Aminoparathion: A highly reactive metabolite of parathion. 1. Reactions with polyphenols and polyphenol oxidase

Article abstract of DOI:10.1021/jf051520m

Spiking of tomato and apple fruits with parathion at different levels of about 1-4 mg/kg irradiation and under simulated sunlight conditions resulted in nearly complete photodegradation within 13 h, but extractable parathion degradation products could not be found in any case. However, after irradiation of an unrealistically spiked apple (134 mg/kg) different photoproducts including aminoparathion (AP) were detectable by HPLC, proving that the hitherto postulated photochemistry of parathion indeed takes place in the fruit cuticle environment. Besides the photoreduction pathway it was shown for the first time that AP is also easily formed by reduction of the primary photoproduct nitrosoparathion with thiols (cysteine, glutathione), while ascorbic acid only leaves hydroxylaminoparathion. In the presence of polyphenols, AP was effectively bound to quinone intermediates formed by both silver oxide and polyphenol oxidases. For pyrocatechol, a disubstituted o-quinone derivative could be isolated as a dark red addition product and structurally be elucidated. However, in the presence of caffeic acid, catechol, naringin, and quercetin, respectively, insoluble dark colored polymers precipitated within 48 h, while in the supernatants AP was not detectable any more. Polymerbound and nonextractable AP was proven by transesterification with sodium ethoxide releasing O,O,O-triethyl thiophosphate which was determined by GC. Additionally, AP itself was a substrate for polyphenol oxidases, resulting in a quinone imine intermediate which in turn reacted with excessive AP yielding deep red colored di- and trimerization products.

Full text of DOI:10.1021/jf051520m

9140 J. Agric. Food Chem. 2005, 53, 9140−9145
Aminoparathion: A Highly Reactive Metabolite of Parathion.
1. Reactions with Polyphenols and Polyphenol Oxidase
BRUNO RUNG AND WOLFGANG SCHWACK*
Institut fu¨r Lebensmittelchemie, Universita¨t Hohenheim, Garbenstrasse 28,
D-70599 Stuttgart, Germany
Spiking of tomato and apple fruits with parathion at different levels of about 1-4 mg/kg irradiation
and under simulated sunlight conditions resulted in nearly complete photodegradation within 13 h,
but extractable parathion degradation products could not be found in any case. However, after
irradiation of an unrealistically spiked apple (134 mg/kg) different photoproducts including amino-
parathion (AP) were detectable by HPLC, proving that the hitherto postulated photochemistry of
parathion indeed takes place in the fruit cuticle environment. Besides the photoreduction pathway it
was shown for the first time that AP is also easily formed by reduction of the primary photoproduct
nitrosoparathion with thiols (cysteine, glutathione), while ascorbic acid only leaves hydroxylamino-
parathion. In the presence of polyphenols, AP was effectively bound to quinone intermediates formed
by both silver oxide and polyphenol oxidases. For pyrocatechol, a disubstituted o-quinone derivative
could be isolated as a dark red addition product and structurally be elucidated. However, in the
presence of caffeic acid, catechol, naringin, and quercetin, respectively, insoluble dark colored
polymers precipitated within 48 h, while in the supernatants AP was not detectable any more. Polymer-
bound and nonextractable AP was proven by transesterification with sodium ethoxide releasing O,O,O-
triethyl thiophosphate which was determined by GC. Additionally, AP itself was a substrate for
polyphenol oxidases, resulting in a quinone imine intermediate which in turn reacted with excessive
AP yielding deep red colored di- and trimerization products.
KEYWORDS: Parathion; nitrosoparathion; hydroxylaminoparathion; aminoparathion; polyphenols; polyphe-
nol oxidase; bound residues
INTRODUCTION
The organophosphorus insecticide parathion (1, Figure 1) is
known to be efficiently photoreduced in plant cuticle environ-
ments, yielding nitroso- (2), (hydroxylamino)- (3), and amino-
parathion (4) (1-4). Due to their high reactivity, it is rather
difficult to detect the reduction products both during in vitro
experiments and, for example, on fruits. However the photore-
duction was unequivocally deduced from the formation of
azoxyparathion (5) and azoparathion (6) (Figure 1) as conden-
sation products both in model experiments and on isolated fruit
cuticles (3). On the basis of the easy addition of nitrosoparathion
to unsaturated fatty acids (3-6), also the formation of fruit
cuticle-bound residues of parathion was recently shown (7).
Directly correlated to sunshine, parathion was readily degraded
during a field experiment on apples, while fruit cuticle-bound
residues were clearly determined by ELISA (8).
