Statistical and chemometric view of the variation in the concentration of selected organophosphates in peeled unwashed and unpeeled washed fruits and vegetables

Accepted Manuscript Statistical and chemometric view of the variation in the concentration of selected organophosphates in peeled unwashed and unpeeled washed fruits and vegetables Shima Behkami PII: DOI: Article Number: Reference:

S0308-8146(19)31328-7 https://doi.org/10.1016/j.foodchem.2019.125220 125220 FOCH 125220

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

2 May 2019 21 July 2019 21 July 2019

Please cite this article as: Behkami, S., Statistical and chemometric view of the variation in the concentration of selected organophosphates in peeled unwashed and unpeeled washed fruits and vegetables, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125220

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Statistical and chemometric view of the variation in the concentration of selected organophosphates in peeled unwashed and unpeeled washed fruits and vegetables Shima Behkami1,2 2

Department of Chemistry, University of Guelph, Guelph Ontario, N1G2W1, Canada 2

Department of Chemistry, University of Najand, Urmia 5719883826, Iran Email: [email protected]

Phone: +14163711647 Abstract Fruits and vegetables play an important role in human nutrition. Study of the contamination sources which result from farming activities is of importance. For this reason, a chitosan– graphene oxide nanocomposite film was prepared and implemented as the extractive phase in thin film microextraction of six organophosphate residues (OPPs) in the samples using highperformance liquid chromatography. The optimized method was validated and the limits of detection (0.7–1.2 µg l-1), limits of quantification (2.3–4.0 µg l-1) and linear dynamic range (2.0–1000.0 µg l-1) were obtained. Principal component analysis revealed clustering of the fruit and vegetable samples based on the selected (OPPs) into two groups of unwashed–unpeeled and peeled–washed. This mapping was further investigated using descriptive method of boxplot. Washing and peeling of the samples, reduced the presence of OPPs to half or one third of interquartile range found in the unpeeled and unwashed samples. Keywords: Organophosphate residues; Thin film microextraction; Chitosan–graphene oxide nanocomposite film; Fruit and vegetable analysis; Clustering

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1. Introduction Fruits and vegetables are a necessary portion of a balanced diet. Among the wide spectrum of fruits, apples are the second highest consumed being available seasonally and geographically. The key components of apple are polyphenols and fibers which have cardioprotective effects on the body (Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014; N. P. Bondonno, Bondonno, Ward, Hodgson, & Croft, 2017) meaning that higher intake of apple reduces heart disorders (Franke, Custer, Arakaki, & Murphy, 2004; Larson, Witman, Guo, Ives, Richardson, Bruno, et al., 2012) as well, apple has positive effects on vascular function (C. P. Bondonno, Yang, Croft, Considine, Ward, Rich, et al., 2012), high blood pressure (N. P. Bondonno, Bondonno, Ward, Hodgson, & Croft, 2017) inflammation (Chun, Chung, Claycombe, & Song, 2008) and lipids (Jensen, Buch-Andersen, Ravn-Haren, & Dragsted, 2009). Tangerine and orange belong to the citrus family. Comparing the two, both are a source of nutrients and flavonoid (Franke, Custer, Arakaki, & Murphy, 2004) except that tangerine has more vitamin A compared to orange and orange has higher vitamin C compared to tangerine. From a nutritional point of view, Vitamin C, helps support connective tissues, including skin, blood vessels and bones (Kalt, Forney, Martin, & Prior, 1999), and can lower the risk of chronic conditions (Xia, Lu, Lu, Liu, Liu, Meng, et al., 2019). Kiwi is composed of many phytochemicals which provide health benefits. Kiwi shows antioxidant activity which is influenced by its bioactive elements inhibiting cancer cell growth (Motohashi, Shirataki, Kawase, Tani, Sakagami, Satoh, et al., 2002). Kiwi is another fruit rich in vitamin C (Nishiyama, Yamashita, Yamanaka, Shimohashi, Fukuda, & Oota, 2004) it is a good source of vitamin E , D, K , Mg and fibers (Collins, 2013). Cucumber has been used as a traditional medicine especially in Asia. It has antioxidant property and contains triterpene glycosides, carotenoids, bioactive peptides, vitamins, minerals, fatty acids, collagens, gelatins, chondroitin sulfates and amino acids, which are used as antimicrobial, anticoagulant and for healing wound (Pangestuti & Arifin, 2018). Tomato is a member of the

