Phototransformation of Amlodipine: Degradation Kinetics and Identification of Its Photoproducts

Article abstract of DOI:10.1371/journal.pone.0109206

Nowadays, monitoring focuses on the primary compounds and does not include degradation products formed during various biological and chemical processes. Transformation products may have the same effects to human health and the environment or sometimes they can be more toxic than the parent compound. Unfortunately, knowledge about the formation of degradation products is still limited, however, can be very important for the environmental risk assessment. Firstly, the photodegradation kinetic of amlodipine was investigated in two experimental conditions: during the exposure to solar radiation and during the exposure to the light emitted by the xenon lamp. In all cases degradation of amlodipine followed a pseudo-first-order kinetics. In the next step, identification of transformation products of amlodipine formed during the exposure to xenon lamp irradiation was performed using ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). As a result sixteen photoproducts were identified, their structures were elucidated and ultimately the transformation pathway was proposed. Fifteen compounds (out of 16 photoproducts) were newly identified and reported here for the first time; some of those compounds were formed from the first photoproduct, amlodipine pyridine derivative. Several analytes were formed only in acidic or basic conditions. Furthermore, the occurrence of amlodipine and its identified degradation products was investigated in environmental waters. Only one out of 16 compounds was found in wastewater effluent. The possibility of the sorption of examined analytes to sewage sludge particles was discussed based on QSAR.

Full text of DOI:10.1371/journal.pone.0109206

Phototransformation of Amlodipine: Degradation
Kinetics and Identification of Its Photoproducts
1
Anna Jakimska *, Magdalena Sliwka-Kaszynska , Piotr Nagorski , Jacek Namiesnik , Agata Kot-Wasik
2
1
1
1
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´
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1 Department of Analytical Chemistry, Faculty of Chemistry, Gdansk University of Technology, Gdansk, Poland, 2 Department of Organic Chemistry, Faculty of Chemistry,
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Gdansk University of Technology, Gdansk, Poland
´
Abstract
Nowadays, monitoring focuses on the primary compounds and does not include degradation products formed during
various biological and chemical processes. Transformation products may have the same effects to human health and the
environment or sometimes they can be more toxic than the parent compound. Unfortunately, knowledge about the
formation of degradation products is still limited, however, can be very important for the environmental risk assessment.
Firstly, the photodegradation kinetic of amlodipine was investigated in two experimental conditions: during the exposure to
solar radiation and during the exposure to the light emitted by the xenon lamp. In all cases degradation of amlodipine
followed a pseudo-first-order kinetics. In the next step, identification of transformation products of amlodipine formed
during the exposure to xenon lamp irradiation was performed using ultra high performance liquid chromatography
quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). As a result sixteen photoproducts were identified, their
structures were elucidated and ultimately the transformation pathway was proposed. Fifteen compounds (out of 16
photoproducts) were newly identified and reported here for the first time; some of those compounds were formed from the
first photoproduct, amlodipine pyridine derivative. Several analytes were formed only in acidic or basic conditions.
Furthermore, the occurrence of amlodipine and its identified degradation products was investigated in environmental
waters. Only one out of 16 compounds was found in wastewater effluent. The possibility of the sorption of examined
analytes to sewage sludge particles was discussed based on QSAR.
´
Citation: Jakimska A, Sliwka-Kaszynska M, Nagorski P, Namiesnik J, Kot-Wasik A (2014) Phototransformation of Amlodipine: Degradation Kinetics and
´
Identification of Its Photoproducts. PLoS ONE 9(10): e109206. doi:10.1371/journal.pone.0109206
´
´
Editor: Ryuichi Morishita, Osaka University Graduate School of Medicine, Japan
Received July 8, 2014; Accepted September 10, 2014; Published October 3, 2014
Copyright: ß 2014 Jakimska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This scientific work (as research project no. 2012/05/N/ST4/01991) has been financially supported within the framework of a grant awarded by the
Polish National Science Centre (in the years 2013–2014). AJ has been financially supported for the preparation of a doctoral dissertation (that includes the research
presented in the manuscript) within the funding of a doctoral scholarship awarded by the Polish National Science Centre on the basis of the decision number
2013/08/T/ST4/00637. The authors also acknowledge Professor Adriana Zaleska for providing access to xenon lamp used during the experiments. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: anik.jakimus@interia.pl
natural conditions (including sunlight, temperature or microor-
ganisms). The degradation process substantially reduces the
potential of a drug to induce a pharmacological effect, however,
Introduction
There are many research which focus on the presence of
pharmaceuticals in the aquatic environment and the removal
TPs may in some cases exhibit similar activity and be more stable
efficiency of these emerging contaminants during wastewater
than the parent compounds [5]. The stability of the compounds in
processes in wastewater treatment plants (WWTP) is limited [1].
