Diflubenzuron Mass Concentration Determination Method in Pesticides Using High-Performance Liquid Chromatography

ABSTRACT

Aims:

Pesticides are actively used in agriculture to protect crops from pests, and accurate determination of the concentration of their active ingredient is critical to ensuring their efficacy and safety. The research aims to develop a methodology that will enable the accurate determination of the mass concentration of diflubenzuron, a widely used active ingredient in pesticide formulations. This is achieved through the use of high-performance liquid chromatography (HPLC).

Methods:

The proposed method for determining the mass concentration of diflubenzuron using HPLC was developed through a series of experiments and optimisation steps. The sample preparation procedure was optimised to extract the maximum amount of diflubenzuron from the pesticide formulation.

Result:

The chromatography conditions were optimised to separate diflubenzuron from other components in the sample matrix. A calibration approach was established for accurate quantification of diflubenzuron in the sample based on peak area measurements. The developed method can be widely used in agriculture, where accurate determination of diflubenzuron concentrations in pesticide formulations is critical for proper application and regulatory compliance. Both regulatory organizations and pesticide manufacturers can use it for batch-release testing and quality control to evaluate the efficacy and safety of pesticide products. In addition, the method can serve as a valuable tool for researchers and analysts working with pesticide residues in crops and environmental monitoring studies.

Conclusion:

The optimised sample preparation, chromatography conditions, and calibration approach provide reliable and reproducible results.

INTRODUCTION

The high sensitivity, selectivity, and adaptability of HPLC make it the most important analytical tool for measuring diflubenzuron in pesticide formulations.[1] It efficiently separates, detects, and measures complex combinations, making it suitable for analyzing diflubenzuron in pesticides. Due to its high accuracy, the ability to handle various sample matrices and analyte concentrations, and the growing demand driven by quality control, regulatory compliance, and research needs in the pesticide industry, HPLC has become the preferred method for the determination of diflubenzuron.[1] Currently, pesticide preparations that have state registration and are included in the list of registered pesticides (as of 2022, 1282 preparations are included in the list) are authorized for use in Kazakhstan. Methodological guidelines for determination of residues of diflubenzuron in apples by HPLC method proposed by the testing laboratory of Scientific and Production Association “TAU” LLP emphasize once again the importance of the method for ensuring standards for pesticide preparations and compliance with regulatory requirements.[2]

Researchers are using HPLC to find out how much of the active ingredient diflubenzuron is in pesticides. The main objective of these studies is to establish reliable techniques for detection and quantification in various samples.[3,4] Manousi et al.[3] and Mei et al.[4] developed and applied an analytical method for the detection of benzoylurea insecticides in apple juice samples that combines monolithic capsule-phase micro-extraction with HPLC-DAD. The method includes optimized adsorption and desorption steps, and its efficiency has been validated. However, further study and validation are necessary for wider use in food safety analysis because the possibility of interference from other substances present in apple juice has not been considered. Zhu et al.[5] investigated the residues of banned pesticides in traditional medicine preparations using QuEChERS and HPLC-QqqqQ-MS/MS methods. They showed that these methods were sensitive and linear but recommended further studies to assess environmental hazards and develop effective purification and recycling methods. The effectiveness of their approach was evaluated by analyzing pesticide residues in traditional remedies, which emphasizes the need for continued research to reduce environmental impact and improve cleaning methods.[5]

Importantly, there is also a lack of studies on the determination of diflubenzuron in food samples, which is important for food safety and regulatory compliance. Therefore, more research is needed to develop sensitive and reliable methods for the analysis of diflubenzuron in food samples. The research aimed to create a solid and approved way to use HPLC to find out the mass concentration of diflubenzuron in pesticide mixtures. The determination of diflubenzuron in pesticide formulations is a challenging task due to various factors, including the complex formulation matrix, the potential influence of other components, and the low concentration of the analyte.

