization for formation of ion at m/z 184 from m/z 199 59

viii

45.
46.

47.
48.
49.
50.

51

52.
53.
54.
55.
56.
57.

58.
59.
60.

61.
62.
63.

64.
65.
66.

Precursor spectrum for ion at m/z 227 ......................................... 61

Rationalization for formation of ion at m/z 227 by a process

involving m/z 242 ....................................................................... 62
Product spectrum of ion at m/z 242 ............................................ 63
Rationalization for formation of ion at m/z 214 from m/z 242 64
Product spectrum for ion at m/z 227 ............................................ 64
Precursor spectrum for ion at m/z 147 ......................................... 65

. Product spectrum for ion at m/z 147 ........................................... 65
Precursor mass spectrum of m/z 171 ........................................... 66
Product mass spectrum of ion at m/z 171 .................................... 66
Rationalization for formation of ion at m/z 171 from 227 ........... 67

Rationalization for formation of ion at m/z 171 from m/z 186 68
Rationalization for formation of ion at m/z 147 from m/z 227 69
A possible rationalization for loss of a methyl group from

m/z 227 ...................................................................................... 70
Product spectrum of m/z 230, deuterated m/z 227 ...................... 71
Rationalization for formation of ion at m/z 212 from m/z 227 72

Rationalization for formation of ion at m/z 199 from the

proposed structures for m/z 227 ................................................. 73
A possible rationalization for ion at m/z 185 from m/z 227 ......... 74
Suggested structures for m/z 227 ................................................. 75
Rationalization for formation of ions at m/z 156 & 128 from

m/z 171 ....................................................................................... 76
Tetralin & l-methyltetralin .......................................................... 77
Structures from literature for an ion having m/z 104 ................... 77
Several of the possibilites for an ion having at m/z 134 ............... 78

ix

67.

68.

69.

70.
71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.
83.

Product mass spectrum for ion at m/z 134 from estrone methyl
ether .......................................................................................... 79

Product spectrum for ion at m/z 134 from 6-methoxytetralin

at 1mtorr collision pressure ........................................................ 8O
Suggested structure for ion at m/z 134 from estrone methyl

ether and likely fragmentations ............................................... 81
Possible formation of ion at m/z 78 from 6—methoxytetralin ........ 82
Suggested structures for ion at m/z 134 from (a) estrone

methyl ether & (b) 6-methoxytetralin ..... . .................................. 83
Product mass spectrum of the CD3- analog for ion having

m/z 134 from estrone methyl ether ............................................ 83
Product mass spectrum of the CD3- analog for ion having m/z

134 from 6-methoxytetralin ..................................................... 84
Suggested structure for m/z 119 from loss of ether methyl in m/z

134 of 6-methoxytetralin ............................................................ 85

Fragmentation process to rationalize the ion at m/z 104 from
m/z 134 in 6-methoxytetralin, and m/z 105 in the CD3- analog 85

Rationalization for a process to from an ion at m/z 91 from

rn/z 134 in 6-methoxytetralin ..................................................... 86
A suggested rationalization for formation of an ion at m/z 78

from the suggested ion at m/z 134 in 6-methoxytetralin ............ 87
Three synthetic steroid structures to add to study ........................ 90
Mass spectrum of estrone ............................................................. 93
Mass spectrum of estrone methyl ether ........................................ 94
Mass spectrum of estrone methyl ether—d3 ................................... 94
Mass spectrum of 6-Hydroxy-tetralin .......................................... 95
Mass spectrum of 6-methoxy-tetralin-d3 ..................................... 95

X

84. Mass spectrum of Michael adduct ............................................... 97

xi

Chapter 1.

Introduction

Mass spectrometry has traditionally been used by organic
chemists as a tool to confirm the identity of a compound. Mass
spectral data is usually combined with NMR and IR data to determine
a structure. Because of the complexity of a mass spectrum, its use is
often limited to the identification of the molecular ion along with only
a few fragmentation patterns, such as loss of water from alcohols at M-

18, or the presence of a tropylium ion at mass-charge (m/z ) 91.

The molecular ion of a pure compound, if present in an E1
spectrum, is found at the highest m/z value in the spectrum with its
isotope peak(s). The molecular weight of the compound under
investigation is determined on the basis this m/z value, and is assumed
to be the molecular weight after electron ionization, which leaves the
compound as a radical cation. The molecular ion, in figure 1, is
shown as the m/z peak at 284 mass units which is the molecular
weight of estrone methyl ether. This spectrum illustrates a typical EI
spectrum of a complex structure. Other than a few major peaks at m/z
160, 186, and 199, the spectrum is composed of a lot of minor
fragmentations, the source of which can be difficult to determine.

 

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50 100 150 290 250

Figure 1. Mass Spectrum and Structure of Estrone Methyl Ether

With the availability of tandem mass spectrometry came an
added dimension of structural information. In tandem mass
spectrometry an ion in the mass spectrum can be isolated and selected
for study. A product scan can be collected on this ion which will give
a mass spectrum of all the fragments produced from this particular ion.
A precursor scan can also be collected which will give all the
precursors of the ion being studied. This allows unambiguous

identification of the origin of particular fragment ions. Knowing

which part of a molecule a particular fragment came from significantly

aids structure determination.

This research involves the use of tandem mass spectrometry,
using a triple quadrupole mass spectrometer (TQMS), to rationalize

the mass spectrum of estrone methyl ether (Figure 1).

Past investigations of this compound have involved the use of
regular mass spectrometry, high resolution mass spectrometry
(HRMS), and an EB double focusing technique called defocused
metastable mass spectrometry. The combined past investigations on
estrone methyl ether provide a solid foundation on which to test the
hypothesis that TQMS will reveal more information about the mass
spectrum of a complex structure than could be determined before this

methodology was available.
Methodology

Interpretation of a mass spectrum is first approached by use of
empirical rules such as Stevenson's rule, or loss of the largest alkyl
group as a radical. These rules can be found in many text books. (1,2)
Problems arise when there are mass values which can not be assigned
by use of the known rules. These must then be rationalized by use of
isotope labeling studies, model compounds or exact mass

measurements.

Information from labeling studies is obtained by comparing the
mass spectrum of the unlabeled compound with that of the labeled
compound. If deuterium is used as the label, any ion fragment that
contains deuterium in place of hydrogen will increase its mass by 1
mass unit for each deuterium.

Model compounds have contributed to mass spectrometry in a
variety of ways. Djerassi used the rigid steroid carbon framework as a
template on which selected functional groups were positioned to
determine their influences on the mass spectrum. Dass and Gross (3)
studied the relative stabilities of various acyclic and three, four, five
and six membered isomeric ions by examining C10H12, ions

produced from a carefully chosen list of compounds.

Exact mass measurements using HRMS give the mass values of
ions to six significant figures. Since the mass of isotopes are not
nominal, the composition of the ions can be distinguished by their
HRMS. For instance, CO, C2H4, N2, and CHZN all have a nominal
m/z value of 28 amu. However, in HRMS they are found to be
27.9949, 28.0313, 28.0062, and 28.0187 respectively.

Djerassi's discussion of the fragment ion at m/z 199 in estrone
methyl ether is a good example of how this information can be used.
(4,5) He combined labeling and modeling to determine that the
angular substituent is not present in this ion, and that carbon number
16 is still attached. Thus, when labeled compounds, 16,16-d2-estrone

 

methyl ether and the 16,16-difluro analog were studied, the peak at
m/z 199 shified to 201 and 235 respectively. This confirmed that the
carbon at position 16 was present in the m/z 199 ion. The fact that the
ion at m/z 199 is present in all of estrone methyl ether's stereoisomers
as well as in the 18-norestrone methyl ether, and is shifted to m/z 185
in the spectrum of 18-nor—13-propyl estrone due to the absence of the
methyl ether functionality, led Djerassi to conclude that the angular
methyl substitutent is not present in this ion. HRMS data were
collected giving C14H150 as the empirical formula for the ion at m/z
199. From this combined information Djerassi proposed a possible
structure and fragmentation pattern for the m/z 199 ion which will be
discussed later.

In an effort to gain more understanding about ion fragmentations
in mass spectrometry, and thereby aid in the interpretation of mass

spectra, new instrumental techniques have been developed.

The first spectrometers originally developed by Dempster used
magnetic analyzers. These were single-focusing instruments in which
positive ions were deflected by a magnetic field. Mass separation was
accomplished on the basis of momentum (p = mv), where m is the
mass of the ion and v the velocity after acceleration. Heavier ions
were diverted into curved paths with a larger radius, and they traveled
slower than lighter ions, which followed a path with a smaller radius

and traveled at faster speeds (Figure 2).

  
 
 

( burn"!
plate

 

Source slit *

From ion
chamber

Figure 2. Magnetic Analyzer (6)

When an ion having a charge ze is accelerated through a voltage
V it acquires a kinetic energy of zeV, where e is the charge of an
electron and z is the number of these charges. Since, kinetic energy is

(1/2)mv2, where m is the mass of the ion, it follows that:

(1/2)mv2 = zeV. (l-a)

 

 

In a magnetic field of strength B, an ion will experience a force
of BzeV. This force will produce an acceleration of r2/r toward the
center of a circular path of radius r. So, from Newtons second law, we

get:
Bzev = mv2/r. (l-b)
If we combine equations (l-a) and (l-b) we get:
m/z = B2r2e/2V. (1 -c)

From (l-c) it follows that at any given magnetic field strength
and accelerating voltage, ions of a given m/z value will follow a
particular path of radius r. These ions at various m/z values can then
be progressively transmitted through the magnetic field, either by
varying B at constant V (magnetic scanning) or by varying V at

constant B (electric or voltage scanning).

