Analysis of Emamectin Benzoate Residues in Cauliflower using HPLC

 

Aiyalu Rajasekaran*, Thangadurai Beneeta, Krishnadas Meeradas, Joy Thomas Sajini and Sandeep Narayanan

Department of Pharmaceutical Analysis, KMCH College of Pharmacy, Coimbatore, Tamilnadu, India.

*Corresponding Author E-mail: rsekaran2001in@yahoo.co.in

 

 

ABSTRACT:

Irrational use of pesticides in vegetables results in residue accumulation, which is a major health hazard which needs to be strictly monitored by Regulatory authorities. Emamectin benzoate is an insecticide which belongs to Avermectin, a family of structurally complex antibiotics produced by a strain of Streptomyces avermitilis. It is the most commonly used insecticides in vegetables like cauliflower, cabbage, tomato The aim of the study is to determine the amount of insecticide Emamectin benzoate present in cauliflower. Cauliflower sprayed with recommended dose and double recommended dose were analyzed for their residual contents. A rapid sensitive HPLC method has been developed and validated for the determination of emamectin benzoate residues in Cauliflower. Reverse phase HPLC system using acetonitrile: ammonium acetate buffer; (50:50) as mobile phase at a flow rate of 1.2 ml min-1 with UV detection at 254 nm was used for quantification of emamectin benzoate. Limit of detection of 2 ng/ml was obtained. Calibration curves that constructed for the analytes spiked into samples followed linear relationships with good correlation coefficients (R2>0.997). Method used permitted the determination of these pesticides in cauliflower at concentration level demanded (MRL) by food and agriculture board. In the analysis, from vegetables treated with emamectin benzoate at double of recommended doses the residual amounts above respective maximum residue limit (MRL) values were found.

 

KEYWORDS: Pesticide residues, emamectin benzoate, cauliflower, HPLC

 


INTRODUCTION:

Cauliflower is one of the several vegetables of Brassica oleracea belong to the family Brassicaceae. It is low in fat, high in dietary fiber contains water vitamin C, possessing nutritional density. The warm and humid climatic condition, increase use of modern high yielding varieties of crops and more use of chemical fertilizers are highly favourable for development and multiplication of pests and diseases. The most common pests that attack cauliflower are worms, aphids and flea beetles. In India, cauliflower cultivators have been using the pesticides frequently to high yield. Control of pests can be achieved by using biological controls such as Geraniums and Borage, the Bacillus thurengensis bacterium, predatory insects or chemical pesticides.One of the widely used pesticides for cauliflower in the Thondamuthur area of Coimbatore, Tamilnadu is emamectin benzoate (Fig.1), also known as, epimethylamino-4"-deoxyavermectin benzoate, is the 4”-deoxy-4”-methylamino benzoate salt derivative of abamectin, a 16-membered macrycyclic lactone produced by the strain of Streptomyces avermitilis.

 

It is chemically synthesized from the natural product mixture abamectin[1]. Its mechanism of action is by blocking interneuron-motor neuron transmission in nematodes by stimulating the release of the inhibitory neurotransmitter GABA [2,3]. It is a white or light yellow crystalline powder mainly used to prevent and control harmful insects like Lepidoptera, Coleoptera, Homoptera, and Mite in many kinds of vegetables, fruit trees, tea, tobacco and cotton.

 

Fig.1. Chemical structure of emamectin benzoate

The overdose of pesticides makes the residue problem, which might pollute our food and be harmful for our health. Several analytical methods have been reported for the analysis of emamectin benzoate in vegetables and fruits [4-10].  This paper describes a simple, rapid, precise, sensitive, accurate and validated HPLC method for quantification of emamectin benzoate residue in cauliflower.

