Simultaneous Determination of Ternary Mixture of Carboxin, Chlorpyrifos, and Tebuconazole Residues in Cabbage Samples Using Three Spectrophotometric Methods

Omer, Shilan A.; Fakhre, Nabil A.

doi:https://doi.org/10.1155/2020/4912762

Journal of Analytical Methods in Chemistry

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Abstract Introduction Experimental Results and Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2020 | Article ID 4912762 | https://doi.org/10.1155/2020/4912762

Simultaneous Determination of Ternary Mixture of Carboxin, Chlorpyrifos, and Tebuconazole Residues in Cabbage Samples Using Three Spectrophotometric Methods

Shilan A. Omer^1,2and Nabil A. Fakhre¹

Academic Editor: Jaroon Jakmunee

Received25 Feb 2019

Revised17 Jun 2019

Accepted04 Jul 2019

Published18 Feb 2020

Abstract

Three simple precise and accurate spectrophotometric methods are developed for simultaneous determination of ternary mixtures of carboxin, chlorpyrifos, and tebuconazole residues in cabbage grown in the experimental field. The first method is a double divisor-ratio spectra derivative that relies on the derivative of ratio spectra and attained through dividing the absorption spectra of the ternary mixture by the sum of standard spectrum of a mixture of two from three components, using methanol as a solvent and measuring CAR at 242 nm, CHL at 236 nm, 276 nm, and 300 nm, and TEB at 226 nm. The second method is a successive derivative of ratio spectra which determined CAR at 256 nm and 258 nm, CHL at 290 nm and 292 nm, and TEB at 226 nm and 228 nm. The third method is a mean centering of ratio spectra where CAR, CHL, and TEB were measured at 306 nm, 280 nm, and 240 nm, respectively. These procedures do not involve any previous separation. The extraction of analytes was carried out by using acetonitrile, and the procedure of purification was fulfilled by dispersive solid-phase extraction with a primary-secondary amine (PSA). The proposed methods showed excellent linearity range for three spectrophotometric methods over the concentration ranges of 1–30 μg/mL, 1–50 μg/mL, and 1–45 μg/mL for carboxin, chlorpyrifos, and tebuconazole, respectively. The analytical characteristics such as detection limit, determination limit, relative standard deviation, and accuracy of the three methods were performed. The limits of detection were in the range of 0.153–0.260 μg/mL for carboxin, 0.137–0.272 μg/mL for chlorpyrifos, and 0.109–0.205 μg/mL for tebuconazole with limits of quantification lower than 0.790, 0.824, and 0.621 μg/mL for CAR, CHL, and TEB, respectively. The recoveries ranged from 87.02% to 94.53% for carboxin, 92.32% to 108.53% for chlorpyrifos, and 87.19% to 98.00% for tebuconazole with relative standard deviations less than 5.91%, 5.99%, and 5.53% in all instances for carboxin, chlorpyrifos, and tebuconazole, respectively. The results obtained from the proposed methods were compared statistically by using one-way ANOVA, and the results revealed that there were no significant differences between three different spectrophotometric methods. The suggested methods can be applied with great success to the simultaneous estimation of carboxin, chlorpyrifos, and tebuconazole residues in cabbage samples.

1. Introduction

Pesticides are greatly applied to keep the crops away from a variety of pests. The main purpose of the use of pesticides is to gain high yield of the agricultural product, but successive and repeated use of certain pesticides have led to their accumulation in plants, animals, sediments, and soils, thus increasing the rate of spreading of contamination in the environment [1]. Recently, the application and production of pesticide through worldwide increased for both agricultural and nonagricultural goals which cause soil, air, surface water, and groundwater pollution that in turn involves significant risk for both environment and human health either directly through exposure to it or indirectly via residues in food and drinking water. Globally, pesticides reached alarming levels in water, air, soil, food production, and biological stuff [2].

