Ozonation of ethinylestradiol in aqueous-methanolic solution: Direct monitoring by electrospray ionization mass spectrometry

Article abstract of DOI:10.1590/S0103-50532010000500004

The ozonolysis of ethinylestradiol (1), a synthetic steroidal estrogen, in an aqueous-methanolic solution was investigated. HPLC-UV analyses revealed that 1 was completely consumed after a 20 min reaction time either at pH 5 or 8. ESI(-)-MS (electrospray ionization mass spectrometry in the negative ion mode) monitoring also revealed the continuous consumption of 1 (detected as [1 - H]^- of m/z 295) concomitantly with the emergence of oxidation products. Chemical structures were proposed for these products based on the data of MS and MS/MS (the m/z values and fragmentation profiles of the anionic specie, respectively). These data, in conjunction with the well-established knowledge about the reactivity of organic molecules toward ozone in aqueous solution, were evaluated and an unprecedented reaction route for the ozonation of 1 could thus be suggested. Hence, the first step in this reaction sequence was ascribed to involve a 1, 3-dipolar cycloaddition of ozone at the phenolic ring of 1 yielding the di-hydroxylated product 2 (detected as [2 - H] ^- of m/z 311). The loss of acetylene as the unique dissociation channel for [2 - H]^- thus confirmed that such hydroxylation occurred at the phenolic ring rather than the acetylenic moiety of 1. Subsequent oxidations were proposed to be the origin of a number of other products, all of them bearing the COOH functionally (this was verified by the characteristic loss of CO₂ during the dissociation of the related deprotonated molecules). The ESI(-)-MS records also revealed notable differences between the reaction conducted at pH 5 and 8, i.e. at slightly acid or basic media, respectively.

Full text of DOI:10.1590/S0103-50532010000500004

J. Braz. Chem. Soc., Vol. 21, No. 5, 787-794, 2010.
Printed in Brazil - ©2010 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Ozonation of Ethinylestradiol in Aqueous-Methanolic Solution: Direct Monitoring
by Electrospray Ionization Mass Spectrometry
Karla M. Vieira, Clésia C. Nascentes and Rodinei Augusti*
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais,
31270-901 Belo Horizonte-MG, Brazil
A ozonólise do etinilestradiol (1), um hormônio sintético esteroidal, numa solução de
água/metanol foi investigada.Análises por HPLC-UV revelaram que o hormônio foi completamente
consumido, tanto em pH 5 quanto 8, após um tempo reacional de 20 min. O monitoramento
por espectrometria de massas com ionização electrospray no modo negativo (ESI(-)-MS)
revelou o contínuo consumo de 1 (detectado em sua forma desprotonada, [1 – H]-, de m/z 295),
concomitantemente com o aparecimento de produtos de oxidação. Estruturas químicas foram
propostas para tais produtos com base nos dados de MS (valores de m/z) e MS/MS (padrões de
fragmentação dos ânions). Estes dados, em associação com conhecimento sobre a reatividade de
moléculas orgânicas frente ao ozônio em solução aquosa, foram avaliados e uma rota reacional
inédita para a ozonização do etinilestradiol foi sugerida.Assim, como primeira etapa nesta sequência
reacional propôs-se uma adição 1,3-dipolar do ozônio sobre o anel fenólico de 1 para gerar o
produto di-hidroxilado 2 (detectado como [2 – H]- de m/z 311).A perda de acetileno como o único
canal de dissociação de [2 – H]- confirmou que a hidroxilação ocorreu no anel fenólico e não na
porção acetilênica de 1. Oxidações subsequentes foram propostas para explicar a formação de
outros produtos, todos eles contendo o grupo funcional COOH (como verificado pela dissociação
das respectivas moléculas desprotonadas através da perda característica de CO2). Os dados de
ESI(-)-MS também revelaram diferenças notáveis entre as reações conduzidas em pH 5 e 8, i.e.,
em meios levemente ácido e básico, respectivamente.