Figure 1. Products formed by the photoreduction of parathion (1)
(nitrosoparathion (2), (hydroxylamino)parathion (3), and aminoparathion
(4)) and the condensation products azoxyparathion (5) and azoparathion
(6), respectively.
that 1 cannot be recovered in high yields from spiked products
as juices after a certain period of time (12-14). Spiking of
grapes, apples, and tomatoes with 1 and then processing into
products such as wine, cider, and ketchup resulted in nearly
complete degradation, wheras only small residues of 4 could
not explain the disappearance of 1 (12-14). For instance, about
85% of 1 added to tomatoes (fortification level: 25 µg/g) was
lost during the processing steps, whereby only 1.7% of the inital
1 were detected together with low levels of 4 and p-nitrophenol
after 6 months of storage (14).
Besides photoreduction, another way is the reduction of the
phenylnitro group by the plant metabolism itself. Suzuki and
Uchiyama determined the formation of 2-4 in spinach homo-
genate fortified with 1 (9-11). Due to this fact and additionally
due to hydrolysis and oxidation reactions, it is not astonishing
* Correspondening author. Tel.: +49 (0)711 459 3979. Fax: +49 (0)-
711 459 4096. E-mail: wschwack@uni-hohenheim.de.
10.1021/jf051520m CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/15/2005

Aminoparathion
J. Agric. Food Chem., Vol. 53, No. 23, 2005 9141
controlled by PrepCon (Preparative HPLC Pump Control, ver 3.0, SCPA
GmbH, Stuhr, Germany), combined with an A0293 variable wavelength
monitor (Knauer, Berlin, Germany) and a column (Nucleosil RP18,
250 × 20 mm, 7 µm, Kronlab, Sinsheim, Germany) including a
precolumn (Nucleosil RP18, 50 × 20 mm, 7 µm). Data aquisition was
performed by a C-R3A Chromatopac integrator (Shimadzu, Duisburg,
Germany). The mobile phase consisted of 10 mM ammonium formate
buffer (pH 4.0) and methanol at a flow rate of 18 mL/min (gradient as
described above for analytical HPLC). The injection volume was 2
mL.
Gas Liquid Chromatography (GLC/FID). GLC was performed using
a PE8600 gas chromatograph (Perkin-Elmer, Rodgau, Germany)
equipped with a flame ionization detector (FID) and a J & W (Folsom,
CA) fused silica capillary column (30 m × 0.32 mm) wall-coated with
DB5 (0.25 µm film thickness). The oven temperature was programmed
to start at 100 °C, followed by an increase of 8 °C/min to 270 °C, held
for 10 min. Injector and detector temperatures were set at 270 °C;
injection (1 µL) was in the split mode (1:5). Helium was used as the
carrier gas with a column head pressure of 80 kPa.
Gas Liquid Chromatography/Mass Spectrometry Analysis (GLC/MS).
GLC/MS was performed on a Finnigan MAT (Bremen, Germany) Ion
Trap 800, 70 eV EI, coupled to a PE8420 gas chromatograph (Perkin-
Elmer, Rodgau, Germany) equipped a J & W (Folsom, CA) fused silica
capillary column (30 m × 0.25 mm) wall-coated with DB5-MS (0.25
µm film thickness). Oven temperature program was given above.
Helium was used as the carrier gas with a column head pressure of 80
kPa.
UV Spectroscopy. UV spectra were measured with a Perkin-Elmer
UV/vis spectrometer Lambda 2 (Perkin-Elmer, Rodgau, Germany).
IR Spectroscopy. IR spectra were measured with a Nicolet Avatar
320 ESP spectrometer (Nicolet, Offenbach aM, Germany) by means
of the ATR technique.
Derivatives of nitrosobenzene or aniline should also be
capable by polyphenols widely present in plants, mostly higher
concentrated in fruit peels as compared to fruit flesh (15). For
example, quercetin and phloretin glycosides appear in apple
peels in the range of 0.2-5 mg/g (16), and about 0.5 mg/g of
quercetin was found in tomato peels (17). Other polyphenolic
compounds such as chalconaringenin, naringenin, naringenin-
7-glucoside, and m- and p-coumaric acids, identified in the fruit
cuticles of tomato cultivars, reached amounts of up to 6% (total
phenolics 7-14%) of the cuticle mass in mature fruits (18).
Polyphenols are also substrates of the so-called enzymatic
browning (19, 20). By the action of polyphenol oxidases quinone
intermediates are formed, which in turn rapidly induce phenol
polymerization processes affording brown colored reaction
products. Additionally it is well-known that not only phenols
but also proteins are involved in these complex processes
(19-21). The nucleophilic amino or mercapto groups of proteins
are quite suitable reaction partners of quinones.
