Determination of chlorfenapyr in leek grown under greenhouse conditions with GC-μECD and confirmation by mass spectrometry.
ABSTRACT A simple analytical method was developed for the determination of chlorfenapyr residues in leeks grown under greenhouse conditions. Residues were extracted by salting out, analyzed by gas chromatography with microelectron-capture detection, and confirmed via gas chromatography-mass spectrometry. The calibration curves were found to be linear with correlation coefficients (r(2) ) in excess of 0.998. The limits of detection and quantification were 0.0015 and 0.005 mg kg(-1) , respectively. For validation purposes, recovery studies were carried out at low and high levels. Yield recovery rates were 87.27-89.64% with a relative standard deviation <6%. A maximum of 0.32 mg kg(-1) of chlorfenapyr residue was detected in leek sample sprayed three times at 7 day intervals until 7 days prior to harvest. The results of this study suggest that chlorfenapyr is acceptable for application in/on leeks under the recommended dosage regimen.
- SourceAvailable from: dss.go.th[Show abstract] [Hide abstract]
ABSTRACT: The association between application rate of a pesticide and its residue in ripe tomatoes was studied. The average residue level (R) of any pesticide in ripe tomatoes remained in quantitative relation to its dose (D), expressed by the following regression equation: R = 0.24 D (mg/kg), where the numerical factor, 0.24, represents the average residue in mg/kg after application of 1 kg active ingredient per hectare with relative standard deviation of 23%. Quantitative association between these 2 factors enables evaluation of greenhouse tomato growers with respect to their observation of Good Agricultural Practice rules and the Plant Protection Act, obligatory in Poland since 1996, and thus may be a reliable basis for the registration of new agrochemicals.Journal of AOAC International 01/2000; 83(1):214-9. · 1.39 Impact Factor
- Biochemical Society Transactions 03/1994; 22(1):244-7. · 3.24 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: In this paper we have developed single drop microextraction (SDME) with modified 1.00 microl microsyringe, followed by gas chromatography with flame photometric detector (GC-FPD) for determination of 13 organophosphorus pesticides (OPPs) in water samples. By using a 1.00 microl microsyringe the repeatability of drop volume and injection were improved, because of using maximum volume of microsyringe and no dead volume. On the other hand, the modification of needle tip caused increasing cross section of needle tip and increasing adhesion force between needle tip and drop, thereby increasing drop stability and achieving a higher stirrer speed (up to 1700 rpm). The method used 0.9 microl of carbon tetrachloride as extractant solvent, 40 min extraction time, stirring at 1300 rpm and no salt addition. The enrichment factor of this method ranged from 540 to 830. The linear ranges were 0.01-100 microg/l (four orders of magnitude) and limits of detection were 0.001-0.005 microg/l for most of analyte. The relative standard deviation (RSD%) for 2 microg/l of OPPs in water by using internal standard was in the range 1.1-8.6% (n = 5). The recoveries of OPPs from farm water at spiking level of 1.0 microg/l were 91-104%.Journal of Chromatography A 02/2006; 1101(1-2):307-12. · 4.26 Impact Factor
Determination of chlorfenapyr in leek grown
under greenhouse conditions with GC‐μECD
and confirmation by mass spectrometry
Md. Musfiqur Rahmana, Jeong‐Heui Choia, A. M. Abd El‐Atyb*,
Jong Hyouk Parka, Ji‐Yeon Parka, Geon‐Jae Imcand Jae‐Han Shima*
ABSTRACT: A simple analytical method was developed for the determination of chlorfenapyr residues in leeks grown under
greenhouse conditions. Residues were extracted by salting out, analyzed by gas chromatography with microelectron‐capture
detection, and confirmed via gas chromatography–mass spectrometry. The calibration curves were found to be linear with
correlation coefficients (r2) in excess of 0.998. The limits of detection and quantification were 0.0015 and 0.005mg kg−1,
respectively. For validation purposes, recovery studies were carried out at low and high levels. Yield recovery rates were
87.27–89.64% with a relative standard deviation <6%. A maximum of 0.32mg kg−1of chlorfenapyr residue was detected in
leek sample sprayed three times at 7day intervals until 7days prior to harvest. The results of this study suggest that
chlorfenapyr is acceptable for application in/on leeks under the recommended dosage regimen. Copyright © 2011 John
Wiley & Sons, Ltd.
