Materials and reagents
Metsulfuron-methyl, chlorantraniliprole, chlorimuron-ethyl were obtained from Pharma Train, Hyderabad, India. KH2PO4 was obtained from Finer Chemical Ltd, Mumbai, India. Acetonitrile was from Molychem, India and HPLC grade methanol and orthophosphoric acid was sourced from Merck, Mumbai, India.
UV–visible spectrophotometer (UV 3000+, Labindia, Mumbai, India), Waters® 2695 HPLC System with separation module, PDA Detector and Empower® software (Waters Corporation, Milford, USA), weighing scale (Afcoset® ER-200A, Hyderabad, India), reversed phase Inertsil® ODS C18 column (4.6 × 250 mm, 5 µ) (GL Sciences, Inc., California, USA), rotary evaporator (Buchi R100, Germany) and pH meter (Adwa-AD 1020, Adwa Instruments, Inc., Szeged, Hungary). All glass apparatus were sourced from Borosil® (Borosil Glass Works Ltd., Mumbai, India).
Experimental design, data analysis and generation of surface plots were performed by using Design Expert® Trial Version 13, State-Ease Inc., Minneapolis, MN, USA.
Chemometrics optimized development of chromatographic parameters
Any developed new experimental methodology is acceptable when it can be performed in considerably less time with the simultaneous saving of material and personal cost. Chemometric tools like Response Surface Methodology (RSM) using desirability function is a good statistical analysis tool for extracting the maximum amount of complex information, for proper optimization of any process technology and determining the values of process parameters at which response reaches its optimum level which can be maximum, minimum or a desired region where the response is stable over a range of parameters [14, 15]. In this research paper D-optimal mixture design approach has been applied for optimizing the RP-HPLC chromatographic conditions for detection of pesticides in stems of Oryza sativa; mobile phase composition, and flow rate were considered as the input variables.
Standard solution preparation
A 50 mg of metsulfuron-methyl, 2 mg of chlorantraniliprole and 50 mg of chlorimuron-ethyl working standard were accurately weighed and transferred into a 100 mL cleanly dried volumetric flask, 7 mL of mobile phase was added, sonicated to dissolve it completely and volume made up to the mark with the same solvent i.e. Stock solution. A 0.6 mL of the above stock solutions were further pipetted into a 10 mL volumetric flask and volume was adjusted up to the mark with mobile phase.
The commonly used herbicide and insecticide together for the rice crop in the east and west Godavari districts of India are DuPont Almix®, a herbicide which contains metsulfuron-methyl and chlorimuron-ethyl, and used for weed management; and DuPont Ferterra®, an insecticide which contains chlorantraniliprole, used for white ear incidence in rice plants. The stems of Oryza sativa were collected from dried agriculture fields of the Rajahmundry region of India, dried, powdered, macerated in ethanol, filtered, and subjected to drying using a rotary evaporator (Buchi R100, Germany). The dried powder served as the test sample. The test samples were dissolved in mobile phase prior to injection into the HPLC system.
RP-HPLC analysis under optimized chromatographic conditions
Analysis was carried out in gradient elution HPLC system (Waters®)with auto-sampler and PDA detector in reversed phase Inertsil® ODS C18 column (4.6 × 250 mm, 5 µ) (GL Sciences, Inc., California, USA) at ambient temperature; the optimized mobile phase composition consisted of phosphate buffer:acetonitrile (85:15) maintained at pH 3.5, the flow rate was kept at 1 mL/min, detector wavelength at 281 nm, injection volume 20 µL and a run time of 15 min. The developed method was validated as per International Conference on Harmonization (ICH) Guidelines for Validation of Analytical Methods . Various parameters were evaluated including linearity, the limit of detection (LOD), the limit of quantification (LOQ), accuracy, precision and robustness as per the ICH guidelines.
Preparation of calibration curve
Appropriate volumes of aliquots (0.2, 0.4, 0.6, 0.8 and 1.0 mL) from standard stock solutions of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl were transferred to different 10 mL volumetric flasks. The volume was made up to mark with the mobile phase to obtain various concentrations of 100, 200, 300, 400 and 500 μg/mL for metsulfuron-methyl and chlorimuron-ethyl; and those of 4, 8, 12, 16 and 20 μg/mL for chlorantraniliprole. The absorbance of resultant solutions was measured at 281 nm. The calibration curve was constructed by plotting the absorbance versus concentration.
Forced degradation studies
The International Conference on Harmonization (ICH) guideline entitled stability tests of new drug substances and products that require stress testing to elucidate the inherent stability characteristics of the active substance . Stress degradation studies on the metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl were done for the proposed method.
From the standard stock solution, 5 mL was transferred into a 50 mL volumetric flask, 1 mL of 5 N HCl was added and kept at 60 ºC for 6 h and then neutralized with 1 mL of 5 N NaOH and diluted to marked volume with mobile phase and mixed properly. The solution was filtered with 0.22 µ syringe filters, suitably diluted and injected into HPLC system and analyzed.
From the standard stock solution, 5 mL was transferred into a 50 mL volumetric flask, 1 mL of 5 N NaOH was added and kept at 60 °C for 6 h and then neutralized with 1 mL of 5 N HCL, mixed properly and volume adjusted with mobile phase. The solution was filtered with 0.22 µ syringe filter, suitably diluted and injected into HPLC system and analyzed.
From the standard stock solution, 5 mL was transferred into a 50 mL volumetric flask, 1 mL of 30% hydrogen peroxide was added and heated to 70 °C for 1 h on a water bath. The flask was removed from the water bath and allowed to cool at room temperature and after volume adjustment with mobile phase was mixed thoroughly. The solution was filtered with 0.22 µ syringe filter, suitably diluted and injected into HPLC system and analyzed.
From the standard stock solution, 5 mL was transferred into a 50 mL volumetric flask, 1 mL of 10% sodium bisulphate was added and heated at 70 °C for 1 h on a water bath. The flask was removed from the water bath and allowed to cool at room temperature, volume adjusted with mobile phase and mixed properly. The solution was filtered with 0.22 µ syringe filter, suitably diluted and injected into HPLC system and analyzed.
The sample solution prepared from the Oriza sativa was injected as described under experimental work. The corresponding chromatogram obtained is noted and the amounts of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl were determined using calibration curves.