With magnetic sector instruments the resolving power is limited
by the spread of translational energies of the ions leaving the ion
source. It was discovered that this problem could be overcome by
filtering the ions through an electric field located before the magnetic
field. By placing a slit between the electric and magnetic sectors, ions
of a closely defined kinetic energy can be selected before mass

analysis. Instruments designed in this way are called double focusing

 

mass spectrometers (Figure 3). These instruments gave much better
resolution than the single focusing instruments.

   

    
 

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1. Source 4. 2nd field free region
2. 1St field free region 5. Magnetic sector
3. Electric sector 6. 3rd field free region

Figure 3. Double Focusing Mass Spectrometer (7)

 

Usually ion fragmentations occur in the ion source, but if an ion
is stable enough to leave the ion source, but not stable enough to make
it to the detector before fragmentation, it is termed a metastable ion. It
was found that these double focusing instruments could be used to
detect and analyze metastable ion decompositions without interference
from the ions in the conventional mass spectrum, by a technique called
metastable defocusing. The mass spectrometer is set so the main ion
beam that would produce the normal mass spectrum is defocused and
therefore not detected. Instead, the ion beam for the decomposition
products from the chosen metastable ion is brought into focus and
detected. A single accelerating voltage scan identifies all precursors
for each fragment ion that is analyzed. For a metastable ion to be
observed it must fragment in a field-free region. The instrument
described above detects ions formed in the first field-free region
between the source and the electric sector, (See figure 3). These ions
are usually observed as low intensity, diffuse peaks. Since metastable
ions generally have low intensity, a precursor or product ion must have
a fairly abundant peak in the mass spectrum for the metastable
transition to be observed. As the mass spectrum becomes more
complex, as they typically are for steroids, the metastable ion peaks
overlap with peaks from normal fragmentations and assignments
become difficult. These were the first MS/MS instruments. They
were termed EB geometry or normal geometry instruments. There
have been many improvements in instrumentation since these early
models.

10

The development of a TQMS by Yost and Enke (9,10) was
accomplished in the late 1970's as a method to perform MS/MS with
the simpler quadrupole mass filter. This instrument contains three sets
of quadrupoles, Q1, Q2, and Q3. In general, a mass-charge value
(m/z) is selected by Q1, and passed into Q2 where the fragments are
formed by collision induced dissociation (CID); the subsequent m/z
values are determined by Q3. Both the m/z values selected in Q1 and
Q3 are determined to 1 amu. This was a major motivation in the
development of a triple quadrupole system. The resolution achieved
by the double focusing instruments, which were the standard for
MS/MS at that time, could not achieve unit resolution for both

precursor ion selection and product ion analysis.

The first and third quadrupoles are used as mass filters (11) by
applying a combination of direct-current (DC) and radio-frequency
(RF) potentials to them. The second quadrupole, passes all ions
without any mass filtering. Only RF potentials are applied to this set
of quadrupoles. This RF potential is used to focus ions that were
scattered by collisions, according to theoretical analysis by Dawson
and Miller. (12,13) A neutral or reactive gas can be introduced into
this chamber during MS/MS operation.

The TQMS used in this study is the Finnigan 700 model (Figure
4). This model has a curved collision chamber, which reduces
chemical noise because neutral species are not transmitted through the
curved quadrupoles. In fact, this model is not actually a TQMS

 

11

because the collision chamber is an octapole, not a quadrupole. The
octapole imparts less angular energy to the ions than a quadrupole.
This means that the energy in the collision chamber more closely
represents the energy delivered to the chamber by the collision gas. It
is also believed to increase transmission efficiency. (14)

conversion
dynode (£2010!)

  

 

 

 

 

lFABl CII £1 electron
I multiplier
. Q1 ; J l_ 1
sample «“F‘r I . ‘
. ca product ion detector

man analyzer

Figure 4. Triple-Quadrupole Mass Spectrometer

The inlet system allows introduction of the sample into the ion
source. There are several different types of inlet systems available to

 

12

the TQMS. These include a direct insertion probe, a GC interface, and
a continous flow bulb inlet system. To use the probe 3 sample is
applied in a small crucible which is inserted into the tip of the probe.
The probe is then introduced thorough a vacuum lock directly into the
ion source. The sample is heated by electrical elements in the probe,
according to programmed instructions. A sample size of a few

micrograms or less is required.

The ion source is the region of the mass spectrometer where ions
are generated. The energy transferred to the vaporized molecules by
the electrons from the filament is usually greater than the ionization
energy. Excess energy remains in the molecular ion, and may serve to
break bonds. Depending on its energy and structure the molecular ion
can remain intact or dissociate. In general, a molecular ion my
dissociate in one of two ways:

(a) M+ >A+ + B

 

(b) M+ >C+ + D

 

The dissociation pathway indicated by (a) produces an odd

electron ion and pathway (b) produces an even electron ion.

Once the sample is in the ion source there are several different
ionization methods available. This instrument can be set up to

perform electron impact (EI), chemical ionization (C1), or fast atom

 

13

bombardment (FAB), for ionization of the sample. The instrument can
scan for positive or negative ions.

After the sample is ionized, there are several scan modes
available. There is, of course, the regular mass spectrum, but also a
precursor scan, product scan, and a neutral loss scan is available to the

user.

A product scan produces a mass spectrum of all the fragment
ions formed from a selected precursor ion. The product scan is
performed by selecting a rf/dc voltage ratio for a specific mass-to-
charge ratio in the first quadrupole. These ions are then passed into
the collision chamber, where collisional dissociation of the selected
ions occurs. The third set of quadrupoles is then scanned, and the
mass spectrum is displayed.

The precursor scan produces a mass spectrum of all the
precursor ions that dissociate to form a particular product ion. This is
accomplished by scanning the first quadrupole, while the third
quadrupole is held constant at a rf/dc voltage ratio for the desired
mass-to-charge value.

A neutral loss spectrum contains all of the parent ion masses that
dissociate to lose a chosen neutral mass. This is accomplished by
scanning the first and third quadrupoles concurrently, but with a mass

difference equal to the selected neutral loss. For example if scanning

14

for a neutral loss of 42, the first quadrupole would be set 42 mass units
higher than the third set of quadrupoles.

Previous Studies of Estrone Methyl Ether

The work done by Djerassi et. a1. twenty years ago laid a solid
foundation for the present project. Djerassi's group studied the mass
spectrum of estrone methyl ether using positive EI mass spectrometry,
high resolution mass spectrometry (HRMS), MS/MS on a EB double
focusing instrument, and deuterium labeling.

In an attempt to understand the fragmentation process and to aid
in determining the structures of ions in the mass spectrum, Djerassi
collected precursor scans on selected ions of estrone methyl ether.
This was done using the EB double focusing instrument described
previously. He collected precursors of ions at m/z 227, 228, 199 and
160. (4) The data are presented in table 1. The precursors are listed in
columns under the ion being studied, which is in bold type at the top
of each column. HRMS data were also reported by Djerassi et al. (4)
These data were also collected by peak matching, using a double
focusing instrument. The data are presented in table 2, and were used
together with other information collected by labeling and analogy with
other steroids to develop the rationalizations for the fragmentation

patterns presented later in this chapter.

 

15

PRECURSOR SCANS FROM DEFOCUSED METASTABLE ION

MASS SPECTROMETRY Table 1. Precursor Scans

 

 

 

 

 

 

 

 

 

191.1. or £7 L110. £2
Precursors 256 242 228 228
Precursors 284 256 214 284
Precursors 284 256 241
Precursors 242 256
Precursors 268 214
Precursors 188

Precursors 175

 

 

 

 

 

 

16

 

 

 

 

 

 

HRMS DA TA
(284); C19H24°2 (241); C17325° (199); C14H15°
(268); C18H200 (228); C16H200 (188); c13nl4o
(256); C18H240 (227); C16H19O (175): clznlso
(242); C17H220 (214); C15H180 (160); C11H22°

 

 

 

Table 2. HRMS data (m/z); reported empirical formula (4.)

Djerassi labeled several sites on estrone methyl ether in order to
study the fragmentation processes in its mass spectrum. He prepared a
16,16-d2; a 6,7—d2, x (where x is on the aromatic ring); a 6,6,7-d3; a
6,6,9-d3; and a 15-d analog.(5.)

From these studies he showed that:
1. m/z 256 (M-28) is represented by two structures;

a. a loss of C-6 and C-7, as ethylene,

 

17

b. a loss of CO from the D ring.

2. m/z 228 and m/z 227 involved loss of hydrogens from carbons
15, and 16.

3. m/z 186 consisted of two different ions.

4. m/z 199 is represented by at least two different structures;

a. the major one incorporates both C-16 hydrogens, with
one of the C-15 hydrogens being transferred to the
neutral fragment,

b. the other(s) do not contain either of the C-1 6 hydrogens.

5. m/z 97 incorporates the C-16 hydrogen atoms.

Djerassi also drew analogies from other spectra in order to
rationalize the spectrum of estrone methyl ether. (4,5,15,&16) These
will be mentioned briefly to complete the literature review, and to
define conclusions that have been applied to the analysis of the estrone
methyl ether mass spectrum.

Djerassi compared the mass spectra of estrone, 18-norestrone
methyl ether, and estrone methyl ether. These all contained a M-lS
peak in their spectra. Estrone (Figure 5) lacks a methyl ether and 18-
norestrone methyl ether (Figure 6) lacks an angular methyl group,

18

whereas estrone methyl ether contains both of these groups. Since he
assumed that the loss of 15 from the molecular ion was a CH3 group
that came from one of these two positions, the data led to the
conclusion that both positions contributed to the M-15 peak in estrone

methyl ether.