The ozonolysis of ethinylestradiol (1), a synthetic steroidal estrogen, in an aqueous-methanolic
solution was investigated. HPLC-UV analyses revealed that 1 was completely consumed after a
20 min reaction time either at pH 5 or 8. ESI(-)-MS (electrospray ionization mass spectrometry
in the negative ion mode) monitoring also revealed the continuous consumption of 1 (detected
as [1 – H]- of m/z 295) concomitantly with the emergence of oxidation products. Chemical
structures were proposed for these products based on the data of MS and MS/MS (the m/z values
and fragmentation profiles of the anionic specie, respectively). These data, in conjunction with the
well-established knowledge about the reactivity of organic molecules toward ozone in aqueous
solution, were evaluated and an unprecedented reaction route for the ozonation of 1 could thus
be suggested. Hence, the first step in this reaction sequence was ascribed to involve a 1,3-dipolar
cycloaddition of ozone at the phenolic ring of 1 yielding the di-hydroxylated product 2 (detected
as [2 – H]- of m/z 311). The loss of acetylene as the unique dissociation channel for [2 – H]- thus
confirmed that such hydroxylation occurred at the phenolic ring rather than the acetylenic moiety
of 1. Subsequent oxidations were proposed to be the origin of a number of other products, all of
them bearing the COOH functionally (this was verified by the characteristic loss of CO2 during
the dissociation of the related deprotonated molecules). The ESI(-)-MS records also revealed
notable differences between the reaction conducted at pH 5 and 8, i.e. at slightly acid or basic
media, respectively.
Keywords: ethinylestradiol, degradation, ozone, reaction monitoring, direct infusion,
electrospray ionization, mass spectrometry
*e-mail: augusti@ufmg.br

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Ozonation of Ethinylestradiol in Aqueous-Methanolic Solution
J. Braz. Chem. Soc.
Introduction
the ESI-generated ions are closely related to that of the
solution.27-29 Electrospray ionization mass spectrometry
(ESI-MS), because of its appealing and unique attributes,
has rapidly becoming an alternative approach suitably
employed to a direct monitoring of a increasing number
of relevant environmental processes.30-40 In these inquiries,
the information provided by the MS (the m/z values of the
ionic species) and MS/MS data (the fragmentation profiles
of a given mass-selected ion) are meticulously evaluated
aiming to propose chemical structures of products (and in
some cases transient intermediates) likely formed in the
condensed phase.41
The identification of by-products arising from
oxidative treatments, which can be more risky than the
own precursors indeed,42 is an important point that must
be taken into consideration for the evaluation of the
overall performance of a given process. Therefore, several
studies have been conducted aiming to identify products
formed by the ozonation of ethinylestradiol (1) in aqueous
medium. For instance, Huber and coworkers18 made use
of LC-MS/MS and GC-MS techniques to identify the
products formed in the reaction of ozone with two model
compounds (bearing the same characteristic functional
groups than 1, i.e., the phenolic ring and the acetylenic
moiety, see Figure 1). Based on these experimental results,
the authors were thus able to propose chemical structures
for possible products arising from the ozonation of 1. In an
analogous report,43 HPLC and GC-MS data showed that 1
reacts with ozone to yield a number of organic acids, such
as malonate and glutarate, which were easily oxidized
further into carbon dioxide. More recently, Dezotti and
coworkers44 performed a systematic investigation on the
reduction of the estrogenic activity of aqueous solutions
of 1 submitted to ozonation. In addition to these studies,
the authors were able to identify, via GC-MS analysis of
derivatized samples, products arising from hydroxylation
at the phenolic ring of 1.