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Solanaceae family and one of the most produced plants and is composed of moisture (95%), carbohydrates (3%), protein (1.2%), total lipids (1%), as well as minerals, such as Ca, P, Zn, K , Mn, Mg and vitamins of A and C plus, riboflavin, pantothenic acid, thiamin pyridoxine and niacin. Moreover, there are phenolic compounds, carotenoids and glycoalkaloids in tomato (Salehi, Sharifi-Rad, Sharopov, Namiesnik, Roointan, Kamle, et al., 2019). Higher consumption of tomato lowers the risk of cancer and cardiovascular diseases (De Stefani, Oreggia, Boffetta, Deneo-Pellegrini, Ronco, & Mendilaharsu, 2000; Salehi, et al., 2019). Zucchini is a vegetable that belongs to the Cucurbitaceae family and is known as a low salt plant, which is low in calories and rich in nutrients. Moreover, zucchini has polyphenolic compounds and is of consumers attention due to its anti-carcinogenic, antimicrobial activities, antioxidant/anti-radical, anti-inflammatory and antiviral benefits (Iswaldi, Gómez-Caravaca, Lozano-Sánchez,

Arráez-Román,

Segura-Carretero,

&

Fernández-Gutiérrez,

2013).

Furthermore, zucchini is composed of caffeic acid and p-coumaric acid, ferulic acid and vanillic acid (Mattila & Hellström, 2007). Overall, due to the nutritional impacts of fruits and vegetables in human health it is necessary to ensure that these food products are free of pesticides and safe enough to consume. Knowing that different pesticide classes are extensively used to reduce the incidence of diseases caused by insects and to increase the high-quality food production there is a need for efficient analytical techniques (Araoud, Douki, Rhim, Najjar, & Gazzah, 2007; Chai & Tan, 2009). Among the pesticides, organophosphorus pesticides (OPPs) are esters and organic acid halides of phosphonic and phosphoric acids (Chambers, Meek, & Chambers, 2010). They may affect the central nervous system through the irreversible inhibition of acetylcholinesterase enzyme (Li, Nagahara, Takahashi, Takeda, Okumura, & Minami, 2002). Moreover, the pesticides residue levels may vary depending on its geographical origin, and so the analysis of fruit and

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vegetable samples for monitoring and determining the pesticide residuals has remained a courtesy purpose. Among all the methods, thin film microextraction (TFME) was carried in this research. In detail TFME is a SPME configuration with increased extraction ability where a flat film plate or a membrane with a high-volume ratio is used as the extractive phase. With this configuration, the extractive phase volume increases, while thickness of extractive phase remains either similar or less than SPME. Moreover, film thickness reduction can lower the equilibrium time. On the other hand, this approach increases the contact surface of the extractive phase and rate of the extraction process (Jiang & Pawliszyn, 2012). Also, in TFME, due to its high surface– to–volume ratio, larger capacity than SPME is available while long equilibration time, like SBSE technique, are not required. In conclusion, according to the type of analytes and the sample matrix, various films with different polarities have been used in TFME. The overall objective of this study was to develop and validate an analytical technique for the extraction and chromatographic analysis of the trace levels of six OPPs in fruits and vegetables. For this purpose, a chitosan–graphene oxide (CS–GO) nanocomposite film was prepared and used as promising extractive phase in thin film microextraction of OPPs in selected fruits and vegetables. Indeed, descriptive analysis (boxplot) and PCA revealed separation of the samples base on the concentration of six OPP’s in two clusters of washed peeled and unwashed unpeeled. 2. Materials and methods 2.1. Reagents and standards The selected OPPs, namely, phosalone, chlorpyrifos, fenthion, profenofos, azinphos-methyl and diazinon, were purchased from Sigma-Aldrich (Milwaukee, WI, USA). A mixture of stock solution containing 1000 mg l−1 of OPPs were prepared in acetonitrile and stored in a 4

refrigerator at 4 °C. The working solutions of these OPPs were prepared from the stock solution. Chitosan (CS) (degree of deacetylation: 85%, Mw: 190,000–310,000 Da) was purchased from Sigma-Aldrich (Milwaukee, WI, USA). Sodium hydroxide (NaOH), graphite, potassium permanganate (KMnO4), hydrogen peroxide (H2O2), sulfuric acid (H2SO4), hydrochloric acid (HCl), acetone, acetonitrile and ethanol were purchased from Merck (Darmstadt, Germany), while HPLC–grade methanol and acetonitrile were provided from Ameretat Shimi (Tehran, Iran). 2.2. Instrumentation A RIGOL L–3000 high performance liquid chromatography (Beijing, China) equipped with a UV–Vis detector and a 20 µl sample loop was used for the analysis of fruit and vegetable samples. The separation was performed using a Welch analytical column XB–C18 5 μm (4.6 mm × 250 mm) with MZ analytical guard column (20 mm × 4.6 mm) packed with the same material under ambient temperature. The solvents used as mobile phase, contained acetonitrile– double distillated water (80:20, v/v) at a flow rate of 1 ml min-1 under isocratic elution mode. The ultraviolet detection monitoring wavelength was set at 220 nm for the all selected OPPs. Furthermore, an ultrasonic bath (Elma Company Germany) was used for the extraction of OPPs from fruit and vegetable samples and preparation of nanocomposite chitosan-graphene oxide film. For desorption of analytes, a vortex (Heidolph Reax Top, Schwabach, Germany) was used. The Scanning electron microscopy (SEM) images were recorded on a TESCAN 149 Mira3 LMU (Kohoutovice, Czech Republic) Instrument. The FT–IR spectra were obtained using an ABB Bomem MB100 (Quebec, Canada). 2.3. Synthesis of chitosan–graphene oxide film nanocomposite Graphene oxide (GO) was synthesized according to Hummer method. To do so, 1.0 g of graphite powder, 13.0 ml of H3PO4 and 120 ml of H2SO4 were poured in a flask and stirred for 5