    

H30 0

Figure 5. Estrone Figure 6. 18-norestrone methyl ether

Djerassi also compared the mass spectra of estrone methyl ether,
16,16-d2 and 15-d estrone methyl ether with estradiol methyl ether
(Figure 7) and its 16,16-d2 and 16,16,17-d3 analogs. A peak at M-57
is observed for all of these compounds, and in the deuterated
compounds there were no mass shifts, indicating that the ion
constituting the M-57 peak didn't contain carbons 15, 16 or 17. This
led to his-conclusion that the ion at m/z 227 in estrone methyl ether

was generated by a loss of ring D.

19

  

H30 0

Figure 7. Estradiol Methyl Ether

The spectra of 18-norestrone methyl ether, estrone methyl ether,
and 18-nor-13-propylestrone (Figure 8) were compared and found to
be very similar, except that the intensities of the M-56 and M-57 peaks
are reversed in the 18-norestrone methyl ether compared to the
spectrum of estrone methyl ether. The presence of a M-57 peak in 18-
norestrone methyl ether demonstrates that transfer of one of the
hydrogens from the angular methyl group to the neutral fragment is
not the only pathway to M-57 . Hydrogens at C-12 and C-8 were
suggested as other alternative transfer species.

 

Figure 8. 18-nor-13-propylestrone

20

A peak at m/z 199 is present in 18-norestrone methyl ether, and
is shifted to m/z 185 in 18-nor-l 3-propylestrone due to the lack of a
methyl ether group. In 16,16-d2 estrone methyl ether, the peak moved
mostly to m/z 201, with a small amount of the ion current remaining at
m/z 199. From this evidence Djerassi concluded that the angular alkyl
group is not present in the m/z 199 ion, but that C-16 remains attached
in the main pathway for its formation.

The essential mechanism proposed by Djerassi (4) for the
formation of the m/z 199 ion is reproduced in figure 9. Djerassi
proposed that all principal peaks above m/z 160 contain rings A and B
intact. He therefore, concluded that m/z 199 contains rings A and B
plus 3 more carbons. One of these carbons he assumed to be 016
from the previously mentioned labeling evidence. The mechanism
involves fragmentation of the 9-11, 13-14, and 16-17 bonds with two
hydrogen transfers. Transfer of the C-15 hydrogen has been verified
by Djerassi using the labeled C-15 analog, the hydrogen transfer from
C-8 has not been verified. Djerassi suggests that there may be
alternative pathways, but he does not present any evidence. (4) The
precursor scan does show several minor precursors, but the molecular

ion is definitely the main precursor.

 

21

 

Figure 9. Proposed mechanism for formation of ion at m/z 199.
(4.5.8.16)

When the mass spectrum of the 15-d analog was obtained the
ions represented by m/z 186 were now distributed between two peaks
of equal intensity-one at m/z 186 and one at m/z 187. This indicates
that there are two distinct ions having m/z 186 in the normal mass
spectrum. One of these retained the isotopic label at carbon number

22

15 and one didn't. Since Djerassi assumed that all ions above m/z 160
contain both rings A and B, he concluded that the other ion
incorporated carbons 11 and 12. Djerassi states that these

mechanisms are tentative (Figures 10 & 11 ). (5)

 

Figure 10. Proposed mechanism for formation of an ion at m/z 186.
(4,538,16)

23

 

Figure 11. An alternative mechanism for formation of an ion at
m/z 186. (4,5,8,16)

According to high resolution measurements, there are two
distinct ions compositions for m/z 256. (8) One of these constitutes
90% of the ion current and arises from loss of C-6 and C-7 as ethylene.
The postulated structure is shown in figure 12. Djerassi believed that
this ion was not involved in the formation of m/z 227 and 228 (8).

The precursor for these ions was suggested to be that shown in figure

 

Figure 12. m/z 256 Figure 13. m/z 256

24

The concerted process generating m/z 228 from estrone methyl other is
shown in figure 14. This was supported by data from 16,16-d2-
estrone methyl ether, 16,16-difluroestrone methyl ether and the 15-d
estrone methyl ether, all of which contained a M-56 peak. The
possibility of this ion being formed by loss of C-6 and C-7 was
therefore eliminated. This ion can also be formed by loss of CO to
form m/z 256, followed by loss of C-16 and presumably C-15, plus the
required number of hydrogens.

   

H3C ¢

Figure 14. Proposed mechanism for formation of ion at m/z 228.
(4,5,8,16)

The peak at m/z 227 requires a more thorough explanation. It is
an even electron ion that involves a hydrogen transfer. The source of

this hydrogen isn't known, but the three most likely mechanisms are

presented in figure 15. The fact that m/z 227 has lost the D ring is
verified by the fact that a peak at m/z 227 is also present in 16,16-d2-
estrone methyl ether, 16,16-difluroestrone methyl ether, 15-d-estrone
methyl ether, estradiol methyl ether, and its 16,16-d2 and 16,16,17-d3
analogs.

The m/z 227 ion is also formed from a m/z 242 ion. Formation
of m/z 242 is believed to be a concerted process from the molecular
ion, with loss of C-16 and 017 as ketene. This is then followed by
loss of a methyl radical to form m/z 227. This methyl loss is believed
to involve the C-15 methylene and a hydrogen, transferred from
another position.

A proposed pathway for the formation of m/z 160 is reproduced in
figure 16. This concerted mechanism is believed to be the main
source of this ion. That this ion is generated by loss of rings C and D
is shown by its appearance in the labeled 16,16-d2 estrone methyl
other mass spectrum. The fact that it is shifted to m/z 163 in the 6,6,9-
d3 analog and to m/z 174 in 1-methyl estrone methyl ether (Figure 17 )
(4) is further verification.

26

"\f‘ H3
“fig“ 311% ..
w *3
o. oo \
oo

H30

1 .
“ T Q.
H300

 

 

Figure 15. Proposed pathways for formation of the ion at m/z
227.(4,5,8,16)

27

  

H3C O

‘1
—' 00

Figure 16. Proposed mechanism for formation of ion at m/z
160.(4,5,8,16)

  

H3C 0
Figure 17. l-methyl-estrone methyl ether
Proposed pathways for the formation of ions at m/z 173 and 174

have not been verified, but the mechansims shown below make sense

in light of the previous discussions (Figures 18 & 19).

 

0CH2

0‘
H300

Figure 18. Proposed mechanism for formation of ion at m/z 17 3.
(4,5)

29

 

Figure 19. Proposed mechanism for formation of ion at m/z 174.

(5)

It is clear from the previous discussion that a lot of work has
already been carried out on estrone methyl ether and related
compounds. As noted earlier, there are still questions remaining about
this work and the mechanisms that were proposed. For instance, as
Djerassi pointed out in the formation of the ion having m/z 227, the
source of the hydrogen transfer is not known. Also, when the
molecular ion fragments to form a m/z 242 fragment ion with
subsequent loss of 15 amu to give a m/z 227 fragment ion, where does
the hydrogen come from for the loss of CH3 ? Furthermore, what is
the structure of m/z 227 ? Instrumental limitations at the time that this
work was done were probably the major factor for the lack of these

results. Much of the work, although expertly done, is inferential.

30

References

1. Silverstein, R. M.; Bassler, G. C.; Morril, T. C., Spectrometric
Identification of Organic Compounds, 5 th ed., John Wiley &

Sons,

2. Mc Lafferty, F. W., Interpretation of Mass Spectra,
University Science Books, 1980.

3. Dass, C.; Gross, M., Org. Mass Spectrom. 20, 35, 1985.

4. Djerassi, C.; Smith, D. H.; Duffield, A. M., Org. Mass
Spectrom., 7, 367, 1972.

5. Djerassi, C.; Wilson, J. M.; Budzikiewic, H.; Chamberlin, J.
W.,Chem. Soc. 84, 4544, 1962.

6. Howe, 1.; Williams, D. H.; Bowen, R. D., Mass Spectrom.
Principals and Applications, 2nd ed., 1981.

31

7. Milne, G., Mass Spectrometry: Techniques and Applications,
Wiley-Interscience, p. 421,197 1.

8. Zaretskii, Z. V., Mass Spectrom. of Steroids, John Wiley &
Sons, 1976.

9. Yost, R. A.;Enke, C. G., J. Am. Chem. Soc. 1978, 100,2274.

10. Yost, R. A.; Enke, C. G.; Anal. Chem. 1979, 51, 1251A.

11. Miller, P.E.; Denton, B. M. J. Chem. Educ. 1986, 63, 517.

12. Miller, P. E.; Denton, M. 3., Int. J. Mass Spectrom. Ion
Processes 1986, 72, 223.

13. Dawson, P. H.; Fulford, J .E., Int. J. Mass Spectrom. Ion
Processes 1982, 42, 195.

14. Syka, J. E. P.; Schoen, A. E., Int. J. Mass Spectrom. Ion
Processes 1990, 96, 97.

15. Smith, D. H.; Djerassi, C.; Buchanan, B. G.; White, W. C.;
Feigenbaum, E.A.; Lederberg, J ., Tetrahedron, 29, 3117,
1973.

32

16. Budzikiewic, H.; Djerassi, C.; Williams, A. E., Structure
Elucidation of Natural Products by Mass Spectrometry Vol.
2.,p. 50-62, 1964.

Chapter II.

6-Methoxy-l-Tetralone and 6-Methoxy-Tetralin
as Model Compounds for Mass Spectrometric
Behavior

It was decided to test the use of two compounds simpler in
structure than estrone methyl ether as model compounds. A
compound used as a model in mass spectrometry should be similar
enough structurally to reflect the compound it is modeling. The model
compound should fragment by the same or similar processes that the
compound that it is modeling follows, and give equivalent fragments.
By use of a simple model compound, pieces of the fiagmentation
processes should become clearer. By using model compounds of
increasing complexity, the interactive fragmentations of complex
molecules should become apparent.

