Hydrolysis and ion-associated partition : The residues were reconstituted in 5 mL of methanol and then hydrolyzed in 4 mL of 4 N KOH solution for 30 min at 30℃ [ECZ→ECZA]. The hydrolysate was added in 40 mL of 2% K
2
HPO
4
, and then adjusted as pH 8.0 (±0.2) with 4 N KOH solution. The adjusted solution was transferred to a 250 mL separatory funnel, followed by liquid-liquid partitioning with hexane (50 mL). After 2 min of vigorously shaking at 300 rpm, the aqueous phase was transferred in a 250 mL round bottom flask and then adjusted as pH 2.0 (±0.2) with 6 N HCl solution. The adjusted solution was transferred to a 250 mL separatory funnel, followed by liquid-liquid partitioning with dichloromethane (50 mL × 2). After 2 min of vigorously shaking at 300 rpm, the organic phases were combined in a 250 mL round bottom flask and filtered through anhydrous sodium sulfate. The organic phase (dichloromethane layer) was evaporated to near dryness by rotary evaporator at 40℃ to a final volume of 5 mL of mobile phase.
- High-liquid performance chromatography analysis
The HPLC system utilized in this study consisted of Shiseido Nanospace SI-2 equipped with fluorescence detector (Japan). Capcell Pak C
18
column (4.6 × 250 mm, 5 μm, Shiseido, Japan) was used to separate the ECZA from sample co-extractives flowed under the isocratic condition with acetonitrile/methanol/DW/formic acid (15/20/65/0.1, v/v/v/v). A 20 μL sample was carried by a mobile phase into a column, which was kept in an oven at 40℃ at flow rate of 1 mL/min. The ECZA was detected at emission wavelength at 330 nm under the excitation wavelength at 300 nm (
Table 2
).
HPLC/FLD parameter for the analysis of ethychlozate
HPLC/FLD parameter for the analysis of ethychlozate
Results and Discussion
- Establishment of instrument optimization
In order to establish the optimum conditions for the analytical method, the physicochemical properties of ECZ were considered (
BCPC, 2012
;
Table 1
). ECZ was possible for HPLC analysis under ion-suppression due to its carboxylic acid existent in the molecule (dissociative property) (
Lee, 2013
). Furthermore, because the indazole-ring in ECZ compound has fluorescence property, it can be measured by using fluorescence detector (FLD). In addition, according to the former information (
Aoki , 2010
; Bohne
et al
., 2007), the analysis of chemical compound containing the fluorophore mainly used HPLC/FLD which the selectivity and sensitivity is excellent. Therefore, in present study, HPLC/FLD was selected as the most suitable analytical instrument.
Meanwhile, the finally acidified ECZA caused the peak tailing under the reconstitution solution of acetonitrile, reflecting its slightly dissociative and acidic properties. Hence, the reconstitution solution was applied to the mobile phase of the ion-suppression condition (formic acid addition), resulting in a considerable increase in the peak symmetry and sharpness of ECZA.
- Establishment of sample preparation procedure
Acetone (
Wong ., 2010
), acetonitrile (
Association of Official Analytical Chemists International, 2010
), methanol (
Tsipi ., 1999
), and ethyl acetate (
Pihlström ., 2007
) have been commonly used as extraction solvents to optimize and improve the pesticide residue analysis. In the present study, acetone was used as the extraction solvent because it was readily separated from water by liquid-liquid separation with non-polar solvents. However, dry samples (brown rice and soybean) are shown to the low extraction efficiency in water-soluble organic solvent due to strong adsorption (
Lee, 2013
). In order to combat the problem, sample extraction process was supplemented to moistness process to increase the extraction efficiency. Additionally, in order to extract all of the compounds (ester and acidic form), the sample was extracted after adding 6 N HCl solution.