30 min at 25°C. Afterwards, 6.0 g of KMnO4 was slowly added to the reaction mixture. The solution was kept at 50 °C for 2 h. Subsequently, the mixture was stirred at this temperature for 12 h. The flask was cooled to 0 °C. Afterwards, 450 ml deionized water was added to the solution and stirred for 15 min. In the next step, 3.0 ml of H2O2 was added and the reaction product centrifuged and washed with deionized water and 5% HCl solution repeatedly. Finally, the product was dried in the oven at 60 °C. Then, the chitosan–graphene oxide (CS-GO) nanocomposite film was synthesized (Golzari Aqda, Behkami, Raoofi, & Bagheri, 2019). A specific quantity of CS (2% w/v) was dissolved in an aqueous solution of acetic acid (1% v/v) at 40 °C. GO was dispersed in aqueous acetic acid (1% v/v) for 30 min using the ultrasonic irradiation. Then the CS solution was added and stirred for 1 h. The solution was sonicated in the ultrasonic bath for 1 h at 60 ° C. Finally, the chitosan–graphene oxide solution was transferred to a petri dish to dry in the air. For crosslinking, CS and GO were placed in an oven at 120 ° C for overnight. 2.4. TFME procedure After preparing the CS–GO film, a piece of 1.5×2.5 cm was cut and employed as the extractive phase. A stainless-steel cotter pin as a keeper was used for handling the extractive phase. The stainless-steel cotter pin was connected to the extraction vessel by a polyethylene holder with inner diameter of the screw cap vial. The prepared extractive phase was conditioned in methanol and water solvents prior to be used in the TFME technique. A volume of 10 ml from the aqueous sample was transferred to the vial containing a magnet. After performing the extraction (~15 min), the CS–GO film was transferred to Eppendorf tube, containing 100 μl of acetonitrile to perform the desorption process by a vortex. 2.5. Sampling and sample preparation In this research a total of 208 samples consisting of apple (32 samples), zucchini (24 samples), cucumber (32 samples), orange (32 samples), tangerine (24 samples), kiwi (32 samples) and 6

tomato (32 samples) were randomly purchased from four different fruit markets. Samples were grouped and analyzed into two groups, namely washed–peeled and unwashed–unpeeled. In both cases the samples were crushed and mixed by a blender to obtain a homogenous mixture. The homogenized sample (10.0 g) was fortified with the standard mixture of 6 OPPs and placed in a beaker containing 10.0 ml of double distillated water and 10 ml of methanol. Then, it was sonicated for 15 min in the ultrasonic bath. The suspension was filtered using a whatman filter paper grade 41 and Buchner funnel and washed with 10.0 ml of double distillated water. Finally, the filtered solution made to 100 ml volume with double distillated water. Eventually, the TFME procedure was implemented using 10.0 ml of the final solution. 2.6. Optimization of extraction procedure Effective parameters associated with the TFME protocol, such as extraction time (10, 15, 20 and 25 min), type of solvent (methanol, ethanol, acetone and acetonitrile), solvent volume (50, 100, 150, 200, 250 and 300 µl), desorption time (3, 5, 7 and 10 min) and sample pH (3, 4, 5, 6, 7 and 8), were optimized. In all the optimization steps, the fortified (100 µg kg-1 from each target analyte) OPPs–free samples were used. 2.7. Method validation In this research, figures of merit, namely, linearity, precision, relative recovery, limits of detection (LODs) and limits of quantification (LOQs), were obtained. Linearity was checked by analyzing the samples (n=3) fortified with the standard mixture of the selected pesticides in a concentration range of 2–1000 µg kg-1 under optimum condition. Precision was determined using the spiked OPPs at three different levels in a day (Intra-day, n=5) and in three different days (Inter-day, n=5) using the blank samples. Moreover, recovery (%) was calculated using the ratio of the target compounds concentration in the fortified real fruit and vegetable samples to the spiked double distillated water. All the analyses were performed in triplicate (n=3). 7