The compounds chosen as model compounds were 6-methoxy-1-
tetralone (Figure 20), and 6-methoxy-tetralin (Figure 21). They were
chosen because they have the same first two rings as estrone methyl
ether, with a methoxy group in the correct position. I believed that the

34

6-methoxy-tetralin would more closely reflect the mass spectrum of
estrone methyl ether because the 6-methoxy-tetralone had a carbonyl
group at carbon number 1. This functional group is known to induce
cleavage on either side of the ketone because of the ease of removal of
a nonbonding electron. However, I believed that it would be worth
observing the effect of the carbonyl group. A comparison could then

be made of the two different processes.

   

Figure 20. 6-methoxy-1-tetralone Figure 21. 6-methoxy-tetralin

The mass spectrum of 6-methoxy-1-tetralone (Figure 22) had a
base peak at m/z 148 and the next largest at m/z 120. This indicated
sequential losses of 28 mass units. Carbon monoxide and ethylene,
both being 28 mass units, were assumed to be the neutral losses. I
suspected that ethylene was lost first because of the ease of cleavage at
the ketone and oxygen's ability to stabilize the positive charge, but it
wasn't known how the resulting fragment would decompose.

1355

 

 

 

 

 

 

 

r001 rc0.0 coo.
Ft.”
001
rv:.0
cow
.01
12%.0
3.1 71.0 00.0
31.0
«‘30 I 69,0 0010 101.0 Jl 161.0
103.1_ 113.9 .0 x .0 1
L,_1_L.III+I_JL._ 1.. 1*” P11 1L
fr *TfTrTV
c0 00 on too v0. ‘40 :00 105

Figure 22. Mass spectrum of 6-methoxy—tetralone.

A product spectrum was collected for the ion at m/z 148 (Figure
23). The loss of 28 amu from the m/z 148 precursor confirms that
there is a mechanism to produce m/z 120 that involves sequential

losses of 28 amu each.

36

f0.”

01
NH

40"

201

 

 

d

 

 

10‘.’ 13
77..2 USA P.’ _ 131.,
(f.1 51 2 ‘5.) P‘SI .Q.’ 9".) ”.0 137v,
F '1 AJ—TJ l; - f A fl

« 00 00 100 1}. - 3‘0 1

 

 

Figure 23. Product spectrum of ion at m/z 148 in 6-methoxy-

tetralone.

A HRMS was then obtained (Table 3), which showed that the
peak at m/z 148 resulted from loss of ethylene, followed by loss of
carbon monoxide to give the peak at m/z 120. The proposed
mechanism is shown in figure 24. Several possible structures can be
drawn for the ion at m/z 120. The cyclopropane ring attached to the
six member aromatic ring would be highly strained, leaving the other
two structures more favored. A statement of which structure is more
correct cannot be made. The ion at m/z 120 could have any one of

these or an entirely different structure.

37

 

   

m/Z 120

Figure 24. Sequential loss of 28 mass‘units from 6-methoxy-
tetralone.

38

HRMS DATA

6-METHOXY TETRALONE

m/z values formula
176.0861 C11H1202
161.0589 C10H902
148.0535 C9H802
120.0569 C8H80
115.0169 ‘ C8H3O
1 15.0604 C9H7
105.0565 C8H9
105.0329 C7HsO

Table 3. HRMS data of 6-methoxy-tetralone

The product spectrum for m/z 120 was then collected to
complete this set of data (Figure 25).

39

 

 

 

 

 

 

 

10°" 91.1 P..‘
«.0
091
00-1 ”‘4
«0-1
904‘ 105.0

20-1

31.9 si.0 01.0 Paras 7’-.° 01.; 0* 33.2 37.0 1.35.01124

L l 1 1 l I
'Y"' vvvvvjvvvvw ,v v- 31 v‘fiV: . . Tev- v . ‘--VVTVCC-

:0 00 70 u n - ,, I - 100 no 120

Figure 25. Product spectrum of 6-methoxy—tetralone, m/z 120.

The product spectrum of m/z 120 showed a loss of 15 mass units
to form an even electron ion at m/z 105. Although HRMS
measunnent of m/z 105 in the original spectrum of 6-methoxy
tetralone showed it to be both C7H50 and C8H9 (See table 3); the
product ion from m/z 120 is most likely C7H50 alone. A possible
methyl loss, accounting for this fragmentation is shown in figure 26.
Whether this rationalization is plausible might be determined
experimentally by labeling the ether methyl group with deuterium and
observing if the peak at m/z 105 shifted.

4O

' CH3
H3C H 2 + H2
m/z 120 m/z 105

Figure 26. Formation of ion at m/z 105 from m/z 120 in 6-

methoxy-tetralone.

A product spectrum was also collected for m/z 161 (Figure 27),

as it was suspected that this peak might represent a separate and minor

 

 

 

 

fragmentation pathway.
100-1 ML? to”
{1.70
00'
60‘
00"
2°— 133.1
10s.: 1r0.9 120.0
55.1 6.3 7.1 .12 9 . . . -
J," ii I :1'r”°l‘l’° .1-.l.‘l‘“‘j‘
. “ 00 1‘0 12r0 T 110 100

Figure 27 . Product mass spectrum of ion at m/z 161 in 6-

methoxy tetralone.

41

It is noteworthy that ions at m/z 148 and 120 were not present in
this spectrum. A peak at m/z 105 was also present in this mass
spectrum. Data collected by HRMS indicated that there were two
different masses for the peak at m/z 105, (See table 3). Many of the
ions represented in the spectrum of 161 are not in the product mass
spectrum of m/z 148. This is indicative of a separate fragmentation
process. A possible rationalization for the formation of this ion at m/z
161 that fits the HRMS data is shown in figure 28.

 

   

H3C 0

m/z161

Figure 28. Possible formation of an ion at m/z 161 from 6-
methoxy tetralone.

42

An alternative pathway for formation of the ion at m/z 161 is
loss of the methyl ether group as a methyl radical. The two largest
peaks in the spectrum of m/z 161 are at m/z 133 and m/z 105.
Possible rationalizations for the formation of ions at m/z 133 and m/z
105 are presented from two resonance forms of a possible structure for
m/z 161 (Figure 29). This process could be tested experimentally by
again exchanging the ether methyl hydrogens with deuterium and
obtaining a mass spectrum, or by collecting a product mass spectrum
ofthe ions at m/z 161, 133, and 105.

   

—-’ <—>
m/z161
(a)
/0
from a
" CZH4 9 — CO C7H5O
mlz 133 mlz 105
i
from b _ .
\ c.
- CO C H
——> / 8 9
m Iz 133 mlz105

Figure 29. Alternative formation of ion at m/z 161, and possible
fragmentation to ions at m/z 133 and 105.

423

The other model compound studied was 6-methoxy-tetralin. In
the mass spectrum of 6-methoxytetralin (Figure 30) the peak at m/z
134, representing a loss of 28 mass units from the molecular ion, is
approximately 80% of the base peak. No other mass values come
close to this abundance.

40‘

40‘

40"

704 "

 

! 13 5 cs 77 18‘ 119 1 136 [A ,
11‘ 132.151] 1:811" Jr: 311' r 1113: -111 11,. P; 11 ill.

«0 00 00 100 120 ‘00 1“

 

 

 

 

 

 

 

f

Figure 30. Mass spectrum of 6-methoxytetralin.

Tetralin, which is the parent hydrocarbon, is known to fragment
by a retro-Diels Alder (RDA) process to give a m/z 104 ion (figure
31).(1)

44

Figure 31. RDA fragment for formation of ion at m/z 104 from

tetralin.

In the case of the 6-methoxy compound, the RDA process gives
an ion having m/z 134. The specific structure of the ion at m/z 134
(mlz 104) is not clearly proven.

A study of isot0pically labeled tetralin was conducted by Stolze
and Budzikiewicz. (2) These authors found that there are two
fragmentation processes, plus a small proportion of complete carbon
scrambling (9-12%). The two processes are a RDA, which is a high
energy process involving the loss of carbons 2 + 3 as ethylene, and a
lower energy process in which a complex rearrangement results in the
loss of carbons 1 +2 (= 3 + 4) as ethylene (Figure 32).

45

l RDA (2+3)

(1 +2) or (3+4)

Figure 32. Two proposed process for fragmentation of tetralin.
A paper by Kuhner & Hesse (3) also reported that tetralin could
fragment in several different ways to give an ion at m/z 104 with the

loss of ethylene. There are three possible structures for this ion listed
in the literature (Figure 33). It is

Figure 33. Structures from literature for m/z 104.

unlikely that the m/z 104 ion has a styrene structure, because the
spectrum of styrene has a major peak at m/z 77 (107 if methoxy is

 

46

attached), and there is only a very minor peak at m/z 77 in the

spectrum of tetralin. No equivalent peak at m/z 107 is found in 6-
methoxytetralin's product spectrum for the ion at m/z 134 (Figure 34).
It is known that the methoxy group is retained in this fragment

because the deuterated compound was prepared, and the mass
spectrum (Figure 35) showed that the peak at m/z 134 was shifted to
m/z 137. This ion could be represented by either of the remaining two

structures. The exact structure has not yet been determined.

".1
.01 . 7.11

103.7
601

“.04

 

2.5 l

 

 

 

119.1

 

112.9

134.1

 

 

'UOOI
0.13

 

Figure 34. Product spectrum of 6-methoxytetralin for ion at mlz

134.

