Development and Validation of a Quick, Easy, Cheap ...

Journal of Chromatographic Science Advance Access published July 16, 2014 Journal of Chromatographic Science 2014;1– 6 doi:10.1093/chromsci/bmu082

Article

Development and Validation of a Quick, Easy, Cheap, Effective, Rugged and Safe Method for the Determination of Imidacloprid and Its Metabolites in Soil Romila Akoijam*, Balwinder Singh and Kousik Mandal Pesticide Residue Analysis Laboratory, Department of Entomology, Punjab Agricultural University, Ludhiana, Punjab 141004, India *Author to whom correspondence should be addressed. Email: [email protected] Received 9 October 2013; revised 10 June 2014

A simplified quick, easy, cheap, effective, rugged and safe method was standardized and validated for the estimation of residues of imidacloprid and its metabolites from different types of soil comprising sandy loam, loamy sand and clay loam soil. The samples were extracted with acetonitrile, clean up by treatment with primary secondary amine sorbent and graphitized carbon. High-performance liquid chromatography (HPLC) is a technique to separate and quantitate residues of polar, nonvolatile and thermolabile chemical compounds, using high-pressure pumps, short and narrow columns packed with microparticulate phases and a detector. The residues were estimated using HPLC equipped with a photodiode array detector system, C18 column. Acetonitrile and water (30 : 70) were used as an isocratic mobile phase at 0.3 mL/min. Imidacloprid and its metabolites presented distinct peaks at retention factors of 4.93 min (6-chloronicotinic acid), 7.91 min (nitroguanidine), 9.12 min (olefin), 11.32 min (nitrosimine), 13.82 min (urea), 15.45 min (5-hydroxy) and 22.47 min (imidacloprid). Consistent recoveries above 80% for imidacloprid and its metabolites were observed when samples were spiked at 0.01, 0.05 and 0.10 mg/kg levels. The limit of quantification of the method was 0.01 mg/kg. The analytical method was validated in terms of parameters including selectivity, linearity, precision and accuracy of the detection system. Introduction Imidacloprid (1-[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imida zolidinimine) is a systemic chloronicotinyl insecticide belongs to a class of chemicals called neonicotinoid (1) (Figure 1). The high insecticidal activity of imidacloprid works by binding to the nicotinergic acetylcholine receptor in the insect nervous system, which interferes with chemical signal transmission in the stimuli (2 –4). The blockage leads to the accumulation of a neurotransmitter, acetylcholine, resulting in excitation, paralysis and eventually death (5, 6). Imidacloprid works similar actions on target and non-target beneficial insects including honeybees, predatory ground beetles and parasitoid wasps (7). However, imidacloprid is ineffective against spider mites and nematodes (8). It is considered a relatively polar material with good xylem mobility and hence is suitable for seed treatment and soil application (9). The low vapor pressure of 1.0  1027 mmHg indicates that this insecticide is nonvolatile. Likewise, the low Henry’s law constant of 6.5  10211 atm m3 per mole indicates that it has low volatility from water. As a result, it is unlikely to be dispersed in air over a large area from volatilization (7). Imidacloprid can persist in soil depending on soil type, pH, use of organic fertilizers and presence or absence of ground cover.