2.8. Statistical analysis Statistical data analysis was conducted using the JMP®, Version SAS Institute Inc., Cary, NC, U.S.A, 2015 software. Principal component analysis (PCA) and boxplot were applied to the data in order to visualize the underlying data structure. To test the difference between the unpeeled–unwashed and peeled–washed samples, student t–test of means having unequal variance and it was noted the two group of samples were significantly different (p<0.05). Significance test was carried out using MS Excel 2013. 3. Results and discussion 3.1. Characterization In order to verify the synthesis protocol, the FT–IR spectra of CS–GO nanocomposite film was recorded. The obtained spectra related to GO, CS, CS–GO nanocomposite film are depicted in Fig. 1 a–c. The assigned absorption band of GO appeared at 3400, 1730, 1250 and 1050 cm -1 which respectively are related to O–H, C=O, C–O–C and C–O stretching vibrations. The comparison of the CS and CS–GO nanocomposite spectra shows that the GO epoxy group (C– O stretching vibration in 1050 cm-1) reacts with the amine group within the CS structure. The primary amine groups (–NH2) are converted into secondary amines (–NH) which show similar pattern. The position of peaks for the C–H (2860 cm-1), C–O–C (1020 cm-1), O–H, N–H (3300– 3600 cm-1) and C=O (1636 cm-1) stretching bonds in –NHCO are similar, while the band at 1565 cm-1 is slightly shifted to 1563 cm-1 with an intensity change. Among the prepared nanocomposites, the surface morphology of CS–GO film (0.4 wt % of GO), the most suitable extractive phase for the current work, was evaluated by SEM (Fig. 1d). The recorded SEM image confirms that the film surface is rough with porous structure.

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3.2. Optimization Chitosan is an amino polysaccharide which is prepared from deacetylation of chitin in basic conditions. This natural polymer, due to its high biocompatibility and low toxicity and possession of –OH and –NH2 in its structure, has been extensively considered as an extractive phase. One scenario to make chitosan amenable as an extractive phase is to produce a film layer from this natural polymer. But due to its low mechanical stability and flexibility, high hydrophilicity and low surface area, it is usually utilized as composite with other materials. Graphene oxide as a filler in the structure of chitosan nanocomposites is used to overcome these problems. To prevent the water solubility of the nanocomposite, thermal crosslinking between GO and CS has been applied (Grande, Mangadlao, Fan, De Leon, Delgado‐ Ospina, Rojas, et al., 2017). By implementing the thermal condition, the epoxy group in graphene oxide reacts with the –OH and –NH2 functional groups in chitosan. In order to make the CS–GO film applicable as extractive phase, contribution of different concentrations of graphene oxide (0.1, 0.2, 0.3, 0.4, 0.5 % wt) were examined and the highest extraction efficiency was achieved at 0.4 %wt. Other influential parameters on the extraction/desorption efficiency, as mentioned in section 3, were followed for the developed TFME technique. Our findings revealed that acetonitrile is the proper desorbing solvent in compared with other solvents (Fig. 2a). Also, the highest extraction was obtained with 100–µl of acetonitrile during a desorption time of 5 min (Fig. 2b, c). As far as the contribution of extraction time and sample are concerned, a duration time of 15 min at pH=6 (Fig. 2d, e) were the most appropriate values. 3.3. Method validation The TFME technique using CS–GO nanocomposite film as extractive phase was applied to investigate the quantitative analysis of OPPs in blank and spiked samples. To obtain figures of merit, all experiments were carried out under optimal conditions including 100–μl acetonitrile 9

as desorption solvent, 5 min desorption time, 15 min extraction time at pH= 6. The linearity for the desired analytes was in the range of 2–1000 µg kg -1 while the regression coefficients were satisfactory (R2> 0.998). The LOD and the LOQ values for the 6 selected organophosphates are in the range of 0.7–1.2 µg kg−1 and 2.3–4 µg kg−1, respectively. To check the precision of the method, the standard deviation percentages for all analytes within intra–day and inter–day was measured as is reported in supplementary Table 1. Moreover, in order to evaluate the repeatability of the extractive phase, the efficiency of five different films were evaluated in one day and the RSD% values were lower than 9.7%. Considering the HPLC mobile phase and applied method on the blank fruit samples, no interference was detected. 3.4. Real samples In order to evaluate the applicability of the method, the prepared CS–GO nanocomposite film was used as the extractive phase in TFME of selected organophosphorus pesticides, such as fenthion, chlorpyrifos, phosalone, profenofos, azinphos–methyl and diazinon in fruit and vegetable samples. The accuracy of the method was tested by spiking the selected organophosphates at different concentrations of 10, 80 and 200 µg kg−1 in each sample. Relative recoveries were found in the range of 83%–102% for the studied analytes in fruits and vegetables samples (supplementary Table 2), indicating the capability of the method to handle samples with complex matrix. Supplementary Fig. 1, shows the chromatograms obtained from TFME–HPLC–UV analysis of a blank fruit sample, a blank fruit sample spiked at 80 µg kg-1 from each organophosphate pesticide, real fruit and vegetable samples before and after spiking (80, 200 µg kg-1) of the analytes.

