Journal of Chromatographic Science 2014;52:624– 628
doi:10.1093/chromsci/bmt090 Advance Access publication July 3, 2013
Determination of Tiadinil and Its Metabolite in Flue-Cured Tobacco
Xuyan Chen1, Kongxiang Zhao1, Zhijin Fan2*, Wutao Mao2, Juanjuan Li2, Xiaotian Ji2, Xuewen Hua2, Guangning Zong2 and Fengyun Li2
1
Tianjin Entry & Exit Inspection and Quarantine Bureau, Tianjin 300461, P. R. China, and 2State Key Laboratory of Elemento Organic
Chemistry, Nankai University, Tianjin 300071, P. R. China
*Author to whom correspondence should be addressed. Email: chenxy04@mails.ucas.ac.cn
Received 24 January 2013; revised 24 April 2013
A novel and sensitive method was developed for the determination of
residues of tiadinil and its metabolite, 4-methyl-l,2,3-thiadiazole-5-
carboxylic acid, in flue-cured tobacco. The pesticides were extracted
with acetone and purified by gel permeation chromatography and
solid-phase extraction. Analysis was performed by ultra-performance
liquid chromatography –tandem mass spectrometry in negative ion-
ization mode. Two precursor –product ion transitions were monitored
for both compounds in the multiple reaction monitoring mode.
Quantification was conducted by using matrix-matched standard
calibration. Recovery values of the proposed method for tiadinil
ranged from 72.5 to 98.2%, with relative standard deviations ranging
from 3.8 to 9.5%; recovery values for 4-methyl-l,2,3-thiadiazole-5-car-
boxylic acid ranged from 75.4 to 103.3% with RSDs ranging from 3.7
to 9.3%. The limit of quantification for both compounds was
0.01 mg/kg. This method is valuable for residual analysis, quality
control and monitoring of tiadinil and its metabolite, 4-methyl-l,2,3-
thiadiazole-5-carboxylic acid, in tobacco.
Introduction
Tiadinil (TDL), N-(3-chloro-4-methyl phenyl)-4-methyl-1,2,3-
thiadiazole-5-carboxamide, is a novel elicitor that was discovered
and developed by Nihon Nohyaku Co. (1). It can be used for the
control of rice blast and other rice diseases by both nursery box
and watering applications. There is a heterocyclic moiety,
1,2,3-thiadiazole, in its chemical structure (Figure 1). The effi-
ciency of TDL is not a result of its direct antifungal activities, but
of inducing systemic acquired resistance to a broad range of
pathogens in plants (1 –5). Yasuda et al. demonstrated that TDL
can induce resistance against tobacco mosaic virus (TMV) and
tobacco wildfire disease (Pseudomonas syringae pv. tabaci)
without antimicrobial activity (2). 4-Methyl-1,2,3-thiadiazole-
5-carboxylic acid (SV-03, Figure 1), which is identified as a me-
tabolite of TDL in rice, can also induce a broad range of disease
resistance activities in tobacco (6). Studies have also shown that
TDL and SV-03 demonstrate systemic acquired resistance for
tobacco against TMV (data not published). TDL and its active
metabolite, SV-03, fulfill all the criteria of elicitors (6, 7), but it
cannot be determined whether the conversion of TDL to SV-03
is necessary for the induction of disease resistance. Recent
studies have discovered that TDL can activate the defense re-
sponse of plants against the herbivorous mite Tetranychus kan-
zawai via an indirect process mediated by plant volatiles that
attract natural enemies (8). TDL has a low risk of developing fun-
gicide resistance due to its original mode of action (2, 5).
Granule formulations of TDL mixed with some insecticides, fun-
gicides and herbicides have been registered and are for sale (1).
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To regulate the use of TDL, a maximum residue level (MRL)
has been established. In Japan, the MRLs of TDL are 1 mg/kg in
brown rice and 0.03 mg/kg in aquatic animals. The MRL for rice
is expressed as the sum of TDL and its two metabolites, SV-03
and 4-hydroxymethyl-1,2,3-thiadiazole-5-carboxylic acid, both
calculated as TDL. The MRL for aquatic animals is expressed as
TDL residue only.
Flue-cured tobacco is one of the most important types of
tobacco. The production of flue-cured tobacco accounts for more
than 80% of the total yield of tobacco in China. Crop rotation with
rice is often applied in the production of tobacco. TDL and its me-
tabolite can be absorbed from soil by tobacco plants. Moreover,
TDL may be registered and used in tobacco production in the
near future as a result of its activity of inducing systemic acquired
resistance against TMV. Until now, few studies have been reported
on the determination of TDL in trace levels. Wang et al. developed
methods for the quantification of TDL in foodstuffs of plant and
animal origins, respectively (9, 10). No research has been reported
on the detection of SV-03. This work developed an ultra-
performance liquid chromatography–tandem mass spectrometry
(UPLC–MS-MS) method combined with gel permeation chroma-
tography (GPC) and solid-phase extraction (SPE) purification pro-
cedures for the determination of residues of TDL and its active
metabolite, SV-03, in flue-cured tobacco.
Materials and Methods
Materials and reagents
Methanol, cyclohexane and ethyl acetate were of HPLC grade
and purchased from Fisher Scientific (St. Louis, MO). Acetone
and acetic acid were from Tianjin Chemical Company (Tianjin,
China). Water for all aqueous solutions was obtained from the
Milli-Q water purification system (Millipore, Billerica, MA).
LC-NH2 cartridges (500 mg, 3 mL) were obtained from Supelco
(Bellefonte, PA). PES syringe filters 25 mm, 0.45 mm were pur-
chased from Whatman (Piscataway, NJ). Flue-cured tobacco
samples were obtained from a local market and confirmed to be
free of residues of the target analyte by UPLC –MS-MS after
sample preparation by the proposed procedures. A weight of
control blank tobacco, sufficient to provide all subsamples
required for each set of experiments, was finely ground and
mixed.
Pesticide standards
Standards of TDL ( purity . 99.0%) and SV-03 ( purity . 99.0%)
were synthesized and purified according to Patent wo9629871
(11) and the reported method (12). Individual stock solutions