The primary breakdown products of imidacloprid in soil are urea and 6-chloronicotinic acid (6-CNA) (10). The majority of toxicity studies have been focused on the parent compound, imidacloprid. The metabolites of imidacloprid, namely olefin and nitrosimine, have greater insecticidal activity than the parent compound (11) while the guanidine metabolite does not possess insecticidal properties, but has a higher mammalian toxicity than the parent compound (12). After soil application or seed treatment, a quick degradation of the active substance was observed after root uptake of the active substance (13). It is a systemic broad-spectrum insecticide and acts as a contact and stomach poison against sucking and some biting insects (rice hoppers, aphids, thrips, whitefly, termites, etc.). It can be applied by soil injection, tree injection, topical application of animal, broadcast foliar, ground application as a granular or liquid formulation or as a pesticide-coated seed treatment (11, 14, 15). With its widespread use in mind, it was intended to standardize the methodology for the estimation of residues of imidacloprid and its metabolites in different types of soil such as sandy loam, loamy sand and clay loam soil. Sarkar et al. (16) studied the persistence and metabolism of imidacloprid in different soils by using traditional methods of extraction and cleanup process of soil. Estimation of residues of imidacloprid in soil with methods using a sonication bath have also been developed (17, 18). The method used by Sarkar et al. (16) studied imidacloprid and urea and olefine metabolites only. To overcome these difficulties, a quick, easy, cheap, effective, rugged and safe (QuEChERS) method was developed and validated for the estimation of imidacloprid and its metabolites in different types of spiked soil samples. The method is based on work done and introduced by Anastassiades and Lehotay (19). The present QuEChERS method provides the quickest, easy, cheap and effective method compared with that of the traditional methods. The QuEChERS method gives high recoveries for a wide polarity and volatility range of pesticides; very accurate (true and precise) results are achieved because an internal standard is used to correct for commodityto-commodity water content differences and volume fluctuations; multiple sample processed within a short time with little solvent usage and waste; a single person can carry out the method without much training or technical skill; very little glassware is used; the method studied imidacloprid and six metabolites, namely, 6-CNA, nitroguanidine, olefine, nitrosimine, urea and 5-hydroxy; the method is quite rugged because extract cleanup is done to remove organic acids; very little bench space is needed and thus the method can be done in a small laboratory and few devices are needed to carry out sample preparation.

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

Figure 1. Chemical structures of (a) imidacloprid, (b) 6-CNA, (c) nitroguanidine, (d) olefine, (e) nitrosimine, (f) urea and (g) 5-hydroxy.

Experimental Standard compounds The technical grade analytical standards of imidacloprid (99.9%) and its metabolites such as nitrosimine (90.6%), olefin (97.9%), urea (99.4%), 6-CNA (98.8%), 5-hydroxy (96.8%) and nitroguanidine (99.0%) were obtained from M/s Bayer CropScience (India), Ltd, Mumbai, India.

Chemicals and reagents Sodium chloride, activated anhydrous MgSO4 and solvents like high-performance liquid chromatography (HPLC) grade acetonitrile were obtained from E. Merck (India) Ltd, Mumbai, India. Sodium sulfate anhydrous was from S. D. Fine Chemicals, Mumbai, India. Primary secondary amine (PSA) sorbent was obtained from Sigma-Aldrich, Mumbai, India. Charcoal decolorizing powder activated was obtained from Qualigens Fine Chemicals, Mumbai, India. All the solvents used were of laboratory grade. All common solvents were redistilled in all glass apparatuses before use. The suitability of the solvents and other chemicals was ensured by running reagent blanks before actual analysis. 2 Akoijam et al.

Instrumentation and apparatus The high-performance liquid chromatograph (Model DGU-2045) was equipped with a reverse-phase, RP, C18 column and a photodiode array (PDA) detector, and dual pumps supplied by M/S Shimadzu Corporation, Kyoto, Japan. The HPLC column, a Luna 5 mm C18 column (250  4.6 mm size, 5.20 + 0.30 mm particle size, 2.20 + 0.30 (90 : 10) particle distribution, 95 + 15A8 pore diameter, 430 + 40 m2/g surface area, ,55.0 ppm metal content, 19.00 + 0.70% total carbon and 3.25 + 0.50 mmoles m22 surface coverage), was obtained from Spincotech Pvt. Ltd, Chennai (India). HPLC was equipped with a LC-20AT pump and a CBM-20A system controller. For instrument control, data acquisition and processing, LC Solution software was used. A good satisfactory separation of peak symmetry was obtained with an isocratic mobile phase comprising of acetonitrile : water (30 : 70, v/v) at a flow rate of 0.3 mL/min. Quantification was achieved with PDA detection at 270 nm based on peak area with a retention factor of 25 min and injection volume of 20 mL. Standard solution A standard stock solution of imidacloprid and its metabolites (1 mg/mL) was prepared in HPLC grade acetonitrile. The

standard solutions required for constructing a calibration curve (2.00, 5.00, 10.00, 15.00 and 20.00 mg/mL) were prepared from stock solution by serial dilution with HPLC grade acetonitrile. All standard solutions were stored at 48C before use.