Carboxin (CAR) which is chemically known as 2, 3-dihydro-6-methyl-oxathiin-5-carboxanilide (Vitavax) is considered a systematic fungicide that has been used to kill pathogenic fungi in the agriculture [3]. The chemical structure of carboxin is shown in Figure 1(a). Carboxin is a type of anilide fungicide that is used broadly at multiple stages of plant growth and during the storage of postharvest to protect the product against rotting. Although it has low mammalian toxicity, the levels of fungicide residues are generally regulated by legislation in order to diminish the risk of consumers’ exposure to the deleterious or dispensable uptake of pesticides [4]. Different analytical techniques were used for the carboxin determination either alone or in combination with other pesticides in food, and environmental samples include spectrophotometric method [3], gas chromatography (GC) [5], gas-liquid chromatography (GLC) [6], thermospray liquid chromatography/mass spectrometry [7], high-performance liquid chromatography (HPLC) [8], and HPLC equipped with UV detector [9].

(a)

(b)

(c)

Chlorpyrifos (CHL) is a crystalline organophosphate insecticide. It is chemically defined as O, O-diethyl-O-3, 5, 6-trichloro-2-pyridyl phosphorothioate insecticide. In agriculture, it remains one of the most widely used organophosphate insecticides [10]. The chemical structure of chlorpyrifos is shown in Figure 1(b). Chlorpyrifos is analyzed by various methods such as spectrophotometry and derivative spectrophotometry [11–15] and spectrophotometry using multivariate calibration methods [16], partial least squares [17, 18], GC [19], GC equipped with electron capture detector (GC-ECD) [20], GC-MS [21], GC-MS/MS [22], and HPLC with UV detection [23–25] was also used for estimation of chlorpyrifos.

Tebuconazole (TEB) is chemically defined as ((RS)-1-p-chlorophenyl)-4, 4-dimethyl-3-(1H-1, 2, 4-triazol-1-ylmethyl) pentan-3-ol. It is utilized to treat pathogenic fungi on the plant in agriculture and on crops [26]. The chemical structure of tebuconazole is shown in Figure 1(c). It was determined by several methods including GC [27], gas chromatography-nitrogen phosphorus detection [28], gas chromatography-flame ionization detector [29], GC-MS [30, 31], and supercritical fluid chromatography (SFC)-MS/MS [32] and mostly determined by HPLC and HPLC-DAD method [33, 34], HPLC-MS/MS [35], liquid chromatography (LC) with UV detection [36], and LC-tandem mass spectrometric detection [37]. In addition, quantitative structure-property relationship (QSPR) models were developed using multiple linear regression, partial least squares, and neural network analyses for determining a number of pesticides including carboxin, chlorpyrifos, and tebuconazole [38].

Different spectrophotometric methods could be used for resolving and determining mixtures which contain two or more active compounds simultaneously without requiring a preliminary separation process such as classical derivative spectrophotometry [39–42]. Furthermore, UV-visible spectrophotometry method could not be used for the determination of chemicals with overlapped spectra, but it might be utilized for the estimation of certain chemicals in various samples [43].

In 1990, Salinas et al. [44] proposed a method for resolving binary mixtures. This method was based on the first derivative of ratio spectra which was attained through dividing the absorption spectra of binary mixtures by the standard spectrum of one of the components. Then, Berzas Nevado et al. [45] expanded the method which was introduced by Salinas from the analysis of binary mixture to ternary mixture by derivative ratio spectra zero-crossing method. This method depends on the measuring of the signal at the zero-crossing point.

Another spectrophotometric method was introduced by Dinç et al. [46–48] for estimation of three compounds in ternary mixtures simultaneously. This method consists of two steps through dividing the absorption spectra of ternary mixtures by the sum of two spectra. Later, the ratio spectrum was derivatized. This method is termed as a double divisor-ratio spectra derivative method which is depending upon the use of the coincident spectra of the ratio spectra derivative gained by the use of a double divisor and measurements at maximum or minimum wavelengths.

Currently, other methods for the determination of ternary mixtures simultaneously were developed by Afkhami and Bahram that lack preceding steps of separation. These methods are successive derivatives of ratio spectra which consist of two steps and depend on the successive derivative of ratio spectra. The other method is mean centering of ratio spectra, this method could be applied successfully for the analysis of binary and ternary mixture simultaneously, and the mathematical clarification of the proposed methods was described in [49, 50]. Recently, pesticides could be determined in binary mixture using mean centering of ratio spectra [51]. Fast, easy, and inexpensive methods are widely used for the determination of pesticide residues in fruits and vegetables involving acetonitrile extraction partitioning and dispersive solid-phase extraction [52]. Hence, this paper tries to establish methods to resolve a ternary mixture of pesticides in cabbage samples and then to compare the results obtained by these three approaches.

