Identification, Synthesis, and Safety Assessment of Thidiazuron [1-Phenyl-3-(1,2,3-thidiazol-5-yl)urea] and Its Metabolites in Kiwifruits

Article abstract of DOI:10.1021/acs.jafc.7b03522

The quality of kiwifruit became worse as a result of the abuse of plant growth regulators (PGRs). The safety of the fruits treated with PGRs also worried consumers. Therefore, the present study analyzed the structure of thidiazuron [TDZ, (1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea)] (1) and its metabolites of biotransformation in kiwifruits using liquid chromatography hybrid ion trap time-of-flight mass spectrometry (LC-IT-TOF-MS). Standard compounds were also synthesized and used for structural identification of those metabolites. In addition, cytotoxicity of TDZ and its metabolites was tested through sulforhodamine B assays against normal Chinese hamster ovary (CHO) cells. Four metabolites were identified. They were 4-hydroxy-thidiazuron (2), 3-hydroxy-thidiazuron (3), thidiazuron-4-O-β-d-glucoside (4), and thidiazuron-3-O-β-d-glucoside (5). Values of IC₅₀ of compounds 1, 2, and 3 to CHO cells were 18.3 ± 1.8, 37.56 ± 1.5, and 23.36 ± 1.59 μM, respectively. Compounds 4 and 5 had no effect on CHO cells.

Full text of DOI:10.1021/acs.jafc.7b03522

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Article
Identification, Synthesis and Safety Assessment of Thidiazuron (1-
phenyl-3-(1,2,3-thidiazol-5-yl)urea) and Its Metabolites in Kiwifruits
Zhiwei Zhang, haihua yang, Zhenhong Gao, Yahong Yuan, Jing Dong, Yuan Wang, and Tianli Yue
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03522 • Publication Date (Web): 16 Nov 2017
Downloaded from http://pubs.acs.org on November 19, 2017
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Identification, Synthesis and Safety Assessment of Thidiazuron
(1ꢀphenylꢀ3ꢀ(1,2,3ꢀthidiazolꢀ5ꢀyl)urea) and Its Metabolites in Kiwifruits
¶
¶
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Zhiwei Zhang,†, ‡, Haihua Yang, †, Zhenhong Gao,† Yahong Yuan,†, §, ζ Jing Dong,Ψ
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Yuan Wang†, Tianli Yue †, §, ζ*
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†College of Food Science and Engineering, Northwest A&F University, Shananxi
712100, China
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‡College of Food Science and Engineering, Qingdao Agricultural University, Qingdao
712100, China
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ΨShimadzu International Trading, Beijing 100020, China
§Laboratory of Quality & Safety Risk Assessment for Agroꢀproducts（YangLing），
Ministry of Agriculture，Yangling, Shaanxi 712100,China
ζNational Engineering Research Center of Agriculture Integration Test(Yangling),
Yangling, Shaanxi 712100,China
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¶The authors contribute equally to this work.
﹡Corresponding author,
Tel: +86ꢀ29ꢀ87092261 and Fax: +86ꢀ29ꢀ87092261; yuetl305@nwsuaf.edu.cn
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■ABSTRACT
Quality of kiwifruit became worse due to the abuse of plant growth regulators
(PGRs). The safety of the fruits treated with PGRs are also worried by consumers.
Therefore, the present study analysed the structure of thidiazuron (TDZ,
(1ꢀphenylꢀ3ꢀ(1,2,3ꢀthidiazolꢀ5ꢀyl)urea)) (1) and its metabolites of bioꢀtransformation
in kiwifruits by using liquid chromatography hybrid ion trap timeꢀofꢀflight mass
spectrometry (LCꢀITꢀTOFꢀMS). Standard compounds were also synthesized and used
for structural identification of those metabolites. In addition, cytotoxicity of
thidiazuron and its metabolites were tested through Sulforhodamine B assays against
normal Chinese hamster ovary (CHO) cells. Four metabolites were identified. They
were 4ꢀhydroxyꢀthidiazuron (2), 3ꢀhydroxyꢀthidiazuron (3), thidiazuron
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ꢀ4ꢀOꢀβꢀDꢀglucoside (4), and thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside (5). Values of IC50 of
compound 1, 2, and 3 to CHO cells were 18.3±1.8 ꢁM, 37.56±1.5 ꢁM, and
23.36±1.59 ꢁM, respectively. Compound 4 and 5 had no effect on CHO cells.
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KEYWORDS: Thidiazuron, Metabolites, LCMSꢀITꢀTOF, Safety Assessment
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■INTRODUCTION
Kiwifruit is rich in vitamin C and a famous fruit all over the world with its
special sweet, sour taste and exquisite flavor. China is the largest producer of kiwifruit
in the world, which occupies about 38 % of the total world amount1
. In recent years,
how to produce more safe and healthy kiwifruit has been raised more attention of
people because of pesticide residues pollution. For example, the phenylurea
herbicides and their metabolites have been studied, such as: diuron in surface water
and ground water2, bioꢀtransformed derivatives of chlortoluron and isoproturon3,
degradation products of chlortoluron4, degradation products of forchlorfenuron5 and
results of phenylurea herbicide photochemical behavior6.