Extraction and cleanup Different types of soil samples such as sandy loam, loamy sand and clay loam soil were used as substrates for standardization of the methodology intended for estimation of imidacloprid and its metabolites. The soil samples were collected from different parts of Punjab, India. The physical characteristics, namely, nature of soil, organic matter, pH and electrical conductivity were ascertained before initiating the experiment. The physicochemical properties of different type of soils used in this study were organic carbon ¼ 0.153%, pH ¼ 8.32 and electrical conductivity ¼ 0.44 dsm21 for sandy loam soil; organic carbon ¼ 0.47%, pH ¼ 7.85 and electrical conductivity ¼ 0.94 dsm21 for loamy sand soil and organic carbon ¼ 0.30%, pH ¼ 8.45 and electrical conductivity ¼ 0.54 dsm21 for clay loam soil. The soil was air-dried under shade, ground, sieved through a 2-mm mesh screen and stored in cloth bags until used. The soil samples were fortified at different levels, i.e., 0.01, 0.05 and 0.10 mg/kg. There were five replications for each treatment.

Methods The QuEChERS method had been performed by taking a representative 15 g sample of soil into a 50-mL centrifuge tube, and 30 mL of acetonitrile was dispensed into all the centrifuge tubes. The samples were well shaken and homogenized at 15,000 rpm (1,680 rcf ) for 2 – 3 min using a Heidolph homogenizer. Sodium chloride (10 g) was added to each sample and shaken vigorously by rotospin for 5 min. The samples were centrifuged using a laboratory centrifuge for 3 min at 2,500 rpm (280 rcf ). The top organic layer (15 mL) from each tube was decanted into another 50-mL centrifuge tube containing 10 g of activated sodium sulfate. It was again shaken using a rotospin for 2– 3 min. The sample extract (6 mL) was transferred to a centrifuge tube containing PSA sorbent (0.15 g), activated anhydrous magnesium sulfate (0.9 g) and graphitic carbon black (0.05 g). The tube was tightly capped and vortexed for 30 s. The tubes were centrifuged for 1 min at 2,500 rpm (280 rcf ). The top extract (4 mL) was transferred into a test tube and concentrated to 2 mL with a rotary evaporator under 358C for further quantification by HPLC.

Results HPLC chromatograms Reversed-phase HPLC, with PDA detection, was shown to be good for determination of imidacloprid and its metabolites because no derivatization step is needed. Chromatographic separation in C18 columns provides good results. The detection at 270 nm offers suitable chromatograms for the quantification of imidacloprid and its metabolites in real samples. Under the chosen conditions, imidacloprid and its metabolites showed retention factors of 4.93 min (6-CNA), 7.91 min (nitroguanidine), 9.12 min (olefin), 11.32 min (nitrosimine), 13.82 min (urea),