Keywords: chlorfenapyr; gas chromatography; leek; greenhouse
Pesticides, including organochlorine pesticides (OCPs), organo-
phosphates (OPs) and nitrogen‐containing herbicides are well‐
known types of environmental contaminants. The use of
pesticides provides benefits for increasing agricultural produc-
tion; however, residues may remain in foods in amounts in
excess of maximum residue limits (MRLs). Bioaccumulation
through the food chain can eventually become a risk to both
animals and humans (Ahmadi et al., 2006). Chlorfenapyr, 4‐
bromo‐2‐(4‐chlorophenyl)‐1‐ethoxymethyl‐5 trifluoro methyl-
pyrrole‐3‐carbonitrile, is a novel broad‐spectrum insecticide/
acaricide used for the control of various species of insects and
mites, including those resistant to carbamate, organophosphate
and pyrethroid insecticides, and also as chitin‐synthesis
inhibitors against pests in cotton, vegetables, citrus and soy
beans (Tomlin, 2000). Various research has been carried out to
identify and estimate its efficacy against insects (Guglielmone
et al., 2000; Kaufman et al., 2001; McLeod et al., 2002; Ahmad
et al., 2003; Rand, 2004).
Chlorfenapyr is a pro‐insecticide that is converted to an active
metabolite in the mid‐gut of insects and mites. Once the formed
metabolite has inhibited oxidative phosphorylation by disrupt-
ing the proton gradient across mitochondrial membranes, the
ability of cells to produce ATP from ADP is affected, ultimately
resulting in cell death and death of the organism (Treacy et al.,
1994; Rand, 2004). This specific acaricide/insecticide generally
has no effect on beneficial insects, including predaceous mites,
which is desirable for the development of new strategies
involving integrated pest management in vegetables (Cao et al.,
Application of pesticides is carried out once or several times
during cultivation. The average residue level of any pesticide in
fruit and vegetables depends primarily on the application rate of
its active ingredient (Sadlo, 2000). Determination of a pesticide’s
fate on crops is very important for good agricultural practice,
and will greatly affect the effectiveness of sprayed pesticides on
plants, the pre‐harvest interval (PHI) and the amounts of residue
on crops at time of harvest (Metwally et al., 1997). However, the
fate of pesticides under normal environmental conditions is
controlled mainly by temperature, humidity and light intensity
(Mabey and Mill, 1978). Therefore, it is not possible to predict
their fate under environmental conditions.
* Correspondence to: Jae‐Han Shim, Natural Products Chemistry Labora-
tory, Division of Applied Bioscience and Biotechnology, College of
Agriculture and Life Science, Chonnam National University, 300
Yongbong‐dong, Buk‐gu, Gwangju 500‐757, Republic of Korea. E‐mail:
Cairo University, 12211‐Giza, Egypt. E-mail: firstname.lastname@example.org
aNatural Products Chemistry Laboratory, Division of Applied Bioscience and
Biotechnology, College of Agriculture and Life Science, Chonnam National
University, 300 Yongbong‐dong, Buk‐gu, Gwangju 500‐757, Republic of
bDepartment of Pharmacology, Faculty of Veterinary Medicine, Cairo
University, 12211‐Giza, Egypt
cPesticide Safety division, National Institute of Agriculture Science and
Technology, Rural Development Administration, Suwon, 441‐707, Republic
Abbreviations used: MRLs, maximum residue limits; OCPs, organochlorine
pesticides; OPs, organophosphates; PHI, pre‐harvest interval; WP, wettable
Biomed. Chromatogr. 2012; 26: 172–177Copyright © 2011 John Wiley & Sons, Ltd.
