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Combination of Pesticides


Combination of pesticides; acetamiprid, flutolanil and etofenprox are usually used for tomato fruits for protecting them against pest infection. Generally, pesticides, residues could be one of the health hazard sources. Two specific simple sensitive chromatographic methods are developed for simultaneous estimation of the concerning pesticides’ residues using simple economic steps of field sample preparation. The first method is HP- TLC method. Hexane: methanol: acetone: glacial acetic acid (8: 2: 0.5: 0.1, by volume) is proposed as a developing system. The second one is RP- HPLC.

Acetonitrile: water (75: 25, v/v) is proposed as a mobile phase. The recommended methods are completely validated regarding ICH guidelines. Their means percentages and standard deviations of accuracy range 100.32± 0.89- 99.27± 0.9. The methods’ repeatability and intermediate precision relative standard deviation percentages range 0.395 & 0.894. They are successfully applied for estimating the pesticides in pure and commercial forms and field samples.


Acetamiprid; flutolanil; etofenprox; HP-TLC; RP- HPLC; validation.


Many plants’ pests and diseases affect food crops. So farmers use the crop specific pesticides to produce abundant high quality crops (Knowles, 2008), (Garau, Angioni, Aguilera Del Real, Russo, & Cabras, 2002).

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Tomato fruits have a high anti-oxidant capacity which reduces the risk of cardiovascular and cancer diseases (Raiola, Rigano, Calafiore, Frusciante, Barone, 2014), (Bhuvaneswari & Nagini, 2005) and contain a variety of vitamins and minerals. Fresh tomato fruits could be a potential source of harmful and toxic pesticide residues (Zork & Maja, 2009). At Egypt, Al-Fayoum governorate, location of the study, tomato fruits are sprayed with three pesticides acetamiprid (ACP), flutolanil (FLL) and etofenprox (ETFP), concurrently, to control attack of insects and fungal growth (Approved Recommendations for the Control of Agricultural Pets, Agricultural Pesticide Committee, Egypt, 2015).

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Acetamiprid (ACP) is N-[(6-chloropyridin-3-yl) methyl]-N’-cyano-N-methylethanimidamide and has PubChem CID 213021, MF: C10H11ClN4 and MW: 222.676g/mol. Flutolanil (FLL) is N-(3-propan-2-yloxyphenyl)-2-(trifluoromethyl) benzamide and has PubChem CID 47898, MF: C17H16F3NO2 and MW: 323.315g/mol. Etofenprox (ETFP) is 1-ethoxy-4-[2-methyl-1-[(3-phenoxyphenyl) methoxy] propan-2-yl] benzene and has PubChem CID: 71245, MF: C25H28O3 and MW: 376.496g/mol. The chemical structures of the compounds are figured out in Figure S1. They are commonly freely soluble in acetonitrile and methanol (Turner, 2017).

The literature survey shows some published methods which determine them individually; spectrophotometric methods for determination of ACP and FLL residues (Val?ria, Sanja, Nata?a & Zsigmond, 2012), (Qin, Shi, Gen-di, Hongboand, & Xiao, 2011), (Gallart, Armenta & Guardia, 2016), HPLC methods for determination of ACP and FLL residues residue (Lazi?, ?unjka, Grahovac, Guzsv?ny, Bagi, & Budakov, 2012), (Mart??nez, Gil Garc??a, Martinez, & Lopez, 2002), (Kikuchi, Sakai, Nemoto, & Akiyama, 2018) and HPLC methods for determination of ETFP residue on vegetables (Jia, Bi, Wang, Qiu, Zhou, & Zhou, 2006), (Sung, Abd El-Aty, Young, Myeong, Mi, & Han, 2014), (Deuk, Hyeok, & Kwon, 2011). In spite of (Hegazy, Abdelfatah, Mahmoud & Elsayed, 2018) has published an HPLC method for determining a limited number of pesticides, they determine pesticides mixture which is different from the concerning mixture. Also, the method is applied to another plant species.