2. Experimental

2.1. Apparatus

Double-beam UV-visible spectrophotometer (Shimadzu, Model UV-1800, Japan) with a fixed 1 nm bandwidth and 1 cm quartz cell was utilized for spectrophotometric measurement, and the computer was connected to a double-beam spectrophotometer in order to record zero-order spectra. All calculations were made using Matlab 6.5 and Microsoft Excel. ANOVA F-test was performed by SPSS.

2.2. Chemicals and Materials

Pesticide standards carboxin (99% purity), chlorpyrifos (99% purity), and tebuconazole (99.9% purity) were provided by Santa Cruz Biotechnology, USA. HPLC-grade solvents including acetonitrile and methanol were provided by Merck (Darmstadt, Germany). Primary-secondary amine (40 μm) (PSA) was supplied by Sigma-Aldrich. Sodium chloride and anhydrous magnesium sulfate were provided by BDH (VWR Chemicals BDH, England); MgSO₄ was heated for activation at 200°C for 4 h and then cooled and stored in a desiccator prior to use.

2.3. Standard Stock Solution Preparation

Stock solutions of carboxin, chlorpyrifos, and tebuconazole pesticide (100 μg/mL) were prepared through dissolving 10 mg of each pesticide in 100 mL methanol and then put in a freezer at −18°C in stained glass-stopper bottles. All stock solutions were stored for less than two months. Working standard solution is prepared daily through diluting the stock solutions in methanol.

2.4. Field Experiment

The field experiment was performed from 20 October 2017 to 10 April 2018 at the Grdarash field which belongs to the College of Agriculture, Salahaddin University, Erbil. For this purpose, cabbage seeds were mixed with carboxin (Vitavax) fungicide in the recommended dose of 3 g/kg seeds and then planted in pots with 5 cm diameter containing sterile soil in greenhouse for 8 weeks and later transplanted to outdoor field. After the plant reaches a certain stage of growth, chlorpyrifos (insecticide) in the recommended dose of 6 mL/5 L water and tebuconazole (fungicide) in the recommended dose of 1.5 mL/5 L water were sprayed foliarly under the supervision of pesticide expert at regular intervals, taken safety period into consideration. The cabbage sampling was conducted on 10 April 2018. The samples were put in sterile plastic bags and transported directly to the laboratory then homogenized and kept in the refrigerator at 4°C for further analysis.

2.5. Sample Preparation

QuEChERS method described by [27] was followed for the extraction of carboxin, chlorpyrifos, and tebuconazole residues from cabbage samples. About 500 g of chopped sample was balanced and homogenized, then weighed 10 g of the prior chopped fresh sample and transferred into a 50 mL Teflon centrifuge tube. Then 10 mL of acetonitrile as extraction solvent was added by using a transfer pipette, and the mixture was shaken for 1 min in an air bath at 22°C to confirm the solvent and entire samples interacted well, and then 4 g of anhydrous MgSO₄ and 1 g sodium chloride were added and vortexed for 1 min instantly; later, centrifugation of the extracts was carried out for 5 min at 5000 rpm; 10 mL aliquot of the higher layer was transported into a 15 mL Teflon centrifuge tube that contains 300 mg PSA and 1.5 g anhydrous MgSO₄ and then vortexed for 1 min and centrifugation at 7000 rpm for 5 min was performed. After that, filtration of the resulting solution was done by using a filter (0.45 μm). Then, the filtrate was transported into a 15 mL tube and cautiously under a stream of nitrogen concentrated to near dryness. Eventually, 5 mL methanol used to redissolve the residues.

3. Application of Methods

3.1. Double Divisor-Ratio Spectra Derivative Method

For CAR to be determined, the stored spectra which contain a different concentrations of CAR in a ternary mixture were divided by the standard spectrum of a double divisor CHL and TEB (10 μg/mL each in methanol) to obtain the ratio spectra. Later, calculation of the first derivative of the ratio spectra using Δλ = 6 nm was performed. The amount of CAR was determined by measuring the amplitude at 242 nm corresponding to a maximum in the first derivative of the ratio spectra in the spectral region selected 220–290 nm and relies merely on the concentration of CAR but not depending on the concentration of CHL and TEB in a ternary mixture.