15.45 min (5-hydroxy) and 22.47 min (imidacloprid) allowing a complete separation of its signal from those of foreign substances present in the samples (Figure 2). Validation of the methods As the quantitative determination of imidacloprid and its metabolites in different types of soil samples is directly related to the evaluation and interpretation of data, a reliable method is required which is reproducible and can be applicable to different samples. The method was fully validated according to analytical method recommendations described in the SANCO guidelines in terms of selectivity, linearity, precision (repeatability and reproducibility) and accuracy for the detection system (20). Selectivity Selectivity of the methods was assessed by comparing the HPLC chromatogram of a set of five blank samples with that of five samples spiked at the limit of quantification (LOQ) level (0.01 mg/g) for HPLC detection. As shown in Figure 2, no peak was detectable at the retention factors of imidacloprid and its metabolites. Linearity The linearity of a method is a measure of range within which the results are directly, or by a well-defined mathematical transformation, proportional to the concentration of analyte in samples within a given range (21). The calibration curves with respect to imidacloprid and its metabolites produce a linear relationship between detector response (y) and analyte concentration (x) (Table I). Limit of quantification As a general rule, residues of imidacloprid and its metabolites were determined by comparison of peak areas of the reference standards with that of the unknown or spiked samples run under identical operating conditions of the instruments employed. LOQ for imidacloprid and its metabolites was worked out on the basis of the response of the nanogram of standard working solution injected as well as the sample weight in milligram injected so that the baseline of the instrument remains stable and no noise is observed. Half-scale deflection was obtained for 2.0 ng imidacloprid, which could be easily identified from the baseline. A 10 g of different types of soil samples such as sandy loam, loamy sand and clay loam soil were extracted, cleaned up and the final volume made to 5 mL and 20 mL of the sample (equivalent to 40 mg soil) when injected did not produce any background interference. Thus, the LOQ was found to be 0.01 mg/kg. Precision The precision of the method was determined by repeatability and reproducibility of the method. Repeatability Repeatability of the developed analysis method was determined by spiking imidacloprid and its metabolites in different concentrations to different soil samples. The within-batch recovery and Determination of Imidacloprid and Its Metabolites in Soil 3

Figure 2. HPLC chromatograms of (a) standards of imidacloprid and its metabolites, (b) control sample of sandy loam soil, (c) spiked sample of sandy loam soil, (d) control sample of loamy sand soil, (e) spiked sample of loamy sand soil, (f ) control sample of clay loam soil and (g) spiked sample of clay loam soil.

relative standard deviation for repeatability (RSDr) of spiked imidacloprid and its metabolites at the levels of 0.01, 0.05 and 0.10 mg/kg are summarized in Table II. The precision (repeatability) in different types of soil samples was in the range from 0.24to 4.40% for imidacloprid and its metabolites (Table II). 4 Akoijam et al.

Reproducibility The reproducibility of this analytical method was determined by analyzing spiked samples under various test conditions (different days). The between-batch recoveries and relative standard deviation for reproducibility (RSDR) investigated at several levels are

given in Table III. The precision (reproducibility) of imidacloprid and its metabolites in three different soil samples ranged from 2.15 to 5.57% (Table III), and all measurements are within 15% at all concentrations (22). Accuracy The recovery tests were carried out on five replicates at each spike level. The average recoveries obtained for imidacloprid

Table I Linearity Equation of Imidacloprid and Its Metabolites Insecticide

Calibration curve

R2

LOQ (mg/kg)

Imidacloprid 6-CNA Nitroguanidine Olefin Nitrosimine Urea 5-Hydroxy

y ¼ 63x þ 48 y ¼ 32x 2 45 y ¼ 98x þ 67 y ¼ 86x þ 53 y ¼ 45x 2 15 y ¼ 15x 2 11 y ¼ 45x þ 44

0.990 0.991 0.991 0.994 0.998 0.994 0.996

0.01 0.01 0.01 0.01 0.01 0.01 0.01

and its metabolites at all concentrations and conditions investigated (Tables II and III) were determined as .80% in all the samples. Discussion To develop a simple, effective and economical reversed-phase HPLC technique by using QuEChERS methodology, several mobile phase compositions were tried. A good satisfactory separation of peak symmetry was obtained with a Luna 5 mm C18 column (250  4.6 mm size) and a mobile phase comprising of acetonitrile : water (30:70, v/v) at a flow rate of 0.3 mL/min to get better repeatability and reproducibility. Quantification was achieved with PDA detection at 270 nm based on peak area with a retention factor of 25 min. The retention factors were found to be 4.93 min for 6-CNA, 7.91 min for nitroguanidine, 9.12 min for olefin, 11.32 min for nitrosimine, 13.82 min for urea, 15.45 min for 5-hydroxy and 22.47 min for imidacloprid (Figure 2). The optimized method was validated as per SANCO guidelines. The result of recovery study for imidacloprid and its