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According to these chromatograms, retention times related to azinphos–methyl, fenthion, phosalone, diazinon, profenofos and chlorpyrifos were 4.2, 6.1, 6.8, 8.4, 8.8 and 10.9 min, respectively. 3.5. PCA analysis Principal component analysis was used for investigation of six OPPs in 208 vegetable and fruit samples in order to show the distribution pattern of selected organophosphates in unwashed unpeeled and washed peeled fruit and vegetables explaining total variance of 75.9%. PC1 is responsible for the separation of two groups of peeled washed and unpeeled unwashed samples (Fig. 3). The group of samples on the right-hand side of the graph are enriched in all the studied OPPs namely diazinon, phosalone, azinphos-methyl, profenofos, fenthion and chlorpyrifos. However, samples of peeled washed located on the left of the quadrant are depleted in the selected OPPs compared to unpeeled unwashed samples. For deeper understanding of the residue doses of the OPPs, boxplot as descriptive method was implemented. 3.6. Box Plot Fig. 4 (a–e) illustrates the box plot of selected fruits (apple, orange, tangerine and kiwi) and vegetables (cucumber, zucchini, and tomato). Fig. 4a shows the boxplot of all the samples analyzed for both peeled washed and unpeeled unwashed. The variation of selected pesticides in unpeeled unwashed group for azinphos-methyl is in the range of (30–180) µg kg−1. Apple with median of 150 µg kg−1 and upper limit of 180 µg kg−1 stands for the highest limit of azinphos-methyl among all the studied samples while its minimum concentration is 92 µg kg−1. Zucchini has the smallest median and lowest limit of 31 µg kg−1 in azinphos-methyl with the maximum of 75.25 µg kg−1. Moreover, the variation in peeled washed samples is between (12– 85) µg kg−1. It was observed that in the peeled washed category the highest upper limit belonged to apple that had a value of 85 µg kg−1 with the lowest limit of 32 µg kg−1. In this group the lowest limit was 12 µg kg−1 in zucchini with a maximum of 30 µg kg−1. From these 11

observations it could be concluded that the concentration of azinphos-methyl was higher in apple among all the other selected samples meaning that this particular pesticide has been used more often by the farmers on the apple trees than other fruits and vegetables studied in this research work. Furthermore, the concentration of azinphos-methyl reduced to half or one third its concentration in peeled washed samples compared to unpeeled unwashed describing that washing affects the concentration of the pesticide. Fig. 4b shows the range of fenthion in unpeeled unwashed samples is (25–130 µg kg−1), where, the range for peeled washed sample is (not detected–40 µg kg−1). In unwashed unpeeled fruits and vegetables, orange has the highest median and as well highest upper limit with a maximum of 130 µg kg−1 and minimum of 32 µg kg−1 demonstrating the widest interquatile range (IQR) among other fruit and vegetable. Moreover, lowest does of fenthion belongs to kiwi that has value of 25 µg kg−1 and upper limit of 60 µg kg−1. In peeled washed group, fenthion dose in orange ranges from 5 µg kg−1 to 40 µg kg−1 and has the highest maximum value while kiwi, zucchini and apple have the smallest minimum value of 5 µg kg−1. From this information it can be concluded that fenthion dose is higher in orange than the other samples both in unwashed unpeeled and peeled washed samples which might indicate that farmers have more frequently used it on this orange tree. Fig. 4c, the range of phosalone in unwashed unpeeled samples is (not detected–80) µg kg−1 while this range for peeled washed samples is (not detected–25) µg kg−1. Tangerine in unpeeled unwashed samples has maximum limit of 80 µg kg−1. While, in peeled washed group, tangerine has the highest upper limit 25 µg kg−1 and Zucchini has the lowest limit among the others with the upper limit of 12 µg kg−1. The IQR of tangerine is wider among all the other studied samples which indicated that the dose of phosalone is higher in tangerine compared to others meaning that this organophosphate is more often used on tangerine trees.