Similarly, for determination of the CHL, the stored spectra which contain various concentrations of CHL in a ternary mixture were divided by the standard spectrum of a double divisor CAR and TEB (5 μg/mL each in methanol) to obtain the ratio spectra. Next, calculation of the first derivative of the ratio spectra was performed at Δλ = 4 nm. The wavelengths at 236 nm, 276 nm, and 300 nm were selected for the estimation of the amount of CHL in the first derivative of the ratio spectra in the spectral region selected 200–320 nm and rely on the concentration of CHL but not depending on the concentration of CAR and TEB in the ternary mixture.

The same idea was applied to determine the TEB; the stored spectra which contain various concentrations of TEB in a ternary mixture were divided by the standard spectrum of a double divisor CAR and CHL (5 μg/mL each in methanol) to obtain the ratio spectra. After that, calculation of the first derivative of the ratio spectra was performed at Δλ = 2 nm. The amount of TEB was determined by measuring the amplitude at 226 nm corresponding to a minimum in the first derivative of the ratio spectra in the spectral region selected 200–250 nm and only dependent on the concentration of TEB but not depending on the concentration of CAR and CHL in the ternary mixture.

3.2. Successive Derivative Ratio Spectra Method

For CAR to be determined, the first derivative of the ratio spectra was attained through division of the recorded absorption spectra of the ternary mixture which contains different concentrations of CAR in the range of 200–350 nm by the standard spectrum of 15 μg/mL of CHL by using Δλ = 2 nm. Next, the second ratio spectra were obtained through division of these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (TEB/CHL) 15 μg/mL for each of TEB and CHL. Then the first derivative of the second ratio spectra was attained at Δλ = 2 nm.

Likewise, to determine the concentration of CHL, the first derivative of the ratio spectra was attained through division of the recorded absorption spectra of the ternary mixture which contains different concentrations of CHL in the range of 200–350 nm by the standard spectrum of 15 μg/mL CAR by using Δλ = 2 nm. Later, the second ratio spectra were obtained through division of these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (TEB/CAR) 15 μg/mL for each of TEB and CAR. Then the first derivative of the second ratio spectra was attained at Δλ = 2 nm.

In the same way to determine the concentration of TEB, the first derivative of the ratio spectra was attained through division of the recorded absorption spectra of the ternary mixture which contains different concentrations of TEB in the range of 200–350 nm by the standard spectrum of 15 μg/mL CHL by using Δλ = 2 nm. Next, the second ratio spectra were obtained through division these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (CAR/CHL) 15 μg/mL for each of CAR and CHL. Then the first derivative of the second ratio spectra was attained at Δλ = 2 nm. The calibration graphs were gained by plotting the amplitudes at 256 nm and 258 nm for CAR, at 290 nm and 292 nm for CHL, and at 226 nm and 228 nm for TEB versus the corresponding concentration of each pesticide.

3.3. Mean Centering of Ratio Spectra Method

For the CAR to be determined, the stored spectra of the ternary mixture which contains different concentrations of CAR were divided by the absorption spectrum of TEB (10 μg/mL), and the obtained first ratio spectra were mean-centered. Next, the second ratio spectra could be attained by dividing these vectors to mean centering of 15 μg/mL CHL/10 μg/mL TEB and then by mean-centering.

Also for the determination of CHL, the stored spectra of the ternary mixture which contains different concentrations of CHL were divided by the absorption spectrum of TEB (10 μg/mL), and the obtained first ratio spectra were mean-centered. After that, the second ratio spectra could be attained by dividing these vectors to mean centering of 10 μg/mL CAR/10 μg/mL TEB and then by mean-centering.

A similar idea was used for TEB determination, the stored spectra of the ternary mixture which contains different concentrations of TEB were divided by the absorption spectrum of CAR (10 μg/mL), the obtained first ratio spectra were mean centered. After that, the second ratio spectra could be attained by dividing these vectors to mean centering of 15 μg/mL CHL/10 μg/mL CAR and then by mean-centering.