the aquatic system plays a major role in determining their
However, the lack of the parent compound in the effluent is not
potential to cause negative effects in the environment. There is a
equal to its total elimination and/or lack of transformation
gap in the knowledge of TPs of various drugs and their impact on
products (TPs). During the wastewater treatment, or later in the
the environment and organisms as well [6]. Consequently, there is
environment itself various stable TPs may be formed under the
a clear need to investigate the environmental fate of different
influence of different factors and reactions. Therefore, it is
pharmaceuticals.
necessary to consider both parent compounds and products of
The compound selected for degradation study was amlodipine -
their transformation which appear in the aquatic environment.
a long-acting calcium channel blocker used to lower blood
The result of the chronic exposure to low doses of pharmaceu-
pressure and to treat anginal chest pain [7]. It is one of the most
ticals and their TPs still remain unknown. Their level in the
often prescribed drugs popularly known as Norvasc [8], and is
environment is not regulated, and thus these compounds are
used in minimum dose of 5 or 10 mg per day depending from the
considered environmentally hazardous [2]. It was found that some
therapy (mono- or combined therapy) [9]. It was observed that
drugs (e.g. ibuprofen) exhibited toxicity in relation to living
among people between 35–64 years about 30% of the US
organisms. Furthermore, it was proved that some of the formed
population and 44% of the European population suffer from
TPs were toxic as well [3,4]. Environmental degradation of
hypertension [10]. Additionally, it was estimated that around 1
xenobiotics depends on their structure as well as weather or
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Phototransformation of Amlodipine
billion people worldwide had hypertension by 2000, and it is
expected that this number would increase to 1.5 billion people by
2025 [11,12]. Even though, amlodipine undergoes oxidative
metabolism [9], it is partially excreted with urine in unchanged
(,6%) form [13] and can be found in e.g. hospital effluents or
wastewaters in up to tens of ng L21 [14]. Furthermore, the
published research showed that amlodipine causes toxic effect such
as inhibition of the regeneration of a hypostome, tentacles and foot
in Hydra vulgaris at the exposure level of 10 mg L21 [15]. The
other study revealed that the main photoproduct of amlodipine, its
pyridine derivative, exhibited a stronger toxic potential than the
parent drug in Ceriodaphnia dubia [16]. However, there is very
little information in the literature on amlodipine and its
transformation products. The compound formed by the aroma-
tization of the ring (pyridine derivative) is the only photoproducts
known and described in the literature so far [16,17,18]. A few
studies were performed for thermal degradation of amlodipine
showing the formation of 3 [19] or 5 main transformation
products [20].
analysis including fragmentation mass spectra of (pseudo-)molec-
ular ions, accurate mass measurements and isotope patterns of
each compound. Finally, the presence of amlodipine and its
transformation products was investigated in various environmental
waters. The application of QSAR allowed the estimation of
possible sorption of target compounds on sewage sludge particles.
Materials and Methods
Reagents
Analytical standards of amlodipine (AML) were purchased from
´
Sigma-Aldrich (Poznan, Poland). LC-MS grade methanol
´
(MeOH) was obtained from Sigma-Aldrich (Poznan, Poland).
Formic acid was from Merck (Warsaw, Poland). Ultrapure water
was obtained from a HLP5 system (Hydrolab, Poland). MeOH
used for extraction were LC grade and were purchased from
Merck (Warsaw, Poland). Individual stock solution of AML at
concentration level of 1 g L21 was prepared in methanol and was
stored at 280uC.
The most important processes which may occur in the aquatic
environment include biodegradation, photodegradation and hy-
drolysis. Biological processes allow only a limited transformation of
pharmaceuticals due to their biopersistance [21]. Biochemical
reactions can occur only in a certain way, whereas photochemical
processes are less predictable, particularly when radicals are
involved, which do not occur in the case of biochemical processes
[21,22]. Therefore, it can be expected that in the aquatic
environment photodegradation will be more important mecha-
nism of pharmaceuticals transformation [23,24], especially in case
of compounds which structure contains aromatic rings, heteroat-
oms and other functional groups that can absorb the sunlight
(direct photolysis) or react with active molecules NOH, 1O2, ROON,
The irradiation experiments
Identification of TPs of AML. Degradation experiments
were performed using a small-scale system that consisted of a
cylindrical photoreactor (25 mL) equipped with quartz window
and cooling system, external light system and optical filters. The
radiation was emitted by 1000W xenon lamp (627H, Oriel) which
transmitted light of wavelength ranged from 250 to 1000 nm.
