Development and Validation of Analytical Method for Determination of Imidacloprid Residues in/on Soil and Water by HPLC

By Kamlesh Vishwakarma¹, Prakash Samnani², and S. Y. Panday²
November 2008

  1. Department of Chemistry, Jai Research Foundation, Vapi, Valvada - 396 108 (Gujarat), India
  2. Department of Chemistry, Faculty of Science, Maharaja Sayajirao University of Baroda, Vadodara - 390 002 (Gujarat), India
Abstract
A rapid, sensitive and reliable HPLC method was developed for the determination of imidacloprid residue in different pH of water and soil. The Validation of analytical method for determination of imidacloprid residues by HPLC. The method consisted of extracting with methanol from soil. The extracts were subjected onto the column filled with florisil column chromatography. The extraction of imidacloprid residues from water with ethyl acetate. Quantification is performed by reversed phase HPLC with UV detector. The limit of quantitation (LOQ) was mg/kg 0.02 in soil and 0.02 mg/L in water. The limit of detection (LOD) was 0.01 mg/kg in soil and 0.01mg/L in water. Recoveries for imidacloprid were 95.18, 94.66, 95.27 and 94.78 % in black, red, sandy loam and clay soil respectively. Recoveries for imidacloprid were 96.86, 86.14, and 92.34 % in pH4, pH7, and pH9 respectively. The detector linearity and the repeatability of the method proved to be very precise.

Key words: High-performance liquid chromatography (HPLC), Imidacloprid residues, soil, water

1 Introduction

The environmental fate for imidacloprid under laboratory conditions and potential effect on biota. Imidacloprid is a chloronicotinyl nitroguanidine insecticides, the IUPAC name is 1-[(6-chloropyridin-3-yl) –n-nitro-4, 5-dihydroimidazol-2-amine. Imidacloprid is a systemic pesticide with physical/chemical properties that allow residues to move into treated plants and then throughout the plant via xylem transport (between leaf surfaces) movement (Buchholz and Nauen, et al. 2002). Imidacloprid is a systemic chloronicotinyl insecticide that enters the target pest via ingestion or direct contact. It acts by disrupting nicotinic acetylcholine receptors in the insect central nervous system. Imidacloprid is used for controlling sucking insects, soil insects, termites, and some chewing insects. It is applied to seeds, soil, crops, and structures, and is used as a topical flea control treatment on domestic pets. Residues of the insecticide then enter the target pest by ingestion or direct contact, disrupting the insect’s nervous system by binding to postsynaptic nicotinic acetylcholine receptors. The disruption of the nervous systems results in modified feeding behavior, paralysis, and subsequent death of the insect (Mullins, et al. 1993).

It has a new mode of action, outstanding biological efficacy, abroad spectrum of activities, low toxicity to warm blooded animals and good plant compatibility. Because of its excellent systemic properties imidacloprid is used as a seed dressing as well as for foliar, soil and stem treatment (Ishaaya and Degheele, 1998). Imidacloprid is rapidly moved through plant tissues after applications, and can be present in detectable concentrations in tissues such as leaves, vascular fluids, and pollen. Many non-target beneficial arthropods such as honeybees, parasitic wasps, and predaceous ground beetles are sensitive to imidacloprid. These organisms may be adversely affected by sub lethal doses of the insecticide, but the effects vary widely depending on application method and route of intake.

Imidacloprid is readily translocated through plant tissues following direct contact. When used as a seed treatment, the insecticide is absorbed by the seedling from the disintegrating seed coat. In a French study, sunflower plants that were seed-treated at a rate of 1.0 mg/seed produced pollen that contained imidacloprid at a concentration of 13.0 ppb (Laurent and Rathahao, 2003). Determination of imidacloprid residue method (Placke-FJ et.al.-l) has also reported (parent compound residue and total residue) in crops is described. For determination of the total residue, the parent compound and all metabolites containing the 6-chloropicolyl moiety are detected as 6-chloronicotinic acid.

Metabolism study of imidacloprid in plants also reported (Rechard Schmuck et al) Honeybees foraging in crop plants seed-dressed with imidacloprid may be exposed to imidacloprid and imidacloprid plant metabolites. Metabolism studies on a large variety of crop plants were reviewed to identify plant metabolites which have a potential toxicological relevance to honeybees. Three different bioassays were conducted to characterize the pharmacological and toxicological profile of imidacloprid and these potentially relevant plant metabolites in the honeybee. The nicotinic acetylcholine receptor (nAChR) was identified as the molecular target of [3H] imidacloprid and some of the tested.

2 Material and Methods

Analytical reference standards of imidacloprid (98.5% purity) obtained from M/s United Phosphorous Limited Mumbai, India. All the other chemical and solvents were used in the study analytical and HPLC grade.

2.1 Fortification of Samples

A representative samples 50g different soil samples viz. Black, red, sandy loam and clay soil were transferred in 250ml Erlenmeyer flask. The soil samples were fortified with imidacloprid at two different fortification LOQ and 10XLOQ Levels, separately. A Volume of 0.5 and 5.00 ml imidacloprid was transferred to each conical flask for 0.02 and 0.20 ppm fortification levels. The control samples were processed similarly after 0.5 and 5.00ml methanol was added.