Table II Recovery Studies (%)a of Imidacloprid and Its Metabolites from Fortified Samples of Sandy Loam, Loamy Sand and Clay Loam Soil (n ¼ 5) Soil samples

Sandy loam

Loamy sand

Clay loam

Level of fortification (mg/kg)

Imidacloprid

Metabolites

a

83.27 + 1.81 (2.17) 82.22 + 1.34 (1.62) 86.34 + 2.08 (2.40) 82.71 + 1.76 (2.12) 87.12 + 0.92 (1.05) 89.09 + 1.47 (1.65) 83.65 + 3.68 (4.40) 87.03 + 2.50 (2.87) 82.99 + 2.19 (2.63)

0.01 0.05 0.10 0.01 0.05 0.10 0.01 0.05 0.10

6-CNA

Nitroguanidine

Olefine

Nitrosimine

Urea

5-Hydroxy

81.79 + 2.90 (3.54) 89.46 + 2.16 (2.41) 91.03 + 0.87 (0.96) 81.78 + 0.79 (0.97) 90.27 + 2.76 (3.05) 98.05 + 1.24 (1.26) 82.93 + 2.01 (2.42) 85.30 + 2.16 (2.53) 96.79 + 1.03 (1.06)

85.62 + 2.11 (2.47) 87.51 + 1.20 (1.37) 81.44 + 0.59 (0.72) 96.42 + 2.13 (2.20) 92.91 + 0.87 (0.93) 99.02 + 1.72 (1.73) 85.37 + 1.17 (1.37) 91.13 + 0.62 (0.68) 88.14 + 2.15 (2.43)

85.16 + 0.79 (0.92) 83.12 + 0.45 (0.54) 84.20 + 1.20 (1.42) 81.70 + 1.18 (1.45) 83.20 + 1.91 (2.29) 87.54 + 0.48 (0.54) 81.69 + 1.38 (1.68) 88.01 + 2.11 (2.39) 99.00 + 3.20 (3.23)

87.62 + 1.79 (2.04) 93.26 + 2.00 (2.14) 98.70 + 3.82 (3.87) 84.64 + 0.91 (1.07) 81.89 + 1.78 (2.17) 92.75 + 1.35 (1.46) 83.81 + 1.80 (2.14) 92.05 + 3.10 (3.36) 93.34 + 2.88 (3.08)

85.97 + 0.86 (1.00) 81.20 + 0.98 (1.20) 90.46 + 1.76 (1.94) 85.70 + 1.87 (2.18) 82.09 + 3.13 (3.81) 94.63 + 0.59 (0.62) 85.01 + 2.09 (2.45) 90.64 + 1.15 (1.26) 97.44 + 0.23 (0.24)

93.76 + 1.41 (1.50) 96.65 + 0.76 (0.78) 89.16 + 1.32 (1.48) 89.05 + 1.20 (1.34) 94.63 + 2.01 (2.12) 96.39 + 1.16 (1.20) 89.20 + 1.41 (1.58) 98.10 + 0.61 (0.62) 86.28 + 2.83 (3.28)

a

Mean + standard deviation of five replicate determinations (repeatability, RSDr, %).