Figure 1. Chemical structures: TDL (A); SV-03 (B).
were prepared by exactly weighing 10 mg of each pesticide, dis-
solving them in 10 mL methanol and storing them in darkness at
–188C. A working standard solution containing both analytes at
different concentrations was prepared from the stock solutions
and stored at 48C. Matrix-matched standard solutions were
obtained by drying appropriate volumes of the working standard
solution under nitrogen and dissolving in blank solutions of flue-
cured tobacco extract.
Sample preparation
Extraction
Five grams of thoroughly homogenized ground sample were
weighed exactly into a 50 mL polypropylene centrifuge tube
and 20 mL of acetone was added. The extraction procedure was
performed under ultrasound for 10 min. After centrifugation at
8,000 rpm for 5 min, the supernatant was transferred to a flask.
The residue was again extracted with 20 mL of acetone and the
supernatant was collected. After evaporation to dryness with
rotary evaporator at 358C, the obtained residue was dissolved in
10 mL of ethyl acetate–cyclohexane (50:50, v/v) and filtered
through a 0.45 mm filter for GPC purification.
Cleanup
An AccuPrep MPS GPC clean-up system (J2 Scientific, Columbia,
MO) nm was applied for purification; this was equipped with a
glass column (300  25 mm i.d.) packed with resin of 200–400
mesh Bio-Beads S-X3 and a DT0001 fixed wavelength detector at
254. Five milliliters of the extract were injected into the system.
The elution was conducted with ethyl acetate–cyclohexane
(50:50, v/v) at a flow rate of 4.7 mL/min. The fraction between
10 and 20 min was collected and evaporated to dryness at 358C
with rotary evaporator. The residue was then dissolved in 2 mL
of acetone for SPE-LC-NH2 purification.
The LC-NH2 cartridge was preconditioned with 5 mL metha-
nol followed by 5 mL acetone. Two milliliters of the previously
obtained residue were directly loaded onto the cartridge. The
flask was rinsed with 2 mL of acetone three times and the risen
portions were loaded onto the cartridge. All of the effluent was
recovered in a flask. The cartridge was subsequently rinsed with
5 mL of methanol, followed by 1 of mL acetic acid, and the efflu-
ent was discarded. Finally, the cartridge was eluted with 5 mL of
methanol and the effluent was collected in the previously used
flask. The collected effluent was consequently evaporated to
dryness with a rotary evaporator at 408C. The obtained residue
was dissolved in 1 mL of methanol– water (1:1, v/v) and filtered
through a 0.45 mm filter before analysis with UPLC– MS-MS.
UPLC–MS-MS analysis
An Acquity Ultra Performance LC-Xevo TQMS system (Waters,
Milford, MA) was employed for analysis. Chromatographic
Table I
Optimized MRM Parameters and Retention Times of TDL and SV-03
Compound Ion mode MRM transitions Cone Collision Retention
voltage (V) voltage (V) time (min)
TDL ESI( –) 266.2/71.0* 28 20 2.05
266.2/238.2 18 15
SV-03 ESI( –) 143.1/71.0* 15 10 0.88
143.1/99.1 10 5
*Labeled transitions were used for quantitative analysis.
separation was accomplished at 258C with an Acquity UPLC BEH
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C18 column (10  2.1 mm, i.d., particle size 1.7 mm; Waters).
The mobile phase contained methanol (A) and water (B). The
linear gradient started at 70% A and was raised to 90% within
2 min. The concentration of A was held at 90% for 1 min before
rapidly returning to the initial conditions in 0.1 min. The column
was re-equilibrated for 0.9 min before the next injection was
conducted. The flow rate of the eluent was 0.3 mL/min. The in-
jection volume was 2 mL for the extracts reconstituted in metha-
nol –water (1:1). The mass spectrometer was set to collect data
in multiple reaction monitoring (MRM) mode using negative
electrospray ionization [ESI( –)] for both compounds. The ioniza-
tion source parameters were optimized according to the follow-
ing parameters: capillary voltage, 2.80 kV; desolvation gas
temperature, 4008C; desolvation gas flow, 800 L/h; cone gas
flow, 50 L/h. Solutions of the individual analytes at 50 mg/mL in
methanol–water were infused at 5 mL/min to optimize the
MRM conditions. Summaries of precursor and product ions,
cone voltages and collision voltages for both analytes are pre-
sented in Table I. Data acquisition and processing were per-
formed by using MassLynx 4.1 with Target Lynx.
Method performance
Confirmation of the identity of target analytes was based on the
ion ratio statistics for the monitored transitions. The relative in-
tensity of the analytes has to be within the maximum permitted
tolerances recommended by the European Union (EU) (13).
Quantification was performed via matrix-matched external
standard calibration curves, which were established with blank
flue-cured tobacco extracts. Accuracy and precision were evalu-
ated through recovery experiments. Six replicates of recovery
tests were conducted at four spiking levels, including the levels
of LOQ and MRL. The limits of quantification (LOQs) were deter-
mined based on a signal-to-noise ratio (S/N ¼ 10) of the confirm-
ation transition, which is less sensitive than the quantification
transition, and the limits of detection (LODs) were calculated by
using S/N ¼ 3.
Results
Method validation
Linearity
Matrix-matched calibration curves were set up with concen-
tration versus peak area by dissolving appropriate volumes of
the working standard solution into blank tobacco extracts,
resulting in six different levels (0, 0.01, 0.1, 1, 4 and 10 mg/kg).
Satisfactory linearity was achieved, with correlation coefficient
Determination of Tiadinil and Its Metabolite in Flue-Cured Tobacco 625
(r 2) higher than 0.99. The calibration curve equations and cor-
relation coefficients were y ¼ 44391x – 1438.8 (r 2 ¼ 0.9992)
and y ¼ 42798x þ 5901.9 (r 2 ¼ 0.9982) for TDL and SV-03,
respectively.
Accuracy, precision and sensitivity
Blank tobacco samples were fortified at four different concentra-
tion levels of 0.01, 0.1, 1 and 10 mg/kg before extraction by
adding appropriate volumes of the working standard solution.
Concentrations of target compounds in fortified samples were
calculated via matrix matched external standard calibration
deviations (RSDs) were between 5.5 and 9.5% for TDL and
between 3.7 and 9.3% for SV-03. Recovery results at different
spiked levels are summarized in Table II. LODs were 0.003 mg/
kg and LOQs were 0.01 mg/kg for both compounds. The results
in Table II show that TDL and SV-03 were determined at
0.01 mg/kg with acceptable recovery and precision.
Application of the method
The proposed method was tested by applying it to the analysis of
10 flue-cured tobacco samples imported or exported from