Received 21 March 2011,Accepted 4 April 2011 Published online in Wiley Online Library: 24 May 2011
(wileyonlinelibrary.com) DOI 10.1002/bmc.1643
On the other hand, cultivation under greenhouse conditions
can eliminate the actions of rain and partially sun on the
reduction of treated pesticides. Thus, it is possible to obtain
accurate information about the persistence of these residues in a
greenhouse. Hence, pesticide residues on crops grown in
greenhouses should be determined to ensure the quality of
Several analytical methods have been described for
chlorfenapyr in different matrices. Multiresidue analysis for
monitoring chlorfenapyr in hops has been published in which a
gas chromatography–mass spectrometry method involved
several steps including derivatization (Hengel and Shibamoto,
2002). In order to omit the time‐consuming derivatization step
in GC analysis, Cao et al. (2005) developed an analytical method
for analysis of chlorfenapyr in cabbage and soil using high‐
performance liquid chromatography coupled with an ultraviolet
detector. However, chlorfenapyr is probably detected in a gas
chromatography–electron capture detector owing to its physi-
cochemical properties, including halogen components (F, Cl, Br)
and low vapor pressure (9.81×10−3mPa, 25°C; IUPAC).
Ditya et al. (2010) reported a gas chromatography with
electron‐capture detection (GC‐ECD) method without derivati-
zation for the determination of chlorfenapyr in chili, cabbage
and soil, and the method demonstrated very high recovery
results of 89–97%. However, the GC‐ECD method for chlorfenapyr
using liquid–liquid extraction and a conventional preparative
column for clean‐up is very time‐consuming. In order to
overcome this problem, we developed a simple and time‐saving
analytical method using salting out, solid‐phase extraction
(SPE) clean‐up, and more sensitive gas chromatography with
microelectron‐capture detection (GC‐μECD) detection to measure
chlorfenapyr in leeks grown under greenhouse conditions. The
treatment parameters, including the PHI, were evaluated to assess
Standard chlorfenapyr (purity 98.0%) was obtained from Dr Ehrenstorfer
(Augsburg, Germany). Acetone, n‐hexane, acetonitrile and sodium
chloride of analytical grade were obtained from Merck (Darmstadt,
Germany). Chlorfenapyr stock standard solutions were prepared in
acetone, and working standard solutions were prepared by serial
dilution of the stock solution using the same solvent. All standard
solutions were stored at −5°C.
Analysis was performed on an Agilent GC model 7890A (CA,USA)
equipped with an Agilent 7683B auto‐sampler and a μECD (63Ni), and
chromatographic separation was performed on an HP‐Ultra 2 capillary
CA, USA), flowing at 2mL min−1with nitrogen gas. A standard split/
splitless injector was used for the split injection mode at a ratio of 30:1 at
270°C with an injection volume of 1μL. The detector was maintained at
300°C with the make‐up gas (N2) flowing at 30mL min−1. The oven
temperature was set to 150°C for 2min, increased to 210°C at 10°C min−1,
held for 1min, then increased to 300°C at 15°C min−1and held for 5min.
Under these conditions, chlorfenapyr presented an average retention
time of 11.6min. An Agilent Chemstation was used for data acquisition.
An Agilent 6890 GC was used to confirm the identity of chlorfenapyr. It
was fitted with a mass‐selective detector Agilent 5973N and was
equipped with an Agilent 7683B auto‐sampler and a split/splitless
injector with an electronic pressure controller. Chlorfenapyr was
separated on a HP‐5MS capillary column (30m×0.25mm, 0.25µm film
thickness, Agilent Technologies, CA, USA). The column oven temperature
was held at 90°C for 1min, then increased to 270°C at 10°C min−1, and
held for 5min. Helium was used as a carrier gas at a flow rate of 1.0mL
min−1. The temperatures of the ion source and quadruple were set at
230 and 150°C, respectively. The temperature of the injector was set to
250°C, and a 2μL portion of the sample was injected in splitless mode.
The mass spectrometer was operated in electron impact ionization
mode (EI, 70eV), and analysis was carried out in selected ion monitoring
(SIM) mode in which two characteristic ions (m/z=59 and 247) for
chlorfenapyr were monitored.
