Structural characterization of photoproducts of pyrimethanil

Full text of DOI:10.1002/jms.3244

JMS letters
Received: 15 March 2013
Revised: 24 May 2013
Accepted: 25 May 2013
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/jms.3244
Structural characterization of photoproducts
of pyrimethanil
Aziz Kinani,a Ahmad Rifai,a,b Sophie Bourcier,a Farouk Jaberb and
Stéphane Bouchonneta*
Introduction
Material and methods
Pyrimethanil is an anilino-pyrimidine fungicide particularly active
against gray mold (Botrytis cinerea) and pear scab (Venturia
inaequalis and Venturia pirina) on grapes, strawberries, tomatoes,
fruits, vegetables and ornamental plants in greenhouses and
open field situation.[1–5] According to the European Food Safety
Authority, pyrimethanil has no evident mutagenic, genotoxic or
carcinogenic potential, but a short-term toxicity study on rats
and mice has shown an increase in liver weight accompanied
by changes in the histopathology of the liver and thyroid.[6] To
avoid these side effects, the concentration of pyrimethanil has
been limited by legislation. The European Commission set the
Maximum Residue Level of pyrimethanil at 10 mg/l in citrus fruits,
5 mg/l in pome fruits, strawberries, table and wine grapes and
0.05 mg/l in tree nuts (EC/600/2010).[7] It is thus important to
increase our knowledge regarding the concentration of
pyrimethanil in environmental matrices, its degradability and
the factors affecting it. One of these factors is the photo
alteration by sunlight, which is known to play a significant role
in the degradation of this compound, due to the prolonged
half-life (77 days approximately) of pyrimethanil in the environ-
ment.[8] Previous studies on the degradation of pyrimethanil
were carried out mainly in a waste water treatment context and
focused on the photo catalytic degradation using various salts
as catalysts. Agüera et al.[9] used TiO2, Vanni et al.[10] and Anfossi
et al.[11] added iron III, Navarro et al.[12] added ZnO and Gomis
et al.[13] used thiopyrylium. Irradiation was carried out using a
mercury or xenon lamp or direct sunlight. These studies have
Pure pyrimethanil (99%) and chromatographic grade solvents
(99.99% purity), methylene chloride (DCM), acetonitrile (ACN)
and formic acid (FA) were purchased from Sigma Aldrich (St
Quentin Fallavier, France). Taking into account the low solubil-
ity of pyrimethanil in water (121 mg lÀ1 at 25 °C), a first solution
was prepared at 100 mg lÀ1 in ACN. This solution was then
diluted to prepare working solutions in water at a concentra-
tion of 5.0 mg lÀ1 (i.e. 5% ACN/95% H2O). This high concentra-
tion was necessary to allow multi stage mass spectrometry
experiments on the photo products of low abundance.
According to the ROMIL technical report, ACN does not absorb
UV light since its transmission at 200 nm is about 100%.[15]
Consequently, ACN is expected not to affect the nature of
the photoproducts obtained during irradiation. This is the main
reason why it has been currently used to dissolve pyrimethanil
in previous studies devoted to photolysis. For instance, Gomis
et al. studied photocatalytic degradation pathways of
pyrimethanil in pure ACN.[13] Photolysis experiments were car-
ried out in a self-made reactor equipped with a high-pressure
mercury lamp (HPL-N 125 W/542 E27 SC; Phillips, Ivry-sur-Seine,
France) delivering radiation at wavelengths ranging from
200 nm to 650 nm. The absorbance spectrum of pyrimethanil
shows an absorption maximum between 260 and 280 nm.[16]
According to manufacturer data, the incident radiation flux
was 6200 lm. The reactor consists in six quartz tubes of
120 ml disposed in a circle around the lamp and immerged
into a sonicator (AL04-12, Advantage-Lab, Switzerland) filled
with deionized water. The solution of pyrimethanil was
degassed with a nitrogen stream prior to irradiation and was
sonicated during irradiation so that free oxygen is not
expected to be dissolved in water in significant amounts.
During experiments, the reactor was cooled by water circula-
tion to maintain a constant temperature of 25 3 °C. For each
experiment, 30 ml of a solution of pyrimethanil was used.