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curves through MassLynx 4.1 with Target Lynx and recovery Tianjin Port. Reagent blank, matrix blank and spiked samples at
values (RE) were calculated as follows: the level of 1 mg/kg and matrix-matched calibration curves
were adopted in each batch of samples to ensure that the system
calculated concentration after preparation was under control. Results showed that neither of the two com-
RE ð%Þ ¼  100%
fortified concentration before extraction pounds were found in these samples.
REs of the proposed method ranged from 72.5 to 98.2% for TDL
and from 75.4 to 103.3% for SV-03. The relative standard
Discussion
Optimization of UPLC– MS-MS parameters
Table II
Recovery and RSD Values The mass spectrometric parameters for both compounds were
automatically optimized through MassLynx 4.1 by direct infusion
Pesticide Fortified Recovery (%) Mean RSD of a solution of standards into the mobile phase via a syringe
level recovery (%) (%)
(mg/kg) 1 2 3 4 5 6 pump. Two MRM transitions with the best responses and colli-
TDL 0.01 83.9 80.8 98.2 89.7 75.0 90.8 86.4 9.5
sion energy and cone voltage were achieved in ESI( –) mode.
0.1 91.6 85.5 88.8 82.5 76.7 86.3 85.2 6.1 Dwell times were set so that 12 data points were acquired in six
1 84.9 81.0 87.2 80.9 87.5 81.1 83.8 3.8 seconds. The acquisition time of each compound was adjusted
10 82.7 72.5 80.1 82.4 76.8 84.3 79.8 5.5
SV-03 0.01 87.3 78.2 103.3 88.6 96.7 92.3 91.0 9.3 according to retention times. With regard to the mobile phases,
0.1 85.0 91.3 86.7 78.3 82.2 75.4 83.2 7.0 methanol increased the ionization of both compounds relative
1 77.6 87.1 84.7 81.4 93.1 84.0 84.7 6.2 to the ionization achieved with acetonitrile. Different aqueous
10 83.9 75.6 82.3 82.5 80.8 79.4 80.8 3.7
phases were tested, including 0.1% formic acid aqueous solution,