Optical filters (UG1, Schott AG) were used for cutting off the IR
irradiation. This equipment was previously applied for identifica-
tion of phototransformation products of three other pharmaceu-
ticals (ketoprofen, ibuprofen and furosemide) in river water
samples [29] and by Go´rska et al. [30] for photodegradation of
phenol.
3DOM*, e2 generated by photosensitizers (indirect photolysis)
aq
[25,26].
The temperature of the samples during the experiment was
maintained at 10uC. The photodegradation experiment was
performed in river water (free from the targets) at three different
pH (3, 10 and no pH adjustment). The concentration of AML was
1 mg L21 since higher initial concentration provides TPs in higher
amount (this increases the possibility of proper identification and
structure elucidation of TPs). 25 mL of aqueous sample containing
AML was stirred magnetically during the irradiation experiment.
Aliquots of about 500 mL were collected during the experiment
every 15 minutes, filtered through a 0.45 mm nylon syringe filter
(Rockwood, USA) and directly subjected to UHPLC-QTOF-MS
analysis for further data evaluation.
Nowadays, investigation of suspected compounds e.g. TPs, their
structure elucidation and/or confirmation requires application of
highly accurate, selective and reliable techniques. To meet the
present expectations, the hyphenated techniques based on liquid
chromatography and high resolution mass spectrometry (LC-
HRMS) have become a powerful tools [27]. Such instruments as
LC- quadrupole time-of-flight mass spectrometry (LC-QTOF-MS)
or LC-Orbitrap-MS provide accurate mass measurements, infor-
mation on isotope pattern of a compound due to their high
resolution and full scan product ion spectra, and thus provide
structural information of the analyte [27,28].
In light of these concerns and the lack of scientific literature on
phototransformation of a widely prescribed drug, amlodipine, the
main aim of this study was to identify transfromation products
formed during the continuous exposure of amlodipine water
solution to UV/VIS irradiation emitted by xenon lamp. Further-
more, studies on photodegradation kinetics with the simulation of
the conditions in natural aqueous waters were undertaken. The
final analysis were performed with the application of LC-QTOF-
MS technique providing reliable results with mass error ,5 ppm.
To the best of our knowledge, this is the most extensive study on
photodegradation products of amlodipine including kinetics and
structure elucidation followed by the proposal of its phototrans-
formation pathway. As a result 16 degradation products were
identified. The first product identified was pyridine derivative of
amlodipine (known in the literature), and the other fifteen were
compounds formed from either the parent molecule or its first
degradation product and were reported here for the first time. The
proposed structures of newly identified compound were elucidated
and verified on the basis of data obtained during LC-QTOF-MS
Degradation kinetic studies. Photodegradation kinetics
were designed in two ways: (1) as a result of an exposure to
xenon lamp light (in order to ensure the independence of the
degradation process from the weather conditions that may affect
the experiment; xenon lamp emits condensed radiation, which
range overlaps with the range of solar radiation), (2) as a result of a
continuous exposure to solar radiation (in order to simulate
conditions similar to those in the natural environment).
Degradation under continuous exposure to solar radiation was
performed for samples characterized by eight different matrices:
wastewater influent and effluent, untreated and treated water,
river water, methanol, ultrapure water for pH 3 and pH 10. For
this purpose, specific matrix was spiked with AML at the
concentration level of 1 mg L21. The samples were kept in glass
vials (25 mL) exposed to natural sunlight; the experiment was
performed during the summer 2013 in order to provide sufficient
sunlight intensity. The aim was to investigate the influence of the
matrix and pH on degradation rate of AML.
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Phototransformation of Amlodipine
Table 1. Photodegradation rate constants (k) and half-lives (t1/2) of AML in various matrices exposed to solar or xenon lamp
irradiation.
