Ethychlozate (ECZ) is a plant growth regulator of synthetic auxin for agricultural commodities (ACs). Accurate and sensitive method to determine ECZ in diverse ACs on global official purpose is required to legal residue regulation. As the current official method is confined to the limited type of crops with poor validation, this study was conducted to improve and extend the ECZ method using high-performance liquid chromatography (HPLC) in all the registered crops with method verification.
ECZ and its acidic metabolite (ECZA) were both extracted from acidified samples with acetone and briefly purified by dichloromethane partition. ECZ was hydrolyzed to form ECZA and the combined ECZA was finally purified by ion-associated partition including hexane-washing. The instrumental quantitation was performed using HPLC/ FLD under ion-suppression of ECZA with no interference by sample co-extractives. The average recoveries of intra- and inter-day experiment ranged from 82.0 to 105.2% and 81.7 to 102.8%, respectively. The repeatability and reproducibility for intra- and inter-day measurements expressed as a relative standard deviation was less than 8.7% and 7.4%, respectively.
Established analytical method for ECZ residue in ACs was applicable to the nation-wide pesticide residues monitoring program with the acceptable level of sensitivity, repeatability and reproducibility.
The pesticides, including plant growth regulators, herbicides, insecticides, and fungicides have contributed much to the quality improvement and the increased production of foods/crops (Park
Plant growth regulators were originated from the development of indole acetic acid (IAA) which is a synthetic auxin for ACs (Spaepen
Among these, Ethychlozate (ECZ), ethyl 5-chloro-3(1
Meanwhile, the Ministry of Food and Drug Safety (MFDS, Korea) has established a tolerance for ECZ residue in 1.0 mg/kg for mandarin and 0.05 mg/kg for other foods (Korea Food Code, 2013). In Japan, MRL of 0.05 mg/kg for ECZ residue are set to 9 food items (MHWL Notification, 2007). In addition, FAO/IAEA has been classified as a normal toxic (FAO/IAEA Record, 2008).
Accurate and sensitive analytical methodology able to determine ECZ in many samples at low level and world-wide officially recognized method is required (Taylor
ACs (ECZ residue-free brown rice, mandarin, pepper, potato, and soybean) were purchased from the local markets in Korea. They were selected as the representative ACs considering their matrix characteristics in an analytical procedure. The samples were homogenized by using a blender, and then kept in a polyethylene container in a freezer at temperature below -50℃.
A pure standard ECZ (certified analytical standard, 98.0%) was purchased from Dr. Ehrenstorfer (Germany). The physicochemical properties of ECZ are shown in Table 1. Analytical-grade hydrochloric acid, potassium hydroxide, potassium phosphate dibasic (K2HPO4), potassium phosphate monobasic (KH2PO4), sodium chloride, and sodium sulfate anhydrous were acquired from Wako chemical (Japan). Acetone, acetonitrile, dichloromethane, n-hexane, and methanol of HPLC grade were supplied by Merck KGaA (Germany). Formic acid of LC-MS grade was obtained from Sigma-Aldrich (USA). All other chemicals and reagents used throughout the study were of analytical grade, unless stated otherwise.
For stock standard solution, ECZ was prepared in acetonitrile at concentration of a 100 μg/mL. The working solutions were prepared via Hydrolysis and ion-associated partition procedures of the sample preparation. First, 5 mL of stock solution was evaporated to near dryness by nitrogen-evaporator (N-EVAPTM111, Oranomation Associates, USA) under a nitrogen steam below 40℃. Subsequently, the residues were conducted by Hydrolysis and ion-associated partition procedures of the sample preparation [ECZ→5-chloro-3(1
Extraction and partition : Precisely 25 grams (± 0.1 g) of the homogenized sample were placed into a 500 mL capped beaker, to which 1 mL of 6 N HCl solution was added to acidify the sample and 80 mL of acetone was added, and the mixture was shaken for 2 min at 300 rpm for extraction (Brown rice and soybean samples were wetted with a 25 mL of the distilled water (DW) for 1 hr before extraction). The extract was filtered by vacuum filtration, and then transferred to a 250 mL beaker. After that, 40 mL of 2% KH2PO4 solution was added and then adjusted as 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% K2HPO4, 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.
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% K2HPO4, 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.
The HPLC system utilized in this study consisted of Shiseido Nanospace SI-2 equipped with fluorescence detector (Japan). Capcell Pak C18 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).
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
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.
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
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).
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
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
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