Thidiazuron (1ꢀphenylꢀ3ꢀ(1,2,3ꢀthidiazolꢀ5ꢀyl)urea, TDZ) (Figure 1), a plant
growth regulator of phenylurea with the ability to enhance plant cell division and cell
expansion7, is applied to increase fruit size and weight8-12. This low dose (0ꢀ10 ppm)
of TDZ increased kiwifruit size without affecting fruit soluble solid content, flesh
firmness or concentration of nonstructural carbohydrates13, 14. In recent years,
presence of TDZ residues and their safety in kiwifruit and the environment have
increasingly caused concern15-17. Toxic properties may persist in metabolites, which
may even exhibit increased toxicity3. Therefore, understanding their degradation and
metabolism is a most interesting goal and challenge in kiwifruit.
In recent years, timeꢀofꢀflight (TOF) mass spectrometry has been applied
successfully to the global identification of target and nonꢀtarget components in herbal
medicine, component analysis, drug metabolite identification and degradation
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studies18-22. LCMSꢀITꢀTOF (Shimadzu) is a type of mass spectrometer that combines
ion trap and TOF (timeꢀofꢀflight) technologies. The instrument possesses some
advantages and functions, such as high speed，high accuracy MSn，formula predictions
software and so on. To check types of formed metabolites, plus detect both expected
and unexpected metabolites, MetID Solution can be used to compare both blank and
treated samples23-25
.
In this study, we used LCMSꢀITꢀTOF to discover the main metabolites of TDZ in
kiwifruit, and in turn, to identify these metabolites by the synthesis standard. Then,
cytotoxicity of TDZ and its metabolites were also evaluated..
■ MATERIALAND METHODS
General. Thidiazuron, 1, (purity>99%, Figure 1), high performance liquid
chromatography (HPLC) grade methanol and acetonitrile were provided by
SigmaꢀAldrich (St Louis, Missouri). Formic acid and ethyl acetate used were of
analytical grade (SigmaꢀAldrich, St Louis, Missouri). HPLCꢀgrade water was
obtained by MilliꢀQꢀPlus ultrapure water system from Millipore (Milford, MA).
Nuclear magnetic resonance spectra were recorded on an AVANCE III, 500 MHz
spectrometer (Bruker, Fällanden, Switzerland). Optical density (OD) was read on
iMark Microplate Reader (BioꢀRad, Richmond, CA). Commercial solvents and
reagents were of analytical grade SigmaꢀAldrich, St Louis, Missouri).
Plant material. The trial was carried out between 2014ꢀ2015, in Xi’an city,
Shaanxi province, China, in a 7ꢀyearꢀold kiwifruit orchard of Hayward from Shaanxi
Bairui Kiwi Fruit Research Institute Co. Ltd(34o03´N, 108o25´E). The vines were
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trained to the pergola system, with 4 m between vines within the row and 5 m
between rows. These experiments were designed according to the standard operating
procedures on pesticide registration residue field trials, issued by the Institute for the
Control of Agrochemicals, Ministry of Agriculture, China26. Fifteen kiwifruit vines
were selected, with uniform vegetative and reproductive characteristics. Fifteen days
after full bloom, all initial fruits of fifteen vines were dipped for 5 s in aqueous TDZ
solution (60 mg/kg), while the fruit of the other five vines were dipped in water only,
as a control set. After ninety days, representative treated and the control samples were
randomly were stored in a refrigerated room under normal atmosphere (T, ꢀ0.5±0.5 oC)
until used.
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Sample preparation. Briefly, 200 g of chopped and thoroughlyꢀhomogenized
sample was extracted ultrasonically with a solvent containing ethyl acetate (200 mL),
anhydrous magnesium sulfate (80 g) , and anhydrous sodium acetate (20 g) for 30 min.
The supernatant of the mixture was paperꢀfiltered and the solid residue was washed
twice with 60 mL of ethyl acetate. Solution were collected and concentrate to less
o
than 2 mL on a rotary evaporator (40 C and 250 mbar). Then the concentrate was
reconstituted in 2 mL of methanol, filtered through a microporous membrane (0.22
o
ꢁm), and stored at −40 C. The sample was prepared according to the method
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described in a previous paper20.
Chromatographic conditions. Liquid chromatography consisted of an LCꢀ20AD
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binary pump, DGUꢀ14A degasser, SILꢀ20AD autoꢀsampler, and CTOꢀ20AC column
oven (Shimadzu, Kyoto, Japan). The analytes were separated on a Shimꢀpack XR
ODS column (2.2 µm 3.0 mm×75 mm Shimadzu). The flow rate, injection volume,
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and column temperature were set to 0.2 mL/min, 20μL, 35oC, respectively. The
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mobile phase consisted of solvent A (acetonitrile) and B (0.1% formic acid in water).
