Stability Indicating HPLC Method for the Determination of Diflubenzuron Insecticide

A new, fast, accurate, reliable and stability indicating HPLC method for the determination of Diflurobenzuron (DFB), in technical and formulation samples in presence of related and degraded impurities has been developed using intersil – 3 stainless steel C. 18 column (5 μm, 250 mm length x 4.6 mm id), acetonitrite: water: 1.4 dioxane (58:39:0.03 v/v) as mobile phase and diphenyl as internal standard. The flow rate of the mobile phase was 2 cm 3 min -1 . Detection was carried out at 260 mm using UV detector. The retention times were 1.55 min, 1.75 min, 6.3 min, 7.9 min and 10.7 min for 2, 6 diflurobenzoic acid, 2, 6 diflurobuzamide, diflurobenzuron, difur (an impurity) and diphenyl (internal standard) respectively. The linearity range of DFB was 0.5 to 15 mg per 100 cm 3 . The LOD and LOQ values for DFB were 0.142 to 0.432 respectively. When various technical and formulated smples were analysed by this proposed method the percentage recoveries were found to be 99.20-101.80% with RSD between 0.01% to 0.45%.

Urea(CAS RN-35367-38-5), is a well-known insecticide used for the control of a wide range of insects, pests and larvae of flies, mosquitoes and locusts. It is also ecto parasiticide. DBF is a chitin synthesis inhibitor that act as an anti-moulting agent, leading to death of larvae and pupae [1].
In this method C-8 (octyl) column, or alternate, mobile phase containing acetonitrile, water and 1:4 Dioxan, in proportion of 45:40:10 and "Linuron" as internal standard are used. However, due to high concentration of 1:4 Dioxan, the column efficiency is reduced and resolution of DFB and related impurity peaks are affected. Also Linuron, the internal standard, used in this method, is a specific agro product, and may not be available readily in expected pure form.
However, in the present method, C-18 (octadecy 1) column and mobile phase consisting of Acetonitrile: Water: 1:4 Dioxan in the proportion of 58:39:03(V/V/V) and Biphenyl is used as an internal standard are used. C-18 columns are readily available and are more economic than C-18(Octyl) columns; content of 1:4 Dioxan is very less in the mobile phase and Biphenyl is cheaply available with simple structure and high purity. Moreover, DFB and its related as well as degraded impurities are very well resolved in this method.

Instrumentation
A high-pressure liquid chromatograph, SHIMADZU, equipped with LC-10 AD pump, SPD-10 AV UV/VIS detector with variable wavelength and Controller Bus Module CBM-10 A was used. A photodiode array detector (Shimadzu SPD M 10 A) was also used for confirming the peak purities.

Chemicals and Solvents
DFB (technical) procured from M/s. Shimac, China with purity of 95% was used as a reference standard. Formulation samples of DFB were procured from market.
Acetonitrile and Dioxan used were of HPLC grade supplied by S D Fine Chemicals and SRL respectively, whereas water used was double distilled prepared in the laboratory.

Stationary Phase
Inertsil-3 stainless steel C-18 column (5 µm; 250 mm length X 4.6. mm i. d.) from GE Science Inc., Japan was used as a stationary phase.

Preparation of Stock Solutions
Stock solution (A) of DFB was prepared by dissolving 100 mg of DFB in 50 cm 3 of 1:4 Dioxan followed by sonication for about 15 minute and diluted to 100 cm 3 with 1:4 Dioxan.
The stock solution (B) of internal standard was prepared by dissolving about 120 mgs of Biphenyl in 50cm 3 of 1:4 Dioxan followed by sonication for about 15 min. and diluted to 100cm 3 with 1:4 Dioxan.

Preparation of Sample Solutions
Various formulation samples namely technical, wettable powder (WP), wettable dispersible granules (WDG) and suspoconcentrate (SC) were accurately weighed equivalent to 10mg of DFB, in duplicate, and transferred to100cm 3 flask containing 50cm 3 of 1:4 Dioxan and sonnicated for 15 minutes and then diluted to 100cm 3 with 1:4 Dioxan. 10 cm 3 of this solution was mixed with 5cm 3 of internal standard (solution B) and diluted to 100cm 3 with mobile phase. This solution was filtered through Whatman no.1 filter paper to obtain clear solution before injection.
In the case of WDG samples, the material was crushed to fine powder in a mortar before weighing and in case of suspo-concentrate formulation (SC) 20cm 3 of water was added to ensure complete dispersion before dissolving in 50cm 3 of 1:4 Dioxan.