47

 

 

 

 

 

10m 100 003.4
1173
XVII ‘
1
91 3
1 3
1 1
30 ‘ ‘ 00 u
5 ‘3 11 3 1 1 9 ‘
II ll 1 1 7
011.1 1.4. . - ' ,E
' ‘1 I. L m 1

 

 

 

Figure 35. Mass spectrum of 6-methoxytetralin-d3

CONCLUSION

The mass spectra of 6-methoxytetralin and 6-methoxy-l-
tetralone were considered in order to explore the potential
effectiveness of simpler model compounds as an aid for studying
compounds that give complex spectra. The 6-methoxy-1-tetralone
proved not to be useful as a model for estrone methyl ether. The
ketone group on the tetralone caused the fragmentation processes to be

 

 

48

driven in a completely different manner than a structure without such
a group, as expected. The 6-methoxytetralin proved to be too small
and simple a compound to accurately reflect the more complex estrone
methyl ether. However, in the next chapter, when this compound's
m/z 134 product ion spectrum is compared with estrone methyl ether's
m/z 134 product spectrum, it aids in the interpretation. It is believed
that this technique, used with compounds, extended by side chains
from carbons 1 & 2, and by analysis with the a three ring structure,
will prove this method to be informative. As it stands, additional

insight into fragmentation processes has been obtained.

Areas For Further Work

The model compound list needs to be extended. The compounds
used in this preliminary study, although giving a place to start, were
too simple to adequately reflect the fragmentation processes of estrone

methyl ether, which is a more complex structure.

The methyl ether hydrogens in 6-methoxy-1-tetralone may be
exchanged for deuterium. This compound could then be used to
further investigate the fragmentations mechanisms. The mass
spectrum of the labeled compound could be used to verify whether
product spectra alone could have been used to elucidate the

mechanism. The ketone group in 6-methoxy—1-tetralone may be

 

49

reduced, and carbon chains introduced to provide models of estrogen
structural fragments (Figure 36).

    

H3C 0

Figure 36. Reduction of ketone group in 6-methoxy—1-tetralone.

Carbons 1 and 2 need to be substituted, building up to a third
ring. This third ring should then be substituted at carbons 12 and 13
to give information about the ions at m/z 227 and 242. This tricyclic
structure would serve as an intermediate model for estrone methyl
ether (Figure 37).

 

H3C G

Figure 37. Tricyclic intermediate structure.

50

The tricyclic compound should be available by a Michael
addition followed by an aldol condensation (Figure 38).

    

Reduction of dorble bond
and ketone

  

Figure 38. Possible formation of tricyclic structure.

51

A similar reaction was accomplished by De Boer. (4) The
Michael adduct was made and confirmed by mass spectrometry
(Figure 39). Separation from by- products and starting material

became a problem in this reaction.

 

 

 

 

 

 

 

 

 

 

14 8199436
1001 . P‘ ‘V‘
rxr04
130
‘F 77 ’f
a 09 ’ ‘ ’ ’
61 \ 11: - 8
l , 1 1 I‘
. +;l >v 'v
s . TIIIIIIII: .. + +

Figure 39. Mass spectrum of Michael adduct from 6-methoxy-1-

tetralone.

The compound should then be reduced to form an unsaturated
ring with an alkyl chain. This would then be a model for the A and B
rings, and may provide insight into the C ring's fragmentation as well,

of estrone methyl ether.

 

52

References

1. Budzikeiwicz, H.; Djerassi, C.; Williams, D, Mass Spectrom.

of Organic Compounds, 1967 .

2. Borchers, F.; Levsen, K.; Stolze, R.; Budzikiewicz, H., Org.
Mass Spectrom. 13, 510, 1978.

3. Kuhne, H.; Hesse, M.. Mass Spectrom. Reviews, 1982, 1, 15.

4. DeBoer, C., J. Organ. Chem, 39, 2427, 1976.

 

Chapter IH

Studies of selected mlz values of estrone methyl
ether

A striking feature of the mass spectrum of estrone methyl ether
is the fairly stable molecular ion which forms the base peak in its
spectrum. Another interesting point is that a number of the fragments
are formed with no oberservable intermediate ions involved in the
decompostion. Yet it takes several steps to rationalize their formation.
This is not meant to imply that the structure actually goes through
each of these steps as a true intermediate, all the movement may
happen as the excited molecule disperses the energy throughout its
structure. Examples of these ions are m/z 199, 160, and 256.

After a review of the literature on estrone methyl ether, its mass
spectrum, and the model compound study, several fragment ions were
selected for further investigation; m/z 199, 227, and 134. Since a new
instrumental technique was being used, some of the previous work
was repeated, both to test the consistency of the experimental results
and to see if any new information could be obtained. Precursor scans
were collected for the selected ions. A comparison of literature values
and data from the TQMS for the selected ions is presented in table 4.

 

54

 

 

 

 

 

Ion m/z Literature Precursors Observed Precursors
199 214, 228, 241, 242, 214, 227, 241, 256,
256, 284 284
134 no data available 162, 176, 206, 230,
256, 284
227 242, 256, 284 242, 243, 255, 256,
269, 284

 

 

 

 

Table 4. Experimentally observed precursors vs. literature precursors

on selected ions.

The precursor scan (Figure 40) for m/z 199 proved similar to
Djerassi's work. The ions at m/z 214 and 227 were listed as precursors
by the TQMS but not by the metastable ion technique used by
Djerassi. Djerassi lists ions at m/z 228 and 242 as precursors, but
these are not observed in the present work. Since Djerassi's instrument
didn't have unit mass resolution, it is questionable whether he could
distinguish between ions at m/z 227 and 228, or 241 and 242. It is
interesting that Djerassi listed both molecular compositions for m/z
256 (C18H24O, & C17H2002) as precursors for m/z 199. (2,3)
These couldn't be distinguished on the TQMS without labeling estrone
methyl ether at carbons 6 & 7.

 

55

The ions at m/z 135, 200, 228, 271, and 272 in the precursor
spectra have been discounted in this study. The ions at the m/z value
135 and 200 are in the range to be the C13 peaks for m/z values 134
and 199 respectively. The ion at m/z 228 is at the very beginning of
the scan, which makes it suspicious. The ions at m/z 271 and 272
represent M-13 & M-12. These are highly unlikely fragments and may
indicate a slight impurity in the sample or instrumental artifacts,

especially since they are low intensity ions.

 

 

 

 

« 0.00
mu m“ '1.”
00-1
004
«a
an
70-1 «f
391.1
211.4 227.2 240.7 2a.: 27.1.1 1
l A l I 1 .~ 1
t - .2 , -
:30 £0 “'0 760 200

Figure 40. Precursor spectrum of ion at m/z 199.

The manner in which the ion at m/z 228 serves as a precursor to
m/z 199 is not easily rationalized. This represents a loss of 29 mass
units, which may be explained as the loss of carbons 6 & 7 plus one
hydrogen. In Djerassi's report (3) he claims that all principal peaks
above m/z 160 contain the A & B rings intact. The ion at m/z 199 is

56

certainty a major ion. Since no supporting evidence is given, the
assumption is made that this information was obtained from spectra
that Djerassi had, but didn't publish, which would rule out the loss of
carbons 6 & 7 to form m/z 199 from m/z 228. This fragmentation
remains a mystery, and appears to confirm the exact mass assigment
problem associated with the metastable ion technique.

Since a product scan had never been done for the m/z 199 ion,
one was collected to add to the literature (Figure 41). This ion was
also chosen because it is a well studied ion, and allowed a firm place

to start the investigation of the mass spectrum of estrone methyl ether.

 

 

 

 

 

 

 

1001 171.0 0000
l 0.00
00-4
001 100.1
100.1
00‘
121.:
00-1 101,1
00.0 71.1 00.0 0 .0 1 1.0 11 u .0 101.1 .0 "14
1-L_1L1L“1-1-1.11 f” 41
W— T v V V r i
00 00 1‘0 Jo 100 100 1B

Figure 41. Product spectrum for ion at m/z 199.

In the event, the product spectrum revealed ions at m/z 184,

171, and 158 as the three most abundant. Rationalizations for the

 

57

formation of each of these three ions fi'om the ion at m/z 199 are
presented. The m/z 199 ion can rearrange to a tropylium ion. A 1,9
hydrogen shift, followed by a charge-induced rearrangement to place
the cation again in a benzylic position, sets the stage for loss of 28
mass units as ethylene to form the m/z 171 (Figure 42).

 

Figure 42. Rationalization for formation of fragment ion at m/z
171 from m/z 199.

58

Fragmentation to an ion at m/z 158, may also proceed by an
initial rearrangement to the tropylium ion structure. This, followed by
a 1,5 hydrogen shift to (c), and a 1,9 hydrogen shifi gives ((1), which is
set up to loose 41 mass units as a C3H5 radical to form the ion at m/z
158 (e) (Figure 43).

 

Figure 43. Rationalization for formation of ion at 15 8 from m/z 199.

 

 

59

The peak at m/z 184 may also be formed from intermediate (c)
in figure 43. Here, instead of a 1,9 H-Shift, an electrocyclic reaction
followed by a 1,9 H-Shift, gives (d), opening of the four-membered
ring in (d), followed by methyl loss then gives (e), m/z 184, as in
figure 44.

   
 
 

Electrocyclic

H30 0

 

Formation of a mlz 184 ion from Djerassi's m/z199

Figure 44. Rationalization for formation of ion at m/z 184 from
m/z 199.

