Experimental design optimization of RP-HPLC method for simultaneous estimation of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl residues in stems of Oryza sativa

The study aims to develop a chemometrics optimized D-optimal mixture design approach assisted RP- HPLC method for the determination of pesticide residues of metsulfuron-methyl, chlorantraniliprole, and chlorimuron-ethyl in the stems of Oryza sativa. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of a pH 3.5 phosphate buffer and acetonitrile in the ratio 85:15. The optimized HPLC method gave a sharp resolution of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl at a retention time of 2.599 min, 3.805 min and 4.661 min receptively. Linearity was observed in the range 100‒500 µg/mL for metsulfuron-methyl (r2 = 0.999), 4‒20 µg/mL for chlorantraniliprole (r2 = 0.999) and 100‒500 µg/mL for chlorimuron-ethyl (r2 = 0.999). The developed method was validated as per ICH guidelines. The proposed chemometrics optimized RP-HPLC method was found to be successful in the resolution of pesticide residues in the stems of O. sativa. The developed method can be applied to routine quantification of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl.


Background
Pesticides and herbicides are used to destroy the insects or other organisms harmful to cultivated plants in modern agricultural practices to enhance the yield and upgrade the quality of the agricultural crops. Nearly 30% of the food production is lost in third world countries due to insects, pests, plant pathogens, weeds, rodents, birds during storage. However, the unregulated and indiscriminate application of pesticides has raised serious concerns regarding environmental pollution and human health issues [1][2][3][4][5][6]. The pesticides ought to be checked for viability, natural, and toxicological tests to get enrolled by the government for legitimate use in determining applications. Sustained accumulation of pesticides in agrarian items is a matter of grave concern for its far-reaching adverse health consequences [2][3][4]. Oryza sativa is the most widely consumed cereal grain as a staple food for a large part of the world's human population, especially in Asia.

Materials and reagents
Metsulfuron-methyl, chlorantraniliprole, chlorimuronethyl were obtained from Pharma Train, Hyderabad, India. KH 2 PO 4 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.

Software
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.

Sample preparation
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 [17]. 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 metsulfuronmethyl, 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 chlorimuronethyl; 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 [17]. Stress degradation studies on the metsulfuronmethyl, chlorantraniliprole and chlorimuron-ethyl were done for the proposed method.

Acid degradation
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.

Alkali degradation
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.

Oxidative degradation
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.

Reduction
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.

Assay
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.

Results
In this research D-optimal mixture design approach was 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. Considering the retention time of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl as three different responses, the software generated ANOVA tables and 3D-surface plots are presented in Tables 1, 2 and 3      The UV spectrum selection of analytes where all three exhibited a sharp response as detector wavelength is given in Fig. 6 and the standard chromatogram obtained (Fig. 7) basing on the chemometrics assisted developed RP-HPLC chromatographic conditions showed proper resolution of the analytes. The chromatogram obtained for the sample solution was shown in Fig. 8 which indicates consistency of the three peaks. The results of system suitability parameters were found within the limits as per ICH guidelines, such as the resolution between samples was not less than 2, theoretical plates were not less than 2000, and tailing factor was less than 2 as shown in Table 4. The optimized HPLC method gave a sharp resolution of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl at retention times of 2.599 min, 3.805 min and 4.661 min respectively. Linearity was observed in the range 100-500 µg/mL for metsulfuronmethyl (r 2 = 0.999), 4-20 µg/mL for chlorantraniliprole (r 2 = 0.999) and 100-500 µg/mL for chlorimuron-ethyl (r 2 = 0.999). The validation and stability parameters of the chemometrics optimized RP-HPLC method are summarized in Table 5. ICH guidelines mandatory oblige the    Table 6 and Fig. 9. For metsulfuron-methyl, highest degradation was found during oxidation (3.83%) and lowest during alkali degradation (1.58%). Chlorantraniliprole degraded most in peroxide (5.19%) and least in alkali (1.05). Finally chlorimuron-ethyl was influenced nearly equal by both acid (10.89%) and peroxide (10.52%) environments.

Discussion
The purpose of this research was to develop and validate chemometrically optimized simple, precise and reliable RP-HPLC method for simultaneous determination of three pesticide residues of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl in stems of Oryza sativa. In earlier studies, chemometrics for the optimization of analytical method development was successfully applied [18,19]. In the present research, the results of the chemometric tools such as response surface methodology and ANOVA were tested for optimization of each factor. The D-optimal mixture design approach has been applied for optimizing the RP-HPLC chromatographic conditions. From the software generated final equations in terms of actual components and actual factors considering retention time of three analytes, positive coefficients were obtained for phosphate buffer and acetonitrile, such as 2.61 and 3.41 for metsulfuron-methyl; 3.80 and 4.91 for chlorantraniliprole; and 4.61 and 5.81 for chlorimuron-ethy respectively. This indicates that the concentrations of phosphate buffer and acetonitrile influence the retention time. Standard protocols of ICH Q2 (R1) guidelines were followed for the analytical method development and validation. The results of system suitability parameters were found within the limits as per ICH guidelines, thus developing precise and accurate results [17]. The developed method was validated for linearity, precision, accuracy and robustness, LOD and LOQ. The RSD values of precision and intermediate precision of developed method were less than 2% which indicates that the method is precise and repeatable. The recovery of three analytes was found in the range of 100 ± 3% which shows that the method is accurate and capable of reproducibility.  Further the LOD values were found between 2.96-3.00, and LOQ values were found between 9.98 and 10.00, and were within the limits. The result of this method was in consonance with reported methods of these pesticides in other combinations in terms of reproducibility, rapid resolution, sensitivity and selectivity [20][21][22].
Results of forced degradation studies of the three analytes showed that the degradation was less than 15% and was within the limits, indicating stability of the analytes under stress conditions. The developed method can be successfully applied to routine quantification of metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl in the food industry since it is reproducible, stable and rapid.

Conclusion
Based on the reported experimental results, this developed chemometrics assisted the new RP-HPLC method for simultaneous estimation of three pesticide residues: metsulfuron-methyl, chlorantraniliprole and chlorimuron-ethyl in stems of Oryza sativa. This method was found simple, accurate, precise and rapid and making it more viable and acceptable and successfully applied for routine analysis in the quality control departments of food industries and government-approved food testing laboratories.