A linear mobile phase gradient started at 90–30% B (0–4min), 30–0% B (4–6 min),
retained for 1 min, then quickly returned to initial 90% B, and maintained 3 min for
column equilibration.
MSn analyses were conducted on an LCꢀITꢀTOFꢀMS (Shimadzu, Kyoto, Japan)
equipped with an electrospray ionization (ESI) source, under the following optimized
operating conditions: positive ion mode, detector voltage at 1.6 kV, curved
o
o
desolvation line (CDL) temperature at 200 C, heat block temperature at 200 C,
nebulizing gas flow of 1.5 L/min, drying gas (N2) pressure of 100 kPa, and scan
ranges of m/z 100–1000 for MS1, m/z 100–800 for MS2 and m/z 100–800 for MS3.
Metabolite analyses were performed by MetID solution, ver. 1.1. Finally, the
Composition Formula Predictor software (Shimadzu) was used to provide chemical
formulae for TDZ and its metabolites.5
Cytotoxicity Assay. TDZ and four metabolites were evaluated for reproductive
cytotoxicity by sulforhodamine B (SRB) assay, using Chinese hamster ovary (CHO)27.
CHO cells were cultured in highꢀglucose DMEM medium, containing 10% (v/v) fetal
bovine serum (FBS), penicillin (100 KU/L) and streptomycin (100 μg/mL), at 37 ℃,
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in a 5% CO2 atmosphere with 95% (v/v) humidity. TDZ and its metabolites were
dissolved in DMSO, then diluted in culture media to the required concentration. The
DMSO content of the final concentration was below 0.1 %(v/v). Cytotoxicity of CHO
cells were determined by the SRB assay performed as described by Zhang Z.5
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Synthesis of the reference standards. The general synthetic routes of the
reference
standards
(4ꢀhydroxyꢀthidiazuron,
3ꢀhydroxyꢀthidiazuron,
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thidiazuronꢀ4ꢀOꢀβꢀDꢀglucoside, thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside) are outlined in Figure
2. Detail synthetic and purification procedures are the same as described by Zhang Z5,
characterization data are given in the supporting information. The NMR data are
showed as follows:
1
4ꢀhydroxyꢀthidiazuron (2) was a colorless solid, yield 71%. H NMR (500 MHz,
MeOD) δ 8.37 (s, 1H, ꢀCHN=N ), 7.15 (d, J = 8.8 Hz, 2H, ArꢀH), 6.73 – 6.61 (m,
2H, ArꢀH). 13C NMR (125 MHz, MeOD) δ, 155.882（ArꢀC）,δ 155.43（ꢀC=O）,
154.42（NꢀCꢀS）, 134.68（ꢀCN=N）, 130.94（ArꢀC）, 124.15（ArꢀC）, 116.67（ArꢀC）.
1
3ꢀhydroxyꢀthidiazuron (3) was a colorless solid, yield 75%. H NMR (500 MHz,
MeOD) δ, 8.42 (s, 1H, ꢀCHN=N), 7.11 (t, J = 8.1 Hz, 1H, ArꢀH), 7.01 (t, J = 2.2 Hz,
1H, ArꢀH), 6.88 (ddd, J = 8.0, 2.0, 0.8 Hz, 1H, ArꢀH), 6.55 (ddd, J = 8.1, 2.3, 0.7 Hz,
1H, ArꢀH). 13C NMR (125 MHz, MeOD) δ, 158.45（ArꢀC）, 154.40（ꢀC=O）, 152.97
（NꢀCꢀS）, 139.70（ArꢀC）, 134.24（ꢀCN=N）, 130.55（ArꢀC）, 111.85（ArꢀC）,
111.70（ArꢀC）, 107.62（ArꢀC）.