Ethinylestradiol (1, Figure 1), a synthetic steroidal
estrogen, is a common component widely used as the active
principle of many contraceptive agents and therapy drugs.1,2
The occurrence of estrogen hormones in natural systems,
such as surface waters, soils and sediments, has becoming
a subject of major concern.3 There are many sources of
the so-called estrogenic pollution including effluents from
municipal and industrial wastewater treatment plants,
livestock wastes, biosolids, septic tanks and landfills.4
Many problems have been related to the existence of these
hormones in natural waters, for instance feminization
of male fishes,5-8 the lower sperm counts in adult males
and an increasing incidence of cancer.9-14 In addition, the
conventional treatment procedures usually employed in
wastewater plants are not capable to completely remove
such compounds from the water that is directly supplied
to the population.4,13,15,16
Figure 1. Chemical structure of the synthetic hormone ethinylestradiol
(1).
Owing to an increasing awareness regarding the
potential risks associated with the release of hormones (and
other persistent pollutants) directly into the environment, the
development of effective treatment technologies to promote
their degradation has becoming a matter of significant
importance.3,17,18 For instance, oxidation processes, which
usually include the use of ozone, chlorine and chlorine
dioxide as effective oxidants, have been widely applied in
treatment facilities not only for disinfection of wastewaters
but also for elimination of undesired micropollutants from
drinking water.3,11,17,19 Among these processes, ozonation
has attracted the foremost attention as ozone has shown
to be a conveniently cheap, clean and powerful oxidant.
As a consequence, ozone has been successfully applied
to remediate several contaminated effluents20-22 and has
been used as an environmental friendly oxidation agent in
many fields.23,24
In this work, direct infusion electrospray ionization
mass and tandem mass spectrometry (ESI-MS and ESI-MS/
MS, respectively) are employed to monitor the ozonation
of 1 in an aqueous-methanolic solution. A very low ozone
flow rate is used aiming to detect and characterize transient
products formed under these mild conditions before their
complete depletion. Based on these experimental data,
and also on the well-known reactivity of ozone toward
organic molecules in water medium, a plausible reaction
route for the degradation of ethinylestradiol (1) by ozone
is proposed. High performance liquid chromatography
(HPLC) is also utilized as an auxiliary technique to verify
whether the substrate degradation can yield residual organic
compounds in solution.
Electrospray ionization (ESI) is well-recognized to
have a remarkable capability to gently transfer ionic
species from the condensed to the gas phase, usually
with no substantial in-source fragmentation.25,26 Most
important, it has been verified that the composition of

Vol. 21, No. 5, 2010
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Experimental
were first isolated within the ion trap and then resonantly
dissociated via collision-induced dissociation (CID) using
helium as the target gas. The relative collision energy was
set to a value at which the product ions were produced
in measurable abundance (varying from 18 to 40%). The
isolation width used in the MS/MS experiments was
2 m/z units.
Chemicals
Ethinylestradiol (98% purity, Organon, São Paulo,
Brazil) and HPLC-grade acetonitrile and methanol (Vetec,
São Paulo, Brazil) were used as received. Milli-Q water
was utilized to prepare all the solutions required. Ozone
was produced by a generator (Eletro-Triozon, Brazil) fed
with synthetic air. The ozone flow rate, determined by the
iodometry titration method, was 20 mmol min-1.
Results and Discussion
Ozonation of ethynilestradiol (1): HPLC monitoring
Ozonation
Firstly, results from control experiments (aqueous-
methanolic solutions of ethynilestradiol continuously
bubbled with air) demonstrated that the hormone was not
consumed at all by molecular oxygen. To verify the rate
of consumption of 1 by ozone in an aqueous-methanolic
solution (at pH 5), as well as the formation of degradation
products, the reaction was monitored by HPLC-UV.
The chromatograms of the initial solution and aliquots
withdrawn after assorted times are shown in Figure 2.
Note that the intensity of the peak related to compound
1 (retention time of ca. 7 min) decreased whereas other
peaks appeared continuously at shorter retention times.
