Emamectin benzoate

The certified reference materials of acephate, chlorpyrifos, dimethoate, emamectin benzoate, imidacloprid, monocrotophos, phenthoate, phorate, profenofos, quinalphos, and triphenyl phosphate (TPP, internal standard) were procured from Sigma-Aldrich Chem. Pvt. Ltd., India, based on the information collected on the types of pesticides used by the farmworkers. The analytical-grade formic acid was procured from Fluka Pvt. Ltd., India, and the other organic solvents, acetonitrile and methanol (pestanal-grade), were obtained from Sigma Aldrich, Merck KGaA Darmstadt, Germany. Salts such as sodium chloride (anhydrous) and sodium sulfate and the analytical-grade reagent ethanol were obtained from Merck, India, and the HPLC column was procured from Agilent Technologies Pvt. Ltd., India. Water was purified with a Millipore Direct Q purification system (Lab Link, Germany).

Emamectin benzoate (98% purity, EMB) was purchased from Sigma-Aldrich Canada. The following were obtained from Sigma–Aldrich (St. Louis, MO, USA): dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS, pH 7.4), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide(MTT), 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA), phenylmethanesulfonyl fluoride (PMSF), bovine serum albumin (BSA), Rhodamine123 (Rh-123), RIPA lysis buffer. All antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).

Emamectin Benzoate (EB) (~93.4%B1a, CAS: 155569-91-8, 98%), 7-benzyloxy-4-trifluoromethyl coumarin (BFC) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich company (St. Louis, Missouri, USA). Abamectin (B1a, CAS: 65195-55-3, 97%) was purchased from Toronto Research Chemicals (North York, Canada). Solvents for LC-MS (ammonium acetate, formic acid and acetonitrile) were obtained from Fisher Scientific (Pittsburgh, Pennsylvania, USA).
A series of gradient concentrations of insecticides diluted from stock solution with 0.1% (w/v) Triton X-100 water were prepared. In each well of the 24-well plate, a liquid artificial diet (~120 μL) was supplied. One hundred microliters of the insecticide solution was added to the dietary surface of every single well after the diet cooled and solidified. A third-instar larva was placed in each well. For each concentration, 24 larvae were treated. Triton X-100 (0.1%, w/v) was employed to process the control group. Two days later, larvae were checked and considered dead if they did not move after gentle prodding. The data were analyzed using DPS 7.05 software (Zhejiang University, Hangzhou, China). When the 95% fiduciary limits of the LC₅₀ values did not overlap, the LC₅₀ values were considered to vary significantly.