Therefore, the aim of the present research was to study if the
reactivity of the parathion reduction product aminoparathion is
a reason for the disappearance of parathion in processed fruits
and vegetables (12-14) and if phenol-bound or conjugated
residues of aminoparathion are formed.
MATERIALS AND METHODS
Reagents. The solvents used were of analytical grade (Merck,
Darmstadt, Germany) and distilled before use. Water was purified by
a Milli-Q 185 plus water purification system (Millipore, Bedford, MA).
Polyphenol oxidase was purchased from Worthington (Lakewood, NJ).
If not otherwise described, all chemicals used were obtained from Fluka
(Steinheim, Germany).
Nuclear Magnetic Resonance Spectroscopy (NMR). NMR spectra
were recordered on Varian Unity Inova-300 and Varian Unity Inova-
500 spectrometers (Varian, Darmstadt, Germany) at 300 and 500 MHz
(1H) as well as 75 and 125 MHz (13C), respectively, at room tem-
perature. Chemical shifts (δ) are given in ppm relative to tetrameth-
ylsilane; s ) singlet, d ) doublet, t ) triplet, q ) quartet, and m )
multiplet, with dd, dt, dq, and ddd denoting a combination.
Irradiation Equipment. The irradiation experiments in solution were
performed using a metal halogenide lamp SOL 500 (Dr. K. Ho¨nle AG,
Planegg, Germany) with a cutoff filter WG295 (Schott, Mainz,
Germany) and water-cooled quartz cuvettes. Fruits were irradiated
employing a suntest CPS+ (Heraeus, Kleinostheim, Germany) with
xenon lamp (250 W/m2, black standard temperature 35 °C, air-cooling).
Synthesis of O,O-Diethyl O-(4-Aminophenyl) Thiophosphate
(Aminoparathion, 4). Sodium tert-butoxide (25 mmol, 2.35 g) and
4-aminophenol (20 mmol, 2.18 g) were supended in 2-butanone (100
mL) and, under stirring, heated to 70 °C. O,O-Diethyl chlorothiophos-
phate (25 mmol, 4.72 g) dissolved in 2-butanone (20 mL) was added
dropwise, and the mixture was held at 70 °C for 2 h. After further
stirring at ambient temperature for 14 h, the reaction mixture was poured
into water (100 mL) and the crude product was extracted with diethyl
ether (50 mL, twice). The combined extracts were washed with sodium
hydroxide solution (0.02 mol/L, 400 mL) and brine (300 mL), dried
over sodium sulfate, and evaporated. The oily residue was purified by
preparative HPLC, yielding 3.72 g (71.3%) of 4 as clear and colorless
oil: UV/vis (methanol) λmax (nm) (log ꢀ) 203 (4.35), 238 (4.02), 292
(3.24); GLC/MS (EI, 70 eV) m/z ) 261 (65%), 233 (17%) [M -
C2H4]+, 205 (24%) [M - C2H4 - C2H4]+, 187 (5%), 125 (100%), 109
Instrumentation and Equipment. High Performance Liquid Chro-
matography (HPLC). A HP 1100 HPLC system was used, consisting
of a degasser, column oven, autosampler, gradient pump, and diode
array detector (DAD) module (Agilent, Waldbronn, Germany). Data
aquisition and processing was performed by HP ChemStation software
(rev A.04.02) with DAD detection wavelengths 235 nm (spectral
bandwidth (SBW) 8 nm), reference 500 nm (SBW 100 nm) and 505
nm (SBW 8 nm), and reference 600 nm (SBW 40 nm). A reversed
phase analytical column (5 µm Eurospher 100-C18 250 × 3 mm,
Knauer, Berlin, Germany) including a precolumn (Nucleosil 5 µm C18,
5 × 3 mm, Knauer, Berlin, Germany) was used at 25 °C. The mobile
phase consisted of 20 mM phosphate buffer pH 4.0 (A) and methanol
(B): gradient % B (t (min)): 60 (0)-80 (12)-90 (20)-90 (28)-60
(31)-60 (35); flow rate 0.5 mL/min; injection volume 10 µL.
