Development of HPLC Based QuEChERS Method for High Recovery of Fungicides From Vegetable Samples

Vegetables are the most essential part of our diet. Several pesticides are used to kill the pests. These pest cause different diseases on vegetable plants. As a result of which they are getting more and more production of vegetables but there are many hazards for humans. To get rid of this problem, a simple QuEChERS method is used which is based on solvent extraction method in which different salts and acetonitrile were used. In this method clean-up process and extraction were important HPLC technique is used to determine the accurate results. Despicable recoveries of fungicides were ranges between 0.63% to 107.68% at spiking levels and at different factors through percentage RSD between 3.6% to 4.7%. LOD of two fungicides were in the range of 0.0021mg/kg to 0.00181mg/kg and LOQ of two fungicides were in the range of 0.0706mg/kg to 0.00606mg/kg. By varying many factors the recoveries of fungicides may vary. The recoveries of two fungicides may increase by increasing temperature, pH and concentration but decrease with the passage of time due to degradation of fungicides. By this QuEChERS method we get different concentration of fungicides in some vegetables but in Methi vegetables they are absent. And determinations and extraction of fungicides were completed by HPLC technique. QuEChERS method is very ecient and good method for determination of pesticides in various vegetables. Hence our experimental results were validated with this ecient method. in Spinach and Fenugreek sample are 65.38, 87.56 respectively in 50 ppm spiking solution. While % age recoveries of Thiophenate Methyl in Fenugreek and Spinach are 107.68 and 73.36 correspondingly which shows positive results. % recovery of Thiophenate Methyl and Azoxystrobin in Spinach is 73.82 and 63.98 while in Fenugreek is 77.32 and 92.42 in 75 ppm spiking solution which are higher values. Recovery % of azoxystrobin in Spinach is 75 and in Fenugreek is 65.42 and of thiophenate methyl in Fenugreek is 74.74 and in spinach is 72.45 which show best results by spiking different concentration of fungicides, we get different vegetables. These results indicate that both pesticides show excellent recoveries in vegetables samples using HPLC based QuChERS method.


Introduction
Azoxystrobin is a wide range pesticide that is used as fungicide for many diseases of crops with different trade names including Amistar, Abound, Quadris, Bankit, and Heritage. It works by constraining mycelial growth, spore germination and mitochondrial respiration. Though, Azoxy shows less toxic effects for bees, birds and mammals and other terrestrial organisms [1]. Azoxystrobin is fungicide that strongly works against various diseases of numerous ornamental and edible crops. A latest study illustrate that azoxystrobin degradation product R234886 can percolate via loamy soils for a long period of time following application of the pesticide [2] and thereby pose a potential threat to vulnerable aquatic environments and drinking water resources [3]. Some diseases controlled or prevented are late blight, rice blast, Septoria, rusts, apple scab, powdery, red spider mite [4] and downy mildew [5]. No numerical proliferation in the gure of animals containing tumor, animals having benign tumors, malignant tumors, single or multiple tumors or metastic tumors in testing individuals administered with azoxystrobin at dose levels of up to 117.1 mg/kg body weight per day for about two years [6]. Azoxystrobin also remained low sub chronic in dermal testing of twenty one days [7]. The study was also established as gross swollenness of the bile duct convoyed by histological alteration. It showed no toxicity of development in the rabbit or rat by dose level of up to and levels displayed to be maternally toxic [7].
Azoxystrobin showed a weak response of clastogenic effect in vitro in mammalian cells at cytotoxic dosages and in vivo it implies no genotoxicity [7].
It is a non-volatile compound and does not leach down, it is highly vulnerable to photolysis. It is not present in groundwater or drinking water because Azoxystrobin does not leach down in soil water [8]. As it does not leach it is very improbable to go into water reservoirs except by direct spray or accidentally.
Though, in ooded anaerobic soils the compound is despoiled with a half-life of about seven weeks during laboratory tests [9]. In plants it is metabolized having complex with more than fteen identi ed metabolites. These metabolites are existing in very less amounts, usually considerably fewer than 5% of the TRR (Total Recoverable Residue).
Thiophenate methyl is class of fungicide which is used for different crops and vegetables to kill different germs causing different disease [9]. Thiophenate methyl has a colorless crystals, [10] having melting point 342° F [11]. It is frugally resolvable in many of organic solvents at 25 °C like Acetone, methanol ,ethyl acetate, dichloromethane, n-octanol ,Xylene, n-hexane. It is somewhat resolvable in many organic solvents [12]. Thiophenate methyl is strongly soluble in methanol, acetone and chloroform [13]. Its solubility in water is 26.6 milligram/liter at 20 °C [14]. When excited to disintegration it releases exact poisonous gases and oxides of nitrogen and Sulphur [15]. It is removed from the body in twenty four hour that gone in muscles afterward 24 hours is mostly abolished within 96 hour [12].
Determination and quanti cation of pesticides from crops is usually done by QuChERS method [16]. The process of QuEChERS method has several steps. This comprises of sample research and abstraction method. At that step samples are mixed regularly [17]. Now different acidic solution and buffers are used to enhance the e ciency of extraction [18]. This is the clean out method of extraction of sample using solid phase extraction method [19].
In this research we propose a multi residue QuEChERS method [20] [21] for the determination of Azoxystrobin and Thiophenate Methyle from two vegetables Spinach and Fenugreek (Methi). Structures of both fungicides is given in Figure S1 (supporting information). Vegetables are crushed and homogenized and treated with acetonitrile in centrifuge tube for extraction [22]. Then sodium sulphate and sodium chloride are added to maintain pH of the sample solution and acetonitrile is added to remove matrix by cleanout method by dSPE. Finally HPLC analysis is carried out [23]. The proposed method is fast, easy and e cient for fungicide analysis with high recovery, low limit of detection and limit of quanti cation [24].
Two types of vegetables Fenugreek (Methi) and Spinach were bought from native market MuzaffarGarh (Pakistan). A blank and spiked sample was prepared for analysis. These vegetables had been eroded by means of water and dried out in sun light