 

60

These fragmentation mechanisms all begin with the same
structure for the ion at m/z 199, which contains carbon 16. Djerassi's
work indicated that there are at least two different structures for m/z
199. One of these structures contains carbon 16 and one doesn’t.
Djerassi stated that most of the ion current at m/z 199 was carried by
an ion which contained carbon 16, and that this ion was formed by a
concerted mechanism from the molecular ion. The precursor scan
collected during this research also showed the molecular ion as the
main precursor of m/z 199.

The ion at m/z 227 was studied next. A precursor scan was
conducted to confum the data from the literature. Then a product
spectrum was collected. The structure of this ion is uncertain, and
there are questions about the source of the hydrogen transfer in its
formation from the molecular ion.

The precursor scan for the ion at m/z 227 (Figure 45) from the
TQMS gave all the m/z values in the literature. However, three more
precursors were observed at m/z 243, 255, and 269. The ion at m/z
269 (M-15) was proposed by Djerassi to occur by loss of methyl from
both the angular methyl and the ether methyl. (3) This ion shows up
as a precursor for the ions at m/z 147 and 171 as well. From an
examination of the deuterated estrone methyl ether we know that the
ions at m/z 171 and 147 both contain the deuterated methyl ether
group; both of the ions m/z values were shifted upward three mass
units. Since the ion at m/z 269 is a precursor for these two ions it also

61

must contain the ether group. However, this ion is also a precursor to
the ion at m/z 227, which contains both the angular methyl and the
ether methyl. This suggests that the loss of 15 mass units may be from
a different source.

210 .2 fit 0‘
100- '0.27

00-
so-
001

20‘ 242.1 255-1 2 .2
J l ““1 r
24 .2 7.: 263.1 egg
1 , r3 , I1L L. .0

I I
220 240 260 280

 

 

 

 

 

Figure 45. Precursor spectrum for ion at m/z 227.

After looking at some of the results a question also came up
about m/z 242. According to the precursor scan of m/z 227, 15 mass
units are lost from m/z 242 to form 227, presumably as a methyl

 

62

radical. The ion at m/z 242 is assumed to be formed from the
molecular ion by loss of ketene, followed by loss of 15 mass units to
form 227 (Figure 46). However, the structure of m/z 242 has not been
confirmed, nor is the structure certain for m/z 227.

 

mlz 227 (a) m/z 227 (b) mlz 227 (c)

Figure 46. Rationalization for formation of ion at m/z 227

by a process involving m/z 242.

613

A product spectrum was collected for m/z 242 (Figure 47). A
loss of 28 mass units to form an ion at m/z 214, (Figure 48) was
observed, as well as a loss of 15 mass units to form an ion at m/z 227.
To rationalize these data a structure for the ion at m/z 242 was
proposed in figures 46 and 48. If this is a correct representation of
m/z 242, loss of a methyl radical would have to involve a hydrogen
transfer. This hydrogen could be transferred from several different
sites to form isomers of the m/z 227 ion in figure 46.

 

 

 

 

 

 

 

 

. 00
‘..1 120 1 F:f01
001
001 203.0
112.1
001
\15-9
220.0
10 .0
2" 10.0.0 1 210.2
01.2 129-9 100.0
' 11.0 00 2 - L
- l
00 100 100 200 230

Figure 47 . Product spectrum of ion at m/z 242.

 

 

64

-02H4
—>

  

 

H3C.

rm2214
Figure 48. Rationalization for formation of ion at m/z 214 from m/z
242.

The product mass spectrum of the ion m/z 227 has two intense
peaks which stand out in the spectrum, (Figure 49) these are m/z 171
and 147. Precursor and product spectra were obtained on both of
these ions in an attempt to better understand the ion at m/z 227
(Figures 50,51,52, & 53).

147.1 ['93
171.1 1.3E
I

100‘

00-1

4°7 51.;
_ ‘ 150.1
00.1 121.0

11 1 0.1
201 3'
55.1 93.2 104.9
I D '
1 .1- in .l A l
I ' ' ' T r T ——v v v

50 100 1gb

 

 

 

 

 

 

 

Figure 49. Product spectrum for ion at m/z 227.

"'1

 

1.01

m

601

00*

' 20+

6H5

881.3

 

 

Figure 50. Precursor spectrum for ion at mlz 147.

91.0

103.3

54.7

 

 

 

00.0 01.1 .0 1 .1 0 .0 09.2 101.;
I if“ 5.? L
. z ,

If]..11z.3

110.5

 

133.2

?33.5 [‘11 101.0

I00)
1'2.»

 

CO.)
0.33

 

 

T v.

‘0 O. 10.

r
12.

Figure 51. Product spectrum for ion at m/z 147.

10.

‘fl 1”.) I...

 

 

 

 

 

0'1
"1 221.2
113.2 111.2 300.2 211i10.1 l;:0.0 001.0 001.0 2qp.0
* a. * .1. r .1. 1. * .1. " .5

Figure 52. Precursor spectrum for ion at m/z 171.

 

 

 

 

1 b 1004
1001 H 1'1.“
l
001
001
120.0
001
100.0
201 100.0
00.0 00.1 00 0 .0 1 .0 110.0 .2 102.0 1 .0
f r ' t v t r t T ' W
60 00 100 100 100 100

 

Figure 53. Product spectrum for ion at m/z 171.

67

The precursor spectrum for m/z 171 also shows m/z 227 as one of its
precursors. A process can be written to show a possible formation of
the ion at m/z 171 from the ion at m/z 227 by using structure (a) and
(b) from figure 46. However, there was no clear pathway from figure
(c) to the proposed ion at m/z 171 (Figure 54).

5 H3

-62”... 1"“ —..:©0

 

1.12H-Sh'fl
1,15H-Shift

6W
22>:

an171

Figure 54. Rationalization for formation of ion at m/z 171 from
mlz 227.

The precursor Spectrum for m/z 171 shows that it is also formed
from ions having m/z 199 and 186, as well as 227, and by several
more minor processes. It has already been shown how 171 could be
formed from the ion at m/z 199. If the ion at m/z 186 is represented as
(a) in figure 55, a hydrogen transfer could produce (b) which can be
rearranged to (c). Loss of a methyl group from (c) generates the m/z
171 ion, d or e (Figure 55).

 

3

H3. Q‘ —> (9‘. ——Hi ©.\ :3]:

 

 
   

(a) “30 (1» 1.30.0)
mlz 186
1— CH3
\ 1,5 H-Shifl
‘__
030/ 0 ~ (00>
H3C “30' “30' (d)
(or) m/z171
H30 0

Figure 55. Rationalization for formation of ion at m/z 171 from
m/z 186.

The other major ion in the product spectrum of m/z 227 is 147.
Its formation can be rationalized by using the structures for m/z 227

as before from (a) or (b), but there is no clear path to form m/z 147
from the structure of m/z 227 represented by structure (c) (Figure 56).

  

  
   
 
 
 
 

H
Q. "" ©é —’
H CO 0
H c- 3 H—CH
H30 m/2227 (a) 3 1 2
1,5 H-Shinl
Fragmentation of
2
} Rearrangemert °
‘— H2 ‘—
with H-transfer
H c H3C
0 /,
3 m/z147 H30

   

1)Reanaggement
H2 2)reto-DA H3C .

H2

Figure 56. Rationalization for formation of ion at m/z 147 from
m/z 227.

70

The ion at m/z 212 in the spectrum of 227 was interesting. This
loss of 15 mass units may have occured from ring C (Djerassi's Rule),
but loss from the ether functionality would result in a highly
conjugated structure (Figure 57). The structure of the ion at m/z 212

was not known.

 

 

ml2212

 

Figure S7. A possible rationalization for loss of a methyl group
to from the m/z 227 ion.

To test whether the mechanism suggested above was plausible
the methyl group of the ether was deuterated. A new product mass
spectrum was obtained of ion m/z 230, (Figure 58) the deuterated
analog of m/z 227.

71

10.1 . 21 .0000
'1 ....

00" 130.0
171.3

00"

201 00.0 12‘. .1 103-0 201.0

AL01.[ 110.2 100 ’ J .1 310.1
001. v 0. ‘3 ‘TLfit AVJ F.1LL i

 

 

 

 

 

 

 

Figure 5 8. Product spectrum of mlz 230, deuterated mlz 227.

The product ion m/z 212 was partially shifted to 215. This
indicates that the loss of 15 mass units occurs both by loss of the ether
functionality, possibly as suggested above, and by a process involving .
retention of this group. That the majority of the ion current is carried
by the ion retaining the ether functionality is reasonable since the ion
at m/z 199, 198, 134, 147, 171, and 159 are also shifted upward three

mass units indicating retention of this ether group.

Alternative mechanisms were then considered to explain the loss
of a methyl radical to form an ion at m/z 212. Again the three

72

possible structures proposed by Djerassi (Figure 46) were considered

as a starting point. Plausible mechanisms for this fragmentation are
formulated for structures (a) and (b) (Figure 59). No similiar
mechanism was conceived for structure (c).

      
 
   

1 )electrocyclic
closure

1)1,2 H-Shifi

——>
2)Electrocyclic
closure

 

Figure 59. Rationalization for formation of ion at m/z 212 from
m/z 227.

73

Possible mechanisms for the fragmentation of m/z 227 to 199
were considered next. Here again fragmentation's from structures (a)

and (b) can be rationalized to provide the structure for the ion at m/z
199. But, no clear pathway could be found from the structure of m/z
227 represented by (c) (Figure 60).

  

H3

12*” ©§ \

H c-
3 ”30' m/z199

“300 (b) 111/2227

Figure 60. Rationalizations for formation of m/z 199 from the
proposed structures for m/z 227.

74

The ion at m/z 185 in the product spectrum of m/z 227 is
interesting. The ion at m/z 227 fragments by a loss of 42 mass units,
presumably as a C3H6 radical, to form an ion at m/z 185. A process
leading to a stable ion at this m/z value could only be conceived from

the structure (b) for m/z 227 in figure 46 (Figure 61).