High Performance Liquid Chromatography/Mass Spectrometry
(LC/MS). LC/MS analyses were performed on a HPLC system (as de-
scribed above), coupled to a VG platform II quadrupole mass spectrom-
eter (Micromass, Manchester, U.K.) equipped with an electrospray inter-
face (ESI). For data acquisition and processing, MassLynx 3.2 software
was used. The mobile phase consisted of 10 mM ammonium formate
buffer (pH 4.0) and methanol. The other conditions were as described
above. MS parameters: ESI+; source temperature 120 °C; capillary
3.0 kV; HV lens 0.5 kV; cone 55 V. For LC analysis, the MS was op-
erated in full-scan mode (m/z 200-1000). For accurate mass deter-
mination (22), data were collected in the multichannel acquisition
(MCA) mode with 128 channels per m/z unit using 11 scans (5 s) with
0.1 s reset time. The resolution was 1530 (10% valley definition). The
samples were dissolved in ammonium formate buffer (0.1 mol/L)/metha-
nol (1 + 1; v/v) containing poly(ethylene glycol) 400 (0.1 µg/µL), poly-
(propylene glycol) 425 (0.1 µg/µL). or poly(propylene glycol) 725 (0.1
µg/µL), respectively, as reference material; the sample concentrations
were similar to that of the reference standards. The solutions were intro-
duced into the ESI source (source temperature 80 °C) at a flow rate of
5 µL/min. With m/z scan ranges 220-350, 325-420, 440-604, and
555-720, five reference peaks could be used for mass calibration.
PreparatiVe HPLC. The preparative HPLC system comprised of two
HD2-200 pumps (Kronlab, Sinsheim, Germany), for gradient elution
1
(63%), 108 (61%), 97 (39%), 80 (65%); H NMR (DMSO-d6, 300
MHz) δ (ppm) 6.80 (m; 2 H), 6.51 (m; 2 H), 5.01 (4-NH2; s; 2 H),
4.12 (dq; 4 H; J ) 7.1/9.9 Hz), 1.25 (t; 6 H); 13C NMR (DMSO-d6, 75
MHz) δ (ppm) 146.28 (d; J ) 1.2 Hz), 140.53 (d; J ) 7.8 Hz), 121.18
(d; J ) 4.5 Hz), 114.25 (d; J ) 1.2 Hz), 64.62 (d; J ) 6.0 Hz), 15.74
(d; J ) 7.1 Hz).
Synthesis of O,O-Diethyl O-(4-Nitrosophenyl) Thiophosphate.
(Nitrosoparathion, 2). Aminoparathion (4) (9.6 mmol, 2.52 g) was
dissolved in 100 mL of methanol and cooled to -18 °C. To this solution
was added a cooled (-18 °C) solution of m-chloroperbenzoic acid (70%,
19.3 mmol, 4.76 g) in 15 mL of methanol stepwise, while stirring.

9142 J. Agric. Food Chem., Vol. 53, No. 23, 2005
Rung and Schwack
After 15 min 200 mL of water was added and the product extracted
twice with 50 mL of diethyl ether. The organic layer was washed with
300 mL of sodium carbonate solution (0.1 mol/L) and 300 mL of brine.
Purification performed by column chromatography on silica gel (25 g)
with petroleum ether/diethyl ether (1:1 v/v) as eluent yielded 637 mg
(24.0%) of 2 as a clear green oil: 1H NMR (DMSO-d6, 300 MHz) δ
(ppm) 8.03 (m; 2 H), 7.51 (m; 2 H), 4.25 (dq; 4 H; J ) 7.1/10.3 Hz),
1.31 (t; 6 H); 13C NMR (DMSO-d6, 75 MHz) δ (ppm) 163.94 (s),
156.01 (d; J ) 7.2 Hz), 121.90 (d; J ) 5.1 Hz), 123.32 (s), 65.58 (d;
J ) 6.0 Hz), 15.72 (d; J ) 7.2 Hz).
Synthesis of O,O-Diethyl O-(4-Hydroxyaminophenyl) Thiophos-
phate. (Hydroxylaminoparathion, 3). To a solution of 2 (0.07 mmol,
19.3 mg) in 3 mL of methanol was added a solution of ascorbic acid
in water (15.4 mg/mL), and the mixture was shaken for 5 min.
Purification performed by preparative HPLC yielded 6.0 mg (30.9%)
of 3 as a clear green oil: UV/vis (methanol) λmax (nm) (log ꢀ) 238
(3.96), 285 (3.08); IR (ATR) ν (cm-1) 3279 (w), 2982 (w), 2929 (w),
2907 (w), 1739 (w), 1597 (w), 1501 (m), 1442 (w), 1390 (w), 1291
(w), 1220 (w), 1200 (m), 1160 (m), 1099 (w), 1013 (s), 918 (s), 816
(s), 781 (s), 721 (w), 689 (w); accurate mass (mean of 8 measurements
( standard deviation) m/z ) 278.0625 ( 0.0007 [M + H]+ (calcd m/z
278.0616 for C10H17NO4PS); 1H NMR (DMSO-d6, 300 MHz) δ (ppm)
6.96 (m; 2 H), 6.80 (m; 2 H), 4.14 (dq; 4 H; J ) 7.1/10.0 Hz), 1.26 (t;
6 H); 13C NMR (DMSO-d6, 75 MHz) δ (ppm) 149.54 (s), 143.07 (d;
J ) 7.8 Hz), 120.86 (d; J ) 4.2 Hz), 113.75 (s), 64.76 (d; J ) 5.7 Hz),
15.75 (d; J ) 7.2 Hz).