LC-MS/MS analysis was performed on a 2690 series HPLC
instrument coupled with a quadrupole time-of-flight ‘Premier’
showed that
a long irradiation time (between 150 and
1400 min, depending on the conditions and catalyst) is required
for total disappearance of pyrimethanil. Degradation of
pyrimethanil in water using the technique of photo-Fenton was
investigated by Sirtori et al. They reported that the addition of
sodium chloride accelerated the reaction and characterized four
chlorinated photo-Fenton by-products.[14] The aim of the present
study was the identification of UV–visible photo transformation
products of pyrimethanil in water. Liquid chromatography
coupled with tandem mass spectrometry (LC-MS/MS) and gas
chromatography coupled with multi stage mass spectrometry
(GC-MSn) were used for analysis, with the aim of covering a large
range of polarities for the detection of the potential transforma-
tion products. Elucidation of the structures of photoproducts
was carried out performing high-resolution measurements and
collision-induced dissociation (CID) experiments. Photolysis
mechanisms have also been proposed to explain the formation
of photo products of pyrimethanil in water.
mass spectrometer equipped with
a Z-spray electrospray
ionization (ESI) source (Waters Technologies, Saint Quentin en
Yvelines, France). Compounds were separated on a T 3.3 μm
analytical column C18 2.1 × 150 mm (Atlantis, France). The
*
Correspondence to: Stéphane Bouchonnet, Laboratoire des Mécanismes
Réactionnels UMR-7651, Ecole Polytechnique, 91128 Palaiseau Cedex, France.
E-mail: stephane.bouchonnet@polytechnique.edu
J. Mass Spectrom. 2013, 48, 983–987
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JMS letters
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Copyright © 2013 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2013, 48, 983–987

JMS letters
HPLC solvents were ACN with 0.1% FA (A) and water with 0.1%
FA (B). 10 μl were injected with a mobile phase flow of 0.2 ml
minÀ1. The following program of linear gradient was applied:
30% of A for 11.0 min and 30–100% of A from 11.1 to
20.0 min. The column was reconditioned with 30% of A from
20.1 to 30.0 min. Ions were recorded in positive ion mode in
the 50–750 domain. The ESI cone and capillary voltages were
set at 20 V and 3 kV, respectively. Extraction cone and ion guide
voltages were set at 3 V and 4 V, respectively. Source and
desolvation temperature were fixed at 120 °C and 450 °C.
Nebulization and desolvation were performed using nitrogen
gas. The cone gas flow was 70 l hÀ1, and the desolvation gas
flow was 700 l hÀ1. For CID experiments, argon was used as
collision gas with a flow of 0.28 ml minÀ1. The electrostatic mirror
was used in the W-mode for accurate m/z measurements. GC-MS
analyses were carried out on a Varian 450GC gas chromatograph
coupled with a Varian 240MS ion trap mass spectrometer; operating
conditions were reported in a recent paper.[17]
Results and discussion
This study is the first to report by-product structures obtained
by UV–visible irradiation of pyrimethanil without added cata-
lyst. Most of the previous studies devoted to pyrimethanil pho-
tolysis used solar irradiation,[18] a neon lamp[19,20] or a xenon
lamp with cutoff of irradiation under 290 nm.[21] Thus, based
on the UV–visible spectra of pyrimethanil (maximum absorp-
tion between 260 and 280 nm[16]), negligible photodegradation
was expected. In the present study, the light has not been
filtered, thus explaining why phototransformation products
were observed.
The relative abundances of by-products were determined
from times ranging from 15 min up to 8 h of irradiation. All
the photoproducts were detected in the solution irradiated
for 3 h; this solution was thus selected to carry out CID and
high-resolution experiments on photoproducts. At this time,
the degradation yield of pyrimethanil, estimated on the basis
of total ion currents, was about 60%. Eight photoproducts were
detected in LC-MS. Retention times, exact m/z ratios of molec-
ular ions and CID main fragment ions are reported in Table 1.
Only compound 1 and aniline in trace amounts were detected
in GC-MS; they were easily characterized owing to the NIST
spectra data base with matching scores greater than 94%.[22]
The first step of the transformation process was assumed to
correspond to the formation of a radical cation by electron
removal, as postulated in previous studies.[17,21,23] The second-
ary amine and all the double bonds of the system constitute
likely ionization sites: electron removal leads in all cases to a
highly delocalized radical cation. In aqueous solution, the
radical cation can be captured by a water molecule. The
resulting species can eliminate a proton to give a solvated free
radical, which can in turn undergo subsequent photoionization
to lead to a cation with an even number of electrons.
Three compounds referred as 6, 7 and 8 were detected with a
molecular weight of 215 amu at retention times of 5.97, 17.70 and
18.06 min, respectively. They likely result from hydroxylation of
pyrimethanil. Under CID, 7 and 8 provided an ion at m/z 77. This
ion, also detected for pyrimethanil, corresponds to the benzene ring
ionized C6H+5. It suggests that the benzene part of the molecule was
not affected in the formation of both compounds 7 and 8. H2O addi-
tion on ionized pyrimethanil followed by proton elimination leads to
J. Mass Spectrom. 2013, 48, 983–987
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JMS letters
Figure 1. Relative abundances of photoproducts as a function of the irradiation time.