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Fig. 4d indicates the range of the concentration for profenofos in selected samples in unwashed unpeeled (not detected-86 µg kg−1) and washed peeled samples (not detected–24 µg kg−1). Apple has the highest upper limit of profenofos in unwashed unpeeled and washed peeled samples. The lowest value belonged to kiwi, zucchini, orange and tangerine while maximum values were respectively 15, 15, 8 and 18 µg kg−1. Fig. 4e showed diazinon concentration in unpeeled unwashed samples is range of (not detected215 µg kg−1) while peeled washed samples are in the range of (not detected–72 µg kg−1). Looking to unpeeled unwashed boxplots, tomato has the highest upper limit of 215 µg kg−1 with minimum of 108 µg kg-1. Lowest limit among all belongs to zucchini and tangerine with maximum values of 50 µg kg−1 and 62 µg kg−1, respectively. In the washed peeled group, tomato and cucumber have the highest upper limit of 74 µg kg−1 with minimum of 37 µg kg−1 and 16 µg kg−1, respectively. Tangerine and zucchini have the lowest lower limit with maximum of 20 µg kg−1and 15 µg kg−1 respectively. The dose of chlorpyrifos ranges from 20 to 75 µg kg−1 in unwashed unpeeled samples and from not detected to 20 µg kg−1in washed peeled samples (Fig. 4f). In unwashed unpeeled samples, zucchini has the highest upper limit among others which is 75 µg kg−1 with an amount minimum of 35 µg kg−1. However, lowest lower limit of 18 µg kg−1 is observed in both tangerine and kiwi. Moreover, orange in peeled washed samples has the highest upper limit of 22 µg kg−1 with minimum of 15 µg kg−1. But the lowest dose of chlorpyrifos is 3 µg kg−1 in zucchini. 3.7. Comparing the results of the current research with other researchers The concentration of pesticides in unpeeled unwashed samples was close to two times the concentration of washed peeled samples as is reported in (Table 1). This explains that washing and peeling reduces the concentration of pesticides in an effective way.

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Looking closely at the results of the analysis for each individual sample, the following was concluded. Starting with kiwi, six OPP’s were detected in the analyzed samples. Among the OPP’s detected in kiwi Diazinon and Azinphos-methyl had the highest concentration while profenofos, chropyrifos, phosalone and fenthion had the lowest. Comparing the results of this research with others, it is observed that Kiwi samples analysed by Bakırcı et al. only contained chlorpyrifos (Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014). The concentration of chloryrifos in this research was within the range reported by Bakırcı et al but in its lower level. Among the OPP’s detected in the apple samples in this research, Azinphos-methyl and diazinon had the highest concentration, while phosalone, fenthion, profenofos and chlopyrifos had the lowest. Compared to the other researchers (Table 1) profenofos concentration in this research was higher compared to Sivaper et al data (Sivaperumal, Anand, & Riddhi, 2015). Diazinon, concentration was higher in this research compared to Pirsheh et al (Pirsaheb, Fattahi, Rahimi, Sharafi, & Ghaffari, 2017) and chlorpyrifos was in the midrange while phosalone in the lower range of Bakirci et al data. (Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014). Moving to tomato, Diazinon and Azinphos-methyl had the highest concentration among other OPP’s while profenofos, phosalone and chlorpyrifos and fenthion had the lowest concentration. Comparing with others (Table 1), diazinon, chlorpyrifos and fenthion concentration were higher in Bidari et al research (Bidari, Ganjali, Norouzi, Hosseini, & Assadi, 2011) and in the range of Bakırcı et al for chlorpyrifos (Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014). In cucumber, Diazinon and azinphos-methyl had the highest concentration whereas chlorpyrifos, phosalone, protenos and fenthion were in the lower range. Comparing the results with other researchers (Table 1), the concentration of diazinon and fenthion in this research was higher compared to Pirshab et al (Pirsaheb, Fattahi, & Shamsipur, 2013) but chlorpyrifos concentration was comparable with Bakırcı et al (Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014) being at the higher range of this work. Tangerine had the highest concentration of azinphos-methyl while diazinon, profenofos,

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chlorprifos, phosalone and fenthion had the lowest. Moreover, the concentration of chlorpyrifos in this research was in the lower range of chlorpyrifos in Bakırcı et al research work (Bakırcı, Acay, Bakırcı, & Ötleş, 2014). Azinphos-methyl and fenthion concentration was the highest among the other OPP’s in orange. Whereas Profenofos, chlorpyrifos, phosalone and diazinon were lower in concentration. Comparing the results in Table 1 the concentration of chlorpyrifos in this research was in the lower range of Bakırcı et al (Bakırcı, Acay, Bakırcı, & Ötleş, 2014) In zucchini, most of the OP’s were within one range with little fluctuation. The concentration of chlorpyrifos was higher in the samples analysed by Bakırcı et al. (Bakırcı, Acay, Bakırcı, & Ötleş, 2014). Overall, the variations of the pesticides could be due to several factors such as the dose of the pesticides used by the farmers as well as the amount of sunshine which the fruit or vegetable received and percent of adsorption of the pesticide by fruit and vegetable (Pirsaheb, Fattahi, Rahimi, Sharafi, & Ghaffari, 2017). 3.8. Comparing the method used in this research with other relevant methods The TFME technique using chitosan–graphene oxide nanocomposite consumes less organic solvent than commercial extractive phase in SPE, molecularly imprinted polymer solid-phase extraction (MIP-SPE), and QuEChERS approaches for extraction of organophosphates in fruit and vegetable samples, which is consistent with green chemistry criteria. Also, a wider dynamic range in compared with other studies is obtained (Table 2). Considering the simplicity of the procedure, the detection limits and recoveries are almost comparable.

