Derivative Spectrophotometric method for Simultaneous Determination the residue of Carboxin, Chlorothalonil and Pyriproxyfen in cabbage samples using Zero-crossing Technique

We developed and validated a spectrophotometric technique for determining Carboxin, Chlorothalonil and Pyriproxyfen residues in cabbage samples that is simple, accurate, sensitive and precise. The approach is based on recording the first derivative curves and utilising the zero-crossing method to compute the estimated values of each component. There are no earlier separation procedures in this procedure. This investigation led to the revelation that the correlation coefficient of the calibration curves was more than 0.9998. The calibration curves for this spectrophotometric approach for Carboxin, Chlorothalonil and Pyriproxyfen are linear in concentration ranges of (1-35) μg/mL, 0.5-7 μg/mL and (1-30) μg/mL, with a relative standard deviation of no more than 5.0%. The proposed approach has been utilised effectively to test Carboxin, Chlorothalonil and Pyriproxyfen residues in cabbage samples concurrently with a high rate of recovery, accuracy and precision.


Introduction
Pesticides are chemicals used to control pests that damage agricultural goods during production, nutrient absorption and storage, resulting in product loss [1].
Carboxylate (CAR) commonly known as Vitavax (2,3-dihydro-6-methyl-oxathine-5carboxanilide) is one of the most popular of several systemic fungicides used in agriculture to eliminate harmful fungi.The chemical composition of carboxin is shown in Figure (1a) [2].Fungicide residue levels in foods are typically regulated to reduce consumer exposure to harmful or unnecessary pesticide ingestion, despite the fact that it has minimal mammalian toxicity [3].For the carboxin determination, many analytical techniques were applied, either alone or in conjunction with other pesticides including gas liquid chromatography [4], highperformance liquid chromatography [5,6] and derivative spectrophotometry [7].It is not poisonous [8].Chlorothalonil is an extensively used pesticide for combating fungi that destroy vegetables, trees, little fruits and other agricultural crops [9].The most widely used analytical methods for locating chlorothalonil residues in various matrices include gas chromatography-mass spectrometry, gas chromatography with electron capture detection, high-performance liquid chromatography with UV detection and high-performance liquid chromatography-diode array detection [10][11][12][13][14][15][16].
Pyriproxyfen (PYR) is poisonous to insects throughout their embryonic, final larval or reproductive stages.Whiteflies, mealworms, scales and thrips are among the pests that Pyriproxyfen is especially efficient against [17].Pyriproxyfen [4-phenoxyphenyl (RS)-2-(2pyridyloxy)-propyl ether] its chemical name.It is a crucial pesticide used to protect plants from whiteflies [18].Pyriproxyfen chemical structure is given in Figure (1c) is a pyridine-based pesticide which is effective on a variety of arachnoid [19].Traditional techniques for detecting pyriproxyfen and other pesticides in agricultural goods such as fruits and vegetables include gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry [20,21].Pyriproxyfen and other pesticides were quantified using high-performance liquid chromatography and HPLC with UV detection [22,23], as well as derivative spectrophotometry [24].The aim of this study is to establish a method to resolve a ternary mixture of pesticides in cabbage samples.The spectroscopic observations were performed using a Shimadzu (UV-1800, Japan) UVvisible double beam spectrometer with a fixed bandwidth of 1 nm and a 1 cm quartz cell.A double beam spectrophotometer linked to a computer was used to record carboxin, chlorothalonil and pyriproxyfen mixed solution zero order spectra as well as data on absorption spectra.Data for Carboxin, Chlorothalonil and Pyriproxyfen with their combination solutions were transformed from zero order spectra to first order derivative spectra using the UV Probe software.

Chemicals and Materials
Santa Cruz Biotechnology in the United States provided 99% pure versions of carboxin, chlorothalonil, and pyriproxyfen.The HPLC solvents acetonitrile and methanol were provided by Merck (Darmstadt, Germany).Sigma Aldrich provided the Primary Secondary Amin (PSA).England).MgSO4 was heated for activation at 400 °C for 4 hours to remove phthalates.It was then kept cold until it was placed in a desiccator.

Standard Stock Solution preparation
Each 10 mg of insecticide were dissolved in 100 ml of methanol to create stock solutions (100 μg /mL), which were then placed in bottles with stained glass stoppers and kept at -18 °C.In less than two months, all stock solutions were kept in storage.Stock solutions are diluted with methanol to form regularly used standard solutions.

Sample preparation
CAR, CHL and PYR residues were extracted from cabbage samples using the modified QuEChERS approach described by Hou et al. [13].After weighing and homogenising 500 g of chopped material, three levels of the standard stock solution were introduced to a 50 mL teflon centrifuge tube containing 10 g of newly weighted sample.10 mL of an extraction solvent composed of toluene (1:1, v/v) and acetonitrile with 5% acetic acid were added using a transfer pipette.The mixture was vortexed for 1 minute after being agitated for 1 minute in an air bath at 22 °C.The liquid was immediately vortexed for 1 minute after 4 g of anhydrous MgSO4 and 1 g of anhydrous sodium acetate were added.The extracts were then centrifuged for 5 minutes at 2500 rpm.After that, the top layer was separated into 10 mL sections and added to a 15 mL Teflon centrifuge tube together with 1.5 g of MgSO4 and 300 mg of PSA.After one minute of vortexing, the samples were centrifuged at 8800 rpm for five minutes.The solution was filtered using a 0.45-m filter and 10 mL of the filtrate was transferred to a 15-mL tube and gradually concentrated with nitrogen until nearly dry.In order to evaluate the pesticide residue, the extract was diluted in methanol according to the recommended protocol using the standard addition method.