Solar irradiation
Matrix
k (day21
)
t1/2 (day)
Wastewater influent
Wastewater effluent
River water
1.87
2.92
2.66
1.79
0.87
0.55
0.32
0.96
0.4
0.2
0.3
0.4
0.8
1.3
2.2
0.7
Untreated water
Treated water
Methanol
Ultrapure water pH 3
Ultrapure water pH 10
Xenon lamp irradiation
k (min21
)
t1/2 (min)
River water
0.08
8.8
doi:10.1371/journal.pone.0109206.t001
Degradation under xenon lamp irradiation was performed only
for river water samples (as well as for the identification of TPs). To
evaluate the degree of degradation of AML, UPLC-QTOF-MS
analysis was performed.
Samples were prepared by solid phase extraction (SPE) with the
application of Oasis HLB cartridges (500 mg, 6 mL; Waters). SPE
cartridges were preconditioned with 5 mL of MeOH and 5 mL of
ultrapure water. 1 mL 0.1 M EDTA/100 mL sample was added
before loading. A volume of 100 mL, 200 mL and 500 mL of
influent, effluent and river water, respectively, were loaded on the
sorbent at a flow rate of 5 mL min21 (if the sample contained solid
particles, it was first filtrated by a cellulose filter). Later, the sorbent
was dried for 15 min. The analytes retained on sorbent were
eluted with 6 mL (362 mL) of MeOH and obtained extracts were
evaporated to dryness in a stream of N2. Dry residues were
redissolved in 1 mL of MeOH/H2O (10:90, v/v) and 5 mL of the
final extract was injected into the UHPLC-QTOF-MS system.
UHPLC-QTOF-MS analysis
Both chromatographic conditions and mass spectrometer
parameters were previously described in other publication [29].
Briefly, analysis was performed on Agilent 1290 UHPLC system
with XTerra MS C18 Column (30 mm62.1 mm; 3.5 mm). The
mobile phase A was ultrapure water with 0.05% FA and B was
MeOH at a flow rate 0.4 ml min21 in gradient elution (95% A to
0% A in 15 min, back to 95% A in 1 min and kept at 95% A for
5 min). The column was maintained at 22uC and the injection
volume was 5 mL.
Results and Discussion
The UHPLC system was coupled to a hybrid quadrupole time-
of-flight (QTOF) mass spectrometer (Agilent 6540 Series Accurate
Mass QTOF-MS) with Dual ESI interface and operated in
positive ion mode. Operation conditions were as follow: sheath gas
temperature 400uC at flow rate of 12 L min21, capillary voltage
4000 V, nebulizer pressure 20 psig, drying gas 10 L min21, gas
temperature 325uC, skimmer voltage 45 V, octopole RF peak
750 V and fragmentor voltage 100 V. Analysis were performed
using MS/MS or TargetMS/MS mode with variable collision
energies (10, 20 or 30 V; however, 20 V was the optimal value for
all the target compounds) and in mass range 50–1000 m/z. The
instrument was operated in the 4 GHz high-resolution mode and
acquisition rate was 1.5 spectra per second. Acquisition data were
processed with Agilent MassHunter Workstation software.
Photodegradation kinetics of AML
The reaction kinetics included the calculation of the degrada-
tion rate and the assessment of the influence of external factors on
this parameter. This was determined based on the dependence of
the product formation/substrate decrease rate on initial concen-
tration of the substrate and the obtained relation allowed
determining the reaction kinetic equation. The degradation
reaction kinetics, i.e. the reaction order, the rate constant and
the half-life were determined for AML for the degradation process
induced by xenon lamp irradiation, as well as the degradation
process caused by continuous exposure to sunlight, for samples
characterized by various matrices. All experiments lasted until the
decrease of AML concentration at least below 10% of the initial
value. The results are presented in Table 1 and in Figure 1a.
The photodegradation of AML in river water induced by the
irradiation with xenon lamp followed a pseudo-first-order kinetic
(Table S1), which was determined by regression analysis, and the
time-based pseudo-first-order rate constant (k) was determined
according to the Eq. (1):
Sample collection and preparation
Aqueous samples collected from the Gdansk area were: influent
´
and effluent water samples from a wastewater treatment plant
(WWTP), and river water from Kacza River exposed to
anthropogenic activity. Samples were collected in pre-rinsed
amber glass bottles, filled to the brim in order to reduce analytes
transition to the gas phase. They were stored at 4uC and analyzed
within 24 h for the presence of AML and identified photoprod-
ucts.
Ct
ln ~{kt
C0
ð1Þ
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Phototransformation of Amlodipine
Figure 1. Kinetic curves for AML obtained for various conditions: exposure to xenon lamp irradiation (a) and exposure to natural
sunlight including eight different matrices (b).
doi:10.1371/journal.pone.0109206.g001
where Ct is the concentration of the compound at the irradiation
time t and C0 is the initial concentration of the analyte.
