The standard for dimethoate and quinalphos were obtained from Sigma-Aldrich Laborchemikalien, Gmbh P O Box-100262 D-30918, Seelze, Germany via Bangladesh Scientific Pvt. ltd. Dhaka, Bangladesh. Standards of both the insecticides contained 99.6% purity. Marketable size of cauliflower, hyacinth bean and eggplant were collected from supervised field trials at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 days after spray (DAS) which (dimethoate and quinalphos) were sprayed with @ 2 ml/L of water. The formulated product of dimethoate was Tafgor 40EC and quinalphos was Kinalux 25EC. The purity of formulated insecticides were tested in the pesticide analytical laboratory and found to be 100% pure.
Extraction, separation and cleanup:
Field collected samples (>250 g) were grounded thoroughly with the meat grinder (Handmixer M-122, Bamix, Switzerland). A sub-sample of 20 g was taken into a wide mouth jar then 100 ml of hexane was added to it. Sodium sulphate (Na2SO4) was also added with sample until water was removed from the sample. The mixture was then macerated with high-speed homogenizer (Ultraturax, IKA T18 basic, Germany) for 2 minutes. The homogenized material was then poured into 250 ml conical flask and placed into the shaker (Orbital Shaking Incubator, Rexmed, Sweden) for 12 hrs continuous shaking. After shaking, the slurry was filtered through a Buchner funnel with suction. The flask and filter cakes were rinsed with 25 ml of hexane each. The filtrate was then transferred into 250 ml round bottom flask and was dried to 5 ml by evaporation using a rotary vacuum evaporator (Laborota-4001, Heidolph, Germany). The concentrated filtrate was then transferred into 500 ml separatory funnel making 10 ml in volume. Around 20 ml methanol was added with 10 ml filtrate and shaked vigorously for 5 minutes. After shaking, the separatory funnel was set on stand and kept undisturbed for 5 minutes. Then the clear part of the solution from the bottom of the separatory funnel was collected in a vial which was then centrifuged at 1200 rpm for 5 minutes (Laboratory Centrifuges, Sigma-3K30, Germany). After centrifuge, supernatant was collected for injection in Gas Chromatography.
Operating condition of GC-FTD:
The concentrated extracts were subjected to analysis by Gas Chromatography (GC-2010 Shimadzu). For Organophosphorus insecticide (dimethoate and quinalphos), FTD (Flame Thermionized Detector) was used. Separation was done by ATTM-1 capillary column (30 m length, 0.25 mm inner diameter and 0.25 μm film thickness). Helium was used as carrier and makeup gas. The injection temperature was 2600C and detector temperature was 2800C, respectively and the column oven temperature was programmed. The column oven initial temperature was 1600C which went upto 2400C following 8 min incremental time. The current was 0.5 pA, Makeup flow was 30 ml/min, H2 Flow was 1.5 mL/min and Air flow was 145 ml/min, respectively. All the injections (2 μL) were done in split mode. The total run time was 12 min. Identification of the analyte in the sample was done by compering the retention time of the corresponding calibration standard and quantification was done by external calibration curves maid with 5 point calibration standard.
Previous to the injection of the sample extract, standard solutions of different concentrations of both pesticide groups were prepared and injected with the above instrument parameters. The samples were calibrated (retention time, peak area etc.) against three to four pointed calibration curves of a standard solution of the concerned pesticide. Each peak was characterized by its retention time. Sample results were expressed in mg/kg automatically by the GC software which represented the concentration of the final volume injected.
Determination of pre-harvest interval:
The amount of residues in all of the collected samples for both the pesticide and every vegetable were calculated following the described procedures. Then the sampling day which was next following MRL was selected. That selected day was chosen as PHI, since the level of residue on that day was below MRL.