Field trial design in a greenhouse
Experiments were conducted in a greenhouse at the experimental fields of
Chonnam National University, Gwangju, Republic of Korea. The experi-
mental area comprised 15 plots, in which a random block scheme was
established with three replicates. In addition, control samples were
cultivated in a separate plot without receiving any treatment with
insecticides. Chlorfenapyr 5% wettable powder (WP; Kyung Nong Co.,
Seoul, Republic of Korea) was diluted 1000 times and sprayed at a.i
0.0125kg 10a−1, which was recommended by the manufacturer. Plants
were sprayed in August and September 2009. The first and second plots
7days; the fourth plot was treated twice at 21 and 14days; and the fifth
plot was treated three times at 21, 14, and 7days prior to harvest (Table 1).
As soon as they were picked, the samples were transferred to the
laboratory where they were chopped and blended. Sub‐samples weighing
approximately 50g each were stored in a freezer pending analysis.
Sample extraction and clean‐up
A 20g leek sample was weighed in a 250mL Erlenmeyer flask, after
which 100mL acetonitrile was added. Subsequently, the mixture was
shaken for 1h on a shaker at 250rpm and filtered through Whatman no.
1 filter paper. The filtrate was transferred to a separatory funnel
containing 15g of NaCl, shaken in a mechanical shaker for 5min, and
Table 1. Spraying schedule of chlorfenapyr 5% wettable powder onto leeks
Treatment frequency Interval prior to harvest (days) Final spraying dateHarvest date
10 September 2009
7 3 September 2009
27 August 2009
3 September 2009
27 August 2009
3 September 2009
Chlorfenapyr residues in leek
Biomed. Chromatogr. 2012; 26: 172–177Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
allowed to stand for 20min. A 50mL portion of the upper layer was
transferred to a round‐bottomed flask, evaporated under 40°C in vacuo,
and then dissolved in 10mL of 2% acetone in n‐hexane. The
reconstituted sample was loaded into an SPE Florisil cartridge (2g,
12mL, Phenomenex, CA, USA), which was conditioned with 10mL of
n‐hexane in advance and then eluted with 10mL of 2% acetone in
n‐hexane. The eluate was evaporated under 40°C in vacuo and
reconstituted in 2mL of 2% acetone in n‐hexane.
Validation of the analytical method
Validation was tested to determine specificity, analytical curves
linearity, limit of detection (LOD) and limit of quantification (LOQ),
and to decide whether or not the method provides adequate precision
and accuracy, associated with recovery. Specificity was assessed by
analyzing the standards of the analyte, blank sample and fortified
sample. The fortified leek sample was prepared at 0.25mg kg−1and
Figure 1. (a) Standard chlorfenapyr at 2mg L−1; (b) untreated leek sample; (c) fortified leek sample at 0.25mg kg−1; and (d) field sample treated
Md. Musfiqur Rahman et al.
Biomed. Chromatogr. 2012; 26: 172–177 Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc
extracted by the method described above. The lowest concentrations of
the working standard solution injected to show signal‐to‐noise ratios of
3 and 10, corresponding to the LOD and LOQ, respectively, were
observed (Ribani et al., 2004, 2007). The analytical curve was constructed
by injecting different concentrations (0.025, 0.05, 0.1, 0.2, 0.5, 1 and
2mg L−1) of standard solutions prepared in pure solvent. The recovery
study was performed by fortifying standard solutions into blank leeks at
two fortification levels of 0.05 and 0.25mg kg−1. The fortified samples
were placed to gasify solvent and then extracted as described above
and analyzed by GC‐μECD. Recoveries were assessed in three replicates
for each level. The precision of the method was evaluated considering
the relative standard deviations (RSD) of the recovery assay.
Results and discussion
Validation of the developed method
analyte were assessed by comparing the chromatograms of the
standard, blank sample and fortified sample (Fig. 1). There were
no interference peaks at the retention time of chlorfenapyr. The
developed method consisted of salting‐out extraction and
GC‐μECD detection and was very selective in enabling analysis
of the analyte in the enormous matrix components.
