Sample Collection Fresh okra fruit samples were collected from different local markets of Dhaka city and transferred to the laboratory for the analysis. The samples were chopped and thoroughly blended to obtain a homogeneous representative sample and stored in a refrigerator at -20°C for further analysis.
Chemicals and Reagents Analytical grade solvents purchased from Merck, Germany such as extra pure n-hexane, ethyl acetate and DCM. H2SO4 (98%, w/w, BDH, U.K), phenol (Merck, Mumbai, India), methanol (99.5%, w/w, Sigma Aldrich), ethanol (99.8% w/v, Sigma Aldrich), NaOH (BDH, U.K) and BF3-CH3OH complex (Merck, Germany) were used during the work. Anhydrous MgSO4, anhydrous Na2SO4 and NaCl of Merck, Germany; florisil from ACROS Organics, USA; Alumina from Merck, Germany; Charcoal from Uni-Chem, China were used. Analytical grade diazinon (97.5% purity), quinalphos (98.4%), chlorpyrifos (99.5% purity), cypermethrin (91.9% purity) and fenvalerate (98.5% purity) purchased from Dr. Ehrenstorfer, Germany were used as pesticides standard.
Instruments The experiment was carried out using oven and furnace (GSM 11/8 Hope Valley, S336RB, England), Analytical balance (model- AL 104, Company- Mettler Toledo, US), Double beam UV spectrophotometer (Model: UV-1800, Shimadzu), Gas chromatograph with a flame ionization detector (GC-FID, Shimadzu-2025), Gas chromatograph with electron capture detector (GC-ECD, Shimadzu-2030), pH meter (Model: Hanna pH 211) and Vortex machine (Cat/Art No 444-1372 (EU), Made in Germany) and Zeeman atomic absorption spectroscopy (GTA 120_AA240Z with PSD auto sampler, Varian, Australia).
Preparation of Pesticides Standard Solutions Primary standard solutions of diazinon, chlorpyrifos, fenvalerate, cypermethrin and quinalphos were prepared separately in 100 mL volumetric flask with n-hexane. For preparation of 100 ppm standard solution 10 mg of each standard compound was taken in 100 mL separate volumetric flask and dissolved in n-hexane and up to marked. After serial dilution the working standard solutions of each of the standards were run into GC-ECD and calibration curves were made.
Extraction and Estimation of Pesticides 10 g homogenized sample and 20 mL ethyl acetate were taken in a 50 mL Teflon tube and vortex for 2 min. 6 g MgSO4 and 1.5 g NaCl were added which then vortex and centrifuged. The supernatant was evaporated in a rotary evaporator. The dried mass was dissolved in 5 ml n-hexane and evaporated to complete dryness. This procedure was repeated for two times. Finally the dried mass was reconstituted in 2 mL of n-hexane. For additional clean-up, a glass column (40 cm long & 12 mm internal diameter) was packed with 10.5 g mixture of alumina, florisil and charcoal (10:10:1) with n-hexane. Then 5 g of Na2SO4 was added to the column and equilibrated with 40 mL n-hexane. The extract in n-hexane was transferred to the equilibrated column and the column was washed with 20 mL of nhexane and eluted with 100 mL of DCM and collected in a round bottom flask. The eluent was concentrated to dryness. The dried mass was dissolved in 5 mL n-hexane and evaporated to complete dryness. This procedure was repeated for two times. Finally the dried mass was reconstituted in 4 mL of n-hexane and transferred to a GC vial. GC-ECD was used for the determination of pesticide residues. A capillary column of 30 m length, 0.25 mm inner diameter, 35mm external diameter and 0.25 μm film thicknesses from Agilent, USA was used. Injector and detector temperature was set at 220 and 290° C, respectively. Column flow rate was 1.0 mL/min, Nitrogen gas was used as career and make up gas, respectively. Injection volume was 1.0 μL, split-splitless mode was set and total program time was 20 min.
Extraction and Estimation of Fatty Acids Sample (10 g) and 30 mL n-hexane were taken in round bottom flask and refluxed in boiling water bath for about an hour and the extract was collected. The procedure was repeated for three times. The extracts were combined and filtered through anhydrous Na2SO4. The filtrate was dried to a constant weight of oil. The oil from each sample was taken in a round bottom flask and 1mL of alcoholic 0.5M NaOH was added to it. Then the flask was shaken well and was kept in hot water bath (95°C) in order to reflux for 30 min. The aqueous layer was acidified with dilute HCl and partitioned with n-hexane. The organic phase was collected and evaporated by rotary evaporator until dryness. 1mL BF3- CH3OH complex was added to the dry mass and was sonicated for 30 sec. Later the sample was heated in hot water bath for 30 minutes and the sample was again dried by rotary evaporator and then 2 mL of n-hexane was added to the flask and was ultrasonicated. The volume was reduced to 0.5 mL and the sample was collected in a sample vial. Then the sample was injected to GC-FID. In GC-FID, separations were performed on HP-5 column (30 m in length, 0.25 mm in diameter and film thickness 0.25 μm). The temperature program in the oven was as followed: 140oC for 1 min (hold) was gradually increased by 7oC/min to 270oC and was again hold for 10 min. Column flow rate of carrier gas (N2) was 2 mL/min. As a fuel for FID, air and nitrogen gases were used. Injector and detector temperature was 280 and 290oC, respectively. Injection volume was 1 μL. The mixtures of standard methyl ester of fatty acids were found to be gave chromatogram with separate peaks in different retention time. Sample was injected into the injector of GC at the same condition, as methyl ester of fatty acid standard and retention time of each fatty acid was compared.
Estimation of Soluble Dietary Fiber The extraction of soluble dietary fiber (SDF) from okra fruit powder was carried out by conventional acid extraction method. Okra fruit powder (10 g) was taken in a flask and 200mL distilled water was added to it. pH of the water adjusted to 2.0 with sulfuric acid. The mixture was stirred for 4 hours at 90oC in a water bath and filtered. The filtrate was concentrated and ethanol was added to a final concentration of 70% and precipitate was obtained. The ethanol precipitation process lasted for 1.0 hours and SDF was collected by centrifugation and dried at room temperature.