MATERIALS AND METHODS

The established approach was subjected to validation to confirm its suitability for routine analysis, namely, in terms of accuracy, precision, linearity, and reliability.[2] The measurement procedure utilizes HPLC with a variable-wavelength UV detector to determine diflubenzuron after dissolving it in acetonitrile and comparing it with a sample extracted from the pesticide “SK (diflubenzuron 480 h/l)” using an acetonitrile–water mixture. Identification and quantification were achieved through retention time and the absolute calibration method, respectively, ensuring selectivity through sample preparation conditions and the chromatography mode.[6] Instruments used include a Waters 1515 liquid chromatograph, a CORTECS®C18+column, a Waters 2489 UV detector, a Waters 2707 autosampler, and auxiliary equipment such as an ultrasonic bath and laboratory scales. Reagents include a standard sample of diflubenzuron, acetonitrile, bidistilled deionized water, filters, orthophosphoric acid, and a sample solvent comprising acetonitrile and a 0.1% solution of orthophosphoric acid in water. All procedures performed in the study were in accordance with the 1964 Helsinki Declaration and its later amendments. A study was approved by the ethics committee of the LLP “TAU Scientific and Production Association” on March 15, 2023.

The use of reagents meeting specified qualifications is permitted, with sampling conducted according to GOST 14189-81 standards. Some preparation samples are kept in a dry, well-ventilated area. The mobile phase is made up of 60 parts acetonitrile and 40 parts 0.1% orthophosphoric acid solution in water. It was made in a certain way, which included degassing.[2] Standard basic solution preparation involves weighing 0.06 g of diflubenzuron standard substance, dissolving it in 40 cm³ acetonitrile in a 100 cm³ flask, and adjusting the volume with acetonitrile to yield a 600 mcg/cm³ solution, stored in a refrigerator after temperature stabilization.[7,8] To make working solutions of diflubenzuron, measuring flasks were filled with the basic standard solution and acetonitrile was added until the concentrations reached 60 mcg/cm³ and 40 mcg/cm³. These solutions were stored in a refrigerator. Prior to experiments, column conditioning was performed using the mobile phase for 45–50 min until stability was achieved. Chromatography conditions involved using a Waters 1515 liquid chromatograph with a Waters 2489 variable wavelength ultraviolet detector, a CORTECS® C18+2.7 μm chromatography column, and operating at a temperature of 22°C with the mobile phase consisting of acetonitrile/0.1% orthophosphoric acid solution at a ratio of 60/40. The injected sample volume was 5 μl, and the retention time for diflubenzuron was approximately 2.8 min.

Five standard solutions of diflubenzuron and five samples of preparation “SC (diflubenzuron 480 g/l)” were prepared for determination, with each undergoing chromatographic analysis. Calibration plotting and quantification were conducted using chromatograph software.[9] A 0.1 g sample of the preparation was weighed, transferred to a 100 cm³ flask, mixed with 40 cm³ 0.1% orthophosphoric acid solution, ultrasonicated for 10 min, and diluted 8 times after temperature stabilization, and then 12.5 cm³ of the resulting solution was transferred to a 100 cm³ flask and diluted further. The solutions were filtered through a membrane filter and chromatographed at least five times. At the same time, at least five times chromatography of the calibration solution lying within the range of linear dependence of the peak area (height) on the concentration of the substance was performed, alternating sequentially the solution of the standard sample and the sample solution of the test drug.[10] The response factor for diflubenzuron (RFi) was calculated using formula (1):

Where S icm– peak space of the standard in the calibration mixture, r.u.; M icm– mass of the standard suspension, g; P i – purity degree of the analytical standard, %. The value of the response factor (RFi) was calculated by averaging the values of single measurements of response factors. The content of diflubenzuron in the SC (diflubenzuron 480 g/l) was calculated by formula (2):

Where Xi – mass concentration of the active substance in the product, g/l; Mi process – mass of the preparation suspension in the analyzed solution, g; Si process – peak area of the active substance in the analyzed solution; RFi – value of the response factor; K = 1000 (conversion factor); p –density of the product, g/cm³.

RESULTS AND DISCUSSION

The method of determining the mass concentration of diflubenzuron in preparative forms of pesticides using HPLC is very important due to its accuracy and high sensitivity. HPLC, which is adept at separating, identifying, and quantifying components based on their physicochemical properties, allows accurate determination of the concentration of diflubenzuron, which is essential for quality control, regulatory compliance, and research. The technical challenges of chromatographic determination of impurities in diflubenzuron solution, especially due to limited impurity profile information, force the use of a “brute force” approach to develop a chromatographic separation. The resolution of peaks of some unknown substances on the chromatogram will increase with increasing efficiency, selectivity, and retention of the chromatographic system (3):

This method is based on the idea that chromatographic resolution is very specific and depends on retention and that retention and resolution can be changed smoothly while using as little time as possible. When the selectivity and efficiency of the chromatographic column are unchanged, the multipliers of the equation related to the corresponding parameters can be conventionally taken as some constant X. Thus, the resolution of the chromatographic system can be simplistically expressed as a function of retention (4):

As can be seen from the graph in Figure 1, the retention contribution to RS resolution increases sharply with a smooth increase in the value of k′ in the range from 0 to 1.