Sample separation and purification were used for ion-associated partition method. ECZ was expected below pKa 3.0 by the existed carboxylic acid in the compound. Therefore, the extract was adjusted below pH 2, and then efficiently separated by a non-polar organic solvent (dichloromethane). According to the official method of MFDS (2013), ECZ residue pesticide was applied only to partial ACs (mandarin and other agricultural commodity). For this reason, the nation-wide pesticide residue monitoring program requesting the analysis of the other ACs has difficulty. Actually, the interfering peak is shown in ECZA retention time of mandarin, pepper, and potato when applying representative samples. In order to resolve the problem, the salts (sodium chloride) were added to increase the ionic strength. This process is suitable for the ion strength control considering the stability of compound in strong acid or alkali conditions. In addition, sample extraction accelerates in removal of impurities.
The ester form of ECZ was demonstrated with a non-polar form, whereas the acidic form of ECZA was demonstrated with the slightly dissociative form. Although ECZ was manufactured as the neutral compound of ethyl ester, because it has quickly hydrolyzed in environment or crops, the acidic form was affected as the practically active component (
Lee, 2013
). For this reason, the analytes of ECZ residues were included together with ECZ and its acidic metabolite altogether. Therefore, this research was measured together based on the calculated method by totally acidic amount reference after transformation of target compound (ester form→acidic form), as suggested by
Lee (2013)
.
An acidic form can be extracted into an organic solvent by suppressing their ionization in an aqueous phase with a buffer of controlled pH or via the addition of acid or base (
Park ., 2011
). In this experiment, in order to suppress the ionization, 2% K
2
HPO
4
solution was added, and then controlled as pH 8.0 (±0.1) using 4 N KOH solution. This adjusted aqueous solution added a non-polar organic solvent (n-hexane), which are efficiently separated by a hydrophilic of the analytes. Subsequently, new aqueous phase was controlled as pH 2 (±0.1), and then added a non-polar organic solvent (dichloromethane), which are separated by a hydrophobic of the analytes.
- Method validation
The selectivity of the analytical method was evaluated via the absence of interfering peaks from co-extractives at the retention time of ECZA. As shown in
Fig. 1
, the typical chromatograms of control and spiked ACs sample were confirmed to the absence of interfering peaks (control samples) as well as good separation of ECZA (spiked samples).
HPLC chromatograms of extracts from agricultural commodities.
A, brown rice; B, mandarin; C,pepper; D, potato; E, soybean; 1, control; 2, for tified with ethychlozate at LOQ of 0.02 mg/kg; 3, fortified with ethychlozate 0.2 mg/kg (A, C, D, and E) and 1.0 mg/kg (B).
The linearity of ECZA working standard solution by an analytical method was conducted via an external standard procedure. The equation of calibration curve was obtained by plotting peak areas in ‘y’ axis against concentrations of ECZA in ‘x’ axis (
Hem ., 2011
), which was y=115.1 × 10
4
x + 0.1935 × 10
4
, with a correlation coefficient (
r
2
) of 0.9999. These values demonstrate that the method's quantification issufficient for Codex Guideline (
r
2
>0.95) (CAC/GL 40, 1993).
The limit of detection (LOD) and limit of quantification (LOQ) were determined based on the standard deviation of blank sample responses (σ) and the slope of the calibration curve (S), which were calculated by multiplying σ/S by 3.3 and 10, respectively (ICH Guideline, 1996). Instrumental LOD and LOQ were determined to be 0.5 and 2 ng, respectively.
MLOQ (Method Limit of Quantitation) is not an instrumental LOQ, but instead is a practical LOQ for the total analytical method. It is usually calculated by using an instrumental LOQ, injection volume, final extract volume, and sample weight in an analytical method (
Lee, 2013
;
Lee ., 2012
). MLOQ value for ECZ was 0.02 mg/kg. MRLs for ECZ of ACs in MFDS are set up in mandarin (1.0 mg/kg) and other agricultural commodity (0.05 mg/kg) (Korea Food Code, 2013). According to the guideline on a residue analytical method in SANCO/825/00, MLOQ is recommended below 0.02 mg/kg if MRL is set up as 0.05 mg/kg (EC Guidance, 2010). Consequently, the proposed MLOQ value is adjudged resonable for determination of MRL of ECZ residue in ACs.