Half-life t1/2 was calculated using Eq. (2) which was derived by
modifying Eq. (1) and replacing Ct with C0/2:
The photodegradation of AML in various matrices induced by
the solar radiation followed pseudo-first-order kinetics as well
(Table S1). The parameters
k and t1/2 were determined
analogously to the xenon lamp experiment and the results are
presented in Table 1 and in Figure 1b. The degradation rate
depended not only on the structure of the compound, the type and
amount of functional groups, but also on the matrix. A significant
increase of degradation rate was observed for samples with
aqueous matrices compared to methanol. This is probably
associated with the radical reactions which occur in aqueous
medium (as a result of exposure to radiation in aqueous samples
reactive hydroxyl radicals are formed and react with the molecule
easily triggering the degradation of the compound). Decreasing the
pH of the water sample (acidic conditions) inhibited the
degradation process since it is a known method of inhibiting
microbiological activity, which limits the decomposition of
compounds.
ln2
t1=2
~
ð2Þ
k
Even though the signal from AML was not observed after
60 min of the exposure to xenon lamp light (t1/2 = 8.8 min), the
experiment was performed for additional 30 min (total irradiation
time was 90 min) in order to degrade the pyridine derivative of
AML, which was created after 15 min of the exposure, and to
identify additional transformation products. The obtained results
are consistent with the literature since the photodegradation
reactions induced by a constant irradiation are usually followed by
the first-order kinetics [31].
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Phototransformation of Amlodipine
The first signal that suggested the occurrence of the degradation
process was a decrease in the AML concentration. However, it
cannot be unequivocally stated that the observed changes in the
amount of the compound in the water samples were a result of its
strict degradation. Therefore, in order to confirm the degradation
of AML, the decrease of the AML concentration followed by the
identification of products formed during the irradiation experi-
ment was observed with the application of UHPLC-QTOF-MS.
formation compounds ranged from 24.5 to 5.3 ppm. The
accurate mass MS/MS spectra for identified photoproducts were
obtained by fragmenting the selected protonated molecule [M+
H]+ at specific m/z value. Based on the fragmentation patterns
observed in the MS/MS spectra the molecular formula and
structure of the products were then tentatively assigned. These
accurate mass measurements of both precursor and product ions
together with the fragmentation pattern of each photoproducts
obtained by UHPLC-QTOF-MS analysis were of high impor-
tance while verifying the structures and elucidating the transfor-
mation pathway of amlodipine transformation. In the main article
only two exemplary MS/MS spectra (for AML 1 and 2) are
presented; the other spectra (for AML 3 – AML 16) are presented
in supplementary materials (Figure S2).
Identification of photoproducts of AML
The identification experiment was performed under xenon
lamp irradiation providing independence of the formation of
degradation products from external conditions (e.g. variations of
temperature or light intensity) which may adversely affect the
identification procedure. Not every wavelength reaches the Earth
at the same intensity and the contribution of the low wavelengths
(below ,250 nm) is limited and dependent inter alia on the
latitude. In the aquatic environment direct and indirect photolysis
occurs simultaneously. The occurrence of direct photolysis
depends on the overlap of the absorption spectrum of the
compound with the wavelength range emitted by the radiation
source, and therefore, compounds with the absorption maxima in
the range of low wavelengths will more probably undergo to
indirect photolysis. However, in the case of a xenon lamp
irradiation the occurrence of direct photolysis is equally possible
because it emits radiation of constant intensity. Amlodipine has
two absorption maxima: 240 and 360 nm (Figure S1) and even
that only the second one overlap the xenon lamp wavelength
range (,250–1000 nm) it was considered sufficient to degrade the
analyte.
The first photoproduct found was amlodipine pyridine deriv-
ative (AML 1, m/z 407.1385). Formation of this product was
explained on the basis of a radical cation intermediate according
to Fasani’s investigation [17]. The structure of this compound was
confirmed by the fragment ions obtained during the MS/MS
fragmentation (Figure 3). Aromatization of the dihydropiridine
moiety is the first step of the amlodipine degradation and this
product seems to be the precursor of all further transformations.
This photodegradation product has been already identified in the
literature [16].
