Monitoring of β-blockers ozone degradation via electrospray ionization mass spectrometry

Article abstract of DOI:10.1590/S0103-50532011000500015

The structures of intermediate products of ozone degradation of different pharmaceutical compounds have been studied. Under the conditions employed, complete ozone degradation of nadolol was achieved after 100 min. The degradation products obtained in aqueous solution were characterized by electrospray ionization mass (and tandem mass) spectrometry (ESI-MS and ESI-MS/MS). The proposed mechanism for degradation, ozone attacks at the aniline amino group giving rise to nitro compounds and further degradation occurs via a series of oxidative processes. Continuous online monitoring by ESI-MS(/MS) with high accuracy mass measurements showed that ozone degradation of atenolol (ATE) and acebutolol (ACE) occurs via mechanisms similar to that of nadolol.

Full text of DOI:10.1590/S0103-50532011000500015

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Article
A
Monitoring of b-Blockers Ozone Degradation via Electrospray Ionization Mass
Spectrometry
Cristina Quispe,a,d Fabiane M. Nachtigall,b Maria Francesca R. Fonseca,b Rosana M.
Alberici,b Luis Astudillo,c Jorge Villaseñor,a Marcos N. Eberlinb and Leonardo S. Santos*,d
aLaboratory of Physical Chemistry, Instituto de Química de Recursos Naturales, Talca University,
PO Box 747, Talca, Chile
bThoMSon Mass Spectrometry Laboratory, Institute of Chemistry, University of Campinas,
13083-970 Campinas-SP, Brazil
cLaboratory of Organic Synthesis and dLaboratory of Asymmetric Synthesis,
Talca University, PO Box 747, Talca, Chile
As estruturas dos produtos de degradação mediada por ozônio de diferentes compostos
farmacêuticos foram estudadas. Uma completa degradação do nadolol foi observada nas
condições empregadas após 100 min. Os produtos de degradação obtidos nas soluções aquosas
foram caracterizados por espectrometria de massas com ionização por electrospray (ESI-MS e
ESI-MS/MS). O mecanismo proposto para a degradação consiste inicialmente do ataque de ozônio
ao grupo amino da anilina formando nitro compostos, seguida de uma série de processos oxidativos.
Monitoramento por ESI-MS(/MS) mostrou que a degradação de atenolol (ATE) e acebutolol (ACE)
por ozônio ocorrem via um mecanismo similar ao do nadolol.
The structures of intermediate products of ozone degradation of different pharmaceutical
compounds have been studied. Under the conditions employed, complete ozone degradation of
nadolol was achieved after 100 min. The degradation products obtained in aqueous solution were
characterized by electrospray ionization mass (and tandem mass) spectrometry (ESI-MS and
ESI-MS/MS). The proposed mechanism for degradation, ozone attacks at the aniline amino group
giving rise to nitro compounds and further degradation occurs via a series of oxidative processes.
Continuous online monitoring by ESI-MS(/MS) with high accuracy mass measurements showed
that ozone degradation of atenolol (ATE) and acebutolol (ACE) occurs via mechanisms similar
to that of nadolol.
Keywords: ozonation, degradation, ESI-MS, pharmaceutical compounds, reaction mechanism
Introduction
Pharmaceuticals enter the aquatic environment
after its ingestion by humans and other animals and
subsequent excretion, either in their intact forms or as
metabolites.6 Wastewater treatment is applied worldwide
but pharmaceuticals may not be efficiently removed,3,7 and
these pharmaceutical residues and degradation products can
be downloaded directly into aquatic environments. Several
studies have stated that the long-term risk to humans from
any single pharmaceutical at sub-mg L-1 levels is negligible,
but the toxicological implications of chronic exposure to
trace contaminants is still unclear.8,9
Concerns about the prejudicial effects to life of
accumulation of pharmaceuticals in the environment has
greatly increased in recent years.1,2 But such accumulation
is governed mainly by the environmental bio- and
photo-transformation3 andthespeedandnatureofthenatural
degradation of these potentially harmful contaminants.4,5
The photolytic degradation of chemicals in the environment
may occur via direct sunlight absorption or by reactions
with strong oxidizing species such as hydroxyl radicals
and singlet oxygen.
Ozonationisanadvancedoxidationprocesswidelyusedin
manywastewatertreatmentplantsfortheoxidationoforganic
contaminantsanddisinfection.Ozone,comparedtoothermost
*e-mail: lssantos@utalca.cl