1
Thidiazuronꢀ4ꢀOꢀβꢀ
D
ꢀglucoside (4) was a colorless solid, yield 65%. H NMR
(500 MHz, MeOD) δ, 8.51 (s, 1H, ꢀCHN=N), 7.41 (d, J = 9.0 Hz, 2H, ArꢀH), 7.12 (d,
J = 9.0 Hz, 2H, ArꢀH), 4.88 (s, 1H, OꢀCHꢀO), 3.92 (dd, J = 12.1, 2.1 Hz, 1H,
ꢀCH2OH), 3.73 (dd, J = 12.0, 5.5 Hz, 1H, ꢀCH2OH), 3.52 – 3.39 (m, 4H ,
ꢀCH2CHOHꢀ, ꢀ CH2CHCHOH, ꢀCH2CHCHCHOH, ꢀOOHCCHOH). 13C NMR (125
MHz, MeOD) δ, 155.96（ArꢀC）, 155.36（ꢀC=O）, 154.13（NꢀCꢀS）, 134.77（ꢀCN=N）,
134.06（ArꢀC）, 122.92（ArꢀC）, 118.51（ArꢀC）, 102.89（OꢀCꢀO）, 78.31 (ꢀCH2CHOHꢀ),
78.13 (ꢀCH2CHCHOH), 75.07 (ꢀCH2CHCHCHOH), 71.54 (ꢀOOHCCHOH), 62.68
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Thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside (5) was a colorless solid, yield 63%. H NMR
(500 MHz, DMSO) δ, 9.54 (s, 1H, ꢀNH), 9.21 (s, 1H, ꢀNH), 8.91 (s, 1H, ꢀCHN=N),
7.27 (s, 1H, ArꢀH), 7.04 – 6.93 (m, 2H, ArꢀH), 6.33 (d, J = 6.8 Hz, 1H, ArꢀH), 5.65
(dd, J = 10.2, 7.4 Hz, 2H, OꢀCHꢀO, OH ), 5.39 (d, J = 5.0 Hz, 1H, OH), 5.26 (d, J =
5.5 Hz, 1H, OH), 4.73 (t, J = 5.6 Hz, 1H, OH), 3.80 (dt, J = 14.8, 7.4 Hz, 1H, ꢀCHOH),
3.74 (dd, J = 9.7, 5.9 Hz, 1H, ꢀCH2OH), 3.50 (q, J = 5.5 Hz, 2H, ꢀ CH2CHOHꢀ, ꢀ
CH2CHCHOH), 3.43 – 3.38 (m, 1H, ꢀ CH2CHCHCHOH), 3.29 (dt, J = 14.7, 7.3 Hz,
1H, ꢀOOHCCHOH). 13C NMR (125 MHz, DMSO) δ, 170.64（ArꢀC）, 163.18
169 （ꢀC=O）, 157.61（NꢀCꢀS）, 141.75（ArꢀC）, 129.14（ꢀCN=N）, 125.09（ArꢀC）,
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108.88（ArꢀC）, 108.61（ArꢀC）, 104.95（ArꢀC）, 94.33（OꢀCꢀO）, 80.64（ꢀCH2CHOHꢀ）,
76.45 (ꢀCH2CHCHOH）, 72.37（ꢀCH2CHCHCHOH）, 69.29（ꢀOOHCCHOH）, 60.69
172 （CH2）.
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■RESULTS AND DISCUSSION
Based on the published findings5, the proposed strategy for identifying and
assessing the safety of TDZ metabolites involves four procedural steps: TDZ
metabolites detected by total ion current (TIC) were recorded, searching for evident
differences between peaks of the blanks and those of treated samples. Obtained MS1
and MSn spectra of differences in peaks were used to deduce TDZ metabolites. The
standard substances were synthesized, and comparison between synthetic standard
substances and TDZ metabolites was performed. Safety of TDZ and its metabolites
were assessed.
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Fragmentations of TDZ. For the fragmentation pattern study, 200 µg/mL TDZ
(1) and four synthetic standard substances of its metabolites (2, 3, 4, 5), prepared in
methanol, were used. Extractꢀion chromatograms of 20 µg/mL TDZ and four synthetic
standard substances (2, 3, 4, 5) appear in Figure 3. The proposed fragmentation
patterns are illustrated in Figure 4. Figure S9 presents accurate mass measurements of
the protonated molecules and fragment ions of TDZ and four synthetic metabolites.
Table 1 shows data for TDZ and synthetic metabolites by LCꢀESIꢀIT/TOFꢀMS
analysis in positive ion mode.
Analysis of metabolites. The treated sample and the control sample were
analyzed by LCꢀESIꢀITꢀTOF/MS. In a search of possible metabolites, targeted data
analysis was carried out with the aid of MetID solution software. Extractꢀion
chromatograms of the treated sample and the control sample are depicted in Figure 5,
while Figure 6 shows mass spectra of 6ꢀ10 in positive ion mode. MSn of metabolite
data from TDZ in positive ion mode, by LCꢀESIꢀITꢀTOF/MS analysis, are presented
in Table 2.
Metabolite 6 (Figure 6a, Table 2，Table 2) eluting at 5.562 min, shows the
predominant protonated molecule ion [M+H]+ at m/z 221.0493 (error, 0.45ppm). 6
yields two main MS2 ions at m/z 127.9922 (error, 7.03 ppm), 102.0127 (error, 6.86
ppm). 6 calculates as C9H8N4OS according to the accurate mass by Formula Predictor
software. The difference of retention time between 1 and 6 was 0.009 min. So,
metabolite 6 was identified as 1.