To create the calibration graph for CAR, CHL, and TEB, mean-centered value was plotted at 306 nm, 280 nm, and 240 nm for three pesticides, respectively, against the corresponding concentration.

4. Results and Discussion

CAR, CHL, and TEB are three pesticides, and their normal UV absorption spectra are completely overlapped in the wavelength range between 200 and 350 nm (Figure 2). As a result, determination of three pesticides in ternary mixture simultaneously is impossible by classical spectrophotometry to resolve a mixture. Therefore, three different techniques of derivative spectrophotometry have been used to reduce interference and resolve the overlapped spectra.

4.1. Double Divisor-Ratio Spectra Derivative Method

In this method, for estimation of the active constituents, the major instrumental parameter conditions required optimization is as follows.

4.1.1. Selection of the Working Wavelength

The selection of the working wavelength in the applied method depends on the first derivative of the ratio spectra of pure compound and its ternary mixture would be coincided in the spectral region corresponding to a maximum point or a minimum point of the wavelength. These coinciding points of the derivative of the ratio spectra can be chosen as working wavelengths for the estimations of the compounds in the ternary mixture. For CAR, the coincident point in the spectral region 220–290 nm corresponding to a maximum (242 nm) was selected as a working wavelength for the determination of CAR in its ternary mixtures (Figure 3). But for CHL we observed that three coincident points in the spectral region 200–320 nm corresponding to a minimum (236 nm), a maximum (276 nm), and a minimum (300 nm) wavelength were suitable for the determination of CHL in its ternary mixtures (Figure 4). Also for TEB the coincident point in the spectral region 200–250 nm corresponding to a minimum (226 nm) wavelength was selected as a working wavelength for the determination of TEB in its ternary mixtures (Figure 5).

4.1.2. Effect of Double Divisor Concentrations

The double divisor was found either by the sum of the absorption spectra of the same concentration of the two compounds in the same ternary mixture or it was gained by preparing the mixed solution of two compounds of the same concentration in the ternary mixture. For this purpose, different concentrations of double divisor were studied. As a result, 10 μg/mL for each of CHL and TEB as a double divisor for determining CAR, 5 μg/mL for each of CAR and TEB for determining CHL, and 5 μg/mL for each of CAR and CHL for determining TEB were found to be proper for the determination of three pesticides.

4.1.3. Effect of the Value of Δλ

For the first derivative of the ratio spectra, Δλ influence was studied. It was noticed that the most relevant value of Δλ which gives optimum recovery is at Δλ = 6, 4, and 2 nm for the determination of CAR, CHL, and TEB, respectively.

The stored spectra of the ternary mixture which contains different concentrations of CAR in methanol in the range of 220–290 nm were divided by the standard spectrum of the double divisor (CHL and TEB, 10 μg/mL for each in methanol) to obtain the ratio spectra (Figure 6(a)). Next the first derivatives of the ratio spectra were plotted with Δλ = 6 nm as displayed in Figure (6(b)). The concentration of CAR was estimated through measuring the signal at 242 nm corresponding to the maximum wavelength.

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(b)

In a similar way, the stored spectra of the ternary mixture which contains different concentrations of CHL in methanol in the range of 200–320 nm were divided by the standard spectrum of the double divisor (CAR and TEB, 5 μg/mL for each in methanol) to obtain the ratio spectra (Figure 7(a)). After that, the first derivatives of the ratio spectra were obtained with interval Δλ = 4 nm. The CHL concentration was estimated through measuring the signal at 236 nm, 276 nm, and 300 nm as demonstrated in Figure 7(b).

(a)

(b)

The stored spectra of the ternary mixture which contains different concentrations of TEB in methanol in the range of 200–250 nm were divided by the standard spectrum of the double divisor (CAR and CHL, 5 μg/mL for each in methanol) to obtain the ratio spectra (Figure 8(a)). Next the first derivatives of the ratio spectra were obtained using Δλ = 2 nm. The TEB concentration was estimated through measuring the signal at 226 nm corresponding to the minimum wavelength as shown in Figure (8(b)).