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4. Conclusions In this research, TFME as an environmentally friendly analytical technique based on chitosan– graphene oxide nanocomposite film was utilized for quantitative analyses of the selected OPPs residues in fruit and vegetable samples. The employed method provides a wide linear range, higher recovery and low detection limits. In general, the method is potentially suitable for screening of fruit and vegetable samples and it can be an alternative to assess these samples exposure to OPPs. As a conclusion, the PCA survey revealed the clustering of samples in to two groups of washed–peeled and unwashed–unpeeled based on the selected OPPs residues. Interestingly, descriptive analysis derived a clear picture of OPP dose in each single fruit and vegetable which is helpful in presenting the highest and lowest dose of the OPPs used in the studied samples.

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Pirsaheb, M., Fattahi, N., & Shamsipur, M. (2013). Determination of organophosphorous pesticides in summer crops using ultrasound-assisted solvent extraction followed by dispersive liquid–liquid microextraction based on the solidification of floating organic drop. Food Control, 34(2), 378-385. Salehi, B., Sharifi-Rad, R., Sharopov, F., Namiesnik, J., Roointan, A., Kamle, M., Kumar, P., Martins, N., & Sharifi-Rad, J. (2019). Beneficial effects and potential risks of tomato consumption for human health: An overview. Nutrition, 62, 201-208. Sanagi, M. M., Salleh, S., Ibrahim, W. A. W., Naim, A. A., Hermawan, D., Miskam, M., Hussain, I., & Aboul-Enein, H. Y. (2013). Molecularly imprinted polymer solid-phase extraction for the analysis of organophosphorus pesticides in fruit samples. Journal of Food Composition and Analysis, 32(2), 155-161. Sivaperumal, P., Anand, P., & Riddhi, L. (2015). Rapid determination of pesticide residues in fruits and vegetables, using ultra-high-performance liquid chromatography/time-offlight mass spectrometry. Food Chemistry, 168, 356-365. Xia, Y., Lu, Z., Lu, M., Liu, M., Liu, L., Meng, G., Yu, B., Wu, H., Bao, X., Gu, Y., Shi, H., Wang, H., Sun, S., Wang, X., Zhou, M., Jia, Q., Xiang, H., Sun, Z., & Niu, K. (2019). Raw orange intake is associated with higher prevalence of non-alcoholic fatty liver disease in an adult population. Nutrition, 60, 252-260. Yang, X., Luo, J., Duan, Y., Li, S., & Liu, C. (2018). Simultaneous analysis of multiple pesticide

residues

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Figure Captions

Fig. 1. The FTIR spectra recorded from (a) GO, (b) chitosan, (c) chitosan–graphene nanocomposite film (d) SEM image of the chitosan–graphene nanocomposite film. Fig. 2. Effect of (a) desorption solvent, (b) volume of desorption solvent, (c) desorption time, (d) extraction time, (e) pH on the extraction efficiency. Fig. 3. PCA of selected peeled washed and unpeeled unwashed fruits and vegetables Fig.4. Box plot of organophosphate pesticide residues in selected fruits and vegetables (µg.kg-1

22

Fig. 1

23

Fig. 2

24

Fig. 3

25

b

c

d

e

f

Profenofos

a

Fig. 4 26

Table1. Comparison of the proposed method with other research works for detection of selected organophosphates in fruit and vegetables

Fruits/Vegetables

Pesticide

Found (mg kg -1)

Orange Apple

Chlorpyrifos Chlorpyrifos Phosalone Chlorpyrifos Chlorpyrifos Chlorpyrifos Chlorpyrifos Chlorpyrifos Diazinon Chlorpyrifos Diazinon Fenthion Diazinon

0.01-0.14 0.01-0.074 0.01-1.59 0.01-0.226 0.57 0.052 0.01-0.43 0.01-0.053 0-0.034 0.-0.035 0.008 ND ND

Diazinon Chlorpyrifos

ND-0.003 ND

Fenthion

ND

Apple Tomato

Profenofos Profenofos

Kiwi

Azinphos-methyl Fenthion Phosalone profenofos Diazinon Chlorpyrifos Azinphos-methyl Fenthion Phosalone Profenos Diazinon Chlorpyrifos

0.03-0.032 0.016-0.161 Unpeeled (µg. kg -1) Peeled (µg. kg -1) 84.75-160.69 47.37-70.81 27.25-59.81 6.75-16.12 35.56-64.56 10.87-18.87 0-50.69 0-15 115.06-180.75 32.68-60.18 20.69-50.12 6.43-13.37 81.8-157 28-59.25 33.06-127.75 7.68-39.37 26.75-54 7.56-14.81 0-23.94 0-7.8 49.8-82.38 14.5-25.06 27.12-63.31 7.75-19.06