Accuracy and precision were conducted via intra- and inter-day analyses (in a single laboratory), and precision was calculated in terms of intra-day repeatability and inter-day reproducibility (
Choi ., 2011
; ICH Guideline, 1996;
Thompson ., 2002
). Accuracy was expressed as a percentage of recovery and precision as a relative standard deviation (RSD). Intra- and inter-day analyses were conducted using the fortified brown rice, pepper, potato, and soybean (no Korea MRL criteria) at three different concentrations of MLOQ (0.05 mg/kg), 2 × MLOQ (0.1 mg/kg), and 10 × MLOQ (0.5 mg/kg) and mandarin (Korea MRL criteria : 0.2 mg/kg) at three different concentrations of MLOQ (0.05 mg/kg), 0.5 × MRL (0.1 mg/kg), and MRL (0.2 mg/kg). Intra-day analysis was conducted in five replicates at each concentration level, whereas inter-day analysis was performed for three consecutive days in triplicate at the same concentrations. The recovery averages of the intra-day experiment ranged from brown rice 87.2-92.9%, mandarin 100.8-105.2%, pepper 89.1-95.3%, potato 82.0-85.9%, and soybean 91.2-102.3%, respectively, and the recovery averages of inter-day experiment ranged from brown rice 86.0-91.7%, mandarin 100.0-102.8%, pepper 88.1-93.2%, potato 81.7-87.1%, and soybean 89.4-100.8%, respectively (
Table 3
). The intra-day repeatability expressed as RSD was less than 8.7% in all ACs, whereas inter-day reproducibility expressed as RSD was less than 7.4% in ACs (
Table 3
). These recovery and RSD values were consistent with the ranges listed in the Codex and SANCO Guideline (CAC/GL 40, 1993; EC Guidance, 2010), and thus the method described herein can be considered excellent as a reliable, reproducible, and accurate routine analytical method. Additionally, it had the tendency to be similar to the results that the existing researcher reports (
Takatsuki , 2002
). Therefore, our newly establishing analytical method for ECZ residue in ACs can be confirmed as the suitable method.
Intra- and inter-day recoveries and RSD of ethychlozate in agricultural commodities
Intra- and inter-day recoveries and RSD of ethychlozate in agricultural commodities
Acknowledgements
This research was supported by a grant (13161MFDS017) from Ministry of Food and Drug Safety in 2013.
2010
Pesticide and industrial chemical residues, In official method of analysis
(18th ed.)
Association of Official Analytical Chemists International
USA
17 -
26
Aoki Y.
,
Kotani A.
,
Miyazawa N.
,
Uchida K.
,
Igarashi Y.
,
Hirayama N.
,
Hakamata H.
,
Kusu F.
2010
Determination of ethoxyquin by high-performance liquid chromatography with fluorescence detection and its application to the survey of residues in food products of animal origin
Journal of AOAC International
93
(1)
277 -
283
Atta S.
,
Ikbal M.
,
Kumar A.
,
Pradeep Singh N. D.
2012
Application of photoremovable protecting group for controlled release of plant growth regulators by sunlight
Journal of Photochemistry and Photobiology B: Biology
111
39 -
49
DOI : 10.1016/j.jphotobiol.2012.03.008
2012
The Pesticide Manual: A World Compendium
(16th ed.)
British Crop Production Council
UK
No. 340, Ethychlozate
441 -
Berdikova Bohne V. J.
,
Hove H.
,
Hamre K.