The trend to analyze a narrow range of pesticides belong certain plant product via specific method has been noticed last years (Abdelfatah, Hegazy, Mahmoud & Elsayed, 2018), (Tiwari, & Asthana, 2012), (Baig, Akhtera, Ashfaq and Asi, 2009). A GC-Mass method is found for the estimation of 186 pesticides including the concerned pesticides (Zhao, 2014). The work aims to estimate the residues of tomatoes crop specific pesticides on the fruits before processing and serving stage.

Materials and methods


HP-TLC method

CAMAG TLC scanner operated by winCATS software, HP-TLC 20 x 20 cm aluminum plates coated with 0.25 mm silica gel 60 F254 (Merck, Germany), rotary flash evaporator (Shanghai Shensheng Biotech limited Co., China), and TV ultra-sonication (Sonix, USA) were used.

RP-HPLC method

1200 infinity series LC connected to 1260 UV – VIS detector with 1260 infinity, and 15 cm x 4.6 mm (i.d 5 µm particle size) Zobrax SB C8 column (Agilent, USA) were used.

Pure samples:

ACP (99.21%), FLL (99.08%) and ETFP (99.18%) were purchased from Sigma-Aldrich (Cairo, Egypt).

Commercial formulations:

Jinx®(ACP; 20 % w/w, Batch No. 309872) , Moncut® (FLL; 25 % w/w, Batch No. 206214) and Primo® (ETFP; 10 % w/w, Batch No. 2046) were purchased from producing companies; Claire Manufacturing Co. (USA), Nichino Inc. (USA) and Shoura Chemicals (Alexandria, Egypt), respectively.

Chemicals and reagents:

Methanol and acetonitrile (HPLC grade) were purchased from Sigma-Aldrich Chemie (GmbH, Germany). Hexane, glacial acetic acid, acetone and sodium sulfate of analytical grade were purchased from El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). De-ionized water was purchased from SEDICO Pharmaceuticals Co. (Giza, Egypt).

Standard solutions

ACP, FLL and ETFP standard stock solution (1000 µg mL-1) and standard working solutions (100 & 10 µg mL-1) were prepared in methanol.

ACP, FLL and ETFP commercial stock solutions (1000 µg mL-1) and working solutions (100 & 10 µg mL-1) were prepared in methanol.

Field sampling:

Sample preparation:

Tomato samples were collected from El-azab district, Al-Fayoum Governorate, Egypt during the winter season (average temperature: 20?C). Samples of three kg of previously sprayed ripe tomatoes by the studied pesticides were collected at three replicates each one was one kg so the total weight for all the samples was three kg. The samples were taken 1, 3, 7 and 10 days after spraying by pesticides regarding FAO/WHO guidelines (FAO/WHO. 1985).

Sample extraction:

1 Kg of sprayed tomato fruits was peeled then the peels were chopped and shake with 20 mL of acetonitrile for 2 minutes in a 50 ml centrifuge tube then 5 g of sodium sulfate were added. The tube was centrifuged for 2 minutes at 6000 rpm. Supernatant extracts were collected, quantitatively, and concentrated to 3 mL under vacuum using a rotary flash evaporator at 28 ?C. All the process of preparation was done thoroughly using clean tools to prevent cross contamination. Samples were kept at fridge to minimize pesticides losses via evaporation or degradation according to recommendation of European Commission.

Calibration curves

HP-TLC method

A series of serial dilutions of ACP, FLL and ETFP in concentrations ranges of 0.10-1.40, 0.01-0.22 & 0.01-0.20 µg mL-1, respectively, were prepared. Samples were applied on HP-TLC plates using 10 µL of each prepared solution using a Camag-Linomat IV applicator.

RP-HPLC method

Different series of serial dilutions of ACP, FLL and ETFP in concentrations ranges of 1.00-14.00, 0.10-2.20 & 0.10-2.00 µg mL-1, respectively, were prepared. Triplicate injections were carried out with injection volume 20 µL.

Cite this page

Combination of Pesticides. (2019, Nov 21). Retrieved from

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