Tangerine Zucchini Cucumber Kiwi Tomato Apple Cucumber Apple Tomato

Orange

Method

Ref

QuEChERS-UPLC-MS/MS & GC-MS

(Bakırcı, Yaman Acay, Bakırcı, & Ötleş, 2014)

DLLME–SFO-HPLC-UV

(Pirsaheb, Fattahi, Rahimi, Sharafi, & Ghaffari, 2017)

UASE-DLLME-SFO-HPLC-UV

(Pirsaheb, Fattahi, & Shamsipur, 2013)

SDLLME-SFO-HPLC-UV

(Pirsaheb, Fattahi, Pourhaghighat, Shamsipur, & Sharafi, 2015)

UASE–DLLME–GC–FPD

(Bidari, Ganjali, Norouzi, Hosseini, & Assadi, 2011)

SPE-HPLC-UPLC/MS

(Sivaperumal, Anand, & Riddhi, 2015)

TFME-HPLC-UV

Present study

27

Apple

Tomato

Cucumber

Tangerine

Zucchini

Azinphos-methyl Fenthion Phosalone profenofos Diazinon Chlorpyrifos Azinphos-methyl Fenthion Phosalone Diazinon Chlorpyrifos Azinphos-methyl Fenthion Phosalone profenofos Diazinon Chlorpyrifos Azinphos-methyl Fenthion Phosalone profenofos Diazinon Chlorpyrifos Azinphos-methyl Fenthion Phosalone profenofos Diazinon Chlorpyrifos

93.81-179.44 28.25-70.5 22.44-55.25 37.88-85.06 49.5-177.75 35.56-55.56 89.19-151.75 42.83-67.12 23.94-40.94 109.19-215.5 28.0-54.75 75.62-126.62 49.88-64.19 37.44-60.56 44.56-62.06 51.69-188.75 23.69-53.56 63.75-131.12 41.31-68.06 42.44-80 0-60 0-65 20.5-38.54 28-75.25 31.33-58.58 0-55 0-60.42 0-45 36.33-73.58

32.62-82.37 7.25-23 6.94-17.25 10.06-22.43 14.5-62.25 9.37-16.31 26.75-51.31 9.87-19.37 6.06-12.44 37.0-71.75 7.87-16.69 27.31-57.69 9.37-22.13 10.25-18.81 10.12-17.37 15.68-73.06 6.87-15.62 17.18-33.12 11.18-20.37 8.03-24.1 0-17 0-20 4.81-12.26 11.16-28.17 6.16-13.92 0-12 0-15.03 0-15 4.08-16.75

28

Table 2. Comparison of the proposed method with other reported methods for the determination of selected organophosphate in fruit and vegetable samples. Desorption solvent (mL)

LOD (µg kg-1)

98-102

10

0.53-0.94

0.01-1.5

(Harshit, Charmy, & Nrupesh, 2017)

87

2.5

1

10-200

(Yang, Luo, Duan, Li, & Liu, 2018)

91-101

6

0.83-2.8

4-200

-

1

0.7

0.01-1

73-115

1

0.42-2.28

2.5-200

Fruit and vegetable

QuEChERS-GC-ECD and UPLCMS/MS QuEChERS-LC–MS/MS

(Sanagi, Salleh, Ibrahim, Naim, Hermawan, Miskam, et al., 2013) (Andrade, Monteiro, Francisco, Figueiredo, Botelho, & Tornisielo, 2015) (Bakırcı, Acay, Bakırcı, & Ötleş, 2014)

87-92

4

1-5

10-250

(Golge & Kabak, 2015a)

Fruit and vegetable

QuEChERS-HPLC-MS-MS

70-120

4

0.8-1.3

10-250

(Golge & Kabak, 2015b))

Fruit and vegetable

SPE-UPLC–MS

74-111

10

0.3-3.8

10-750

Fruit and vegetable

TFME-HPLC-UV

92-106

0.1

0.5-1.5

2-800

(Sivaperumal, Anand, 2015)) Present research

Matrices

Extraction method

Fruit and vegetable

SPE-HPLC-DAD

Fruit

QuEChERS-HPLC-MS

Fruit and vegetable

MIPSPE-UPLC-M

Vegetable

QuEChERS-HPLC-MS/MS

Fruit and vegetable

Recovery (%)

29

LDR (µg kg-1)

References

&

Riddhi,

Highlights

  

Variation in the concentration of six organophosphate residues is measured using HPLC. Residue of six organophosphates were detected in selected fruits and vegetables. PCA enabled clustering of peeled unwashed and unpeeled washed fruits and vegetables.

30