2007
Simultaneous quantitative determination of the synthetic antioxidant ethoxyquin and its major metabolite in Atlantic salmon (Salmo salar, L), ethoxyquin dimer, by reversed-phase high-performance liquid chromatography with fluorescence detection
Journal of AOAC International
90
(2)
587 -
597
Choi J. H.
,
Mamun M. I. R.
,
Abd El-Aty A. M.
,
Park J. H.
,
Shin E. H.
,
Park J. Y.
,
Cho S. K.
,
Shin S. C.
,
Lee K.B.
,
Shim J. H.
2011
Development of a single-step precipitation cleanup method for the determination of enrofloxacin, ciprofloxacin, and danofloxacin in porcine plasma
Food Chemistry
127
(4)
1878 -
1883
DOI : 10.1016/j.foodchem.2011.02.027
Do J. A.
,
Lee M. Y.
,
Cho Y. J.
,
Kang I. H.
,
Kwon K. S.
,
Oh J. H.
2013
Development and validation of an analytical method for pyrimisulfan determination in agricultural commodities by LC-MS/MS
Analytical Science and Technology
26
(2)
154 -
163
DOI : 10.5806/AST.2013.26.2.154
Giannakoula A. E.
,
Ilias I. F.
,
Maksimović J. J. D.
,
Maksimović V. M.
,
Zivanović B. D.
2012
The effects of plant growth regulators on growth, yield, and phenolic profile of lentil plants
Journal of Food Composition and Analysis
28
(1)
46 -
53
DOI : 10.1016/j.jfca.2012.06.005
Hem L.
,
Choi J. H.
,
Park J. H.
,
Mamun M. I. R.
,
Cho S. K.
,
Abd El-Aty A. M.
,
Shim J. H.
2011
Residual pattern of fenhexamid on pepper fruits grown under greenhouse conditions using HPLC and confirmation via tandem mass spectrometry
Food Chemistry
126
(4)
1533 -
1538
DOI : 10.1016/j.foodchem.2010.11.147
Kim H. Y.
,
Yoon S. H.
,
Park H. J.
,
Lee J. H.
,
Gwak I. S.
,
Moon H. S.
,
Song M. H.
,
Jang Y. M.
,
Lee M. S.
,
Park J. S.
,
Lee K. H.
2007
Monitoring of residual pesticides in commercial agricultural products in Korea
Korean Journal of Food Science and Technology
39
(3)
237 -
245
Lee H.
,
Kim E.
,
Moon J. K.
,
Zhu Y. Z.
,
Do J. A.
,
Oh J. H.
,
Kwon K.
,
Lee Y. D.
,
Kim J. H.
2012
Establishment of analytical method for cyazofamid residue in apple, mandarin, korean cabbage, green pepper, potato and soybean
Journal of the Korean Society for Applied Biological Chemistry
55
(2)
241 -
247
Lee S. M.
,
Kim J. Y.
,
Kim T. H.
,
Lee H. J.
,
Chang M. I.
,
Kim H. J.
,
Cho Y. J.
,
Choi S. W.
,
Kim M. A.
,
Kim M. K.
,
Rhee G. S.
,
Lee S. J.
2013
Establishment of analytical method for dichlorprop residues, a plant growth regulator in agricultural commodities using GC/ECD
Establishment of analytical method for dichlorprop residues
32
(3)
214 -
223
Min Z. W.
,
Hong S. M.
,
Yang I. C.
,
Kwon H. Y.
,
Kim T. K.
,
Kim D. H.
2012
Analysis of pesticide residues in brown rice using modified QuEChERS multiresidue method combined with electrospray ionization-liquid chromatography-tandem mass spectrometric detection
Journal of the Korean Society for Applied Biological Chemistry
55
(6)
769 -
775
DOI : 10.1007/s13765-012-2153-y
Omeroglu P. Y.
,
Boyacioglu D.
,
Ambrus Á.
,
Karaali A.
,
Saner S.