Synthesis of O,O,O-Triethyl Thiophosphate (TETP). According
to the method described by Wettach et al. (7), the preparation of TETP
was performed from sodium ethoxide (11 mmol: 0.26 g of Na in 6
mL of ethanol) and O,O-diethyl chlorothiophosphate (8 mmol, 1.50
g). However, instead of heating the mixture was stirred at ambient
temperature for 14 h. Then the reaction mixture was poured into water
(50 mL) and extracted three times with 20 mL of diethyl ether. After
being dried over sodium sulfate and evaporation, without further
purification, 1.24 g (78.2%) of pure TETP was obtained as colorless
clear liquid: GLC/MS (EI, 70 eV) m/z ) 198 (76%), 170 (9%) [M -
C2H4]+, 153 (8%), 143 (19%), 142 (17%) [M - C2H4 - C2H4]+, 126
(26%), 121 (80%), 115 (50%), 114 (52%), 109 (33%), 97 (61%), 93
(72%), 81 (28%), 65 (100%).
Synthesis of O-(4-{[6-({4-[(Diethoxyphosphorothioyl)oxy]phenyl}-
amino)-3,4-dioxo-1,5-cyclohexadien-1-yl]amino}phenyl) O,O-Diethyl
Thiophosphate (7). Aminoparathion (4) (0.54 mmol, 140 mg) and
pyrocatechol (0.54 mmol, 59 mg) were dissolved in dried diethyl ether
(6 mL), and silver oxide (1.07 mmol, 248.6 mg) was added. After the
mixture was stirred for 30 min at ambient temperature, the solids were
filtered off and the solvent was evaporated. The crude residue was
purified by preparative HPLC and yielded 9.3 mg (5.5%) of 7 as a
dark red, strongly viscous liquid: UV/vis (methanol) λmax (nm) (log ꢀ)
224 (4.24), 262 (4.13), 303 (4.14), 433 (3.62); IR (ATR) ν (cm-1) 3277
(w), 2981 (w), 2916 (w), 2870 (w), 1741 (w), 1608 (m), 1585 (m),
1557 (m), 1504 (s), 1491 (s), 1407 (m), 1391 (m), 1336 (w), 1291 (w),
1197 (s), 1161 (s), 1097 (m), 1012 (s), 919 (s), 818 (s), 791 (s), 756
(m), 742 (m), 708 (m); LC/MS (ESI+) m/z (relative intensity) ) 627.07
(100%), 628.13 (29.2%), 629.12 (15.2%), 630.11 (3.8%), 631.10
(1.0%); accurate mass (mean of 6 measurements ( standard deviation)
m/z ) 627.1146 ( 0.0006 [M + H]+ (calcd m/z 627.1154 for
C26H33N2O8P2S2); 1H NMR (DMSO-d6, 300 MHz) δ (ppm) 7.22 (m; 8
H), 5.75 (s; 2 H), 4.21 (dq; 8 H; J ) 7.0/10.2 Hz), 1.30 (dt; 12 H; J )
0.6 Hz); 13C NMR (DMSO-d6, 75 MHz) δ (ppm) 147.25 (d; J ) 7.8
Hz), 121.56 (d; J ) 4.8 Hz), 97.24 (s), 65.04 (d; J ) 5.7 Hz), 15.73
(d; J ) 6.9 Hz). Note that some carbon signals are not detectable due
to rapid keto-enol tautomerisms of the ortho-quinoid ring system.
Synthesis of O-[4-({6-[(Diethoxyphosphorothioyl)oxy]-3-imino-
4-oxo-1,5-cyclohexadien-1-yl}amino)phenyl] O,O-Diethyl Thiophos-
phate (8), O-(2-Amino-3-oxo-3H-phenoxazin-7-yl) O,O-Diethyl Thio-
phosphate (9), and O-(4-{[3-Amino-4-({4-[(diethoxyphosphorothioyl)-
oxy]phenyl}amino)-6-oxo-2,4 -cyclohexadien-1-ylidene]amino}phenyl)
O,O-Diethyl Thiophosphate (10). To a solution of polyphenol oxidase
(PPO) in phosphate buffer pH 6.5 (25.5 mg/500 mL ) 74460 U/L;
PPO activity, 1460 U/mg of dry weight) a methanolic solution of 4
(93.3 mg/5 mL, 0.36 mmol) was added. The mixture was stirred
vigorously for 70 h and extracted with diethyl ether (200 mL, four
times). After drying over sodium sulfate and solvent evaporation, 8-10
were isolated by preparative HPLC. Whereas 8 was only stable in
solution and completely decomposed during isolation, 9 and 10 were
individually obtained in yields of 8.1 mg (11.9%) and 4.6 mg (6.2%),
respectively.