1,0}

mlz 227 b

H30

1 5 1+8th

w 1) 'IC”3 CH2 30.3 .Q
2) Electrocyclic

°'°‘"° mlz185
”30*1130. ! H2:1—-. H30 0
mlz 185

Figure 61. A possible rationalization for ion at m/z 185
from m/z 227.

75

The rationalizations presented here suggest that structure (c) can
be eliminated from the list of possibilities for the ion at m/z 227. It is
possible that both (a) and (b) exist or they may inter convert somehow
inside the instrument. (Figure 62).

 

Figure 62. Suggested two structures for m/z 227.

The product mass spectrum of m/z 171 was compared with that
of the corresponding deuterated ion now found at m/z 174. It was
observed that the product ion peaks at m/z 156 and 128 were not
shifted, indicating a loss of the ether methyl group. To form the m/z
156 ion a loss of 15 mass units from m/z 171 ( or 18 mass units from
m/z 174) is required. There have been two structures proposed for
m/z 171. Both of these have, at most, one hydrogen per carbon atom.
A loss of a methyl radical from any place in the structure other than
the methyl ether would be difficult to rationalize. Loss of the methyl
group from either structure leaves a highly conjugated system. If the
loss of a methyl radical is followed by loss of carbon monoxide in
structure (a), an azulene radical-cation is possible for the m/z 128 ion.

If the loss of a methyl radical is followed by loss of carbon monoxide

76

in structure (b), naphthalene radical-cation is formed and observed at
m/z 128 (Figure 63). The peaks at m/z 156 and 128 are of very low
intensity in the regular mass spectrum and wouldn‘t normally be
examined. It's only the unique ability of tandem mass spectrometry to
give product spectra that allowed these observations to be made.

”171(3) mlz156 mlz128
we”: -CH3 0”:
H3C' mlz171(b) "1,2128

Figure 63. Rationalization for the formation of ions at m/z 128
and 156 from m/z 171.

The ion at m/z 134 had not been studied by Djerassi and was
chosen as a new ion for study. This was, in part, because the model
compound 6-methoxy-tetralin has its base peak at m/z 134. The
questions are; whether the ions at m/z 134 are the same in estrone
methyl ether and 6-methoxytetralin, whether the ions have the same
fragmentation behavior, and whether product ion analysis will aid in
the investigation and provide more information and insight than were

available before.

77

The ion represented by m/z 134 is believed to occur by a RDA
fragmentation pathway, based on studies of tetralin and 1-
methyltetralin (Figure 64). (4) A RDA fragment was found in tetralin,
but when a methyl group was placed on carbon-1 a different
fragmentation pathway resulted, and the RDA process became a minor
pathway (4%).

H3
1

O.

(a) (b)
Figure 64. (a) Tetralin and (b) l-Methyltetralin.

Three structures were found in the literature that have been

proposed for this ion, omitting the methoxy group (Figure 65). (1)

Figure 65. Structures from literature for an ion having m/z 104.

78

The ion at m/z 134 is almost certainly C9H100, and could have
a variety of structures. Some of the possibilites are presented below in
figure 66.

 

 

 

 

 

Figure 66. Several of the possibilites for an ion having m/z 134.

Product mass spectra were collected on both compounds for m/z
134 (Figures 67 & 68); both contained the same major ions 119, 104,
and 91. The biggest difference in the two spectra obtained at the same
collision pressure appears to be the intensity of the peak at m/z 7 8. In
the mass spectrum of 6-methoxytetralin it is very large, and it is
almost absent in estrone methyl ether. Another attempt to collect
data for the product ion at m/z 134 in the estrone methyl ether
spectrum gave slightly different results. A collision pressure of 0.5
mtorr was used instead of 1 mtorr as used in the previous spectra. I

was unable to maintain an acceptable vacuum at any higher collsion

79

pressure. This resulted in very little fragmentation. The same major
peaks were observed except the mass values at m/z 105 and 106 were
absent in the lower collision pressure spectrum, and a mass value of
m/z 104 was present in their place. The origin of this mass difference
might be a result of a mass error, or a difference in fragmentation
processes do to the difference in collision energies. This is not being
examined at this point. Instead, the mass value at m/z 78 in the two
compounds spectra will be considered.

1"! ”9.) 00¢

100.3
01.0 |
00" '

30‘

 

 

 

 

 

 

10.2 91.0 .1
0:10 00‘: 10'0 [31.1 Pa [0110 - TipJ .- .0
03 i 1? 110 ,

 

81

Figure 67. Product spectrum for ion at m/z 134 from estrone
methyl ether at lmtorr collision pressure.

 

 

(30

01.2 0002
‘°.1 ' 70.10

001 70.1

110.2
100.2 110.1

001_

30"

65 11 10“,‘

53* , nah JE'VJJ | °' ‘1"

0‘ v I f :“

 

 

 

 

 

 

 

 

Figure 68. Product spectrum for ion at m/z 134 from 6-
methoxytetralin at lmtorr collision pressure.

The most significant precursor ions for m/z 134 from estrone
methyl ether are the molecular ion, m/z 284, and m/z 256 (M-28).
Since m/z 134 is an odd electron ion it would have to involve a
rearrangement to be formed from the molecular ion. There are two
ions found at m/z 256, and without labeling carbons 6 & 7, it's not
possible to know which one is its precursor. However, Djerassi
determined that the structure for m/z 256 (C18H24O) carries 90% of
the ion current at this m/z value. If this structure is used as the
precursor for the ion at m/z 134, a different structure from the reto-
Diels-Alder ion is formed. This structure cannot easily account for

the formation of an ion at m/z 78 (Figure 69). Other precursor ions

 

 

81

noted in Table 4 are extremely weak in the estrone methyl ether
spectrum and perhaps may be discounted. The m/z 272 ion may be an
artifact, as already noted, since M-12 fragmentations are improbable.

107‘

 

 

 

 

m/z134

.V
- c H
111/278 2 7 kczm

e j
\ .
H30 H30-

111/2105 111/2106
Figure 69. Suggested structure for m/z 134 from estrone methyl ether

 

and likely fragmentations.

The spectrum obtained at higher argon gas pressures, figure
67(a), reveals the ions suggested in figure 69 at m/z 105 and 106. The
spectrum obtained at the lower gas pressure in figure 67 (b) shows only
the ion at m/z 119 as a significant fragmentation ion. This ion is a loss
of 15 mass units and is probably a methyl radical. If the mass
assigments in the deuterated mass spectrum are correct this ion is
shifted two mass units upward. This is unexpected and difficult to
explain. If this mass assigment is off, it's more likely to be one mass

 

 

 

unit rather than two and would then represent retention of the ether

methyl.

If 6-methoxytetralin fragmentated by a retro-Diels Alder process
to form an ion at m/z 134, an ion at m/z 78 could be easily rationalized

as shown in figure 70.

 

l—CH3OC—CH
0 7‘
\

mh78

Figure 70. Possible formation of an ion at mlz 78 from 6-
methoxytetralin.

There appear to be significant differences that suggest that the ion
from estrone methyl ether is (a) and the ion from 6-methoxytetralin is
(b) as shown in figure 71. For this difference to be confirmed, mass

 

 

83

spectra of the CD3-labeled analogs, must be added to the data for
comparsion (Figures 72 &73).

. ‘1' ‘1
)0»... 0
H300 (a) 2 H300 \ ) H3C

(b

 

 

 

 

 

 

" Figure 71. Suggested structures for ion at m/z 134 from; (a) estrone
methyl ether and (b) 6-methoxytetralin.

100.: 0000
1001 F0.00

00"
60‘
00"

20*
94 10

56.5 6 .0 69.4 70.0 QL.6 99.1 10 .
‘ v

 

 

 

 

r-q
pa

120.0 131.5
:1 l
I f

. r I
60 00 100 120

l

 

Figure 72. Product mass spectrum of the CD3-analog for ion having
m/z 134 from estrone methyl ether.

 

 

84

 

 

 

 

 

 

 

 

190‘11 1000
10°“ 1.10
00‘
00.0
60‘
90,9.
0od
66.9
20‘ 1 | 110.9
101.9
50.0 0 .0 90.0 103 109.0 121.9 130
' .2 121.1
1159'? 112-9". .1 9‘1 :1 11 102T 1””‘110‘1 I1 - -
010 00 100 120

Figure 73. Product mass spectrum of the CD3-analog for ion having
m/z 134 from 6-methoxytetralin.

The significant product ions from m/z 134 obtained from 6-
methoxytetralin, are: 119, 104, 91, and 78. To form the ion at m/z 119
a fragmentation involving loss of a methyl radical must occur. The
peak at m/z 119 remains at m/z 119 in the CD3-analog, so the ether
methyl is lost (Figure 74).

 

 

85

Figure 74. Suggested structure for m/z 119 from loss of ether methyl
in mlz 134 of 6-methoxytetralin.

The fragmentation process to form the ion at m/z 104 appears to
involve loss of CHZO, perhaps as a four-centered cyclic mechanism as

shown in figure 75. In the CD3-analog this ion would shift to mlz
105.

 

 

 

Figure 75. Fragmentation process to rationalize the ion at m/z 104
from m/z 134 in 6-methoxytetra1in, and m/z 105 in the CD3-analog.

The loss of 43 mass units to form an ion at m/z 91 can be CH3CO
or C3H7. The former is more likely because the 10 hydrogens in the

ion at m/z 134 are spread out over more than four carbon atoms, and

 

 

loss of C3H7 would require alot of hydrogen transfer. Also, loss of
CH3CO is more easily rationalized as shown in figure 76. However,
this mechanism implies that m/z 91 should remain a strong ion in the
CD3-analog. A small ion is found here, but a larger m/z 95 peak is
present. This makes no sense because 91 + 3 = 94.