These data thus indicate that degradation products, likely
with higher polarity than 1 as they eluted faster from the
non-polar (C18) chromatographic column, were formed in
this medium.46 In addition, the peak related to precursor 1
greatly decreased after 20 min. At last, similar HPLC data
regarding the ozonation of 1 in an aqueous medium were
achieved in previously-reported studies.3,10,17,20
Because of the very low solubility of 1 in pure water,
a mixture of methanol/water (3:7 v/v) was used as solvent
in all the experiments. Methanol is a convenient solvent
as it is completely miscible in water and has a very
low reactivity toward ozone.45 Hence, to a solution of 1
(100 mg L-1, 338 mmol L-1) in methanol/water 3:7 v/v
(20 mL), at pH 5 (the natural pH of such solution) or 8
(achieved by the addition of NH4OH 1 mol L-1), an ozone/
air gas stream was continuously bubbled. Aliquots of the
reaction solution were withdrawn at assorted times and
immediately analyzed.
Analytical methods
HPLC-UV
Chromatographic separation was performed in an
SPD-M10A VP instrument (Shimadzu, Kyoto, Japan)
using a Hypersil C18 column (250 mm long, 4.6 mm i.d.,
5 µm particle size) and acetonitrile/H2O (2:3) as mobile
phase. The isocratic elution was maintained at a flow rate
of 1 mL min-1 for a 10 min chromatographic run. The
injection volume was of 20 µL and UV detection carried
out at 210 and 280 nm.
ESI-MS monitoring and ESI-MSn structural evaluation
Figure 3 displays representative ESI mass spectra in the
negative ion mode, ESI(-)-MS, of an aqueous-methanolic
solution of ethinylestradiol (1) obtained after 0, 20, 100 min
of ozone bubbling. The mass spectra in the positive ion mode
(not shown) were also gathered but the results were not as
clarifying as in the negative ion mode. Because of that,
only the ESI(-)-MS are presented herein. Hence, in Figure
3a, which displays the ESI(-)-MS of the initial solution of
1 at 100 mg L-1, an intense and prominent ion of m/z 295,
corresponding to [1 – H]- (the deprotonated form of 1),
is observed. Its mass-selection and fragmentation upon
collision-induced dissociation (CID) yielded a product ion of
m/z 269 (not shown) formed via acetylene loss. Note that the
relative intensity of the ion [1 – H]- decreased considerably
as the reaction proceeded whereas new and intense
anions of m/z 301, 311, 325, 335, 343, 359, 375 emerged
continuously and concomitantly (Figures 3b and 3c).
ESI-MS and ESI-MS n
The analyses were conducted in a LCQFleet
(ThermoScientific, San Jose, CA) mass spectrometer
bearing an electrospray ionization (ESI) source and
operating in the negative ion mode. Mass spectra were
obtained as an average of 50 scans, each one requiring 0.2 s.
The aliquots were directly infused into the ESI source at a
flow rate of 25 µL min-1 by means of a 500 mL microsyringe
(Hamilton, Reno, NV). ESI source conditions were as
follows: heated capillary temperature of 285 °C, sheath gas
(N2) flow rate at 20 (arbitrary units), spray voltage of 5.0 kV,
capillary voltage of -38V, tube lens offset voltage of -110V.
For the ESI-MS/MS experiments, the precursor ions

790
Ozonation of Ethinylestradiol in Aqueous-Methanolic Solution
J. Braz. Chem. Soc.
Figure 3. ESI(-)-MS monitoring of the ozonation of ethinylestradiol (1)
conducted in an aqueous-methanolic solution at pH 5. Aliquots were
collected at the following reaction times: (a) 0 min; (b) 20 min; (c) 100 min.
Figure2. HPLC-UV(210nm)chromatogramsoftheinitialsolutionof 1(a)
and of aliquots taken after different times of ozonation: (b) 20 min; (c) 100
min. Note the appearance of new chromatographic peaks with retention
times shorter than that of 1.
These data thus indicated a continuous consumption of
1, as also verified in the previously described HPLC-UV
data, with the concomitant formation of oxidation products.