Metabolite 7 (Figure 6b, Table 2) eluting at 4.242 min, shows the predominant
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protonated molecule ion [M+H]+ at m/z 237.0441 (error, 0 ppm). 7 yields three main
MS2 ions at m/z 127.9922 (error, 7.03 ppm), 110.0609 (error, 8.18 ppm), 102.0127
(error, 6.86 ppm). 7 calculates as C9H8N4O2S according to the accurate mass by
Formula Predictor software. The protonated molecule at m/z 237.0441 is 15.9949 Da
higher than the protonated molecule of TDZ (m/z 221.0492) corresponding to addition
of oxygen. In order to deduce the site of hydroxylation on 7 (m/z 221.0492), we
compared the MSn data of 7 with the corresponding data for TDZ. The MS2 ions of 7
at m/z 127.9922 and 102.0127 form through neutral loss of C6H7N and C7H5NO2;
therefore, the most likely hydroxylation position is located on the benzene ring. Based
on the results of the above analysis, standards (2, 3) of possible metabolites were
synthesized. As can be seen in Figure S9 (Figure 6b), 7 shares with 2 the common
MS1 ion at m/z 237.0441, MS2 ions at m/z 127.9922/102.0127 and m/z 110.0609 (error,
8.18 ppm). We conclude that 7 and 2 share the same fragment pathway. The retention
time difference for 7 and 2 is 0.002 min. Therefore, 7 is identified as 2
(4ꢀhydroxyꢀthidiazuron).
Metabolite 8 (Figure 6c, Table 2) eluting at 4.735 min, shows the predominant
protonated molecule ion [M+H]+ at m/z 237.0428 (error, ꢀ5.48 ppm). 8 yields three
main MS2 ions at m/z 127.9903 (error, ꢀ7.81 ppm), 110.0607 (error, 6.36 ppm),
102.0115 (error, ꢀ4.90 ppm). Using the accurate mass by the Formula Predictor
software, 8 calculates as C9H8N4O2S. The protonated molecule at m/z 237.0428 is
15.9936 Da higher than the protonated molecule of TDZ (m/z 221.0492). Based on the
analysis result for 7, the most likely hydroxylation position for 8 is at the benzene ring.
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The retention time difference for 8 and 3 is 0.005 min. Therefore, 8 is identified as 3
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(3ꢀhydroxyꢀthidiazuron).
Metabolite 9 (Figure 6d, Table 2) shows the protonated molecular ion [M+H]+ at
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m/z 399.0969 with mass error of 0 ppm, retention time 2.892 min. 9 yields four main
MS2 ions at m/z 237.0423 (error, ꢀ7.59 ppm), 127.9914 (error, 0.78 ppm), 110.0608
(error, 7.27 ppm), 102.0127 (error, 6.86 ppm). The fragment ion at m/z 237.0423 leads
to a MS3 product ions at m/z 102.0117 (error, ꢀ2.94 ppm). In comparison with the
protonated molecule of 2 (3), 9 represents an increase of 162.0528 Da (C6H10O5). 9
calculates as C15H18N4O7S, according to the accurate mass by Formula Predictor
software. Thus, we deduce that 9 is a glycosylated metabolite of 2 (3). Comparing the
data of 9 and the standard substances (4, 5), the retention time difference for 9 and 4
is 0.009 min. Therefore, 9 is identified as 4 (thidiazuronꢀ4ꢀOꢀβꢀDꢀglucoside).
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Metabolite 10 (Figure 6e, Table 2), shows the protonated molecular ion [M+H]+
at m/z 399.0967 with mass error of ꢀ0.5 ppm, and representing an increase of
162.0537 Da (C6H10O5), compared to the spectra of MS2 at m/z 237.0430. The
fragment ion at m/z 237.0430 (error, ꢀ4.64 ppm) leads to three MS3 product ions at
m/z 127.9908 (error, ꢀ3.91 ppm), 110.0604 (error, 3.63 ppm), 102.0112 (error, ꢀ7.84
ppm). 10 calculates to be C15H18N4O7S, and thus the glycosylated metabolite of 2, 3.
As can be seen in Table 1, Table 2, the retention time error is 0.008 min between 5
and 10. Therefore, 10 is identified as 5 (thidiazuronꢀ3ꢀOꢀβꢀ
Except for four metabolites of TDZ (4ꢀhydroxyꢀthidiazuron(2),
3ꢀhydroxyꢀthidiazuron(3), thidiazuronꢀ4ꢀOꢀβꢀDꢀglucoside(4), and
Dꢀglucoside).
thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside(5) were synthesized, which are meta or para
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derivatives, we have tried our best and used different methods to find out and
synthesize ortho substituted derivatives, but no results are available until now. These
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derivatives may be discovered and synthesized by other researchers in future studies.
