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Research Detail

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M.D.H. Prodhan*
Pesticide Analytical Laboratory, Entomology Division, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh

S.N. Alam
Pesticide Analytical Laboratory, Entomology Division, Bangladesh Agricultural Research Institute, Gazipur, Bangladesh

Determination of organochlorine pesticide residues in shrimp is very important to ensure the consumer’s safety and to fulfill the importer’s demand. Therefore, a simple and efficient multiple organochlorine pesticide residues analytical method using quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction technique and Gas Chromatography coupled with Electron Capture Detector (ECD) has been developed and validated for the determination of 19 organochlorine pesticides (α- BHC,   δ- BHC, β- BHC, γ- BHC, Heptachlor, Aldrin, Heptachlor Epoxide,  γ- Chlordane, α- Chlordane, α-  Endosulfan, 4,4 DDE, Dieldrin, Endrin, 4,4 DDD, β- Endosulfan, 4,4 DDT, Endosulfan sulphate, Methoxychlor, and Endrin Ketone) in shrimp. The method was validated by evaluating the accuracy, precision, linearity, limit of detection (LOD) and limit of quantification (LOQ). The average recoveries of the selected pesticides ranged from 84% to 106% with RSDr ≤ 14% in four fortification levels of 0.05, 0.1, 0.2 and 0.3 mg kg-1. The linearity was ≥ 0.996 for all of the selected pesticides with matrix matched calibration standards. The LOD ranged from 0.003 to 0.009 mg kg-1 and the LOQ was 0.05 mg kg-1. This method was applied successfully for the residue analysis of 40 shrimp samples collected from different regions in Bangladesh.

  Shrimp, Organochlorine pesticide residues, Modified QuEChERS extraction, GC-ECD, Method validation
  
  
  
  Animal Health and Management
  Pesticide, Shrimp

The present study was initiated to develop and validate an analytical method for the determination of 19 organochlorine pesticide residues in shrimp and to monitor OCPs residues status in shrimp in Bangladesh.

Chemicals and reagents  Reference standards of Organochlorine Pesicide Mix (α- BHC, δ- BHC, β- BHC, γ- BHC, Heptachlor, Aldrin, Heptachlor Epoxide, γ- Chlordane, α- Chlordane, α-  Endosulfan, 4,4 DDE, Dieldrin, Endrin, 4,4 DDD, β- Endosulfan, 4,4 DDT, Endosulfan sulphate, Methoxychlor, and Endrin Ketone) were obtained from SIGMA-Aldrich, Germany through SF Scientific, Dhaka, Bangladesh. Analytical grade Acetonitrile (MeCN), methanol, Sodium chloride (NaCl), anhydrous magnesium sulphate (MgSO4) and Primary Secondary Amine (PSA) were also obtained from SIGMA-Aldrich, Germany through SF Scientific, Dhaka, Bangladesh. Sample preparation procedures  The quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction technique, which was first introduced by Anastassiades et al. (2003), is widely used for the extraction and cleanup of food matrices. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method modified by Prodhan MDH et al. (2015) was used for the extraction and clean-up of organochlorine pesticide residues from shrimp matrix. The method is described below: Ten gm of properly homogenized eggplant sample was taken in a 50ml screw-capped polypropylene centrifuge tube and 10 ml acetonitrile (MeCN) was added into the centrifuge tube. The centrifuge tube was closed properly and shaken vigorously for 30 sec. by vortex mixer. Then 4g anhydrous MgSO4, 1g NaCl were added into the centrifuge tube and it was shaken by vortex mixer for 1 minute. Afterwards, the extract was centrifuged for 5 min at 5000 rpm. An aliquot of 3 ml of the MeCN  layer was transferred  into a 15 ml micro centrifuge tube  containing 600 mg anhydrous MgSO4 and 120 mg Primary Secondary Amine (PSA). The content of the centrifuge tube was thoroughly mixed by vortex for 30 sec. and centrifuged for 5 minutes at 4000 rpm. After centrifuge, a 1 ml supernatant was filtered by a 0.2 µm PTFE filter, and then it was taken in a clean HPLC vial for injection. Preparation of pesticide standard solution Mixed pesticide standard stock solutions of α- BHC, δ- BHC, β- BHC, γ- BHC, Heptachlor, Aldrin, Heptachlor Epoxide, γ- Chlordane, α- Chlordane, α-  Endosulfan, 4,4 DDE, Dieldrin, Endrin, 4,4 DDD, β- Endosulfan, 4,4 DDT, Endosulfan sulphate, Methoxychlor, and Endrin Ketone were prepared in hexane: toluene (50:50) at a concentration of 200 mg l-1 and stored at -200C until use. An intermediate mixed standard solution of 10 mg l-1 in acetone was prepared from the mixed standard solution of 200 mg l-1. Then working standard solutions of 0.5, 1.0, 2.0, 3.0, and 5.0 mg l-1 in acetonitrile were prepared by transferring the appropriate amount from 10 mg l-1 intermediate mixed standard solution into five separate 5-ml volumetric flasks. Preparation of matrix matched calibration standard solution Matrix matched calibration standards were prepared by adding 100 µl of the mixed pesticide standards working solutions of 0.5, 1.0, 2.0, 3.0, and 5.0 mg l-1 and 900 µl of the blank extract to reach the final concentrations of 0.05, 0.10, 0.2, 0.3 and 0.5 mg l-1, respectively. Calibration standards in acetonitrile having the same concentrations as in the matrix matched calibration standards were also prepared. All the standard solutions were kept in a freezer at -200C until use. A typical chromatogram containing 19 organochlorine pesticides prepared with matrixmatched calibration standard. Operating condition of GC A Gas Chromatograph (GC-2010 Shimadzu) coupled with Electron Capture detector (GC-ECD) was used for the identification and quantification of selected organochlorine pesticides. Separations were done by RTX-CL capillary column (30 m long, 0.25 mm i.d. and 0.25 μm film thicknesses), nitrogen was used as carrier (column flow 1.5 ml/min.) and make up gas as well. The injector and detector temperatures were set to 250 °C and 330 °C, respectively and the column oven temperature was programmed, which was started from 180 °C and went up to 220°C with incremental rate of 5 °C (12 min hold), then it raised to 260°C with incremental rate of 5°C. All the injections (1 μl) were done in spit mode. The total run time was 28 min. Identification of the analyte in the samples was done by comparing the retention time of the corresponding matrix matched calibration standard and quantification was done by external calibration curves maid with 5 point matrix matched calibration standard.

  SAARC J. Agri., 16(1): 81-93 (2018)
  DOI: http://dx.doi.org/10.3329/sja.v16i1.37425
Funding Source:
1.   Budget:  
  

The described method in this study is an efficient and effective multi-residue analytical method using GC-ECD for the determination of 19 organochlorine pesticide residues in shrimp. A very good accuracy and precision was found for all analysts using this proposed method. The average recoveries ranged from 84 to 106% with RSD r ≤ 14% and RSDR ≤5%, thus fulfilling the requirement set by SANTE document number SANTE/11945/2015 for accuracy and precision (European commission 2015). Ninteen organochlorine pesticides were incorporated in this method that helps the scientist/ analysts for quick determination of multiple pesticide residues in shrimp. In addition, this analytical method was applied successfully to monitor the selected organochlorine pesticide residues in shrimp in Bangladesh. Thus the proposed method can be used successfully to monitor multiple organochlorine pesticide residues in shrimp.

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