2.2 Extraction and Sample Clean-Up

A volume of 100 ml methanol was added into the Erlenmeyer flask at different fortified soil samples were placed onto orbital shaker for 30 minutes. After shaking, the solutions were filtered into the round bottom flask of 500 ml capacity through whatman filter paper No. 1. The residual cake was extracted twice. The methanol extract were collected, pooled and concentrated to dryness using vacuum evaporator at 40°C. The residue re-dissolves in methanol. The concentrated soil samples were cleaned by column chromatography. A glass column was packed with adsorbent in between two layer of anhydrous sodium sulphate. the cleanup of soil samples process florisil was used as adsorbent. Column was pre- conditioned with methanol and concentrated extract were loaded onto top of the column and eluted with 100ml acetonitrile. Eluate were concentrated a using rotary vacuum evaporator and residue re-dissolve in 5ml acetonitrile and final volume was made upto the mark with acetonitrile (10ml).

3 Extraction of Water Samples

The fortified water samples were extracted with ethyl acetate. After extraction the samples are filtered and evaporated to the dryness using a rotary vacuum evaporator. The residue re-dissolves in 5ml acetonitrile and final volume was made upto the mark with acetonitrile (10ml).The analysis of imidacloprid in soil and water using reverse phase HPLC technique was used for quantitative analysis. A Shimadzu LC-2010 AHT with LC-solution softer ware, C-18 column, Phenomenex, 25cm length x 4.6 mm i.d. and 0.5mu particle size, Mobile phase A: 0.01% (v/v) acetic acid in water(60). Add 0.1 mL acetic acid and dilute to 1 litre with water. Mobile phase B: acetonitrile (40) at 1ml flow rate and detector set a 252 nm λmax was used for analysis imidacloprid statdard showed sharp peak at 4.93 minute under the described HPLC condition.

4 Results and Discussion

The linearity of the detector response was tested for imidacloprid, in solvent and in Matrix over the range of 0.02 to 5.00 mg/kg. A very precise linear relation between the injected amount and the resulting peak area was observed over the entire range with correlation coefficients between 0.999. (Ishii-Y et. al) has also reported HPLC method developed for the determination of imidacloprid residue in 9 kinds of crops and soil. The method consisted of extracting with acetonitrile/water (80:20 v/v), prewashing of the concentrated extracts with cyclohexane and alkaline solution, silica gel column chromatography, and finally reversed-phase HPLC. The recoveries of imidacloprid were 75-109%. The limits of determination of the method were 0.005, 0.01 and 0.02 mg/kg for crops, rice straw and soil, respectively. The accuracy and precision of the method was evaluated on the basis of the recoveries obtained for fortified samples soil and water. The limit of quantitation (LOQ) was 0.02 mg/kg for imidacloprid in soil and 0.02 mg/L in water. The limit of detection (LOD) was 0.01 mg/kg for imidacloprid in soil and 0.01 mg/L in water. Recoveries for imidacloprid were 95.18, 94.66, 95.27 and 94.78 % in balck soil red soil sandy loam and clay soil. The recoveries for imidacloprid were 96.86, 96.14 and 92.34 % in water pH4, pH7 and pH9. The method for imidacloprid recovery was also reported (Ralf et al). The repeatability of the method was determined for each analyte by running a set of five recoveries each at two different fortification levels for selected matrices. The resulting mean recovery rates ranged from 79 to104% with relative standard deviations between 0.8 and 15.3%. These data demonstrate the excellent sensitivity, selectivity and precision of the method. Similar results were found in present method validation for different soil and waters. The repeatability of the method was determined for each fortification levels by running a set of five recoveries each different fortification levels for selected matrices. The % RSD was the resulting mean recovery rates ranged from 94.66 to 95.27% in soil with relative standard deviations between 1.21 to 3.37%.

The % RSD was the resulting mean recovery rates ranged from 92.34 to 96.86% in water with relative standard deviations between 1.66 and 3.23%.These data demonstrate the excellent sensitivity, selectivity and precision of the method.

Table 1 · Accuracy (% Recovery) and Precision (%RSD) of Imidacloprid in Soil
Substrates Fortification of LOQ and 10× LOQ levels in mg/kg %Recovery Mean %Recovery SD %RSD
Black soil 0.02 97.20 95.18 1.18 1.21
0.20 93.16 2.24 2.40
Red soil 0.02 97.25 94.66 1.86 1.91
0.20 92.07 3.10 3.37
Sandy loam soil 0.02 97.00 95.27 1.97 2.03
0.20 93.54 3.01 3.22
Clay soil 0.02 96.35 94.78 1.31 1.36
0.20 94.60 2.23 2.36
Table 2 · Accuracy (% Recovery) and Precision (%RSD) of Imidacloprid in Water
Substrates Fortification of LOQ and 10× LOQ levels in mg/kg %Recovery Mean %Recovery SD %RSD
Water pH4 0.02 97.60 96.86 1.63 1.67
0.20 96.11 2.21 2.30
Water pH7 0.02 96.30 96.14 1.60 1.66
0.20 95.98 3.10 3.23
Water pH9 0.02 95.00 92.34 1.25 1.31
0.20 89.67 2.34 2.61

Fig 1 · Linear Dynamic Range Data of Imidacloprid
Fig 1

Data for Linearity Determination of Imidacloprid in Solvent
Concentration (mg/L) Peak Area Mean Peak Area % Variation
0.02 649 645 647.00 0.62
0.05 1628 1629 1628.50 0.06
0.25 8174 8170 8172.00 0.05
1.00 32541 32505 32523.00 0.11
5.00 165310 165493 165401.50 0.11
Regression parameters Imidacloprid standard in solvent
Slop: (b)33096.66
Y-axis intercept: (a)-159.78
Correlation coefficient: (r)0.999

References

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