Cytotoxicity. Cytotoxic action is expressed as the value of 50% inhibition
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concentration. Cytotoxicity to CHO cells from high to low was: 1 (IC50=18.3±1.8
ꢁM), 3(IC50=23.36±1.59 ꢁM), 2(IC50=37.56±1.5 ꢁM), while 4 and 5 exhibit no
activity in CHO. Hydroxylation reduced cytotoxicity of TDZ, whereas glycosylation
resulted in loss of cytotoxicity. TDZ improve fruit size and weight, increase farmer
income, but produce some hazards to food safety. As a researcher, we ought to breed
new varieties instead of spraying in solution of plant growth regulator.
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Supporting Information Available:
1H, 13C NMR spectra of 2, 3, 4, 5 are provided in electronic supporting information.
This information is available free of charge via the Internet.
The supporting information consists of the following information:
Figure S1. 1H NMR spectra of 4ꢀhydroxyꢀthidiazuron(2)
Figure S2. 13C NMR spectra of 4ꢀhydroxyꢀthidiazuron(2)
Figure S3. 1H NMR spectra of 3-hydroxy-thidiazuron (3)
Figure S4. 13C NMR spectra of 3-hydroxy-thidiazuron (3)
Figure S5. 1H NMR spectra of thidiazuron ꢀ4ꢀOꢀβꢀ
Figure S6. 13C NMR spectra of thidiazuron ꢀ4ꢀOꢀβꢀ
Figure S7. 1H NMR spectra of thidiazuronꢀ3ꢀOꢀβꢀ
Figure S8. 13C NMR spectra of thidiazuronꢀ3ꢀOꢀβꢀ
Dꢀglucoside (4)
Dꢀglucoside (4)
Dꢀglucoside(5)
D
ꢀglucoside(5)
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Figure S9. Mass spectra of the synthetic standards in positive ion mode
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Funding
Investigation and product safety assessment of plant growth regulators in fruits and
vegetables (GJFP201601403);
Research on key technology of safety hazard factor identification control in shaanxi
fruit and vegetable food (2016KTCQ03ꢀ12);
Qingdao Agricultural University High ꢀ level Talent Fund (6631115045)
Notes The authors declare no competing financial interest.
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11. PerezꢀBarraza, M. H.; OsunaꢀEnciso, T.; SantiagoꢀCruz, M. D. J.; AvitiaꢀGarcia,
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13. Famiani, F.; Battistelli, A.; Moscatello, S.; Boco, M.; Gardi, T.; Proietti, S.;
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currentꢀyear fruit size and production in Actinidia deliciosa without decreasing return
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16. Shao, J. L.; Fan, J. L.; Lin, T.; Yang, D. S.; Zou, Y. H.; Wang, L.; Liu, H. C.,
Determination of 5 plant growth regulators residues in fruits and vegetables by high
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Dispersive liquidꢀliquid microextraction for the determination of three cytokinin
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Residual determination of clothianidin and its metabolites in three minor crops via
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screening strategies for organic contaminants, residues and illicit substances in food,
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22. Hernández, F.; Grimalt, S.; Pozo, Ó. J.; Sancho, J. V., Use of ultraꢀhighꢀpressure
liquid chromatography–quadrupole timeꢀofꢀflight MS to discover the presence of
pesticide metabolites in food samples. J Sep Sci. 2009, 32, 2245ꢀ2261.
23. Liang, Y.; Hao, H.; Xie, L.; Kang, A.; Xie, T.; Zheng, X.; Dai, C.; Hao, K.; Sheng,
L.; Wang, G., Development of a systematic approach to identify metabolites for
herbal homologs based on liquid chromatography hybrid ion trap timeꢀofꢀflight mass
spectrometry: genderꢀrelated difference in metabolism of Schisandra lignans in rats.
Drug Metab Dispos. 2010, 38, 1747ꢀ1759.
24. Liang, Y.; Xiao, W.; Dai, C.; Xie, L.; Ding, G.; Wang, G.; Meng, Z.; Zhang, J.;
Kang, A.; Xie, T., Structural identification of the metabolites for strictosamide in rats
bile by an ion trapꢀTOF mass spectrometer and mass defect filter technique. J
Chromatogr B. 2011, 879, 1819ꢀ1822.
25. Liu, Y.; Kou, Y.; Xue, M.; Xu, Y.; He, L.; Ruan, J.; Liu, K., Structural elucidation
of in vivo metabolites of phencynonate and its analogue thiencynonate in rats by
HPLCꢀESIꢀMSn. Talanta. 2010, 82, 1200ꢀ1211.
26. Wang, Y., Standard Operating Procedures on Pesticide Registration Residue Field
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27. Bertheussen, K.; Yousef, M. I.; Figenschau, Y., A new sensitive cell culture test
for the assessment of pesticide toxicity. J Environ Sci Heal B. 1997, 32, 195ꢀ211.
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FIGURE CAPTIONS
Figure 1. Structures of thidiazuron and synthetically prepared metabolites of
thidiazuron (numbers of the compounds refer to Table 1).
