Chlorpyrifos, (Fig.1) is an organophosphorus insecticide, which is chemically named as thiophosphoric acid O, O′- diethyl ester-O″-(3, 5, 6-trichloro-pyridin-2-yl phosphorothioate) ester (Radišić et al., 2009).
In market it is available under numerous trade names, i.e., Dursban, Lorsban, Pyridane, Pyrinex, Silrifos etc. It is a white or colorless crystal which has a slightly skunky odour, similar to that of rotten eggs or garlic. Chlorpyrifos is insoluble in water but it is soluble in organic solvents like ethyl acetate, acetone, benzene, chloroform, methanol, diethyl ether at room temperature (Radišić et al., 2009).
Chlorpyrifos is the common name for the chemical 0,0-diethyl 0-(3,5,6-trichloro-2-pyridinyl)-phosphorothioate. Chlorpyrifos is used as an insecticide to control many different kinds of pests, including roundworms, termites, mosquitoes etc. Some products which contain chlorpyrifos are especially used in agriculture industry for the treatment of fruits and vegetables (Blakely et al., 2014; Pujeri et al., 2015; Karabasanavar & Singh, 2012). Fruits and vegetables being highly nutritious are produced for local consumption as well as for export purposes. In order to produce higher and better yield, a large amount of insecticide is used by farmers during the entire period of growth (Blakely et al., 2014; Pujeri et al., 2015; Karabasanavar & Singh, 2012). In a study of seven aerobic soils ranging in texture from loamy sand to clay, with soil pH values from 6.0 to 7.4, the soil half life for radiolabeled chlorpyrifos ranged from 11 to 141 days. However, studies have found chlorpyrifos in soils for over one year following application. Since a huge amount of insecticide is used so their irrational and continual use becomes the reason of accumulation of insecticide residues in the primary agriculture products. The continuous use of these insecticides increases the possibility that residues of these compounds could be found in some fruits and vegetables, thus making this matter a public health and sanitary defense issue (Blakely et al., 2014; Pujeri et al., 2015; Karabasanavar & Singh, 2012). Also, being an organophosphorus insecticide, chlorpyrifos acts as poison if it is touched, inhaled, injected or eaten in any manner (Eaton et al., 2008; Watts, 2013). Chlorpyrifos affects the nervous system. On exposure to chlorpyrifos, it moves to all the parts of the body through metabolic pathways and when the body tries to break it down into its metabolites it gets converted to another form which is called chlorpyrifos oxon. This oxon further binds permanently to the enzymes, which controls the messages that travel between the nerve cells (Eaton et al., 2008; Watts, 2013). Nerves and muscles do not respond correctly when this oxon binds with too many of the enzymes. This causes the body to make more enzymes so that normal nerve function can resume. The body can break down and excrete most of the unbound chlorpyrifos in feces and urine within a few days. Chlorpyrifos that finds its way into the nervous system may stay there much longer. Chlorpyrifos is principally excreted in the urine (Eaton et al., 2008; Watts, 2013).
For human beings to remain healthy, it is important to include fruits and vegetables in their diet for most of the actions taking place in our body. Even though these fruits and vegetables are healthy but they are equally prone to pests and diseases which attack them during their time of production as well as storage thus, degrading their yield and quality (Blakely et al., 2014; Pujeri et al., 2015; Karabasanavar & Singh, 2012). So, to prevent these issues farmers use high amount of pesticides and other products. Even though the use of pesticides has increased the quality and quantity of fruits and vegetables but it has also affected the life of its consumers by causing a large number of health issues to the human beings. Although a large amount of pesticides gets removed from the human body in the form of urine and fecal matter but, still some pesticides (especially chlorpyrifos) are very persistent and can remain in human body for a long term (Eaton et al., 2008; Watts, 2013).
Detailed survey of literature for chlorpyrifos revealed that various techniques have been discovered and used for the assay of chlorpyrifos residue in fruits and vegetables samples. These techniques include TLC (Tewari, 1976), HPLC with UV detection (High Performance Liquid Chromatography) (Richard et al., 2006; Sajjad et al., 2009; Cozma et al., 2011; Devendra et al., 2011; Barkat et al., 2012; Paranthaman et al., 2012; Shailendra et al., 2012; Alamgir et al., 2013; Tordzagla et al., 2013), Liquid chromatography-tandem Mass spectrometery (Steven et al., 2005; Rohan et al., 2012), High Performance Thin Layer Chromatography (HPTLC) (Iqbal et al., 2007; YueY et al., 2008; Akkad & Schwack, 2012; RouhollahD et al., 2012), Gas chromatography–Mass spectrometry(GC-MS) (Paranthaman et al., 2012; Steven et al., 2005; Tomas et al., 2012),Gas Chromatography with Electron Capture detection (GC-ECD) (Devendra et al., 2011; Paranthaman et al., 2012; Mohammad et al., 2010; Subhash et al., 2010), Spectrophotometry (Venugopal et al., 2012), Reflectance near-infrared spectroscopy (Umesh et al., 2012), Chemiluminescence assay (Aifang et al., 2008), immunoassay (Gabaldón & Maquieira, 2007) and Capillary electrochromatography, (Weimin et al., 2010) but in present study an attempt has been made to analyze chlorpyrifos by Thin Layer Chromatography-Flame Ionization Detection technique (TLC-FID) and to validate the method. TLC-FID is a technique which combines the advantages of TLC with the possibility of quantitation using FID. (Cebolla et al., 1998; Bharati et al., 1993; Stephens et al., 1998; Jiang et al., 2008; Ranny, 1987) The separation is made with the TLC Method on chromarods instead of TLC plates and the detection of chlorpyrifos is done with a FID. To separate and identify chlorpyrifos poison, standard solution was prepared and spotted on chromarods with micro dispenser and the rods were made to run in the Hexane: Acetone solvent system and was afterwards subjected to TLC-FID instrument/ IATROSCAN MK-6 s, after which the chromatograms were generated. (Cebolla et al., 1998; Bharati et al., 1993; Stephens et al., 1998; Jiang et al., 2008; Ranny, 1987)Only few methods have been reported for the determination of pesticide residue in leafy vegetables. The methods adopted so far include HPLC with UV detection, (Barkat et al., 2012; Paranthaman et al., 2012; Shailendra et al., 2012) Capillary Chromatography (Weimin et al., 2010) and Gas Chromatography with electron capture detector. (Subhash et al., 2010; Hussain & Samia, 2010)
The current “Joint Meeting on Pesticide Residues” (JMPR) that comprises the WHO Core Assessment Group and the FAO Panel of Experts on Pesticide Residues in Food and the Environment is responsible for reviewing pesticide toxicological data and estimating Acceptable Daily Intakes (ADI), acute reference doses (ARfDs). JMPR has fixed the ADI (per day per kg body weight) for Chlorpyrifos as 0.01 mg/kg (Bhushan et al., 2013).