The endogenous compounds interfering with the
For most chromatographic analyses, a linear relation was
observed between the area (y) and concentration (x) of the
analyte. This can be expressed as a linear regression equation
(y= a + bx), and the corresponding calibration curve of
chlorfenapyr was y=75710−980x in the range of 0.025–2mg
L−1. The linearity was excellent with a correlation coefficient of
r2=0.9986. The residual concentrations of chlorfenapyr in the
treated samples were determined via the calibration curve
Recovery and precision
The extraction efficiency of the analytical procedure was
evaluated via recovery experiments conducted in triplicate using
the fortified blank leek samples at two different fortification
levels (0.05 and 0.25mg kg−1). The precision reflects the variation
in results when repetitive analyses were carried out under the
same conditions. The numerical value used for precision was the
RSD. Recoveries were measured by comparing peak areas of
the spiked sampleswith external standards inacetone. The mean
recoveries were 89.64 and 87.27% with RSD less than 6%
(Table 2). Although the recovery rates acquired through the
developed method were slightly lower than those of Ditya et al.
(2010) (89–97%), the current method was accurate and precise
enough to produce at least 87.27% recovery and 4.46% RSD.
Field‐incurred residues and pre‐harvest intervals
The developed method was applied to the treated leek samples.
The leek samples were analyzed according to the methodology
described above. The highest residual concentrations were
found in the samples treated three times and was decreased
with treatment twice (14 and 7days and 21 and 14days) and
treatment once (7 and 14days). The chlorfenapyr residues after
application 14 and 7days pre‐harvest (PHI, the time between
the last pesticide application and harvest of the treated crops)
to the leeks are given in Table 3, and were less than the MRLs
[3mg kg−1, KFDA (2008); 1mg kg−1, EPA (2003)]. Chlorfenapyr
residues below their MRLs were successfully determined
through the developed method.
Confirmation of the positive treated sample
In order to avoid a false‐positive result, the existence of
chlorfenapyr in the field‐treated sample was verified via mass
Table 2. Validation of the analytical method for chlorfenapyr in leeks
Recovery (%) Mean (RSD%) LOD (mg kg−1) LOQ (mg kg−1)
Table 3. Residues of chlorfenapyr in leeks
Spray frequency Interval before
Chlorfenapyr residues (mg kg−1) MRL (mg kg−1)
123Mean (RSD %)
ND, Not detected. KFDA, Korea Food and Drug Administration (2008). EPA, United States Environmental Protection Agency (2003).
Chlorfenapyr residues in leek
Biomed. Chromatogr. 2012; 26: 172–177 Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
spectrometry. For optimization of the MS parameters, the
standard chlorfenapyr was monitored in full‐scan mode in the
range m/z 50–450. Although the range of m/z should be
reduced to the smallest appropriate scope so as to increase the
sensitivity of the full scan, the range of m/z was set based on its
molecular mass ([M·+]=407) and the main fragmented ion (m/z
59). It is better to test three or four ions for the target compound
in the matrix and then choose two ions that are more sensitive
and show less interference in the final SIM mode. Therefore, in
the mass spectrum of chlorfenapyr shown in Fig. 2, m/z 59, m/z
137, m/z 247 and m/z 328 were tested, with m/z 59 and 247
finally chosen, and the positive treated leek samples were
confirmed in SIM mode.
In this study, a fast, easy and effective method was described for
the analysis of chlorfenapyr in leek using GC‐μECD in combina-
16.00 18.00 20.00
6.008.00 10.00 12.0014.00
Figure 2. Selected ion monitoring mass chromatogram obtained using a HP‐5MS fused silica capillary column for (a) standard chlorfenapyr (10ppm),
(b) EI mass spectrum of chlorfenapyr from mass library and (c) mass chromatogram of treated leek sample.
Md. Musfiqur Rahman et al.
Biomed. Chromatogr. 2012; 26: 172–177 Copyright © 2011 John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/bmc
tion with GC‐MS. Salting out method for extraction and SPE
cartridge using solvent of optimum polarity fraction for clean‐up
with GC‐μECD analysis gave this experiment good repeatability
(RSD) and accuracy (recovery) within a short time. Thus, the
proposed methodology was rapid and selective with a simple
sample preparation procedure, and could be used for the
convenient and effective determination of chlorfenapyr in leek
samples. Therefore, application of chlorfenapyr at the recom-
mended level to leek is safe from crop protection and
environmental contamination points of view.