As represented in Scheme 1, product 2, detected in its
deprotonated form of m/z 311, was proposed to be formed
via a hydroxylation at the position 2 of the phenolic ring
of 1. In a previous report,43 compound 2 was identified as
one of the initial products resulting from the ozonation of
1 in aqueous medium. In this reaction pathway, the ozone
attack was proposed to occur exclusively at the phenolic
ring of 1 as its second reactive group, the ethynil substituent,
exhibits a considerably lower reactivity toward O3.18
This assumption was confirmed by the fragmentation of
[2 – H]- which yielded an intense product ion of m/z 285
arising from the loss of acetylene (Figure 4). Moreover, on
the basis of previous studies18,47 other isomers of 2, with the
second hydroxyl group connected at positions 1 and 4 of
the phenolic ring of 1, were also possibly formed.
Figure 4. ESI tandem mass spectra (MS/MS) of the anion [2 – H] - of
m/z 311.
The hydroxylated compound 2 (and its positional
isomers), which has a high reactivity toward O3 than the
own precursor 1, could be quickly oxidized further to
yield an unstable quinone-type intermediate11 (not shown
in Scheme 1) that underwent sequential oxidation and

Vol. 21, No. 5, 2010
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791
ring cleavage48,49 to produce the muconic acid derivative 3
(detected as [3 – H]- of m/z 343, Figure 3).
Fromintermediate3anumberofensuingtransformations
(Scheme 1) were proposed to take place. Hence, 3 was
oxidized further to yield 4 (detected as [4 – H]- of m/z 335,
Figure 3) via the release of four molecules of H2. The driving
force of such conversion, certainly induced by ozone (a
strong oxidizing agent), was probably the formation of an
Scheme 1. Reaction pathways proposed for the reaction of ethinylestradiol (1) with ozone in an aqueous-methanolic solution at pH 5. Ions quoted under
the structures are those detected by ESI(-)-MS. Note that the initial O3 attack at the position 3 of the phenolic ring of 1 lead to the formation of product 2.
O3 could also attack at positions 1 and 4 of such ring but the structures of the resulting products (positional isomers of 2) are not displayed herein.

792
Ozonation of Ethinylestradiol in Aqueous-Methanolic Solution
J. Braz. Chem. Soc.
extensive p-electron conjugation system that surely play a
role in the stabilization of compound 4.Actually, it has been
reported that stabilized compounds (with similar conjugated
systemsthan4)canbepromptlyformed viadehydrogenation
processes promoted by oxidizing agents, such as molecular
oxygen and ozone.50 In an parallel pathway, it was proposed
that 3 could react with O3 to yield the epoxide 5 (detected
as [5 – H]- of m/z 359, Figure 3). Such process probably
proceeded via the well-known Crigee mechanism,51 which
postulates that the attack of O3 on the (available) ethinyl
moietyof3couldresultintheformationofaprimaryozonide
(not shown in Scheme 1) that could quickly decompose to
yield 5. Following this reactional sequence, it was plausibly
suggested that ozone could react at the carbon-carbon
double bond of the epoxide ring of 5, probably via the
Crigee mechanism, to yield the aldehyde 6 (detected as its
deprotonated form of m/z 375, Figure 3). Compound 6 could
then undergo further oxidation and subsequent elimination
of 2 molecules of CO and H2O to yield product 7 (detected
as [7 – H]- of m/z 301, Figure 3). Finally, intermediate 3
could undergo loss of water to yield product 8 (detected as
[8 – H]- of m/z 325, Figure 3).
fractions, obtained as the ratio between the absolute intensity
of a given ion and the sum of the absolute intensities of the
whole set of ions as measured from a certain mass spectrum,
comprise a convenient approach to monitor the changes in
the relative concentrations of compounds 1-8 as the reaction
proceeded. Hence, these results revealed that after 20 min
reaction time a notable degradation of 1 was achieved
(a similar finding was accomplished by the HPLC-UV
data as indicated in Figure 2). Note also that compound
2 reached its maximum relative concentration at roughly
20 min and decreased afterwards. Furthermore, product 4
was found to be predominant after 100 min reaction time
which possibly denotes its superior resistance towards
ozone attack in comparison to the other products. Finally,
the relative concentration of the other compounds (3, 5,
6, 7 and 8) experienced smaller (but perceptible) changes
during the reaction course.