Xenon lamp irradiation of AML solution caused after 15
minutes formation of further seven new products. Formation of
AML 2 (m/z 389.1722) is postulated to occur from AML 1 by loss
of chlorine atom and hydroxylation of benzene ring by hydroxyl
radicals present in the aqueous environment under photolysis [34],
and its MS/MS spectrum together with the structures of each
fragment ion are presented in Figure 4. In the mass spectrum of
product AML 3, the most important fragment is the m/z
371.1613, which corresponds to the elimination of the chlorine
atom and loss of molecular hydrogen from the AML 1. The
structure of m/z 357.1461 (AML 4) may corresponds to the
aromatized amlodipine molecule after elimination of chlorine
atom and methyl group resulting formation of lactone fragment.
Another photoproduct detected after 15 minutes of the
exposure of water solution to xenon lamp is AML 5. This product
with the m/z value 348.1008 may correspond to the derivative
after loss of 2-aminoethenol fragment from AML 1 compound.
The most important fragment in the mass spectrum of product
AML 6 is m/z 330.1344 and may correspond to the disconnection
of chlorine atom and etheneamine from the AML 1 product.
Compound AML 7 with the m/z value 339.1351 was probably
formed by dehalogenation, oxidation and elimination of formal-
dehyde molecule from AML 1 product, whereas AML 8 (m/z
343.1298) results by loss of methyl substituent and transformation
of the methyl ester to carboxylic acid derivative, followed by
dehydrogenation.
The identification of photoproducts was performed only in river
water samples exposed to xenon lamp radiation. This matrix was
chosen for several reasons: river water is representative for the
aquatic environment, it is a source of drinking water, a place of
wastewater discharges and popular recreational place for people.
Identification of photoproducts was performed by UHPLC-
QTOF-MS analysis of river water sample spiked with AML
exposed to a xenon lamp radiation and collected at specified
intervals. Firstly, (pseudo-)molecular ions in the full scan acquisi-
tion mode which occurred during the experiment and could
respond to probable photodegradation products were selected.
Later, the analysis in TargetMS/MS mode was performed in
order to obtain the fragmentation spectra of selected compounds.
As a result, it was possible to verify the pre-selected compounds
and elucidate the structures of photodegradation products. In
order to provide reliable results acceptable accuracy mass
measurement error was below 5 ppm for (pseudo-)molecular ions
and 10 ppm for fragment ions. This analytical approach, including
specific identification evidence (e.g. MS, MS/MS, reference
standard, experimental data, etc.) as indicated by other authors
[32,33], is a safe way to very accurate identifications and helpful in
elucidating the structure of ‘predicted’ compounds, in particular
when the analytical standard is not available and the analyte is not
present in available databases [33].
Compound AML 9, which structure corresponds to the AML 3
byproduct after elimination of the furanylmethyl group (m/z
298.1074), was identified in the sample taken after 30 minutes of
continuous exposure of the amlodipine solution. Further irradia-
tion of amlodipine (45 min) revealed formation of another
degradation product (AML 10, m/z value 284.0917). This
compound was assumed to be the result of elimination of methyl
substituent from AML 9 photoproduct.
Xenon lamp irradiation of the studied AML resulted in the
formation of new chromatographic peaks corresponding to
transformation products generated during the photolysis process.
Xenon lamp irradiation of AML produced a further sixteen new
products (AML 1–16), and fifteen of them are reported here for
the first time. All photoproducts had lower molecular weights than
the parent compound and photoinduced dimerization was not
observed. Detailed results presenting the identified compounds are
listed in Figure 2. The experimental masses (m/z) deviation from
the theoretical molecular masses for protonated [M+H]+ trans-
In the mass spectrum of product AML 11, the most important
fragment is the m/z 329.1142, which may corresponds to the
hydrolysis of ethyl ester of AML 4 photoproduct.
The irradiation of a sample containing amlodipine in a water
solution acidified to the pH 3 led to the formation of another
compound AML 12. This product with the m/z value 381.1212
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Phototransformation of Amlodipine
Figure 2. Degradation products of amlodipine identified during the irradiation of river water samples by xenon lamp light.
doi:10.1371/journal.pone.0109206.g002
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Phototransformation of Amlodipine
may correspond to amlodipine derivative after hydrolysis of ethyl
ester.
wastewater effluent (average peak area 6 SD (n = 4) = 1,1E+
0562,0E+04). These results can be interpreted in various ways.
Amlodipine as a parent compound is rarely detected in surface
water and wastewater samples [35,36]; however, it was detected in
hospital effluents and in wastewaters from WWTPs at an average
concentration level ranged from 36.5 to 93.9 ng L21 [14].