Table III Recovery and RSD Values of Spiked Samples of Sandy Loam, Loamy Sand and Clay Loam Soil with Imidacloprid and Its Metabolites at 0.01 mg/kg Level. Compound

Imidacloprid

6-CNA

Nitroguanidine

Olefin

Nitrosimine

Urea

5-Hydroxy

Day

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Sandy loam

Loamy sand

RSDR %

Clay loam

Recovery %

RSDr %

Recovery %

RSDr %

Recovery %

RSDr %

Sandy loam

Loamy sand

Clay loam

83.27 85.78 91.05 81.79 86.89 90.83 85.62 82.76 87.17 85.16 90.72 82.68 87.62 82.00 91.27 85.97 87.13 93.18 93.76 84.15 87.10

2.17 3.29 4.67 3.54 1.08 3.60 2.47 4.89 3.10 0.92 1.99 3.13 2.04 3.15 1.19 1.00 2.87 1.73 1.50 4.08 2.65

82.71 89.09 85.88 81.78 84.29 90.01 96.42 90.11 87.23 81.70 88.74 84.09 84.64 93.33 91.23 85.70 82.95 86.43 89.05 86.10 92.55

2.12 1.89 3.01 0.97 1.69 3.80 2.20 2.76 0.90 1.45 3.67 4.80 1.07 3.45 2.11 2.18 3.06 3.73 1.34 4.46 2.98

83.65 87.19 88.79 82.93 86.51 90.02 85.37 81.98 90.14 81.69 89.50 82.94 83.81 89.56 81.93 85.01 82.38 89.97 89.20 87.61 93.75

4.40 2.75 3.98 2.42 4.68 2.32 1.37 4.07 4.64 1.68 2.57 3.81 2.14 4.03 2.23 2.45 1.47 1.36 1.58 4.24 2.07

4.58

3.71

3.03

5.23

4.93

4.09

2.62

5.15

4.76

4.76

4.20

4.94

5.35

5.04

4.66

4.36

2.15

4.48

5.57

3.60

3.52

RSDr, relative standard deviation (repeatability); RSDR, relative standard deviation (reproducibility).

Determination of Imidacloprid and Its Metabolites in Soil 5

metabolites in the three different types of soil ranging from 81.20 to 99.02% (sample spiked at 0.01, 0.05 and 0.10 mg/kg levels) suggested the good accuracy of the method. The precision of the proposed method was carried in terms of the repeatability and reproducibility. The low % RSD values of repeatability and reproducibility reveal that the proposed method is precised. The absence of interference peak indicates that method can be used for routine analysis of soil samples in monitoring program.

Conclusion A specific, simplified, quick, cost-effective reversed-phase HPLC method by using QuEChERS has been developed and validated. The method gives accurate and precise results for the estimation of residues of imidacloprid and its metabolites from different types of soil comprising sandy loam, loamy sand and clay loam. The consistent recoveries ranging from 81.20 to 99.02% for imidacloprid and its metabolites were observed when samples were spiked at 0.01, 0.05 and 0.10 mg/kg levels. The LOQ and limit of detection of the method were worked out to be 0.01 and 0.003 mg/kg. The present QuEChERS method is effective and provides the quickest, easy and cheap method compared with that of the traditional methods. The method was developed, validated and tested for spiked samples, and further evaluation of the method using real samples could be conducted. Therefore, the method can be applied for studying the monitoring of imidacloprid and its metabolites in soil.

Acknowledgments The authors are thankful to the professor and Head, Department of Entomology, Punjab Agricultural University, Ludhiana, India, for providing the necessary research facilities.

References 1. Leicht, W.; Imidacloprid—a chloronicotinyl insecticide; Pesticide Outlook, (1993); 4: 17– 21. 2. Abbink, J.; The biochemistry of imidacloprid; PflanzenschutzNachrichten Bayer, (1991); 44: 183– 195. 3. Muchembled, C.; Development of insecticidal treatment in beet; Pflanzenschutz-Nachrichten Bayer, (1991); 44: 175– 182. 4. Elbert, A., Nauen, R., Leicht, W.; Imidacloprid, a novel chloronicotinyl insecticide: biological activity and agricultural importance. In Ishaaya, I., Degheele, D. (eds). Insecticides with novel modes of action. Springer, Berlin, (1998), pp. 50– 73, 288. 5. Matsuda, K., Buckingham, S.D., Kleier, D.; Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors; Trends in Pharmacological Sciences, (2001); 22: 573– 579. 6. Millar, N.S., Denholm, I.; Nicotinic acetylcholine receptors: targets for commercially important insecticides; Invertebrate Neuroscience, (2007); 7: 53– 66.

