Bull Environ Contam Toxicol (2011) 87:653–656

DOI 10.1007/s00128-011-0421-x

Determination of Hymexazol in Cucumber and Soil Samples

by Derivatization Using GC-FPD
Dali Sun • Li Li • Ran Ji • Wei Li • Huochun Ye •

Yijun Wu • Chenglan Liu

Received: 1 June 2011 / Accepted: 19 September 2011 / Published online: 30 September 2011
Ó Springer Science+Business Media, LLC 2011

Abstract A sensitive and effective analytical method for (Fig. 1) recommended for use to against various diseases in
the determination of hymexazol in cucumber and soil cucumber such as damping-off, anthracnose, blight, dry-
samples by gas chromatography with a flame photometric rot, phytophthora blight that caused by fungus, which lead
detector was developed. This method was validated with great losses of cucumber yields. Because hymexazol is a
fortified at three different levels of 0.2, 1.0 and 5.0 mg/kg. systemic fungicide, translocated to most plant tissues, there
Average recoveries obtained from cucumber and soil is potential risk to consumer health if the pesticide is still
samples at three fortified levels were 94.0%–107.8% with present at harvest (Qian et al. 2011). Therefore, it is
relative standard deviations (RSDs) of less than 11.4%. imperative to set up an effective and efficient analytical
Limits of quantification (LOQ) in cucumber and soil were method to evaluate and monitor hymexazol residues. The
0.2 mg/kg. The method was successfully applied to deter- maximum residue limits (MRLs) in Japan for hymexazol in
mine hymexazol in real samples of cucumber and soil cucumber is 0.5 mg/kg. However, no MRLs have been set
under open fields. by China and the WHO/FAO.
There are limited reports available in literature about the
Keywords Hymexazol
Cucumber
Soil
Derivatization analytical methods of hymexazol. The difficulty in hym-
exazol analysis results from its high polarity and spectral
properties. Its maximum-absorption is at 200 nm, which
Cucumber is an important vegetable crop in China, results in severe solvent interference during HPLC analy-
which suffers yield losses due to diseases. Hymexazol, sis. Furthermore, hymexazol is unstable at high tempera-
3-hydroxy-5-methoxzaolum, is an effective fungicide ture, making direct GC analyzing unsuitable. Pilar et al.
(2008) developed a method for the analysis of multi-resi-
dues of oxazole fungicides including hymexazol in wines
and juices by UPLC with two novel sample preparation
procedure (i) stir bar sorptive extraction (SBSE) and (ii)
membrane-assisted solvent extraction (MASE). Tamura
D. Sun
H. Ye
C. Liu (&)
et al. (2008) reported a method for the determination of
Key Laboratory of Natural Pesticide and Chemical Biology,
Ministry of Education, South China Agricultural University, hymexazol in agricultural products by GC-NPD with a
Guangzhou 510642, People’s Republic of China high-polarity capillary column and highly deactivated inlet
e-mail: liuchenglan@scau.edu.cn liner. Their results showed that the recovery of hymexazol
from spiked agricultural products ranged from 65.0% to
L. Li
W. Li
Y. Wu
State Key Laboratory of Integrated Management of Pest Insects 84.7%, and the limits of detection (LOD) was 0.02 mg/kg.
and Rodents Institute of Zoology, Chinese Academy of Sciences, The present work was carried out to establish a simple and
Beijing 100101, People’s Republic of China efficient analytical method for determination of hym-
exazol. Hymexazol was applied in cucumber and soil to
R. Ji
Beijing Research Institute of Chemical Industry, SINOPEC, evaluate its dissipation and residue levels under field con-
Beijing 100013, People’s Republic of China ditions, and afford evidence for registration in China.

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654 Bull Environ Contam Toxicol (2011) 87:653–656