Figure 2. Synthetic reagents, conditions, and route of thidiazuron metabolites (a).
80 ℃, 8 h, (b) BBr3, CH2Cl2, ꢀ30 ℃, 12 h, (c) TBAB, K2CO3, CHCl3, 50 ℃, 10 h, (d)
NH3·H2O, CH3OH, rt. ,8 h.
Figure 3. Extracted ion chromatograms of the synthetic standards of thidiazuron
metabolites (numbers of the compounds refer to Table 1).
Figure 4. The fragmentation pattern of (A) 1, (B) 2 and 3, (C) 4 and 5 (numbers of
the compounds refer to Table 1).
Figure 5. Extracted ion chromatograms of thidiazuron and its metabolites in the
treated and control kiwifruit samples (numbers of the compounds refer to Table 2
Figure 6. Mass spectra analysis of thidiazuron and its metabolites in kiwifruits in
positive ion mode (a, 6; b, 7; c, 8; d, 9; e, 10; numbers of the compounds refer to
Table 2).
).
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Table 1 Composition, retention rime and mass spectral fragmentation dataa of
synthetically prepared metabolites of thidiazuron
RTb
Theoretical
Elemental
[M+H]+
(m/z)
Error
(ppm)
ꢀ1.81
NO.
1c
MSn
(min)
5.553
Value (m/z) compositions
221.0492
C9H8N4OS
1ꢀMS1
221.0488
127.9913
102.0120
94.0651
C3H3N3OS
C2H3N3S
C6H7N
1ꢀMS2
1ꢀMS2
1ꢀMS2
2 MS1
2 MS2
2 MS2
2 MS2
3 MS1
3 MS2
3 MS2
3 MS2
127.9919
102.0115
94.0657
4.69
ꢀ4.90
6.38
ꢀ6.33
ꢀ6.25
5.45
6.86
ꢀ6.75
4.69
ꢀ3.63
ꢀ5.88
ꢀ2.00
ꢀ4.22
3.13
2.73
2.94
2.34
ꢀ2.73
ꢀ6.86
ꢀ4.26
ꢀ2.53
0.78
7.29
6.86
ꢀ3.91
4.54
5.88
2
3
4
4.240
4.740
2.883
237.0441
127.9913
110.0600
102.0120
237.0441
127.9913
110.0600
102.0120
399.0969
237.0441
127.9913
110.0600
102.0120
127.9913
110.0600
102.0120
399.0969
237.0441
127.9913
110.0600
102.0120
127.9913
110.0600
102.0120
C6H8N4O2S
C3H3N3OS
C6H7NO
237.0426
127.9905
110.0606
102.0127
237.0425
127.9919
110.0596
102.0114
399.0961
237.0431
127.9917
110.0603
102.0123
127.9916
110.0597
102.0113
399.0952
237.0435
127.9914
110.0608
102.0127
127.9908
110.0605
102.0126
C2H3N3S
C6H8N4O2S
C3H3N3OS
C6H7NO
C2H3N3S
C15H18N4O7S 4 MS1
C6H8N4O2S
C3H3N3OS
C6H7NO
4 MS2
4 MS2
4 MS2
4 MS2
4 MS3
4 MS3
4 MS3
C2H3N3S
C3H3N3OS
C6H7NO
C2H3N3S
C15H18N4O7S 5 MS1
5
3.325
C6H8N4O2S
C3H3N3OS
C6H7NO
5 MS2
5 MS2
5 MS2
5 MS2
5 MS3
5 MS3
5 MS3
C2H3N3S
C3H3N3OS
C6H7NO
C2H3N3S
465
466
467
a The instrumentation: LCMSꢀITꢀTOF, conditions: positive ion mode; b RT = retention time; c1,
thidiazuron; 2, 4ꢀhydroxyꢀthidiazuron; 3, 3ꢀhydroxyꢀthidiazuron; 4,
thidiazuronꢀ4ꢀOꢀβꢀDꢀglucoside; 5, thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside.
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Table 2 Composition, retention rime and mass spectral fragmentation dataa of
thidiazuron metabolites in kiwifruits
RTb
Theoretical
Elemental
[M+H]+
(m/z)
Error
(ppm)
0.45
NO.