0H2

/.—‘ - CH3CO /.

\ ”2
\

0

H3

Figure 76. Rationalization for a process to form an ion at m/z 91 from
m/z 134 in 6-methoxytetralin.

The ion at m/z 78 is a particularly troublesome ion to explain. The
plausible mechansim shown in figure 70 involves a loss of C3H4O (56
mass units), including the methyl ether group. However, the CD3-
analog clearly shows that this ion is shifted upward 3 mass units to
m/z 81. The alternative loss of C 4H8 can be ruled out, because there
are only 10 hydrogens and one of the deuterium atoms from the CD3-
analog would have to be lost. The only alternative that has been
found, is that the 56 mass units are accounted for by CO and C2H4.
This clearly requires a rearrangement of the ether methyl group. A
possible mechanism for m/z 134 in 6-methoxytetralin fragmentating to

 

 

87

form an ion having m/z 78 with retention of the methyl group in
shown in figure 77 .

 

 

 

 

 

 

 

 

 

mh1m5

O —l lmmfl —-| 1&9» /.\‘—l 3+

”30 mlz134
< 0300) "3

11,5 H-Shifl

 

—|: _|;
0 ._ o. ._ ‘11
l

H
H2 2 H3

 

 

 

mh78

Figure 77 . A suggested rationalization for formation of an ion at m/z
78 from the suggested ion at m/z 134 in 6-methoxytetralin.

We can't prove that the mechanism of figure 77 is happening, but
it is clear that product ion analysis of this m/z 134 ion has disclosed

aspects of the ion fragmentation reactions that are entirely unexpected.

Conclusion

The analysis given above leads to the conculsion that product
ion analysis reveals new and unexpected information concerning
fragmentation process. The analysis also points out the difficulty in
rationalizing these unexpected results.

Additional insight has been gained about the ions at m/z 199,
227, 242, 212, 147, 171, 185, 134, 78, and 214 and this has been
added to existing data. Possible structures have been suggested for the
ions at m/z 242, 171, 174, 134, 227, and 212.

The deuterated and regular product spectra were observed for
m/z 171. The fact that the peaks at m/z 156 and 128 had lost the
methyl from the methyl ether was observed. This led to a different
fragmentation pattern than had been considered in previous work.

The ion at m/z 134 was studied by a model compound, 6—
methoxytetralin. From comparison of the two product spectra,
possible structures were determined for the m/z 134 ion for each

compound.

From this work it has been shown that approaching the study of

complex molecules, with reference to simpler model compounds, can

 

provide additional information and insight into ion reactions.
Evidence from this preliminary study indicates that the TQMS
combined with model compounds and selected labeling is a
worthwhile approach for the fragmentation patterns of complex
structures.

Areas For Further Work

In estrone methyl ether the carbons at position 6 & 7 could be
labeled to provide insight into the formation of the ions at m/z 199 and
m/z 134. There is still uncertainty about the M-15 ion at m/z 269.
Djerassi had proposed that it was formed both by loss of the angular
methyl and the ether methyl. The fact that it is listed as a precursor for
mlz 227 which contains both of these methyl groups raises some doubt
about the previous assumption. The change in mass value for the peak
at m/z 105 to m/z 104 in the product spectra of the mass value m/z
134 in estrone methyl ether should be investigated.

What is observed in the mass spectrometer is very dependent on
the collision energy and gas pressure. For these studies we want the
gas pressure and collision energy low enough that there are no second
order products. Studies should be done in order to find the best
conditions for the data to be collected .

Information on rearrangements would also come out of this
work. The fact that an ion is formed in an intra-molecular process may
be proved by showing its relative abundance doesn't vary with the
pressure.

Estradiol (17 alpha & 17 beta) could be added to the study as
well as estriol. This would complete the list of naturally occurring
estrogenic hormones. Three more steroid structures which are not
naturally occurring could be added to the list of model compounds, if a
way for them to be made was found (Figure 78).

 

Figure 78. Three synthetic steroid structures to add to study.

91

References

l. Kuhne, H.; Hesse, M., Mass Spectrom. Reviews, 1, 15, 1982.

2. Budzikeiwicz, H.; Tetrahedron, 21, 1855, 1965.

3. Djerassi, C.; Wilson, J. M.; Budzikeiwicz, H.; Chamberlin, J.
W., J. Am. Chem. Soc. 84, 4544, 1962.

4. Djerassi, C.; Williams, D.,H.; Budzikiewicz, H., Mass
Spectrometry of Organic Compounds, p. 68, 1967.

5. Stolze, R.; Budzikiewicz, H. Org. Mass Spectrom. 13, 25,
1978.

 

 

Chapter IV

Experimental Methods
Mass Spectrometry

The mass spectra were obtained on the TQMS, Figinnan model
700. The solids probe was used in E1 mode with 70 eV.

Tandem Mass Spectrometry

MS/MS spectra were obtained on the TQMS, Figinnan model
700. Argon was used as the collision gas; the electron multiplier was
set at 15sz and the dynode at 5V‘s. All spectra were run in positive
EI mode.

HRMS

HRMS were obtained at the Michigan State University Mass
Spectrometry Facility which is supported, in part, by a grant (DRR-
00480) from the Biotechnology Research Technology Program,
National Center for Research, National Institutes of Health. The
samples were run on a JOEL JMS-AXSOSH.

Methylation of Estrone

 

 

93

The methylation was accomplished according to published
methods. (1,2,3). Estrone (0.4mmole) was dissolved in 95% ethanol
(10ml) by heating. The reaction mixture was removed from heat and
dimethyl sulfate (42u1) and 10% NaOH (0.2ml) were added by
alternate addition. The mixture was then refluxed under argon. The
reaction was followed by TLC, (Rflestrone) = 0.56) & (Rf(estrone
methyl ether) = 0.66) with hexane : ethyl acetate (3:7) as the
developing solvent. The reaction mixture was washed with 2 N HCl
and then extracted into ether. The methylated product was confirmed
by mass spectrometry. The molecular ion of estrone is at a m/z value
of 270, (Figure 79). After the phenolic group was replaced with a
methoxy group a mass spectrum revealed the molecular ion at a m/z
value of 284 (Figure 80). The product was recovered in 91% yield
after workup.

‘5 m (3.1.!)

"'1

m‘ All.

 

 

 

 

 

 

Figure 79. Mass Spectrum of Estrone.

gh‘ 104.1 80..
10.!

0.).

00‘

00‘ l".1

2'1

 

 

 

 

    

i. 8.. 33. 1.. . 8“

Figure 80. Mass Spectrum of Estrone Methyl Ether.
Deuteration of Estrone Methyl Ether

The procedure for the methylation of estrone was used. DMS-d6
was used in place of DMS. The product was confirmed by mass

spectrometry (Figure 81).
It.” 3011? _.:D::
00*
00*

302.1

 

 

 

 

 

90 100 450 300 :90

Figure 81. Mass Spectrum of Estrone Methyl Ether-d3.

 

 

95

Methylation and Deuteration of 6-Hydroxy Tetralin

6-Hydroxytetralin was converted to 6-methoxytetralin-d3 by the

same procedure as stated above for estrone methyl ether using DMS-

d6. The‘product was confirmed by mass spectrometry, figure 82 & 83.

10.1

”‘1

 

 

1-1

x731

 

 

 

 

 

 

 

Figure 82. Mass spectrum of 6-Hydroxy-Tetralin.

 

 

 

 

 

 

at “10.01
1“
1C?
,
It, .‘
’ C9
“rut m J
1T! 9.9184
103
“‘~
[’6‘
11‘- ' m ' 2h ‘

 

 

Figure 83. Mass spectrum of 6-Methoxy-Tetralin d3_

96

Michael adduct from 6-methoxy-1 -tetralone

A published procedure that was performed on a similar
compound was used (4). 6-Methoxy-1-tetralone (0.03 moles) and 1,5-
diazabicaycolo (4.3.0) non-S-ene (DBU) (0.06 moles) were placed
under argon vapors saturated with methyl vinyl ketone (0.06 moles).
The reaction was followed by thin layer chromatography (TLC) using
1:1 hexane-ethyl acetate as the developing solvent. Workup was done
by washing with 1% HCl, extraction with CH2C12 and drying the
organic layer over Na2S04.

There were at least 9 spots for which Rf values could be
calculated. The material was chromatographed with hexane:ethyl
acetate starting with 100% hexane, ending with 100% ethyl acetate.
The Michael adduct came off the column at the 30:20 ml hexanezethyl
acetate fraction. The Michael adduct was still impure with six spots
still remaining in this fraction. A mass spectrum was collected for this
fraction which showed a molecular ion at m/z 246 (Figure 84) which
is assumed to be the Michael adduct. The TLC data was 6—methoxy—l-
tetralone Rf=0.56, and Michael adduct Rf=0.48. The product was

recovered in 67% yield (impure).

 

 

9W7

 

 

1
first
1 o
7" ’
s .9 " ‘ ‘ ’
0: us a
1 1
L1: an '1llllllr' ‘ '

I89...

 

Figure 84. Mass Spectrum of Michael adduct.

 

References

1. Fieser & Fieser, Reagents for Organic Synthesis Vol. 1, 293,
1967.

2. Gundy, J .; James, B. G; and Pattenden, Tet. Let.Vol. 9, 757,
1972.

3. Org. Syn. Coll. Vol. 4, 837, 1963.

4. J. Organic Chem, Vol. 39, 2427, 1974.

 

 

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