Ahmad M, Iqbal Arif M and Ahmad Z. Susceptibility of Helicoverpa
armigera (Lepidoptera: Noctuidae) to new chemistries in Pakistan.
Crop Protection 2003; 22: 539–544.
Ahmadi F, Assadi Y, Milani Hosseini SMR and Rezaee M. Determination of
organophosphorus pesticides in water samples by single drop
microextraction and gas chromatography–flame photometric detec-
tor. Journal of Chromatography A 2006; 1101: 307.
Cao Y, Chen J, Wang Y, Liang J, Chen L and Lu Y. HPLC/UV analysis of
chlorfenapyr residues in cabbage and soil to study the dynamics of
Ditya P, Das SP, Sarkar PK and Bhattacharyya A. Degradation dynamics of
chlorfenapyr residue in chili, cabbage and soil. Bulletin of Environ-
mental Contamination and Toxicology 2010; 84: 602–605.
EPA. Federal Register 2003; 68(187): 55519.
Guglielmone A, Volpogni MM, Scherling N, Cobeñas MM, Mangold AJ,
Anziani OS, Ioppolo M and Doscher M. Chlorfenapyr ear tags to
control Haematobia irritans (L.) (Diptera: Muscidae) on cattle.
Veterinary Parasitology 2000; 93: 77–82.
Kaufman PE, Rutz DA, Doscher ME and Albright R. Efficacy of chlorfenapyr
(AC 303630) experimental pour‐on and CyLence formulations against
naturally acquired louse infestations on cattle in New York. Veterinary
Parasitology 2001; 97: 123–129.
KFDA. MRLs for Pesticides in Food. Korea Food and Drug Administration:
Republic of Korea, 2008; 93.
Kurz MHS, Gonçalves FF, Adaime MB, da Costa IFD, Primel EG and
Zanella R. A gas chromatographic method for the determination of
the fungicide chlorothalonil in tomatoes and cucumbers and its
application to dissipation studies in experimental greenhouses.
Journal of the Brazilian Chemical Society 2008; 19: 1129–1135.
Mabey W and Mill T. Critical review of hydrolysis of organic compounds
in water under environmental conditions. Journal of Physical and
Chemical Reference Data 1978; 7: 383–407.
McLeod P, Diaz FJ and Johnson DT. Toxicity, persistence, and efficacy of
spinosad, chlorfenapyr, and thiamethoxam on eggplant when
applied against the eggplant flea beetle (Coleoptera: Chrysomeli-
dae). Journal of Economic Entomology 2002; 95: 331–335.
Metwally M, Osman MS and Al‐Rushaid R. A high‐performance liquid
chromatographic method for the determination of cypermethrin in
vegetables and its application to kinetic studies after greenhouse
treatment. Food Chemistry 1997; 59: 283.
Rand GM. Fate and effects of the insecticide‐miticide chlorfenapyr in
outdoor aquatic microcosms. Ecotoxicology and Environmental Safety
2004; 58: 50.
Ribani M, Bottoli CBG, Collins CH, Jardim ICSF and Melo LFC. Validação
em métodos cromatográficos e eletroforéticos (Validation for
chromatographic and electrophoretic methods). Quimica Nova 2004;
Ribani M, Collins CH and Bottoli CBG. Validation of chromatographic
methods: Evaluation of detection and quantification limits in the
determination of impurities in omeprazole. Journal of Chromato-
graphy A 2007; 1156: 201.
Sadlo S. Quantitative relationship of application rate and pesticide
residues in green house tomatoes. Journal of AOAC International
2000; 83: 214–219.
Tomlin CDS (ed.). The Pesticide Manual: A World Compendium, 12th edn.
British Crop Protection Council: Surrey, 2000; 154.
Treacy M, Miller T, Black B, Gard I, Hunt D and Hollingworth RM.
Uncoupling activity and pesticidal properties of pyrroles. Biochemical
Society Transaction 1994; 22: 244–247.
Chlorfenapyr residues in leek
Biomed. Chromatogr. 2012; 26: 172–177Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/bmc