Methods
Sampling
Grape samples were collected from the local fruit and vegetable market around Chandigarh, India. The samples were subjected to refrigeration and analysis within a week of collection. All samples were freshly extracted.
Materials
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Acetone, E. Merck
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Hexane, E. Merck
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Ethyl Acetate, E. Merck
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99% pure Chlorpyrifos Standard, Accustandard Inc.
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Deionized Water
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Sodium Sulfate, Anhydrous
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Grape
Measurement conditions for TLC FID
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Principle of Separation: Thin Layer Chromatography with the use of Chromarods (a special rod coated with a thin layer adsorbent)
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Detection: Hydrogen Flame Ionization Detector (FID) MK-6(s)
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Detection Time: 25 s/scan
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Hydrogen Flow Monitor: Electronic flow meter (digital display)
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Air Flow Monitor: Air flow meter (float type)
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Chromarod Holder: Available for loading 10 Chromarods
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Measuring Modes: Normal scan/Blank scan
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Power: AC 100,120,220&240 V, 50/60 Hz
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Power Requirement: ~50 VA
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Temp. /Humidity: 10~350 C/20–80 RH
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Dimensions: ~520x430x265mm MK-6 s~520x430x260
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Weight: ~25Kg MK-6 s: ~23Kg
Extraction of Chlorpyrifos residue from grapes
The efficiency of ethyl acetate extraction method was not tested within this paper as it had been proven to be suitable for a very wide range of pesticide–commodity combinations. The chlorpyrifos pesticide was extracted from grapes samples with optimized extraction method (Hussain & Samia, 2010). 100 g of grapes were macerated with 10–15 g sodium sulfate (anhydrous) and 15 g of sodium bicarbonate in a pestle and mortar to make a fine paste. After maceration, the sample was extracted in 100 mL ethyl acetate at room temperature on mechanical shaker for one hour. The extract (pH 8) was filtered through Teflon filter 0.45 μm and the procedure was repeated by washing the remaining sample 2–3 times with ethyl acetate and concentrated on rotary evaporator. The final volume was made 5 mL with ethyl acetate in a glass flask and 20 μL of 5% formic acid in ethyl acetate solution were added to maintain pH 5–5.5,where most acid and base labile pesticide are sufficiently stabilized. The temperature during extraction was maintained between 25 and 33 °C to obtain good extraction efficiency and the temperature was not allowed to exceed 33 °C.When deep frozen samples were processed the mixture of sample homogenate and the extracting solvent was kept in a water bath at 30oCto reach the specified temperature range.
Sample preparation
Standard stock solution was prepared by accurately weighing 0.1000 g of standard chlorpyrifos and dissolving it in ethyl acetate and making the volume 100 mL in a volumetric flask.
Working standard solutions of different concentrations were prepared (that is, 25 μg/mL, 50 μg/mL, 75 μg/mL, 100 μg/mL) by diluting the standard stock solution accordingly. Both the stock and working standard solutions were stored in a refrigerator.
TLC-FID instrumentation
Preparation of mobile phase
The mobile phase was prepared by adding 54 ml of hexane in 6 ml of acetone (9:1, v/v) (Barkat et al., 2012). The mobile phase was poured in the developing chamber and was lined with filter paper and left as such for 30 min to make the chamber saturated with the vapors of mobile phase.
General requirements
S-III chromarods were used for performing chromatographic separation. Samples were applied using a Micro dispenser (DRUMMOND). Hydrogen and air flow were 160 mL/min and 2–2.5 mL/min respectively.
Procedure
Set of 10 chromarods was previously assembled in a frame. IATROSCAN MK-6 s instrument was started and the rods were subjected to blank scan twice to initially activate the chromarods for further processing. The activated chromarods were kept on chromarod holder. Afterwards, the working standards of different concentrations and grape sample were applied using a micro dispenser. The chromarods were kept in the previously prepared solvent system kept in the development tank and was left as such for development. Chromarods were sequentially passed through H2 flame in the IATROSCAN FID for peak quantitation at 25 s/scan.