Semipreparative enantioseparation of methamidophos by HPLC-UV and preliminary in vitro study of butyrylcholinesterase inhibition

Abstract

Many chiral pesticides are introduced into the environment as racemates, although their pesticidal activity is usually the result of preferential reactivity of only one enantiomer, while the other enantiomer may have toxic effects against nontarget organisms. Methamidophos (O,S-dimethyl phosphoramidothioate), a chiral compound, is an insecticide widely used in agriculture in both developed and developing countries. However, this pesticide has a high toxicity not only to targeted insects but also to human and animals. In the present study, the enantiomers of methamidophos were enantiomerically separated by a semipreparative chiral liquid chromatography at the multimilligram scale on a polysaccharide-based chiral stationary phase and a preliminary evaluation of their in vitro inhibition of plasma butyrylcholinesterase (BChE) of hens was performed. In the present study, our first effort was to resolve the racemic mixture of methamidophos and to that end reversed-phase, normal-phase, and polar organic elution conditions were investigated in four different polysaccharide-based chiral phases. The best performance was achieved on a cellulose tris(3,5-dimethylphenylcarbamate) phase under normal phase. This chromatographic condition allowed the separation of 225 mg of methamidophos enantiomers with a high degree of chiral purity (>98%) in a short analysis time. Significant differences were found between the concentration that causes 50% of enzyme inhibition (IC50) of the three isoforms of methamidophos. (-)-Methamidophos showed an IC50 approximately three times larger than the (+)-enantiomer for plasma BChE of hens.

Comparative in vitro study of the inhibition of human and hen esterases by methamidophos enantiomers

“…Sodium dodecyl sulfate (SDS), paraoxon, bovine serum albumin (BSA), Coomassie Brilliant Blue G-250, Histopaque-1077, tris(hydroxymethyl) aminomethane, ethylenediaminetetraacetic acid (EDTA), phosphoric acid 85%, acetylthiocholine (ACTh) and 5,5 -dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma, St. Louis, MO, USA; mipafox and phenyl valerate were obtained from Oryza Laboratories, Inc., Chelmsford, MA, USA; sodium citrate and triton X-100 were purchased from Rhiedel-de Haën, Hannover, Germany; 4-aminoantipyrine, potassium ferricyanide, and dimethylformamide were purchased from Merck, Darmstadt, Germany; heparin 25,000 IU/5 ml was obtained from Roche, Rio de Janeiro, Brazil; Deltametrin (K-otrine ® ) was obtained from Bayer Cropscience Ltd., Rio de Janeiro, RJ, Brazil; and piperazine citrate (Proverme ® ) was purchased from Tortuga Agrarian Zootechnical Company, São Paulo, Brazil. The analytical standard (±)-methamidophos was obtained from Sigma, St. Louis, MO, USA, and the enantiomeric separation was conducted according to the method described by Emerick et al (2011). The enantiomers of methamidophos were obtained with 99.5% of optical purity for the (+)-methamidophos and 98.3% of optical purity for the (−)-methamidophos.…”

Occupational Evaluation of Federal Highway Police Officers Exposed to Cholinesterase Inhibiting Insecticides

“…Acetylcholinesterase in erythrocytes and BChE in plasma sample were analyzed according to Ellman et al 20 with some modifications, as described elsewhere. 20,[22][23][24][25][26][27] In short, 1875 μL of 0.1 M phosphate buffer (pH 8.0), 75 μL of DTNB (10.3 mM prepared in 0.1 M phosphate buffer pH 7.0), 225 μL of plasma (diluted in water 1/142) or erythrocytes samples (diluted in water 1/600), and 225 μL of acetylthiocholine (10.7 mM prepared in 0.1 M phosphate buffer, pH 8.0) were added to the cuvette. Afterward, the kinetic program of the UV/VIS spectrophotometer was started with four absorbance readings, at 450 nm, with 1 minute of interval between each one.…”