Compound 8: UV/vis (methanol) λmax (nm) 233, 300, 486; LC/MS
(ESI+) m/z (relative intensity) ) 535.14 (100%), 536.10 (25.2%),
537.06 (13.3%), 538.06 (2.8%), 539.06 (0.7%); accurate mass (mean
of 5 measurements ( standard deviation) m/z ) 535.0902 ( 0.0016
[M + H]+ (calcd 535.0891 for C20H29N2O7P2S2).
Compound 9: red crystals, UV/vis (methanol) λmax (nm) (log ꢀ) 237
(4.06), 437 (3.99); IR (ATR) ν (cm-1) 3445 (m), 3336 (m), 2980 (w),
2931 (w), 1712 (w), 1650 (w), 1584 (s), 1568 (s), 1504 (m), 1466 (s),
1449 (s), 1415 (s), 1400 (m), 1367 (m), 1312 (w), 1287 (m), 1245 (s),
1181 (s), 1163 (s), 1139 (m), 1118 (s), 1040 (m), 1012 (s), 959 (s),
897 (s), 868 (s), 852 (s), 843 (s), 812 (s), 793 (s), 765 (s), 741 (s), 696
(s), 659 (s); LC/MS (ESI+) m/z (relative intensity) ) 381.07 (100%),
382.11 (19.1%), 383.08 (7.3%), 384.09 (1.2%); accurate mass (mean
of 5 measurements ( standard deviation) m/z ) 381.0667 ( 0.0007
1
[M + H]+ (calcd 381.0674 for C16H18N2O5PS); H NMR (DMSO-d6,
500 MHz) δ (ppm) 7.73 (d; 1 H; J ) 8.8 Hz), 7.32 (dd; 1 H; J )
2.5/1.7 Hz), 7.21 (ddd; 1 H; J ) 1.5 Hz), 6.79 (NH2; s; 2 H), 6.37 (s;
1 H), 6.35 (s; 1 H), 4.24 (dq; 4 H; J ) 7.1/10.3 Hz), 1.31 (dt; 6 H; J
) 0.7 Hz); 13C NMR (DMSO-d6, 75 MHz) δ (ppm) 180.45 (s), 149.61
(d; J ) 7.5 Hz), 148.77/148.07/147.49 (s), 142.36 (d; J ) 1.0 Hz),
131.57 (d; J ) 1.8 Hz), 129.08 (d; J ) 1.0 Hz), 118.53 (d; J ) 5.1
Hz), 108.56 (d; J ) 5.4 Hz), 103.99 (s), 98.69 (s), 65.56 (d; J ) 6.0
Hz), 15.88 (d; J ) 7.2 Hz).
Compound 10: dark red, very viscous liquid, UV/vis (methanol)
λmax (nm) (log ꢀ) 233 (4.81), 268 (4.68), 350 (4.73), 532 (3.96); IR
(ATR) ν (cm-1) 2978 (w), 2929 (w), 2782 (w), 2701 (w), 1711 (m),
1574 (s), 1505 (s), 1374 (s), 1344 (s), 1257 (m), 1201 (s), 1159 (m),
1098 (w), 1020 (s), 924 (s), 820 (m), 791 (m), 760 (m) 694 (m); LC/
MS (ESI+) m/z (relative intensity) ) 626.14 (100%), 627.16 (30.6%),
628.10 (14.1%), 629.15 (3.6%), 630.12 (0.8%); accurate mass (mean
of 6 measurements ( standard deviation) m/z ) 626.1311 ( 0.0010
1
[M + H]+ (calcd 626.1313 for C26H34N3O7P2S2); H NMR (DMSO-
d6, 500 MHz) δ (ppm) 9.14 (NH; s; 1 H), 7.40/6.94 (m, m; 2 H, 2 H),
7.25/7.23 (m, m; 2 H, 2 H), 6.53 (NH2; s; 2 H), 5.67 (s; 1 H), 5.49 (s;
1 H), 4.21 (dq; 8 H; J ) 7.0/10.2 Hz), 1.31/1.30 (t; 12 H); 13C NMR
(DMSO-d6, 75 MHz) δ (ppm) 180.65 (s), 154.34/150.20/146.80 (s),
147.45, 135.66 (d; J ) 1.8 Hz), 147.11/146.48 (d, d; J ) 7.8 Hz),
125.26/121.79 (s, s), 121.60/121.31 (d, d; J ) 4.8 Hz), 96.46/89.03
(s), 65.06/64.98 (d, d; J ) 6.0 Hz), 15.77/15.74 (d, d; J ) 7.2 Hz).