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920
Monitoring of b-Blockers Ozone Degradation via Electrospray Ionization Mass Spectrometry
J. Braz. Chem. Soc.
common oxidizing reagents such as H2O2 or chlorine, is more
efficient in pollutant degradation and less harmful to most
living organisms.10 In water, ozone can oxidize contaminants
via direct selective reactions11 such as ozone addition to
doublebondsortheinitiationofchainradicalreactionsbythe
productionoffreehydroxyradicals(equations1-4). Hydroxy
radicals are generated from ozone decomposition, which is
catalyzed by the presence of hydroxyl anions or by traces of
other substances, such as transition metal cations.
data on substrate degradation rates and mineralization and
to investigate the presence of residual organic compounds
in the aqueous solutions.24
Experimental
Chemicals
Nadolol (NAD), atenolol (ATE) and acebutolol (ACE)
were purchased from Sigma. All the solvents were of
HPLC grade and purchased from Merck and used without
purification. Doubly distilled water was used to prepare
all the solutions in the experiments. Ozone was generated
using a laboratory ozone generation (OZOCAV) ozonizer
fed with air/oxygen.
O3 + H2O→ 2 HO· + O2
O3 + OH- → O2·– + HO2·
O3 + HO· → O2 + HO2·
O3 + HO2· → 2 O2 + HO·
(1)
(2)
(3)
(4)
Degradation procedures
The decomposition of ozone in aqueous solutions
is known to proceed via the chain reactions shown in
equations 1-4.12 Ozone therefore indirectly attacks organic
compounds since such reactions are faster than direct
O3 attack. In previous studies,13,14 we described the O3
interaction with pharmaceutical compound nadolol (anti-
hypertensive). In this study, we report the characterization
via electrospray ionization mass spectrometry (ESI-MS)15,16
of the oxidation intermediates and products of degradation
of nadolol generated during O3 oxidation. Furthermore, the
O3 degradation of two other b-blockers compounds atenolol
and acebutolol was also studied.
The ozonation reactions were performed in a batch glass
reactor thermostated at 25 °C. The reactor was charged with
100 mL of the drug aqueous solutions (1.6 × 10-4 mol L-1)
and fed with 1.2 × 10-4 mol L-1 of ozone at a constant
flow of 46 1 mL min-1. The ozone concentration was
monitored measuring the absorbance at 254 nm, using the
molar extinction coefficient of ozone of 2900 L mol-1 cm-1.
Aliquots (1.0 mL) were taken at several reaction times
and promptly analyzed by ESI-MS(/MS) and HPLC. All
samples were protected from light and refrigerated at 4 °C
prior to analyses.
ESI-MS (and its tandem version ESI-MS/MS) has
been used extensively to monitor the composition and
degradation of organic compounds in the environment.17,18
ESI-MS(/MS) with its unique characteristics has become a
major technique to elucidate reaction mechanism especially
in aqueous solutions via the detection and identification of
reactants, products, and intermediates,19,20 even the short-
lived ones that occur at very low concentrations.21,22
Gas chromatography-mass spectrometry (GC-MS) has
often been the technique of choice for reaction screening
and product elucidation.23 However, GC-MS is an
offline technique that demands extraction and sometimes
derivatization pre-steps, transient, most polar, or relatively
unstablecompoundsmaybemissed.Wehavethenperformed
ESI-MS(/MS) monitoring of the O3 advanced oxidation of
nadolol, atenolol and acebutolol in water to search for all
intermediatesandproductsofthisenvironmentallyimportant
process. These products are the ones most likely to be found
in treated wastewaters.We also used high performance liquid
chromatography(HPLC), ultravioletspectroscopy(UV), and
total organic carbon (TOC) analyses to collect additional
Analytic methods
Drugs concentrations were monitored using a Perkin
Elmer Series 200 chromatograph HPLC system equipped
with a UV-Vis detector.A Merck-Chromolith Performance
RP-18 column (4.6 mm × 100 mm) was used with
H2O:MeOH (70:30, v/v) mobile phase at a flow rate of
0.6 mL min-1. Water was previously acidified to pH 3 using
aqueous solution of HCl. Organic acids were quantified
with a transgenomic ORH-801 column (6.5 mm × 300 mm)
using 5.0 × 10-3 mol L-1 sulphuric acid as mobile phase at
a flow rate of 0.8 mL min-1.
Ultraviolet (UV) absorbance measurements were
performed using a HP 8453 (Agilent) spectrophotometer
equipped with a quartz cell with a 1.0 cm path length.
Absorbances were measured with baseline correction,
scan rate of 200-600 nm, and integrate time of 0.5 s and
intervals of 1 nm.
Total organic carbon (TOC) experiments were carried
out in a TOC 5000A (Shimadzu) instrument at 680 °C using
a platinum catalyst.