6c
MSn
(min)
5.562
Value (m/z) compositions
221.0492
C9H8N4OS
6ꢀMS1
221.0493
127.9913
102.0120
237.0441
127.9913
110.0600
102.0120
237.0441
127.9913
110.0600
102.0120
399.0969
237.0441
127.9913
110.0600
102.0120
102.0120
399.0969
237.0441
127.9913
110.0600
102.0120
127.9913
110.0600
102.0120
C3H3N3OS
C2H3N3S
6ꢀMS2
6ꢀMS2
7 MS1
7MS2
7 MS2
7 MS2
8 MS1
8 MS2
8 MS2
8 MS2
127.9922
102.0127
237.0441
127.9922
110.0609
102.0127
237.0428
127.9903
110.0607
102.0115
399.0969
237.0423
127.9914
110.0608
102.0127
102.0117
399.0967
237.0430
127.9918
110.0603
102.0121
127.9908
110.0604
102.0112
7.03
6.86
0
7
8
9
4.242
4.735
2.892
C6H8N4O2S
C3H3N3OS
C6H7NO
7.03
8.18
6.86
ꢀ5.48
ꢀ7.81
6.36
ꢀ4.90
0
C2H3N3S
C6H8N4O2S
C3H3N3OS
C6H7NO
C2H3N3S
C15H18N4O7S 9 MS1
C6H8N4O2S
C3H3N3OS
C6H7NO
9 MS2
9 MS2
9MS2
9 MS2
9MS3
ꢀ7.59
0.78
7.27
6.86
ꢀ2.94
ꢀ0.50
ꢀ4.64
3.91
2.73
0.98
ꢀ3.91
3.63
ꢀ7.84
C2H3N3S
C2H3N3S
C15H18N4O7S 10 MS1
10
3.317
C6H8N4O2S
C3H3N3OS
C6H7NO
10 MS2
10 MS2
10 MS2
10 MS2
10 MS3
10 MS3
10 MS3
C2H3N3S
C3H3N3OS
C6H7NO
C2H3N3S
474
475
a The instrumentation: LCMSꢀITꢀTOF, conditions: positive ion mode; b RT = retention time;
476
477
c 6, thidiazuron; 7, 4ꢀhydroxyꢀthidiazuron; 8, 3ꢀhydroxyꢀthidiazuron; 9,
thidiazuronꢀ4ꢀOꢀβꢀDꢀglucoside; 10, thidiazuronꢀ3ꢀOꢀβꢀDꢀglucoside.
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479
480
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Figure 1. Structures of thidiazuron and synthetically prepared metabolites of
485
486
487
488
489
490
491
492
493
494
495
496
497
498
thidiazuron (numbers of the compounds refer to Table 1).
1,
4ꢀhydroxyꢀthidiazuron; 3, 3ꢀhydroxyꢀthidiazuron; 4, thidiazuron ꢀ4ꢀOꢀβꢀ
5, thidiazuronꢀ3ꢀOꢀβꢀ ꢀglucoside.
thidiazuron
(TDZ,
(1ꢀphenylꢀ3ꢀ(1,2,3ꢀthidiazolꢀ5ꢀyl)urea));
2,
D
ꢀglucoside;
D
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H
H
H
H
S
S
N
N
NCO
N
N
S
(a)
(b)
N
H3N
N
N
O
O
N
N
R1
3
R
N
R1
D1(2)/ D2(3)
R
4
R2
R2
C1 C2
/
B
A1/ A2
H
H
S
N
N
N
H
H
S
N
N
OAc
(c)
O
N
O
N
OH
O
O
AcO
AcO
O
N
E1
O
HO
HO
(d)
F1(4)
OAc
H
H
N
S
N
OH
H
H
N
S
N
N
N
O
N
O
OAc
N
OH
O
O
O
AcO
AcO
O
HO
HO
E2
F2(5)
OAc
OH
A1, C1 : R1=OMe, R2=H
A2, C2: R1=H, R2=OMe
D1(2): R3=OH, R4=H
D2(3): R3=H, R4=OH
500
501
Figure 2. Synthetic reagents, conditions, and route of thidiazuron metabolites:
502
503
504
505
506
507
508
509
510
511
512
513
514
(a) 80 ℃, 8 h, (b) BBr3, CH2Cl2, ꢀ30 ℃, 12 h, (c) TBAB, K2CO3, CHCl3, 50 ℃, 10 h,
(d) NH3·H2O, CH3OH, rt. ,8 h.
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519
Figure 3. Extracted ion chromatograms of the synthetic standards of thidiazuron
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523
524
525
526
527
528
529
530
metabolites (numbers of the compounds refer to Table 1)
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533
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535
Figure 4. The fragmentation pattern of (A) 1, (B) 2 and 3, (C) 4 and 5 (numbers of the
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539
compounds refer to Table 1)
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543
Figure 5. Extracted ion chromatograms of thidiazuron and its metabolites in the
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546
547
548
549
550
551
552
553
554
treated and control kiwifruit samples (numbers of the compounds refer to Table 2).
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557
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559
560
561
562
563
564
a
d
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566
567
568
569
570
571
572
573
574
575
576
577
b
578
579
580
581
582
583
584
585
c
e
Figure 6. Mass spectra analysis of thidiazuron and its metabolites in kiwifruits in
positive ion mode（a, 6; b, 7; c, 8; d, 9; e, 10; numbers of the compounds refer to
Table 2）.
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TOC Graphic
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