Although, amlodipine is a common drug for lowering blood
pressure, it is metabolized [9] and excreted in unchanged form at
very low level. This might be the main reason of the non-detection
of amlodipine as parent compound in tested samples. Even when
AML is present in very small quantities its non-detection may be
related to the insufficient sensitivity of applied techniques.
Exposure of this drug solution to xenon lamp light in the water
at pH 10 caused formation of four photoproducts. Compound
AML 13 and AML 14 originated from amlodipine after
aromatization (AML 1); however, AML 15 and AML 16 were
formed directly from amlodipine. The most important fragment in
the mass spectrum of product AML 13 is the ion of m/z 379.1035,
which corresponds to hydrolysis of ethyl ester from the parent
molecule. The structure of m/z value 37.1261 (AML 14) may
correspond to AML 1 product after transformation of the methyl
ester group to aldehyde moiety. In the mass spectrum of product
AML 15, the most important fragment is the m/z 366.1107, which
may correspond to the elimination of etheneamine fragment from
the AML. The final compound detected in this solution (AML 16,
m/z value 352.0936) was probably formed by hydrolysis of methyl
ester from the AML 15 molecule.
However, there is a possibility that both parent compound and
TPs are partially adsorbed on the sewage sludge particles, which
are known to sorb certain organic compounds on their surface
[37]. The physical-chemical properties of AML and its TPs were
calculated using quantitative structure-activity relationships
(QSAR) (EPI Suite) [38], and the results are presented in Table 2.
It can be noticed that all values of octanol/water partition
coefficient, log Kow, are lower than 4, which indicate high
solubility in water of most of the examined compounds (except for
AML 5 which log Kow was 4.47). The calculations of soil
adsorption coefficient, log Koc, gave more specific results
suggesting low or negligible sorption to sewage sludge and
moderate or rapid migration to ground water for most of the
compounds. The exception was observed for AML 5 (log
Irradiation of amlodipine solution by the xenon lamp has been
terminating after 90 minutes. No signals originate from AML was
recorded, whereas the presence of several degradation products
were still detected in the examined solution.
On the basis of the degradation products identified in this
experiment the hypothetical amlodipine degradation pathway was
suggested, and schematically shown in Figure 5.
The occurrence of AML and its photoproducts in
environmental waters
K
oc = 3.44) indicating its moderate sorption do sludge particles,
and it was also observed in STP model, which estimated its
removal by sewage sludge on a level of 54%. An increased sorption
was noticed for AML 15 and AML 16 as well, however, it was
considered as insufficient to conclude that the sorption process was
the most dominant one in their removal.
The presence of amlodipine and identified compounds of its
phototransformation was investigated in wastewater (influent and
effluent) and river water in order to estimate the actual pollution of
the environment. The analysis was performed using UHPLC-
QTOF-MS system in TargetMS/MS mode. Confirmation of the
presence of degradation products was done by using information
obtained during the analysis and MS/MS fragmentation of
(pseudo-)molecular ion (compliance of retention time (deviation
,2%), mass accuracy of (pseudo-)molecular ion (,5 ppm) and
minimum two fragment ions (,10 ppm), and relative intensity of
fragment ions (deviation ,5%)).
Although, the predicted removal of AML and its photoproducts
through the sorption to sewage sludge particles was estimated on
the level below 6% for most of the analytes, the presence of AML
in the sludge was reported previously [36]. In order to determine
the actual degree of contamination of the environment it would be
valuable to perform further research on the presence of the parent
compound together with its TPs on various stages of wastewater
treatment and in ground water, where many organic contaminants
may migrate resulting in its contamination.
Photodegradation products of amlodipine were not detected in
the tested samples, except for AML 2 which was present in the
Figure 3. MS/MS spectrum of AML 1 with proposed structures of each fragment ion.
doi:10.1371/journal.pone.0109206.g003
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Phototransformation of Amlodipine
Figure 4. MS/MS spectrum of AML 2 with proposed structures of each fragment ion.
doi:10.1371/journal.pone.0109206.g004
The occurrence of only the photoproduct AML 2 may suggest
that the amlodipine was present in the wastewater; however, it
Conclusion
The photodegradation of amlodipine was investigated using
UHPLC-QTOF-MS. The degradation kinetics of AML was
determined for two types of experiments: with the exposure of
the samples to solar radiation, and with the application of xenon
lamp radiation. It was observed that all the reactions follow a
pseudo-first-order kinetics, and the time-based pseudo-first-order
rate constants (k) and half-lives (t1/2) were determined for both
degraded or migrated to other parts of the environment e.g.
ground water. Ongoing research confirms that tracking the fate of
not only amlodipine but also other pharmaceuticals in the aquatic
environment wider the knowledge of the environmental pollution.