(a)

(b)

4.2. Successive Derivative Ratio Spectra Method

The stored spectra of the ternary mixture which contains different concentrations of CAR in the range between 200 and 350 nm were divided by a standard spectrum of 15 μg/mL of CHL, and the ratio spectra were obtained. Then the first derivative of these ratio spectra was attained by using Δλ = 2 nm. Next, the second ratio spectra were obtained through dividing these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (TEB/CHL) 15 μg/mL for each of TEB and CHL. Then the first derivative of the second ratio spectra was attained at Δλ = 2 nm (Figure 9). The CAR concentration was estimated through measuring the signal at 256 nm and 258 nm of CAR in the presence of CHL and TEB.

In a similar way, the stored spectra of the ternary mixture which contains different concentrations of CHL in the range between 200 and 350 nm were divided by a standard spectrum of 15 μg/mL of CAR, and the ratio spectra were obtained. Then the first derivative of these ratio spectra was attained by using Δλ = 2 nm. Later, the second ratio spectra were obtained through dividing these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (TEB/CAR) 15 μg/mL for each of TEB and CAR. Then, the first derivative of the second ratio spectra was attained at Δλ = 2 nm (Figure 10). The CHL concentration was estimated through measuring the signal at 290 nm and 292 nm of CHL in the presence of CAR and TEB.

The stored spectra of the ternary mixture which contains different concentrations of TEB in the range between 200 and 350 nm were divided by the standard spectrum of 15 μg/mL of CHL, and the ratio spectra were obtained. Then the first derivative of the ratio spectra was attained by using Δλ = 2 nm. Next, the second ratio spectra were obtained through dividing these vectors (the first derivative of the ratio spectra) by the first derivative of the ratio spectra (CAR/CHL) 15 μg/mL for each of CAR and CHL. Then the first derivative of the second ratio spectra was attained at Δλ = 2 nm (Figure 11). The TEB concentration was estimated by measuring the signal at 226 nm and 228 nm of TEB in the presence of CAR and CHL.

The most important factor needed for optimization of reliable determination of the three pesticides in the ternary mixture is as follows.

4.2.1. Effect of Divisor Concentration

The influence of divisor concentration on the selectivity of the method and analytical parameters such as slope, intercept, detection limit, and correlation coefficient of the calibration equation was studied. For this purpose, various concentrations of divisors have been examined (1, 3, 5, 10, 15, 20, and 30 μg/mL) for each of TEB and CHL for carboxin determination, CAR and TEB for chlorpyrifos determination, and CAR and CHL for tebuconazole determination. Markedly altering the divisors concentration had a considerable impact on the method selectivity. Hence, 15 μg/mL of each of CAR, CHL, and TEB was used as divisors.

4.2.2. Selection of the Working Wavelength

The minimum or maximum of the successive derivative ratio spectra with respect to wavelength was used for the construction of calibration graph.

Another parameter that should be optimized is the amount of Δλ and its impact on the derivation of ratio spectra. For this purpose, different Δλ was studied, and it was noticed that the value of Δλ has no significant effect on the ratio spectra derivative method; therefore, Δλ = 2 nm was used.

4.3. Mean Centering of Ratio Spectra Method

The stored spectra of the ternary mixture which contains different concentrations of carboxin were divided by the standard spectrum (10 μg/mL) of TEB, and the first ratio spectra were attained and then mean-centered. Mean centering of the ratio spectra was gained in the range between 220 and 320 nm. After that, the second ratio spectra were attained through dividing these vectors to mean centered of (15 μg/mL CHL/10 μg/mL TEB) and then mean-centered as clarified in Figure (12). The concentration of CAR was estimated through measuring the signal at 306 nm corresponding to the maximum point.

In the similar way, the stored spectra of the ternary mixture which contains various concentrations of chlorpyrifos were divided by the standard spectrum (10 μg/mL) of TEB, and the first ratio spectra were attained then and mean-centered. Mean centering of the ratio spectra was gained in the range between 250 and 320 nm. Later, the second ratio spectra were attained through dividing these vectors to mean centering of 10 μg/mL CAR/10 μg/mL TEB and then mean-centered as shown in Figure (13). The concentration of CHL was estimated through measuring the signal at 280 nm corresponding to the maximum point.