2012
An overview on steps of pesticide residue analysis and contribution of the individual steps to the measurement uncertainty
Food Analytical Methods
5
(6)
1469 -
1480
DOI : 10.1007/s12161-012-9396-4
Park J. Y.
,
Choi J. H.
,
Abd El‐Aty A. M.
,
Kim B. M.
,
Park J. H.
,
Choi W. J.
,
Shim J. H.
2011
Development and validation of an analytical method for determination of endocrine disruptor, 2, 4‐D, in paddy field water
Biomedical Chromatography
25
(9)
1018 -
1024
DOI : 10.1002/bmc.1559
Peterson G. E.
1967
The discovery and development of 2, 4-D
Agricultural History
41
(3)
243 -
254
Pihlström T.
,
Blomkvist G.
,
Friman P.
,
Pagard U.
,
Österdahl B. G.
2007
Analysis of pesticide residues in fruit and vegetables with ethyl acetate extraction using gas and liquid chromatography with tandem mass spectrometric detection
Analytical and Bioanalytical Chemistry
389
(6)
1773 -
1789
DOI : 10.1007/s00216-007-1425-6
Sharma D.
,
Nagpal A.
,
Pakade Y. B.
,
Katnoria J. K.
2010
Analytical methods for estimation of organophosphorus pesticide residues in fruits and vegetables: A review
Talanta
82
(4)
1077 -
1089
DOI : 10.1016/j.talanta.2010.06.043
Spaepen S.
,
Vanderleyden J.
,
Remans R.
2007
Indole-3-acetic acid in microbial and microorganismplant signaling
FEMS Microbiology Reviews
31
(4)
425 -
448
DOI : 10.1111/j.1574-6976.2007.00072.x
Takatsuki S.
,
Nemoto S.
,
Sasaki K.
,
Toyoda M.
2002
Determination of ethychlozate and its degradation product in fruits by HPLC and LC/MS
Journal of the Food Hygienic Society of Japan
43
(1)
30 -
34
DOI : 10.3358/shokueishi.43.30
Taylor M. J.
,
Hunter K.
,
Hunter K. B.
,
Lindsay D.
,
Le Bouhellec S.
2002
Multi-residue method for rapid screening and confirmation of pesticides in crude extracts of fruits and vegetables using isocratic liquid chromatography with electrospray tandem mass spectrometry
Journal of Chromatography A
982
(2)
225 -
236
DOI : 10.1016/S0021-9673(02)01610-2
Thompson M.
,
Ellison S. L. R.
,
Wood R.
2002
Harmonized guidelines for single-laboratory validation of methods of analysis (IUPAC Technical Report)
Pure and Applied Chemistry
74
(5)
835 -
855
Tsipi D.
,
Triantafyllou M.
,
Hiskia A.
1999
Determination of organochlorine pesticide residues in honey, applying solid phase extraction with RP-C18 material
Analyst
124
(4)
473 -
475
DOI : 10.1039/a809724k
Wang K. S.
,
Lu C. Y.
,
Chang S. H.
2011
Evaluation of acute toxicity and teratogenic effects of plant growth regulators by Daphniamagna embryo assay
Journal of Hazardous Materials
190
(1)
520 -
528
DOI : 10.1016/j.jhazmat.2011.03.068
Wong J. W.
,
Zhang K.
,
Tech K.
,
Hayward D. G.
,
Krynitsky A. J.
,
Cassias I.
,
Schenck F. J.
,
Banerjee K.
,
Dasgupta S.
,
Brown D.
2010
Multiresidue Pesticide Analysis of Ginseng Powders Using Acetonitrile-or Acetone-Based Extraction, Solid-Phase Extraction Cleanup, and Gas Chromatography−Mass Spectrometry/Selective Ion Monitoring (GC-MS/SIM) or−Tandem Mass Spectrometry (GC-MS/MS)
Journal of Agricultural and Food Chemistry
58
(10)
5884 -
5896
DOI : 10.1021/jf903851h