 

MATERIALS AND METHODS:

Sample material:

Cauliflower grown in two different areas of Thondamuthur area in Coimbatore, Tamilnadu was selected before harvesting the samples. Cauliflower bearing plants sprayed with the recommended dose (1.5 L h-1 ) and double recommended dose (3.0 L h-1) were collected at the same day 6 h after spraying emamectin benzoate. Commercial samples of cauliflower were also collected from different markets (Singanallur, Coimbatore). The analysis conducted from March 2010 to May 2010.    

 

Chemicals and reagents

HPLC grade acetonitrile, ethyl acetate was purchased from E. Merck. Ultra high pure water obtained from Milli-Q water purification was used. Technical grade pesticide standard emamectin benzoate was obtained from Tamilnadu Agricultural University with a purity of 95-99%. The standard was stored at a temperature of -5ºC. All samples were filtered through Millipore membrane filter of 0.2 microns and solvents were filtered through 0.45 microns before injection into the column. Anhydrous sodium sulphate for residues analysis was maintained at 300ºC overnight.

 

Preparation of standard stock solution:

Standard stock solution were prepared by dissolving the standard emamectin benzoate in mobile phase (acetonitrile: ammonium acetate buffer (50:50)) to get a concentration of 10 µg/ml. From the stock solution series of dilutions of concentrations 25, 50, 75, 100, 125, 150 ng/ml were prepared and stored at 4ºC in the dark. These solutions were used for the preparation of calibration curve using the procedure described in section 2.4.2.

 

Extraction

Sample pretreatment

Edible part of cauliflower (untreated (standard), recommended dose treated, double dose treated and commercial sample) (75 g) were made into small pieces and homogenized by a kitchen blender and kept in a freezer by wrapping with airtight polythene bag at a temperature below -15ºC [11]. The above mixture was added to anhydrous sodium sulphate and mixed well.                      

 

Preparation of standard solution for calibration curve

Standard solutions of concentrations 25, 50, 75, 100, 125, 150 ng ml-1 were added to 2 g each of the untreated sample mass prepared (section 2.3.1) and mixed well. 5 ml of ethyl acetate was added to each of these masses and shaken well. Then it was passed through Whatman No.1 filter paper and the filtrate was collected. The ethyl acetate layer was separated and evaporated to dryness. 5 ml of mobile phase was added to each residue and mixed well, filtered again through membrane filter (0.2 micron) and the filtrate was injected into the chromatographic system. Calibration curve was plotted using the peak area generated against concentration of pesticides spiked.

 

Preparation of sample solutions

Cauliflower sprayed with emamectin benzoate (recommended dose and double dose) was collected from farm, commercial sample purchased from local market were washed and treated as in section 2.4.1. Five ml of ethyl acetate was added to each of these masses and shaken well. Then it was passed through Whatman No.1 filter paper and the filtrate was collected. The ethyl acetate layer was separated and evaporated to dryness. 5 ml of mobile phase was added to each residue and mixed well, filtered again through membrane filter (0.2 micron) and the filtrate was injected into the chromatographic system.  The amount of emamectin benzoate present in all the samples was calculated from regression equation.

 

HPLC system

HPLC having Diode Array Detector manufactured by Shimadzu Prominence was used for separation and quantification of pesticides. Separation was performed on reversed phase C18 column (5 µ, 25 cm x 4.6 mm I.D.) and protected by pre-column RP C18 (Phenomenex Inc, Torrace, CA, USA).  Samples were injected using a Rheodyne injector (772i). The working condition of HPLC was binary gradient, mobile phase was acetonitrile: ammonium acetate buffer; (50:50), flow rate 1.2 ml min-1, injection volume was 20 µl and the wavelength of the Diode Array Detector was fixed at 254 nm for the residual analysis of emamectin benzoate.

 

Identification and quantification

The retention time of the compound to be identified was compared with respect to the technical grade reference standard (Fig.2-4). A calibration curve was plotted from chromatographic experiment using standard solutions for quantitative determination (Table 2).