On the other hand, the stored spectra of the ternary mixture which contains various concentrations of tebuconazole were divided by the standard spectrum (10 μg/mL) of CAR, and the first ratio spectra were attained and then mean-centered. Mean centering of the ratio spectra was gained in the range of 220–320 nm. Next, the second ratio spectra were attained through dividing these vectors to mean centering of 15 μg/mL CHL/10 μg/mL CAR and then mean-centered as shown in Figure (14). The concentration of TEB was estimated by measuring the signal at 240 nm corresponding to the maximum point.

The main parameter that should be studied for successful application of the mean centering of ratio spectra method could be summarized as a follows.

4.3.1. Effect of Divisor Concentration

The study of divisor concentration that has a considerable impact on the method selectivity and analytical parameters such as slope, intercept, detection limit, and correlation coefficient of the calibration equation was tested. To achieve that, various concentrations for each of CAR, CHL, and TEB (3, 5, 10, 15, 20, 25, and 30 μg/mL) were examined. The best results were obtained upon using 10 μg/mL of TEB and 15 μg/mL of CHL for estimation of CAR and 10 μg/mL each of CAR and TEB for quantification of CHL. For determination of TEB, 10 μg/mL of CAR and 15 μg/mL of CHL were found to be the best divisor.

4.3.2. Selection of the Working Wavelength

To estimate the wavelength, zero-crossing point technique can be used where the interference of the other two components was eliminated, although the maximum and minimum of peaks could be used as a working wavelength. Furthermore, in this method, the signal-to-noise ratio was enhanced through removing the derivative steps.

5. Validation of Proposed Methods

5.1. Calibration Graph and Statistical Data

The statistical data of different calibration graphs were obtained by three different spectrophotometric methods, including double divisor-ratio spectra derivative (DDRSD), successive derivative of ratio spectra (SDRS), and mean centering of ratio spectra (MCRS) methods. The linearity range of the three proposed methods over the concentration ranges was 1–30 μg/mL for CAR, 1–50 μg/mL for CHL, and 1–45 μg/mL for TEB in the ternary mixture which was assessed by analyzing various mixtures of CAR, CHL, and TEB. However, the high value of the regression coefficients which is larger than 0.999 indicates that the calibration graphs had a good linearity. The limit of detection and limit of quantification of the proposed methods were found and calculated according to the equation mentioned in [53] and the main results are shown in Table 1. The accuracy and precision of the proposed methods were evaluated at three different concentrations of each pesticide in the ternary mixture (five replicate measurements), and then the percentage recovery and percentage relative standard deviations were calculated at all three concentration levels. The results rely on the value of the %R and %RSD, the percentage recovery was found to be in the range of 95.03% to 104.89%, and the good percentage recovery indicates a good accuracy, and the main results are shown in Table 2. It is clear that the three methods can be applied to resolve the three pesticides in the ternary mixture with good accuracy and precision. All the three methods are quick, nondevastating, and cheap, and they do not need costly solvent and reagents; also poison and ozone-harming organic solvents and polluting reagents are not required [54].

5.2. Application of the Methods

In this study, three proposed methods were applied for the estimation of CAR, CHL, and TEB in real cabbage samples. The real fresh cabbage samples spiked at three different concentration levels (1, 15, and 30 μg/mL) for carboxin, (1, 30, and 50 μg/mL) for chlorpyrifos, and (1, 20, and 45 μg/mL) for tebuconazole with five replications per level. The mean percentage recoveries of three pesticides in real cabbage samples are shown in Table 3. In all circumstances, the mean percentage recoveries were greater than 87.02%, 92.32%, and 87.19% with a relative standard deviation less than 5.91%, 5.99%, and 5.53% for carboxin, chlorpyrifos, and tebuconazole, respectively, and the good recoveries suggested that the proposed methods have a good accuracy. One-way ANOVA test was used to compare the results between the three proposed methods statistically. For this purpose, Snedecor’s F-values were computed and compared with the standard tabulated value . The similar computation processes were repeated for each pesticide. Table 4 shows that the calculated or experimental F-values are less than the tabulated values F-values in the analysis of variance, indicating that there was no significant difference between the three methods. To keep food safe and secure for consumers health, maximum residue limit (MRL) has been set by the European Union (EU) Food Safety Authority for these pesticides. The amount of carboxin, chlorpyrifos, and tebuconazole residues in cabbage samples was less than the EU-MRL (1 mg/kg) [55], so these three pesticides have no risk for consumer health. The three spectrophotometric methods were successfully applied with the aid of standard addition method for simultaneous determination of the residue of each CAR, CHL, and TEB in real cabbage samples, and the main results are summarized in Table 5.