Figure 2. Mechanisms postulated for the formation of compounds 1 to 8 from irradiated pyrimethanil.
the free radical referred as A in Fig. 2. Subsequent photoionization of
A leads to a cation with an even number of electrons on which water
addition followed by proton elimination leads to two isomeric struc-
tures with MW = 233 amu. These compounds were not detected in
this work, but they were reported in a previous study devoted to
the photo catalytic degradation of pyrimethanil.[11] Water elimination
from each of these structure leads to compounds 7 and 8 (Fig. 2). 7
corresponds to the only structure which can eliminate CO in mass
spectrometry, while 8 can eliminate HO. and H2O, leading to a stable
final state in both cases, and also H2CO (see Table 1). A cyclic struc-
ture has been postulated for 6 given its short elution time in compar-
ison with 7, 8 and pyrimethanil. Indeed, Hamilton reported that the
elution times in reverse-phase HPLC are correlated to the number
and the size of the rings and that cyclic structures elute before their
acyclic analogues.[24] In mass spectrometry, the pseudo molecular
ion 6H+ eliminates CO, as 7H+ does. Furthermore, Fig. 1 shows that
the formation of 6 follows the disappearance of 7, thus suggesting
that 6 likely arises from degradation of 7. A mechanism involving
photo ionization of 7 followed by cyclization and water molecule ad-
ditions has been postulated for the formation of 6 (see Fig. 2, right
hand).
Given that kinetics data for compound 4 (213 amu) match
those for 6 (see Fig. 1) and that 4H+ also eliminates CO, 4 was
assumed to result from dissociation of 7 by a mechanism
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J. Mass Spectrom. 2013, 48, 983–987

JMS letters
[2] A. Navalon, A. Prieto, L. Araujo, J. L. Vilchez. Determination of
pyrimethanil and kresoxim-methyl in green groceries by headspace
solid-phase microextraction and gas chromatography-mass spec-
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[3] X. H. Sun, Y. F. Liu, Z. C. Tan, Y. Y. Di, H. F. Wang, M. H. Wang. Heat ca-
pacity and enthalpy of fusion of pyrimethanil laurate (C24H37N3O2).
J. Chem. Thermodyn. 2004, 36, 895.
[4] X. Yu, L. Pan, G. Ying, R. S. Kookana. Enhanced and irreversible
sorption of pesticide pyrimethanil by soil amended with biochars.
J. Environ. Sci. (China) 2010, 22, 615.
[5] H. Zhao, Y. K. Kim, L. Huang, C. L. Xiao. Resistance to thiabendazole
and baseline sensitivity to fludioxonil and pyrimethanil in Botrytis
cinerea populations from apple and pear in Washington State.
Postharvest. Biol. Tec. 2010, 56, 12.
analogous to that postulated for the formation of 6 (Fig. 2, right
hand). The formation of compound 2 (MW 177) may be also ratio-
nalized by a mechanism following photoionization of 7, in com-
petition with that postulated for the formation of 6 and
involving a ring aperture induced by the positive charge (Fig. 2,
right hand).
Structures B and C in Fig. 2 result from a water molecule
addition on ionized pyrimethanil followed by proton elimina-
tion. Compound 1, also characterized in GC-MS (see above),
results from elimination of the cyclohexa-2,4-dienone radical
from B, while the loss of the radical pyrimidine radical from C
leads to aniline; both compounds have been detected in a
previous study.[9] Isomeric forms of B and C have also been
considered; they are not displayed in Fig. 2 as they lead,
according to the mechanisms postulated above, to molecules
which are not detected.
Kinetics data show that 3 (eluted at 3.3 min) is among the first
compounds that are formed under irradiation. Cyclization of ion-
ized pyrimethanil followed by proton elimination leads to a free
radical, whose subsequent photoionization followed by hydride
addition leads to 3. The structure postulated for 3 is in good
agreement with the consecutive losses of CH3CN, CH4 and CN ob-
served under collisional activation of the ion 3H+. Kinetics data
(see Fig. 1) show that compound 5 seems to be produced from
3. Furthermore, given their very close retention times (3.30 min
for 3 vs 3.34 min for 5) and their difference of two mass units
(MW = 199 for 3 vs. MW = 197 min for 5), 5 was postulated to result
from dehydrogenation of 3 after photoionization of the latter
according to the mechanism depicted in Fig. 2.