Incubation of Aminoparathion with Polyphenols and PPO. To a
solution of 10-40 µmol of caffeic acid, catechol, pyrocatechol,
quercetin, or naringenin, respectively, in 0.25 mL of acetone were added
50 µL of a methanolic solution of aminoparathion (45.3 g/L), 3.75 mL
of water, and 1 mL of an aqueous PPO solution (1 g/L, 1460 U/mL).
Under occasional shaking the mixture was incubated at 25 °C for 24
h. After addition of 1 mL of methanol the mixture was centrifuged
(3000 min-1), the supernatant analyzed for aminoparathion by HPLC,
and the precipitate washed four times with about 1 mL of water and
lyophilized.
RESULTS AND DISCUSSION
Photoreduction of parathion 1 was postulated as stepwise
reduction of the nitro group (Figure 1) (1-3). Although 3 and
4 have not been found individually, their evidental formation
was proven by the condensation products 5 and 6, respectively.
During irradation of 1 in the presence of 2-propanol (1 g/L) as
model for hydroxylated cutin acids, now 3 and 4 could be
identified by means of HPLC, LC/MS, and comparison with
synthesized standards. After 6 h of irradiation, 3 increased to 4
mol % of the initial parathion, while 4 reached 0.25 mol %
only. Contrarily, after irradiation of 1 in cyclohexene (1 g/L)
as model for olefinic cuticle constitutents, only 5 and 6 could
be determined (data not shown).

Aminoparathion
J. Agric. Food Chem., Vol. 53, No. 23, 2005 9143
Figure 2. HPLC chromatogram of a fruit extract obtained after irradiation
of an apple fortified with parathion (1, 134 mg/kg) and irradiated for 5 h
(Suntest CPS+).
To examine the photochemical behavior of 1 on authentic
plant material, an unrealistically high fortified apple (134 mg/
kg) was irradiated, first. After 5 h of irradiation, the apple was
extracted with methanol and a mixture of cyclohexane, diethyl
ether, and butanone (50 + 45 + 5, v/v/v). The initial content
of 1 was decreased to 111 mg/kg, and 2 and 4-6 could be
detected in the extracts by means of HPLC (Figure 2). As on
fruit surfaces 4 can only be photochemically formed by
photoreduction of 3, the absence of 3 in the extracts can be
traced back to further reactions of 3 during the concentration
steps performed before analysis. Second, irradiation experiments
with tomatoes and apples were repeated at three lower fortifica-
tion levels, which also will be reached in the field. For spiking,
apples and tomatoes were dipped for 6 h in aqueous solutions
of parathion as described by Wettach et al. (7), yielding initial
levels of 0.70, 1.89, and 4.50 mg/kg for apples and 0.89, 1.28,
and 3.79 mg/kg for tomatoes, respectively. After an irradiation
time of 13 h more then 90% of the initial 1 levels could not be
extracted from the fruits, anymore (Figure 3). Due to the fact
that none of the known photoproducts were found by HPLC,
the disappeared 1 had to become cuticle-bound, which was
shown in former studies on enzymatically isolated cuticles by
immunochemical methods (7).
Suzuki and Uchiyama described the reduction of 1 by spinach
homogenate, yielding 2-4, depending on an electron transport
system (9-11). Although the authors identified the substances
by specific reactions, they never isolated the metabolites nor
specified constituents accountable for the reduction. Only the
initial step, the reduction of 1 into 2, was described as a
cytochrome-dependent reduction. In model systems we could
show that further reduction of the nitroso compound 2 is
independent from complex plant electron transport systems. In
the presence of ascorbic acid or thiols such as cysteine and
glutathione, 2 was easily reduced yielding 3 and 4, respectively.
The total conversion ensued in less than 7 h (Table 1). Ascorbic
acid, cysteine, and glutathione are common constituents of plants
and reach, for instance, up to 83, 30, and 17 mg/100 g of fresh
weight, respectively, in tomatoes (23). Therefore, it was shown
for the first time that the formation of 3 and 4 in plants does
not necessarily require light or enzymes but is simply a result
of reducing agents present naturally, accompanied by increasing
reactivity and further reactions with different plant constituents
as polyphenols, for example.