Figure 2. Full scan mass spectra: TDL standard (A); SV-03 standard (B).

626 Xuyan et al.

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Figure 3. MRM chromatograms: TDL standard (A); SV-03 standard (B).

Figure 4. GPC elution of: blank flue-cured tobacco extract (A); standard solution of TDL (B); standard solution of SV-03 (C).

10 mM ammonium acetate solution, 0.1% ammonium hydroxide Optimization of purification procedures

and pure water. No modifiers showed good results for both ana- Tobacco is rich in proteins, lipids, carbohydrates, pigments and
lytes; therefore, pure water was chosen. Full scan mass spectra organic acids. The challenges associated with the determination
and MRM chromatograms of TDL and SV-03 standards are shown of both compounds in flue-cured tobacco at trace levels are sub-
in Figures 2 and 3. stantial as a result of the complexity of the matrix. GPC was used

Determination of Tiadinil and Its Metabolite in Flue-Cured Tobacco 627

to remove the high molecular interferences in the study. The
separation efficiency of GPC was investigated. Five milliliter stan-
dards of TDL, SV-03 and the extract of blank flue-cured tobacco
were injected into the GPC system. Elution was conducted with
ethyl acetate–cyclohexane (50:50, v/v) at a flow rate of 4.7 mL/
min. As shown in Figure 4, the coextracts in tobacco eluted in
5 –20.5 min, whereas TDL and SV-03 eluted in 11.5 –19.5 and
10.5 –17 min, respectively. The collection of a fraction between
10 and 20 min guaranteed excellent recovery values of target
analytes. Interferences in this fraction were removed by subse-
quent SPE purification. The purification effects of GBC, C18,
Florisil, SAX, PSA and LC-NH2 were compared. Poor recovery
made the application of GBC impossible because of its strong ab-
sorption. The separation of TDL and SV-03 from interferences
could not be achieved by either C18 or Florisil cartridges as a
result of the weak absorption to target analytes and the interfer-
ence. Regarding the SAX cartridge, SV-03 could not easily be
eluted from it owing to its strong absorption to anions; mean-
while, interferences could not effectively be removed.
Acceptable purification was achieved by both PSA and LC-NH2
cartridges and there was no obvious difference in the recovery
of TDL. The recovery of SV-03 was much better with the
LC-NH2 cartridge than the PSA because of the weaker absorp-
tion of LC-NH2 to anions than PSA.
Conclusions
A sensitive method was developed for the determination of TDL
and SV-03 residues in flue-cured tobacco. A combination of GPC
and SPE was used for sample purification. The purification effi-
ciency of the GPC system and different cartridges were carefully
optimized, including GBC, C18, Florisil, SAX, PSA and LC-NH2.
Analysis was completed in 4 min as a result of the application of
UPLC –MS-MS. Satisfactory recovery and RSD were achieved. The
LOQ was 0.01 mg/kg for both analytes. This method is a valuable
measurement for residues of TDL and SV-03 in tobacco for
quality control and monitoring. Ten flue-cured tobacco samples
imported or exported from Tianjin Port were analyzed. Neither
of the two compounds was found in these samples.
Acknowledgments
This study was funded in part by grants from the National Natural
Science Foundation of China (20872071), the Tianjin Natural
Science Foundation (10JCZDJC17500), the National Key Project
for Basic Research (2010CB126105), the National Key Technology
Research and Development Program (2011BAE06B02) and the
628 Xuyan et al.
Foundation of Achievements Transformation and Spreading of
Tianjin Agricultural Science and Technology (201002250),
Tianjin Key Technology Research and Development Program
(11ZCGYNC00100).
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