Procedure for Calibration
Into a series of 100cm 3 flasks, varying amount of stock solution A (1 to 16cm 3 ) were taken and 5 cm 3 of internal standard solution (B) was added. The contents were diluted up to the mark with the mobile phase. 20 µl of each solution was injected into the column and peak area ratios were recorded for all the Chromatograms. Calibration curve constructed by plotting peak area ratio (Y-axis) against the amount of DFB in mg/cm 3 (X-axis) and the linear relationship was evaluated by calculation of regression line by the method of least squares.

Assay Procedure
Each of the sample solution, prepared as above in duplicate, was injected in the column and the peak area was recorded as described in calibration procedure.

Chromatography
The mobile phase comprising of Acetonitrile: water: 1:4 Dioxan in the proportion of 58:39:03 was selected because it was ideal to resolve DFB, Difur (an impurity) and Biphenyl (internal standard) with retention times of 6.3 min., 7.90 min. and 10.7 min. respectively (Figure 1). Also the impurities, formed due to degradation, namely 2, 6 difluorobenzamide and 2, 6 Difluoro Benzoic acid are also well resolved from DFB ( Figure 2). Solutions of DFB, Difur and Biphenyl in 1:4 Dioxan gave maximum absorption at 259 nm, 262 nm and 248 nm respectively. 260nm was selected for detection, because at this wavelength all the three components gave measurable absorbance (Figure 3). Since 1:4 Dioxan content in the mobile phase was minimum, the column performance was not affected and the results obtained were highly reproducible. The peak purities of DFB Difur and Biphenyl were confirmed using PDA detector. The chromatographic parameters of the system are give in Table-3.

Linearity, Limit of Detection and Limit of Quantification
The plot of peak area ratios versus the respective concentration of DFB was found to be linear in the range form 0.5 mg to 15 mg/100cm 3 (Table-2). The limit of detection (LOD) and limit of quantification (LOQ) were calculated by using the equations, LOD = 3.3 x σ/S and LOQ = 10 x σ/S Where, 'σ' the noise elimination, is the standard deviation of the blank responses (six injections) and 'S' is the slope of corresponding calibration curve of DFB. LOD and LCQ values were found to be 0.142 and 0.432 respectively

Assay
Contents of DFB found in the commercial brand of samples by the present method are as shown in the Table-3.
The low values of RSD indicate that the method is precise and accurate.

Accuracy and Precision
To study the accuracy and precision of the present method, the recovery experiments were performed by standard addition technique. Four different levels of standards were added to pre-analyzed samples and each level was repeated thrice. The percentage recoveries in the samples were in the range of 99. 4

Stability Indicating Ability of the Method
In a series of five volumetric flasks of 100 cm 3 capacity, 10 mgs of DFB were taken and 5 ml of 1 N NaOH were added to each flask and kept aside for undergoing alkali hydrolysis. After every 24 hrs. one of the flasks were treated with 1 N HC 1 to neutralize to pH 7 and the solution was diluted up to the mark with mobile phase and 20 µl of each were then injected in the HPLC column and the present procedure was followed to analyze DFB. It was observed that the decomposed neutralized solution gave a number of low retention products, the percentage of which significantly increased from 1 to 5 days as shown in Table-5. The impurities decomposition products at retention times 1.55 min. and 1.75 min. were confirmed to be 2, 6 Difluorobenzamide and 2, 6 Difluoro Benzoic acid by spiking the standards of these compounds however, other impurities/products could not be identified due to non-availability of standards.
These results indicate that a present method is a stability indicating method since the method can be not only for assay of DFB but also the impurities like Difur, 2, 6 Difluoro Benzoic acid, 2, 6 Difluorobenzamide without interference.