Figure 5. Proposed photodegradation pathway of amlodipine.
doi:10.1371/journal.pone.0109206.g005
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Phototransformation of Amlodipine
Table 2. Various physical-chemical properties of AML and its photodegradation products, and their estimated removal with
sewage sludge based on QSAR (EPI Suite).
b
Compound
log Kow (estimated)a
log Koc
Removal by sludge (STP model), %
AML
2.07
2.72
1.60
1.94
0.42
4.47
2.36
1.55
20.07
2.16
1.62
21.13
20.41
1.18
2.61
3.53
3.24
2.30
2.36
1.91
1.93
1.09
3.44
1.86
2.00
0.82
2.17
1.87
20.47
20.29
0.81
2.14
2.29
1.43
5.57
3.8
AML 1
AML 2
AML 3
AML 4
AML 5
AML 6
AML 7
AML 8
AML 9
AML 10
AML 11
AML 12
AML 13
AML 14
AML 15
AML 16
1.91
2.10
1.77
53.93
2.65
1.89
1.76
2.32
1.92
1.76
1.76
1.82
3.35
13.55
8.19
aOctanol-water partition coefficient (criteria: log Kow ,1 highly soluble in water (hydrophilic);.4 not very soluble in water (hydrophobic)).
bSoil organic carbon-water partitioning coefficient (criteria: log Koc ,1.5 negligible sorption to sludge, rapid migration to ground water; 1.5–2.4 low sorption to sludge,
moderate migration to ground water; 2.5–3.4 moderate sorption to sludge, slow migration to ground water; 3.5–4.4 strong sorption to sludge, negligible to slow
migration to ground water).
doi:10.1371/journal.pone.0109206.t002
experiments. The identification of degradation products was the
main aim of this research, and it was performed with the
application of xenon lamp irradiation in order to independent the
experiment from the external conditions, which may influence the
experiment, and to provide a constant light source with a
wavelength range overlapping the natural sunlight wavelength.
As a result sixteen transformation products were identified, and 15
TPs are new compounds which structure was elucidated and
reported here for the first time. It was discovered that the
degradation process do not stop after the formation of the first
photoproduct (amlodipine pyridine derivative), but it is progress-
ing and reveal the existence of various compounds, some of them
are created from the first photoproduct. It was also discovered that
some TPs are formed in acidic (pH 3) or basic (pH 10) conditions.
As a result, transformation pathway of amlodipine was proposed.
Finally, the occurrence of AML and its photoproducts was
investigated in various water samples (wastewater influent and
effluent, river water). Only AML 2 was detected in four
wastewater effluent samples. Performing the QSAR proved that
sewage sludge adsorption is not a dominant process in AML
removal (except for AML 5, which sorption was estimated on level
of ,54% in STP model). In general, the values of log Kow were
below 4 indicating high solubility in water of most of the
compounds, and the results for log Koc suggested low or negligible
sorption to sewage sludge and moderate migration to ground
water. Further research on the presence of AML and its TPs at
various stages of wastewater treatment and in ground water should
be performed.
UHPLC-QTOF-MS is a very powerful tool for the identifica-
tion of TPs in the aqueous environment providing accurate mass
measurements, MS/MS fragmentation or isotope profiling which
are of a high significance while identifying degradation products
and elucidating/verifying their structure. To the best of our
knowledge this is the most extensive study on amlodipine
photodegradation published so far.
Supporting Information
Figure S1 Absorbance spectrum of amlodipine.
(TIF)
Figure S2 MS/MS fragmentation spectra of amlodipine
and its 16 photoproducts identified by UHPLC-QTOF-
MS.
(TIF)
Table S1 Detailed data used for photodegradation
kinetics calculations (initial concentration was treated
as 100%).
(DOCX)
Author Contributions
Conceived and designed the experiments: AJ AKW. Performed the
´
experiments: AJ. Analyzed the data: AJ MSK PN AKW. Contributed
´
reagents/materials/analysis tools: AJ JN AKW. Wrote the paper: AJ MSK
PN AKW.
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Phototransformation of Amlodipine
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