The mass-selection and fragmentation of the above-
mentioned anions, i.e., [3 – H]-, [4 – H]-, [5 – H]-, [6 – H]-,
[7 – H]-, and [8 – H]-, yielded product ions arising mainly
from loss of CO2 (44 Da) and other small molecules
(Table 1). These data are thus consistent with the proposed
structures for the reaction products since all of them bear
the COOH functionality and the loss of CO2 is a very
characteristic dissociation channel for carboxylates.52
In Figure 5, the fraction of each anion, i.e., [1 – H]-,
[2 – H]-, [3 – H] -, [4 – H] -, [5 – H] -, [6 – H] -, [7 – H] -
and [8 – H] -, is plotted against the reaction time. These
Figure 5. Fractions of the anions [1 – H]-, [2 – H]-, [3 – H]-, [4 – H]-,
[5 – H] -, [6 – H] -, [7 – H] - and [8 – H] - as a function of reaction time.
Each fraction was calculated as the ratio between the absolute intensity
of one of such ions and the sum of the absolute intensities of the whole
set of ions.
Table 1. Main product ions arising from the dissociation of the anions
[3 – H]-, [4 – H] -, [5 – H] -, [6 – H] -, [7 – H] - and [8 – H] - detected in
the ESI(-)-MS of an aqueous-methanolic solution of ethinylestradiol (1)
submitted to an ozone stream
pH influence
Precursor ion (m/z)
[3 – H]- (343)
Product ions (m/z; relative abundance %)
[3 – H – CO2]- (299; 100)
[3 – H – H2O]- (325 ; 10)
[3 – H – C2H2]- (317; 7)
It has been reported that at higher pH (basic medium)
the decomposition of ozone with the formation of hydroxyl
radicals is favored whereas at acidic medium ozone tends
to react via 1,3 dipolar cycloaddition. Because of that,
products formed in basic or acidic media are usually quite
distinct.53,54 Hence, the ozonation of 1 was also conducted
at pH 8 to verify whether the products obtained would
be similar to those detected at pH 5 (as displayed in
Scheme 1). The ESI(-)-MS of the aliquots withdrawn after
successive reaction times are displayed in Figure 6. Note
that these data revealed that the same sort of anions (with
a remarkable exception: the anion [4 – H] - of m/z 335)
[4 – H]- (335)
[5 – H]- (359)
[6 – H]- (375)
[4 – H – CO2]- (291; 100)
[4 – H – H2O]- (317; 40)
[5 – H – CO2]- (315; 100)
[5 – H – H2O]- (341; 20)
[6 – H – CO2]- (331; 100)
[6 – H – H2O]- (357; 30)
[7 – H – CO2]- (257; 100)
[8 – H – CO2]- (281; 100)
[7 – H]- (301)
[8 – H]- (325)

Vol. 21, No. 5, 2010
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793
were also detected at pH 8. However, unlike the reaction
conducted at pH 5 for which compound 4 shown to
be the predominant specie after 100 min reaction time
(Figures 3 and 5), at pH 8 the major product obtained after
identical reaction time was found to be 3. These divergent
results thus seem to corroborate the expected distinct
behavior that ozone can exhibit depending on the solution
pH.53,54
Acknowledgments
The authors are grateful to the Brazilian sponsor
agencies, CNPq and FAPEMIG (EDT PRONEX 479/07)
for the financial support.
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Received: August 5, 2009
Web Release Date: January 28, 2010