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Quispe et al.
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ESI-MS(/MS) data were collected using a high
resolution hybrid quadrupole (Q) and orthogonal time-of-
flight (TOF) mass spectrometer (Q-Tof, Micromass UK)
with constant nebulizer temperature of 50 °C. The ESI
source and the mass spectrometer were operated in the
positive ion mode, and the cone and extractor potentials
were set to 40 and 10 V, respectively, with a scan range of
m/z 50-1000. Samples were infused into the ESI source
at flow rates of ca 15 mL min-1 via a microsyringe pump.
ESI-MS/MS experiments were carried out by selection of a
specific ion in Q1 and by performing its collision-induced
dissociation (CID) with argon in the collision chamber.
The product ions MS analysis was accomplished with the
high-resolution (ca. 5.000) and high accuracy (ca. 10 ppm)
orthogonal TOF analyzer.
Results and Discussion
Degradation of nadolol
Our investigation began with HPLC monitoring for the
O3 degradation of nadolol (NAD) in an aqueous solution at
a concentration of 1.6 × 10-4 mol L-1 at pH 3 or 9, with O3
concentration at 1.2 × 10-4 mol L-1 (Figure 1). Regardless
the pH (acidic or basic), more than 90% of NAD was
found to be degraded after 60 min at similar rates. TOC
analyses were then performed to evaluate the mineralization
extent, which was found to be only 20% according to TOC
removal. This result suggests that NAD was completely
consumed after 60 min of O3 degradation, but desirable
mineralization to CO2, H2O and NH3 was far for completion.
UV monitoring of O3 degradation (Figure 2) also indicated
complete NAD consumption, but little information is
provided of the nature of the degradation products.
The UV spectra of Figure 2i show three characteristic
absorption bands of nadolol at 203, 217 and 277 nm. After
Figure 2. UV spectra of (i) an aqueous solution of nadolol at
1.6 × 10-4 mol L-1, (ii) an aqueous solution of atenolol at 1.9 × 10-4 mol L-1,
(iii) an aqueous solution of acebutolol at 1.5 × 10-4 mol L-1 and aliquots
of the reaction mixture withdrawn after 60 min (iv) and 160 min (v) of
ozone treatment respectively.
O3 treatment, the UV spectra show that intensities of these
absorption bands were greatly reduced, suggesting nadolol
was degraded, but not totally mineralized with persistent
organic intermediates.
Figure 1. HPLC monitoring of the ozonation of nadolol at pH 3 () and
pH 9 ().

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Monitoring of b-Blockers Ozone Degradation via Electrospray Ionization Mass Spectrometry
J. Braz. Chem. Soc.
ESI-MS(/MS) monitoring
of 2 afforded hydroxylamine [3 + H]+ by loss of isobutene
as noted through the ion of m/z 92, as depicted in Figure 3d.
The ion of m/z 236 (Figure 3d) was likely formed
by aromatic ring opening, which produced a series of
degradation products detected as the ions of m/z 378,
342 and 376. Furthermore, ESI-MS was able to gently
and efficiently fish directly from the solution to the gas
phase for MS analysis also an increasingly abundant ion
of m/z 236. This ion was characterized as aminotriol 9
(Figure 5). Figure 4c shows the ESI-MS/MS spectra of
intermediate [9 + H]+ that dissociated by loss of isobutene
(C4H8) producing the fragment ion of m/z 180. That loss is
indicative of tert-butyl group in the molecule, as observed
in Figure 4.Another interesting fragmentation pathway was
observed for [9 + H]+ by loss of C4H8O2, which structure
could be realized as 2-hydroxytetrahydrofuran.
Two major mechanisms can be considered for O3
treatment. The first one assumes O3 addition to double
bonds, whereas the second considers indirect reaction in
water via hydroxyl radicals (equations 1-4). Formation
of lower and higher masses intermediates was observed.
Considering the lower mass compounds, Figure 5 presents
a proposed route of degradation based on the ESI-MS(/MS)
data, whereas Figure 6 presents proposed pathways
Figure 3 displays representative ESI(+) mass spectra
of nadolol aqueous solution (pH 9) monitored after 60,
100 and 160 min from ozone treatment. Similar spectra
(not shown) were acquired when using solutions at pH 3.
The spectra at zero irradiation time (Figure 3a) detected,
as expected, an intense ion of m/z 310 corresponding to
protonated NAD. The ESI-MS/MS (Figure 4a) for collision
induced dissociation of protonated NAD of m/z 310 shows
a series of major and structurally diagnostic fragment ions
arising from the loss of isobutene (m/z 254), H2O plus
isobutene (m/z 236), 2 × H2O plus isobutene (m/z 218) and
NH3 plus 2 × H2O and isobutene (m/z 201).
The degradation process was monitored by intervals of
20 min during 160 min of reaction. Illustrative spectra are
displayed in Figure 3. The degradation of nadolol of m/z 310
was clearly perceived as well as the formation of organic
intermediates.After 60 min of ozonation (Figure 3b), three
additional and increasingly abundant ions of m/z 92, 148
and 236 were detected. The ion of m/z 148 (Figure 3d),
which indicates the loss of a subunit of 161 Da (from
nadolol of 309 Da), was characterized as the protonated
form of dihydroxylamine 2 (Figure 5). Further degradation
Figure 3. ESI(+) Mass spectra acquired for aliquots of the reaction mixture after O3 treatment of nadolol at pH 9 for (a) 0 min; (b) 60 min; (c) 100 min
and (d) 160 min.