6. Comparison of the Methods

A comparison has been made between the results obtained by the proposed methods for the determination of carboxin, chlorpyrifos, and tebuconazole in real cabbage samples in the standpoint of linearity, LOD, LOQ, percentage recovery, and percentage relative standard deviations with other reported methods (Table 6). The table illustrates that the proposed methods have a good linearity, % recovery, and %RSD, indicating that these methods could be successfully applied for determination of the residues of these three pesticides in cabbage samples. Furthermore, the derivative spectrophotometric results were compared to one of the multivariate techniques, namely, partial least squares regression (PLS-2). For this purpose, a calibration set for the ternary pesticides mixture CAR, CHL, and TEB was constructed using the collected thirty-five spectral data as independent variables and the concentrations of the pesticide in the mixture as dependent variables. The recorded spectra in the wavelength range of 200–350 nm with a sampling interval of 2 nm were arranged as row matrix. The matrix was organized into pairs, and each absorbance matrix is paired with its corresponding concentration matrix and was used as a training set to build up a PLS-2 model using Origin Pro 2018 SR1. By using the cross-validation technique for evaluating estimation or performance for calibration, the concentration of the sample left out was predicted. The process was repeated as the number of samples [56]. Figure (15) shows a plot of the root mean PRESS against the number of factors for PLS-2 model for the determination of pesticides in their mixture. The calibration model for PLS-2 was confirmed with the thirty-five mixtures of the training set. The predictive capability of PLS-2 was examined for simultaneous determination of CAR, CHL, and TEB in each mixture (Table 7). The statistical t-test for the difference between the two population means and F-test for the difference in the two variances were used to compare the result of the cited pesticides obtained by the suggested derivative techniques with those of thirty-five selected ternary-pesticide mixture samples analyzed via the established PLS-2 model in concentration ranges up to 30 μg/mL, 50 μg/mL, and 45 μg/mL for carboxin, chlorpyrifos, and tebuconazole, respectively (Table 8).

7. Conclusion

In this work, three sensitive and reliable spectrophotometric methods including (DDRSD, SDRS, and MCRS) were used for the determination of three pesticide (carboxin, chlorpyrifos, and tebuconazole) residues simultaneously in a ternary mixture of cabbage samples without preliminary separation process. Standard addition technique can be easily used in the proposed methods and the matrix effects can be removed. Therefore, the three proposed methods can be applied successfully for resolution of the ternary mixture and do not require complicated or expensive instruments. In addition, it is rapid and low-cost method when compared with other methods such as chromatographic methods. Also, we used the QuEChERS method for extraction of real cabbage sample because it is very simple and inexpensive as well as it involves minimum steps with a very short time in comparisons with other extraction methods and provides good recoveries and precision. The methods developed showed satisfactory validation parameters in terms of linearity, LOD which is lower than 0.260, 0.272, and 0.205 μg/mL for CAR, CHL, and TEB, respectively, and LOQ which is lower than 0.790, 0.824, and 0.621 μg/mL for CAR, CHL, and TEB, respectively. The efficiency of the proposed methods can be illustrated by the mean percentage recovery values which were between 87.02% and 108.53% with %RSD lower than 6%. According to the statistical analysis, no significant differences between the three proposed methods were detected. As a result, all the three proposed methods have a good accuracy, precision, and sensitivity and are typically suited for the estimation of carboxin, chlorpyrifos, and tebuconazole residues in real cabbage samples.

Data Availability

All data are included within the manuscript.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors are grateful to the Chemistry Department, College of Education, Salahaddin University, Erbil, Iraq, for their kind support during this work.

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Copyright © 2020 Shilan A. Omer and Nabil A. Fakhre. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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