Figure 3. Extractable parathion (1) from tomatoes (i) and apples (ii) before
and after irradiation (13 h, Suntest CPS
(a c).
+) at different fortification levels
−
Table 1. Yields of (Hydroxylamino)parathion (3) and Aminoparathion
(4) during Reduction of Nitrosoparathion (2) with Ascorbic Acid,
Cysteine, and Glutathione (Data for Single Experiments)
react
reacn time (h)
2 (mol %)
3 (mol %)
4 (mol %)
cysteine
0.5
7.0
24.0
0.5
7.0
24.0
0.5
4.4
2.0
1.4
75.3
4.0
0.8
1.5
1.4
2.3
nda
nd
nd
nd
nd
69.0
71.6
72.3
2.1
37.0
60.6
nd
glutathione
nd
ascorbic acid
88.9
88.7
27.6
7.0
24.0
nd
nd
a nd: <0.5%.
as intermediates. The addition of nucleophiles, such as amines,
to these R,â-unsaturated carbonyl intermediates is well-known
by the formation of melanin, and therefore, the same reactivity
should be expected for 4. This hypothesis was first tested by
oxidation of pyrocatechol with silver oxide in the presence of
4. Already after a few minutes the color changed to red and 7
(Figure 4) could be isolated from the reaction mixture. To
check, if 7 is also formed during oxidation of pyrocatechol by
PPO, the PPO of potatoes was used exemplarily. Therefore, the
polyphenols were removed from potatoes as described by
Schaller (24), and pyrocatechol and 4 were incubated together
with the obtained polyphenol-free potato powder (25). As
expected, the color of the incubation turned to red and 7 could
be identified by HPLC, demonstrating that the reaction of 4
with pyrocatechol is also catalyzed enzymatically.
Polyphenols such as catechol, quercetin, flavanols, or antho-
cyanidins, ubiquitously occur in plant materials. Catalyzed by
polyphenol oxidase (PPO) polyphenols polymerize to mostly
dark brown macromolecules with ortho-benzoquinoid structures
Various authors described that not only phenolic compounds
but also aromatic amines such as aniline serve as substrates for
PPO, yielding ortho-benzoquinone imines (26, 27). Therefore,
the suitability of 4 as PPO substrate was tested by adding a

9144 J. Agric. Food Chem., Vol. 53, No. 23, 2005
Rung and Schwack
action of PPO. To finally test authentic plant material, we spiked
self-made apple juice with 4 (22 mg/L) and analyzed the juice
after a storage time of 2 months, whereafter free 4 could not be
determined anymore.
Conclusion. Disappearance of parathion from fruits and
vegetables in the field will mainly be caused by photoreduction
of the phenyl nitro group. However, due to the high reactivity
of the parathion reduction products, they cannot be found as
extractable residues. Besides the formerly known ene-type
addition of nitrosoparathion to cuticular lipids, different PPO-
catalyzed reactions of aminoparathion with or without polyphe-
nols give rise for the formation of hitherto unknown parathion
residue species which also cannot be analyzed by common
methods of residue analysis. It is principally to be expected that
the reaction types found for parathion are also transferable to
other nitroaryl pesticides.
Figure 4. Structure formulas of products formed by PPO-catalyzed
reactions of 4 (R ) −OPS(OC2H5)2).
Table 2. Recoveries of Pure Aminoparathion (4) and
Polyphenol-Bound 4, Determined as Triethyl Thiophosphate (TETP)
after Treatment with Sodium Ethoxide
conjugate
recovery as TETP (%)
ABBREVATIONS USED
aminoparathion
caffeic acid
Catechol
naringenin
pyrocatechol
quercetin
53.1 (
57.0 (
34.6b
34.6b
35.5b
21.7b
±
±
2.0%)a
2.5%)a
ATR, attenuated total reflection; PPO, polyphenol oxidase;
TETP, triethyl thiophosphate.
ACKNOWLEDGMENT
We thank S. Reeb, Dr. J. Conrad, and Dr. B. Vogler (Institute
of Chemistry, University of Hohenheim) for NMR measure-
ments and PD Dr. M. O. Lederer (Institute of Food Chemistry,
University of Hohenheim) for the GLC/MS measurements.
a n
)
4. b Data for single experiments.
methanolic solution of 4 to a phosphate buffered solution (pH
6.5) of PPO. After 24 h at ambient temperature, three red
reaction products (8-10, Figure 4) could be isolated. The
formation of 7-10 clearly shows that 4 coming into contact
with plant material can be transformed into a couple of hitherto
unknown products explaining the absence of 4 in residue
analyses.
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Received for review June 27, 2005. Revised manuscript received
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