Development of Cost Effective HPLC Based Analytical Methods for Quantitative Determination of Selected Pesticide Formulations

Abstract

Many pesticides are carcinogenic chemical substances those have proved to be lethal for human. Therefore, the application of these chemicals should be controlled and claimed percentage of active content in pesticide formulations available in market should be maintained. In order to investigate presence of correct claimed percentage in all formulations, accurate, reliable and low cost methods are significantly required. Considering the importance of these methods, a research work was proposed to develop analytical method for simultaneous quantitative determination of active content in pesticide formulations. For all the studies, HPLC1-UV and HPLC2-UV were used. Seven methods were developed and validated by following ICH guidelines. In which dimethomorph and chlorothalonil; azoxystrobin, fludioxonil and metalaxyl-M; mesotrione, atrazine, benoxacor and s-metolachlor; bromoxynil heptanoate, bromoxynil octanoate and MCPA; clopyralid, tribenuron and fluroxypyr; imidacloprid; and fipronil, chlorfenapyr and pyriproxyfen as method 1, 2, 3, 4, 5, 6, 7 respectively were subjected to develop the simultaneous determination of mentioned pesticides. For optimization of each method, mobile phase, flow rate, stationary phase and wavelength were suitably selected. For all the methods except method 1, flow rate was 1.0 mL/min. For method 1: selected flow rate was 1.2 mL/min. For method 2 and 5: stationary phase was RP–18 end capped (5 μm, 4.6 mm x 25 cm) while, for rest of all methods, selected stationary phase was Beckman C-18 (5 μm, 4.6 mm x 15 cm). For all these methods, a mixture of acetonitrile and water was used as mobile phase. These mobile phase solvents were pumped on binary gradient mode with different ratio for different methods. The mobile phase composition (acetonitrile : water) for methods 1 and 7 was 80 : 20, for method 2; 50 : 50, for methods 3 and 6; 70 : 30 and for methods 4 and 5 was 85 :15. At the last, wavelengths were selected for each method for optimum chromatographic response of analytes. For which detection wavelength 230 nm, 220 nm, 212 nm and 270 nm; were selected for methods 1, 2, 5, 6, respectively and 260 nm for methods 3, 4 and 7. Finally, validation of all the developed methods was initiated following ICH guidelines implementing several parameters such as: system suitability test, linearity, LOD, LOQ, precision, accuracy and robustness. The system suitability test for all the developed methods were reported by investigating I asymmetry, capacity factor, and number of theoretical plates of all analytes for each method. Linearity of each developed method was determined by plotting calibration curve between chromatographic response and concentration of analyte. The correlation coefficient was found to be greater than 0.996 for all newly developed methods. LOD & LOQ were investigated based on standard deviation of chromatographic response and their respective slope from calibration curve data. For the validation of precision of methods, interday and intraday analysis of formulation were performed. The percent RSD were less than 2.0 for intraday analysis and less than 3.0 for interday analysis for all the methods. Accuracy for all the newly developed methods was checked by inter laboratory comparison (ILC) test among different laboratories. The Z-score values were calculated for each method in order to report the performance of all laboratories. The z-score values of all developed methods were exceptionally well in comparison with other participated laboratories. Robustness of the all methods were checked by making some deliberate changes in flow rate, mobile phase composition and wavelength for each newly developed method. No considerable change in response of analytes was observed upon above mentioned changes. This proved the robustness of all newly developed methods. Consequently, all the proposed methods can suitably be used for simultaneous quantitative determination of all studied pesticides in formulations for investigating active contents