 

Recovery

The degree of efficiency of the extraction procedure employed was studied. Untreated cauliflowers were spiked with known concentration of the standard emamectin benzoate solution and extraction and clean- up were performed as mentioned above. The concentration of the pesticide in the final extract was calculated (Fig. 5).

 

Statistical analysis

External calibration and recovery tests were performed. The residue results were the means from three replicates of treatment and all data’s were calculated using regression equation.

 

RESULTS:

Selectivity

Under described chromatographic conditions, the retention time of emamectin benzoate standard benzoate was found to be 5.4 minutes (Fig.2).  No endogenous interfering peak appeared at the retention times of the compounds of interest.

 


 


 

Fig.2. Chromatogram of emamectin benzoate reference standard (50 ng/ml)

 

Fig.3. Chromatogram of sample sprayed with recommended dose of emamectin benzoate.

 

Linearity

The calibration curves were linear over the range of 8-128 ng mL-1. The correlation coefficient was 0.997. Limit of detection and Lower limit of Quantification The LOD was found to be 0.0022 mg kg-1 . The LOQ was 0.0029 mg kg-1 with coefficient of variation of 0.6 % and accuracy of 99.99 %. (Table 1) 

 

Table 1. Chromatographic performance of the developed HPLC method.

Parameters

Values

Retention time 

5.4

(min)

Theoretical plates  (N)

5039

HETP 

23.72

K’ factor 

3.7

R2    

   >0.997

LOD 

 0.0022 mg kg-1

LOQ

  0.0029 mg kg-1

Peak purity index

 

Fig.4. Chromatogram of sample sprayed with double the recommended dose of emamectin benzoate.

 

Fig.5. Chromatogram of the commercial sample showing peak for emamectin benzoate.

 

Precision and Accuracy

The Intra-day accuracy ranged between 99.99% and 100.05% with precision of 0.6% and 0.3%. The Inter-day accuracy ranged between 99.98% and 100.01% with precision of 0.50% and 0.78%.

 

Recovery (extraction efficiency) from plant mass

The extraction efficiency of emamectin benzoate at the concentrations of 25, 50 and 75 ng mL-1 was found to be 100.06%, 99.96% and 99.81% with precision of 0.46%, 0.23% and 0.14% respectively.

 

Stability

In laboratory studies, emamectin benzoate was found to be stable in aqueous hydrolysis (acid, neutral and alkaline) under dark conditions.

 

Ruggedness and robustness

The ruggedness and robustness of the methods were studied by changing the experimental conditions. No significant changes in the chromatographic parameters were observed when changing the experimental conditions (operators, instruments, source of reagents and column of similar type) and optimized conditions (pH, mobile phase ratio and flow rate).

 

System suitability

System suitability parameters such as column efficiency (theoretical plates), resolution factor and peak asymmetry factor of the optimized methods were found satisfactory.

 

Table 2. Amounts of residues detected in cauliflower samples treated with emamectin benzoate

Dose

MRL (ppm)

Residue found* mg kg-1

Recommended dose

0.5

0.064

Double of the Recommended dose

0.116

Commercial sample

   0.026-0.031

*average of 5 determinations

 

DISCUSSION:

Emamectin benzoate is rapidly degraded under sunlight and gives two products namely, 8a-oxo-4‘‘-deoxy-4‘‘-(epi-methylamino)avermectin B1a (MAB1a) benzoate and MAB1a-10,11-14,15-diepoxide with low residual content of undegraded parent compound [12]. The HPLC method was developed and validated and the emamectin benzoate content in cauliflower which was sprayed with recommended dose of emamectin benzoate was determined to be 0.064 mg kg-1, the sample which was sprayed in double that of recommended dose was found to be 0.116 mg kg-1, while commercial sample collected from the market contained between 0.026-0.031 mg kg-1. These results indicated that the analytical method is suitable to measure emamectin benzoate concentrations in food, fruits and vegetables.