6 Akoijam et al.

7. Fossen, M.; Environmental fate of imidacloprid. (2006) http://www. cdpr.ca.gov/docs/emon/fatememo/imidaclprdfate2.pdf (accessed June 9, 2011). 8. Tomlin, C.D.S.; The pesticide manual: a world compendium; 14th ed. British Crop Protection Council, Surrey, England, (2006), pp. 598– 599. 9. Horowitz, A.R., Ishaaya, I.; Biorational insecticides—mechanisms, selectivity and importance in pest management programs; In Horowitz, A.R., Ishaaya, I. (eds). Insect pest management—field and protected crops. Springer, Berlin, Heidelberg, New York, (2004), pp. 1– 28. 10. Rouchaud, J., Gustin, F., Wauters, A.; Imidacloprid insecticide soil metabolism in sugar beet field crops; Bulletin of Environmental Contamination and Toxicology, (1996); 56: 29 –36. 11. Nauen, R., Tietjen, K., Wagner, K., Elbert, A.; Efficacy of plant metabolites of imidacloprid against Myzus persicae and Aphis gossypii (Homoptera: Aphididae); Pesticide Science, (1998); 52: 53– 57. 12. Tomizawa, M., Casida, J.E.; Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors; British Journal of Pharmacology, (1999); 127: 115– 122. 13. Sur, R., Stork, A.; Uptake, translocation and metabolism of imidacloprid in plants; Bulletin of Insectology, (2003); 56: 35– 40. 14. Bai, D., Lummis, S.C.R., Leight, W., Brer, H., Sattelle, D.B.; Actions of imidacloprid and related nitromethylene on cholinergic receptors of an identified insect motor neurone; Pesticide Science, (1991); 33: 197– 204. 15. Schmuck, R., Nauen, R., Ebbinghaus Kintscher, U.; Effects of imidacloprid and common plant metabolites of imidacloprid in the honeybee: toxicological and biochemical considerations; Bulletin of Insectology, (2003); 56: 27 –34. 16. Sarkar, M.A., Roy, S., Kole, R.K., Chowdhury, A.; Persistence and metabolism of imidacloprid in different soils of West Bengal; Pest Management Science, (2001); 57: 598–602. 17. Bonmatin, J.M., Moineau, I., Charvet, R., Fleche, C., Colin, M.E., Bengsch, E.R.; A LC/APCI-MS/MS method for analysis of imidacloprid in soils, in plants, and in pollens; Analytical Chemistry, (2003); 75: 2027– 2033. 18. Baig, S.A., Akhter, N.A., Ashfaq, M., Asi, M.R., Ashfaq, U.; Imidacloprid residues in vegetables, soil and wa´ter in the southern Punjab, Pakistan; Journal of Agricultural Technology, (2012); 8: 903– 916. 19. Anastassiades, M., Lehotay, S.J.; Fast and easy multiresidue method employing acetonitrile extraction/partitioning and ‘dispersive solidphase extraction’ for the determination of pesticide residues in produce; Journal of AOAC International, (2003); 86: 412– 431. 20. SANCO. Safety of the food chain: chemicals, contaminants, pesticides. European Commission Health and Consumer Protection Directorate-General, (2013). Sanco/12571/2013. 19 November 2013, rev.0. 21. Francotte, E., Davatz, A., Richert, P.; Development and validation of chiral high-performance liquid chromatographic methods for the quantification of valsartan and of the tosylate of valinebenzyl ester; Journal of Chromatography B: Biomedical Sciences and Applications, (1996); 686: 77 –83. 22. Roger, C.; Validation of chromatographic methods in biomedical analysis viewpoint and discussion; Journal of Chromatography B: Biomedical Sciences and Applications, (1997); 689: 175– 180.