Sample Preparation
Samples of spinach and Fenugreek were kept in sun light for some days for water evaporation. These vegetables were dried, crushed and ltered the powder from 25 meshes and put the vegetables in distinct envelope. Weighed up 1.0 gram of each crushed vegetable and placed into 15 mL centrifuge tube and saturated with 4 ml of deionized water for 2 minutes. 7 ml of various working solutions were prepared, agitated and vortexed strongly for 3 minutes. Then put 7 ml analytical grade ACN in every centrifuge tube and agitated for 3 min. Then put 1.0 g sodium chloride and 4 gram anhydrous Na 2 SO 4 in every tube and shaken for 3 minutes. Then centrifuged in TGL-16 high speed centrifugal device at 4000 rpm for 5 mints.
Detached the upper ACN layer and added 0.05 gram activated carbon, 0.150 gram Na 2 SO 4 and centrifuged again at 4000 rpm for 7 mints. Acetonitrile organic layer was strained by Whatman lter paper and HPLC analysis was carried out. Then solutions of various concentrations 10, 20, 30, 40 and 50 (ppm) of Azoxystrobin and Thiophenate methyl, are analyzed by HPLC (Fig. 2). Peaks under the area of two fungicides are determined and calibration curves for both fungicide standards are plotted (Fig. 3). A straight-line graph is obtained with correlation coe cient (R 2 ) of 0.9990, 0.9998. By using the Y value of peaks under the area in straight line equation the determined amount in mg/kg of azoxystrobin and thiophenate methyl in Spinach and Fenugreek sample is obtained. After separate analysis of each standard solution of both pesticides, mixture of each concentration is analyzed to check the effect of one pesticide on the other and results are presented in Fig S2 (supporting information).
Obtained RSD, LOD and LOQ for Azoxystrobin are 3.6%, 0.00211 and 0.0706 respectively. Similarly, RSD, LOD and LOQ for Thiophenate Methyl are 4.7%, 0.00181 and 0.00606. The detailed information is given in Table 1.

Quchers Method Validation
The removal of fungicides and method authentication is based on QuChERS method which is very simple and easy method to determine the fungicide residue in vegetables without internal standard. Different concentration such 50, 75 and 100 ppm of both pesticides are spiked in vegetable samples. Aacetonitrile is used as solvent for extraction of fungicides and water is used for the soaking of samples.
Chromatograms showing the retention times and peak areas for Thiophenate Methyl and Azoxystrobin at 50, 75 and 100 ppm spiking concentrations in Spinach and Fenugreek are shown in Fig. 4 and Fig. 5 respectively.
Extraction e ciency of the method for both pesticides is given in  not simply precise and extra well-organized as well as saves currency, chemical reagents and period. This method is very sensitive in extraction of two pesticides in different vegetables at parts per million.

List Of Abbreviations
"Not Applicable" in this section.

Declarations Compliance with Ethical Standards
Authors declare no con ict of interest in this study. All the authors contributed equally to this research. The corresponding author designed the study and revised the manuscript. The manuscript is submitted with the approved consent of all authors.

Consent for Publication
"Not Applicable" in this section.

Availability of data and materials
The complete data that support the ndings of this study are openly available in the online library of Higher education commission Pakistan (HEC).

Competing interest
The authors whose names are listed immediately below certify that they have NO a liations with or involvement in any organization or entity with any nancial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-nancial interest (such as personal or professional relationships, a liations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.