O and 4 mL of 2 N HCl were added to the aqueous layer, and

N then the pH was adjusted below 2. The mixtures were
CH extracted by liquid–liquid partition with 75 mL of ether
3
(25 mL 9 3 times), shaking for 2 min each time. The ether
OH fractions were combined and filtered through anhydrous
sodium sulphate. One milliliter of 1% PEG was added to
Fig. 1 The chemical structure of hymexazol
the extracts and the volume reduce to 1 mL by evaporation
at 40°C, and then mixed with 0.05 mL of 25% K2CO3
Materials and Methods aqueous solution and 2 mL of 0.2% DECTP acetone
solution. After incubation for 30 min at 45°C, the mixtures
Hymexazo1 standard (purity at 98%) was provided by the were evaporated to near dryness at 40°C and redissolved in
Quality Supervision and Inspection Center of Pesticide 2 mL of methanol.
(China). A stock solution of hymexazol (1,000 mg/kg) was Field trials including the dissipation and final residue
prepared in acetone, and working standards were obtained experiments were carried out at Beijing, China during
after further diluted to the required concentration. The 2010. The experiment field consisted of three replicated
stock and working standard were stored in dark at 4°C. O, plots with each plot being 20 m2. Another three plots were
O-diethylthiophosphoryl chloride (DECTP) with purity at sprayed with water and maintained as controls. During the
98% (Acros Organics Co. USA) was dissolved in acetone experiment period, cucumber plants received routine hor-
(0.2 mL DECTP was added to 100-mL volumetric flask). ticultural treatment. In order to study the dissipation trends
Macrogol-400 (PEG) (First Reagents Factory of Shanghai of hymexazol in cucumber and soil, 65% wettable power
China) was dissolved in acetone (1 g PEG-400 was added (WP) formulation of hymexazol was sprayed on cucumber
to 100-mL volumetric flask). Acetone, sodium hydrogen at the dosages of 50 mg active ingredient per plant.
carbonate, sodium chloride, hydrochloric acid, potassium Cucumber and soil samples were collected on 0 (2 h after
carbonate, ether and sodium sulphate anhydrous were of spraying), 1, 2, 3, 4, 5, 7, 10, and 14 days. In order to study
analytical grade and were purchased from Beijing Chemi- the final residues of hymexazol, 25 mg active ingredient
cal Reagents Company (China). Sodium sulfite was added per plant (low dosage) and 50 mg active ingredient per
in ether overnight and redistilled. Anhydrous sodium sul- plant (high dosage) were sprayed on cucumber. Cucumber
phate was baked at 650°C for 4 h before use. and soil samples were collected randomly from each
An Agilent technologies 4890 GC system equipped with treatment plot at 7, 14 and 21 days after the hymexazol
a flame photometric detector (FPD) under phosphorus application. Soil was sampled to a depth of 0–10 cm in
mode was used with a HP 1701 capillary column each plot using a tube auger. Control samples were
(30 m 9 320 mm 9 0.25 lm). Temperatures of injector obtained from control plots. All samples were put into
and detector were held constant at 250 and 230°C, polyethylene bags and transported to the laboratory where
respectively. The column-oven temperature was 180°C. they were chopped, thoroughly mixed, and divided into
High purity nitrogen was used as carrier gas and makeup sub-samples. The sub-samples were kept at -20°C until
gas with flow rates of 1.2 and 15 mL/min, respectively. analysis.
Hydrogen and air were used as detector gases at 75 and
100 mL/min, respectively. Injections were carried out in
the splitless mode, and the injection volume was 2 lL. Results and Discussion
Twenty grams of cucumber or soil were extracted with
50 mL acetone in a 250-mL conical flask with stopper, and Acetone, dichloromethane, and methanol were tested as
shaken for 30 min on an oscillator. The extracts were extraction solvents, and acetone was the most efficient.
transferred to another 150-mL flat bottom flasks containing Different amount of acetone (30, 50, 70 mL) was also
1 mL of 1% PEG acetone solution, and then the flat bottom studied and the results indicated that the volume of 50 mL
flasks were washed twice with 20 mL acetone. The mix- was the most suitable. Because hymexazol has relatively
tures were evaporated to near dryness in a rotary evapo- high volatility and adsorptivity, 1 mL of 1% PEG acetone
rator at 40°C, washed with 40 mL of 2% NaHCO3 aqueous solution was added as protective agent to prevent the loss
solution (20 mL 9 2 times), and then transferred into a of hymexazol during evaporation steps.
250-mL separating funnel to (Eight grams of NaCl were The derivatization reaction proved to be a good method
added for cucumber samples firstly) The residues were to convert hymexazol into a more stable compound with
extracted by liquid–liquid partition with ether two times at lower polarity. According to the structure of hymexazol,
the same volume of 25 mL. The organic layer was dis- we hypothesized that a hydroxyl should be relatively
carded. Twenty milliliter of 20% NaCl aqueous solution easy to react with phosphorus oxychloride. Some studies

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O
Quantification was performed by comparing sample
S
N O K2CO3 N peak areas to a standard curve derived from hymexazol-
O O O
CH3 O
P
P S spiked samples at eight different concentrations. For
OH
Cl
CH3 O
chromatographic procedures, a relation could be observed
between detector response (y) and analyte concentration
(x). The stock solution of hymexazol (1,000 mg/kg) was
Fig. 2 The reaction procedure of derivatization diluted stepwise with acetone to make a series of standard
solutions (2.0, 4.0, 10.0, 16.0, 20.0, 40.0, 100.0, 160.0 mg/
kg). One milliliter of the standard solutions was added to
y = 12573x2 + 39391x + 15091 20 g of cucumber or soil samples. The standard series
R2 = 0.9995
obtained were 0.1, 0.2, 0.5, 0.8, 1.0, 2.0, 5.0, 8.0 mg/kg,
correspondingly. Spiked-samples were treated following
Area

the sample preparation and detection mentioned above.