[6] EFSA. http://www.efsa.eu.int, 2006.
[7] E. COMMISSION. Vol. 9, Journal officiel de l'Union Européenne, 2010.
[8] INERIS. Pyrimethanil
- N° CAS 53112-28-0. Normes de qualité
environementale 2011.
[9] E. A. Ana Aguera, A. Tejedor, A. Fernandez-Alba. Photocatalytic pilot
scale degradation study of pyrimethanil and its main degradation
products in waters by means of solid-phase extraction followed by
gaz and liquid chromatography with mass spectrometry detection.
Environ. Sci. Technol. 2000, 34, 1563.
[10] A. Vanni, F. Fontana. Photodegradation of Pyrimethanil induced by
iron(III) in aqueous solutions. J. Environ. Monit. 2003, 5, 635.
[11] L. Anfossi, P. Sales, A. Vanni. Degradation of anilinopyrimidine
fungicides photoinduced by iron(III)-polycarboxylate complexes.
Pest Manag. Sci. 2006, 62, 872.
[12] S. Navarro, J. Fenoll, N. Vela, E. Ruiz, G. Navarro. Photocatalytic
degradation of eight pesticides in leaching water by use of ZnO
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[13] J. Gomis, A. Arques, A. M. Amat, M. L. Marin, M. A. Miranda. A mechanis-
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[14] C. Sirtori, A. Zapata, S. Malato, A. Agueera. Formation of chlorinated
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Conclusion
[15] M. Ashley. ROMIL technical report. Cambridge GB-CB5 9QT. www.
romil.com, 2006.
In conclusion, eight photo products were identified by HPLC-MS/
MS. Among them, compound 1 was the only one which was also
detected by GC-MS, with traces amounts of aniline. With the ex-
ception of compounds 1 and 7, the structures proposed for pho-
toproducts on the basis of mass spectra interpretation have not
been reported in previous studies.
[16] V. Feigenbrugel, S. Le Calve, P. Mirabel. Molar absorptivities of 2,4-D,
cymoxanil, fenpropidin, isoproturon and pyrimethanil in aqueous
solution in the near-UV. Spectrochim Acta A 2006, 63, 103.
[17] S. Coffinet, A. Rifai, C. Genty, Y. Souissi, S. Bourcier, M. Sablier, S.
Bouchonnet. Characterization of the photodegradation products of
metolachlor: structural elucidation, potential toxicity and persis-
tence. J. Mass Spectrom. 2012, 47, 1582.
[18] I. Oller, W. Gernjak, M. I. Maldonado, L. A. Perez-Estrada, J. A.
Sanchez-Perez, S. Malato. Solar photocatalytic degradation of some
hazardous water-soluble pesticides at pilot-plant scale. J. Hazard.
Mater. 2006, 138, 507.
[19] A. Vanni, F. Fontana, A. Cignetti, M. Gennari. Behaviour of
pyrimetranil in soil: Abiotic and biotic processes, Pesticide in air,
plant, soil & water system. Proceedings of the XII Symposium Pesticide
Chemistry, Piacenza, Italy, 4-6 June 2003 pp. 233-238.
[20] A. Vanni, L. Anfossi, A. Cignetti, A. Baglieri, M. Gennari. Degradation
of pyrimethanil in soil: Influence of light, oxygen, and microbial
activity. J. Environ. Sci. Health B 2006, 41, 67.
[21] T. Katagi. Photodegradation of pesticides on plant and soil surfaces.
Rev. Environ. Contam. Toxicol. 2004, 182, 1.
Yours,
AZIZ KINANI, AHMAD RIFAI, SOPHIE BOURCIER, FAROUK
JABER AND STÉPHANE BOUCHONNET
aLaboratoire des Mécanismes Réactionnels UMR-7651, Ecole
Polytechnique, 91128 Palaiseau Cedex, France
bLaboratoire d'Analyse des Pesticides et des Polluants Organiques -
Commission Libanaise de l'Energie Atomique - Centre National de
Recherches Scientifiques, Beirut, Lebanon
[22] NIST/EPA/NIH. NIST Mass Spectral Library. National Institute of
Standard and Technology. 2005, DC.
[23] D. O. J. S. Miller. Photolysis of polycyclic aromatic hydrocarbons in
water. Water Res. 2001, 35, 233.
[24] R. J. Hamilton. Blackie Academic and Professional. Thomson Science:
New york, 1998.
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J. Mass Spectrom. 2013, 48, 983–987
Copyright © 2013 John Wiley & Sons, Ltd.
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