Calibration Graph and Zero-crossing method
Three sets of calibration flasks, each having a capacity of 10 mL and containing a mixture of CAR, CHL and PYR diluted to volume with methanol, were used to create a sufficient stock

Application of the methods
The suggested method was employed in this study to determine the CAR, CHL and PYR in genuine cabbage samples.Five replications of each concentration level were injected into the genuine fresh cabbage samples: (1.0, 20, 35) μg/mL for CAR, (0.5, 4.0, 7.0) μg/mL for CHL and (1.0, 15, 30) μg/mL for PYR.The relative standard deviation of three pesticides in real cabbage samples was less than 5% for CAR, CHL and PYR, indicating the accuracy of the proposed technique.The European Union (EU) food safety authority has established a maximum residual limit (MRL) for some pesticides in order to guarantee that food is safe for consumer health.The quantity of CAR, CHL and PYR residues in cabbage samples that were either grown or procured from the market were lower than the EU-MRL (1 mg/kg), and PYR was not found in the samples of cabbage that were procured from the neighbourhood market .
Therefore, none of these three pesticides pose a risk to the health of consumers.The residue of each CAR, CHL and PYR was simultaneously determined using the spectrophotometric technique and the standard addition method in actual cabbage sample.The main conclusions are provided in (Table 3).

Figure 1 :
Figure 1: The chemical structure of (a) Carboxin (b) Chlorothalonil (c) Pyriproxyfen BDH supplied anhydrous sodium acetate and magnesium sulphate (VWR Chemicals BDH, solution for the samples.In the first sequences, CHL and PYR concentrations are fixed at (1 and 5) μg/mL, respectively, whereas CAR concentrations range from (1 to 35) μg/mL.The second sequences had fixed CAR and PYR concentrations of 5 μg/mL each and variable CHL concentrations of (0.5-7) μg/mL.The third sequences had constant CAR and CHL concentrations of 5 and 1 μg/mL, respectively, together with variable PYR concentrations of (1 to 30) μg/mL.On a white background, absorption spectra between (200 and 350) nm were visible.The computer application SHIMADZU UV Probe Data System converts zero-order spectra of CAR, CHL and PYR in ternary combinations into first-derived spectra in the (200-350) nm range (Version 2.43).Figures(2,3) depict the overlap spectra (zero, first order) for the three insecticides in the ternary combination (3).

Table 1 P
displays the analytical characteristics and statistical information of the calibration curves of the suggested method for each pesticide with an estimated CAR, CHL and PYR, including the linear range of the calibration graph, correlation coefficients, LOD, LOQ and relative standard deviation.According to the table, the calibration curves for the suggested technique have strong correlation coefficient values that are greater than 0.999, which indicates that the calibration curves are linearly consistent.Lower LOD and LOQ values show how sensitive the suggested techniques are.The precision and accuracy of the proposed first derivative spectrophotometric technique based on the zero-cross methodology for the simultaneous measurement of CAR, CHL and PYR were also investigated.The relative standard deviation percentage (RSD%) for each pesticide was less than 5% based on the results of five replicate measurements of three different CAR, CHL and PYR concentrations and the recovery percentage (Recovery%) was greater than 95.04%, demonstrating the suggested approach's acceptable precision and accuracy.The results are shown in the table(2).In the (200-350 nm) region, the usual UV absorption spectra of CAR, CHL and PYR completely overlap (Figure2).As a result, it is impossible to use normal spectrophotometry to separate a mixture and detect three pesticides at once in a ternary mixture.Therefore, interference has been reduced and overlapping spectra have been cleaned up using the zero-crossing method of derivative spectrophotometry.

Discussion 1 .
Normal absorption spectra of Carboxin, Chlorothalonil and Pyriproxyfen solution CAR, CHL and PYR UV absorption spectra, as well as their combination, were measured against methanol as a blank reagent.Figure (2) depicts the absorption spectra of Carboxin and Pyriproxyfen solutions at 10 µg/mL and Chlorothalonil solution at 4.0 μg/mL, respectively.The CAR, CHL and PYR spectrums clearly overlapped one another, as demonstrated.As a result of substantial overlapping and spectral interference, identifying each of these chemicals under investigation based on their usual absorption spectra when present in the same solution is extremely challenging.

Figure 3 :
Figure 3: First derivative spectra of 10 μg/mL each of Carboxin and Pyriproxyfen and 4 μg/mL of Chlorothalonil

Table 1 :
The statistical parameter for determination of carbon, chlorothalonil, and pyriproxyfen by proposed methods