 

CONCLUSION:

Cauliflower growers have been using the pesticides frequently to have the high yield. But the overdoses of pesticides make the residue problem, which might pollute our food and environment. Appropriate use of pesticides, in particular to the vegetables and other edibles to be addressed in India and other countries. Frequent and systematic monitoring programs to be implemented to control the use of pesticides in vegetables and fruits. In order to remove residual effect of pesticides which are toxic, the recommended dose of postcodes is to be informed well in advance to the farmers so that the residual pesticides in vegetables will be lower than the recommended dose. A simple and rapid method was provided to detect the concentrations of EM in parts per million levels which can be extended for the routine analysis of EM in vegetables.

 

REFERENCES:

1.     Volker Jungmann, István Molnár, Philip E. Hammer, D. Steven Hill, Ross Zirkle, Thomas G. Buckel, Dagmar Buckel, James M. Ligon, J. Paul Pachlatko, 2005. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes: Appl Environ Microbiol71(11) 6968-6976.

2.     Fanigliulo A, Sacchetti M., Emamectin benzoate, 2008. New Insecticide Against Helicoverpa armigera: Mass Spectrom. Rev, 73(3)651-3

3.     McKellar, Q.A., Benachaoui, H.A., 1996. Avermectins and milbemycins. J. Vet. Pharmacol. Ther. 19, 331– 351.

4.     M Kuster, M Lopez de Alda, D Barcelo, 2000, Analysis of pesticides in water by liquid chromatography-tandem mass spectrometric techniques: Mass Spectrom. Rev, 25(6)900 – 916.

5.     A Kaihara, Kimihiko Yoshii, Yukari Tsumura, Yumiko Nakamura, Susumu Ishimitsu, and Yasuhide Tonogai, 2002, Simultaneous Determination of Azimsulfuron, Flazasulfuron and Halosulfuron-methyl in Grains, Seeds, Vegetables and Fruits by HPLC: J.Health Sci, 46(5) 336–342.

6.     S Islam, Nazneen Afrin, Mohammad Shawkat Hossain, Nilufar Nahar, Mohammad Mosihuzzaman, Mohammad Iqbal Rouf Mamun, 2009. Analysis of some pesticide residues in cauliflower by high performance liquid chromatography: Am. J. Environ Sci, 5 (3): 325-329,

7.     C. M. Torres, Y. Picó and J. Mañes, 1996, Determination of pesticide residues in fruit and vegetables: J. Chromatogr. A,754(1-2) 301-331.

8.     Hirotaka Obana, Masahiro Okihashi, Kazuhiko Akutsu, Yoko Kitagawa, Shinjiro Hori,2002, Determination of Acetamiprid, Imidacloprid, and Nitenpyram Residues in Vegetables and Fruits by High-Performance Liquid Chromatography with Diode-Array Detection: J. Agric. Food Chem, 51 (9)2501–2505,

9.     Chai Mee Kin, Tan Guan Huat, Asha Kumari, Malays. 2006, Application of solid-phase microextraction for the determination of pesticides in vegetable samples by gas chromatography with an electron capture detector: J.Chem, 8(1) 067 – 071.

10.   Zhang Y, Wu Y, Hu J, Wang H, Pan C, Liu F, 1997, Determination of emamectin benzoate residue in vegetables by high performance liquid chromatography with fluorescence detection: Se Pu. 26(1):110-2.

11.   Amechi C. Chukwudebe, J. Agric. Immobility of Emamectin Benzoate in Soils :1996, Food Chem.,  45 (10)4137–4146.

12.   Mushtaq, M.; Chukwudebe, A.C.; Wizesinki, C.; Allen, L. R. S.;  Luffer-Atlas, D.; Arison, B.H., (1998). Photodegradation of emamectin benzoate in aqueous solution, J. Agric. Food Chem. 46,  1181–1191.

 

 

 

 

Received on 13.06.2013          Modified on 01.07.2013

Accepted on 07.07.2013         © RJPT All right reserved

Research J. Pharm. and Tech. 6(10): October 2013; Page 1157-1160