Figures 3 and 4 showed the standard curve of hymexazol in
cucumber and soil samples.
The efficiency of the analytical method was evaluated
Concentration (mg/kg) by spiking hymexazol into cucumber and soil samples at
0.2, 1.0, and 5.0 mg/kg with five repetitions at each level.
Fig. 3 The standard curve of hymexazol in cucumber at the
concentrations ranged from 0.1 to 8.0 mg/kg The recovery of hymexazol from cucumber and soil sam-
ples ranged from 94.0% to 107.8% and the RSD range was
8.1% to 11.4% (Table 1).
y = 10713x2 + 46102x + 9963.1
R2 = 0.9996
The recovery and accuracy of the our analytical method
were within the acceptable parameters described in the
Area

Guideline on Pesticide Residue Trials issued by the Min-

istry of Agriculture of the People’s Republic of China,
2004.
Limit of detection (LOD) was obtained as signal-to-
noise (S/N) ratios of 3, and limit of quantification (LOQ)
Concentration (mg/kg)
was defined as the minimum of the spiked samples. The
Fig. 4 The standard curve of hymexazol in soil at the concentrations presence of potential interference in the chromatograms
ranged from 0.1 to 8.0 mg/kg from the analyzed samples was monitored by running
control blank samples in each calibration. Blank samples
reported that organic phosphate could be obtained by the were extracted and derivatized by the same procedure. The
reaction of phosphorus oxychloride and hydroxyl (Argauer LOD by this method on our apparatus was 1 9 10-10 g and
1969, Butler and Madonough 1968). Figure 2 showed the LOQ for hymexazol in cucumber and soil was 0.2 mg/kg.
reaction procedure of derivatization. The obtained hym- The method was validated with three spiked levels
exazol derivative contained a phosphorus element and which showed good accuracy and sensitivity. It was suc-
responded well on FPD, which indicated this procedure cessfully employed for the determination of hymexazol in
suitable to be analyzed by GC-FPD. This reaction could be cucumber and soil samples collected from treated fields.
easily conducted with relatively high productivity, few The chromatograms of hymexazol in cucumber and soil
reagents and no special instrument. samples were shown in Figs. 5 and 6. Compared to others

Table 1 Recovery and RSD of hymexazol in samples at different levels

Sample Fortified Recovery (%) RSD (%)
level (mg/kg)
1 2 3 4 5 Average

Cucumber 0.2 102.8 88.3 91.6 86.0 112.0 96.1 11.4

1.0 104.2 99.1 81.9 90.5 107.6 96.7 10.8
5.0 101.8 100.7 95.7 86.7 85.6 94.0 8.1
Soil 0.2 97.1 100.0 112.2 110.0 119.7 107.8 8.6
1.0 99.6 91.4 110.9 86.1 106.7 98.9 10.4
5.0 107.9 89.4 89.5 87.6 107.1 96.3 10.7

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656 Bull Environ Contam Toxicol (2011) 87:653–656

FPD2 B, (EML70510\SIG20003.D) The residue dynamics of hymexazol in soil was shown

counts
140000
in Fig. 7. The initial concentration of hymexazol in soil
130000 was 6.4 mg/kg after 2 h of the application. Five days after
120000 application, the dissipation rate of hymexazol was 89%.
110000 The dynamic regression equation and the half-life of
100000
hymexazol in soil was C=16.0218e-0.3667T, T1/2= 1.9 days.
0.094

90000
The dissipation of pesticides in soil was affected by variety

8.473
80000
70000 of complex physical, chemical and biological processes,
60000 including sorption–desorption, volatilization, chemical and
50000
biological degradation, uptake by plants, run-off and
0 2.5 5 7.5 10 12.5 min
leaching under field conditions (Zhang and Cooper 1996).
Fig. 5 Chromatogram of hymexazol in cucumber (0.2 mg/kg) Hymexazol residues in cucumber were lower than the
Japanese maximum residue limits (MRLs) of 0.5 mg/kg on
7, 14 and 21 days after the treatment of this pesticide. With
FPD2 B, (EML70410\SIG20007.D)
respect to hymexazol, the cucumbers would be considered
counts
safe to human consumption.
220000
200000
Acknowledgments This work was supported by grants from the
180000
Institute for the Control of Agrochemicals, Ministry of Agriculture of
160000
the People’s Republic of China.
140000
120000
8.356

100000
80000 References
60000
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0 2.5 5 7.5 10 12.5 min
carbamate pesticides after hydrolysis and chloroacetylation.
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Fig. 6 Chromatogram of hymexazol in soil (1.1 mg/kg)
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bar sorptive extraction and membrane-assisted solvent extraction
Residues (mg/kg)

for the ultra-performance liquid chromatographic determination

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Fig. 7 Dissipation of hymexazol in soil
of three pesticides in different soils. Arch Environ Contam
Toxicol 30:15–20
studies, in which UPLC or GC-NPD was used to analysis
hymexazol, this method showed satisfied recovery and
precision, high sensitivity and low LOD and LOQ.

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