Figure 1.

Graph of the function k′

For retention values up to k′=1 in chromatography, the system contributes 0.5 to the realized resolution, implying that half of its resolving power is utilized when the critical peak pair elutes with a retention time twice the dead time. The brute force method employs a high-elution-force mobile phase to weakly retain substances, facilitating their chromatographic peak separation. Gradually increasing retention values allows for the resolution of critical peak groups. A technical diflubenzuron sample was used as a model for blind separation of related impurities, with 20 μl of its solution chromatographed using a highly efficient column: 4.6*250 mm filled with 3 μm spherical sorbent particles. The system’s back pressure with water as the eluent is approximately 27 MPa [Figure 2].

Figure 2.

Chromatogram of diflubenzuron test solution

When using reversed-phase HPLC for diflubenzuron, a polar compound, we need to use lipophilic grafted sorbents like octadecylsilyl silica gel as a stationary phase. End-capped silica gel reduces the effects of defined compounds that contain nitrogen on the gel. A flow rate of 0.8 ml/min mitigates system back pressure. The new chromatographic conditions make it possible to control the composition of diflubenzuron in both quality and quantity. They also separate it from related compounds with a retention time of 4.5 minutes. A calibration curve was made with diflubenzuron standards ranging from 1 to 100 μg/ml. It showed a linear relationship with a correlation coefficient of 0.9998, and it had a limit of detection (LOD) of 0.05 μg/ml and a limit of quantification (LOQ) of 0.1 μg/ml, showing that it was very sensitive. Sample analysis used to validate the method showed that it worked correctly, with diflubenzuron yields ranging from 95% to 103% and an RSD of less than 2%, which means the method was very accurate. The method works with different types of preparations, such as emulsifiable concentrates (ECs) and wettable powders (WPs). It can handle diflubenzuron mass concentrations of 25 to 250 μg/ml and 10 to 100 μg/ml, respectively. It is a reliable way to find diflubenzuron in pesticide formulations.[2]

The study confirms the HPLC method’s accuracy, sensitivity, and efficiency in measuring diflubenzuron concentrations in pesticide formulations, offering significant advantages for quality control and environmental monitoring. Despite diflubenzuron’s low hazard to humans and mammals, its potential toxicity to aquatic invertebrates underscores the importance of maintaining safe environmental levels. The method’s high sensitivity enables precise determination even at low concentrations, crucial for regulatory compliance and quality assurance.[11,12] While the method’s reliability is evident under various chromatographic conditions, further validation is needed for its applicability to matrices beyond pesticide formulations and other active ingredients. Therefore, broader investigation is required to ascertain its suitability for diverse analytes and matrices, highlighting the method’s potential impact on sustainability and health.

In addition to the HPLC method, other techniques exist for determining diflubenzuron concentrations in pesticides. Gas chromatography–mass spectrometry (GC/MS) uses gas chromatography to separate diflubenzuron from pesticide components before mass spectrometry detection. Stringhini et al.[13] and Dong et al.[14] optimized pesticide residue analysis in tomato and peach samples using the QuEChERS extraction method combined with ultrahigh performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). This method, known for its simplicity and cost-effectiveness, detects diflubenzuron and its metabolites in fruits and vegetables at very low concentrations, although it requires several hours for sample preparation and analysis.

The UV spectrophotometry method measures the absorbance of diflubenzuron at a specific wavelength, determining its concentration by comparing the optical density of the sample with that of a standard solution. Pecev-Marinković et al.[15] proved that a kinetic spectrophotometric method could be used to find diflubenzuron residues by making the best choices about the solvent, wavelength, and sample preparation. Successfully applied to water and baby food samples after solid-phase extraction, this method offers a simple, cost-effective alternative to HPLC and GC/MS. The LC-MS/MS method uses liquid chromatography to separate diflubenzuron from pesticide parts and tandem mass spectrometry to find it. It is very sensitive.

Wang et al.[16] developed conditions for diflubenzuron detection in vegetables using ultra-HPLC and tandem mass spectrometry, ensuring specificity even in complex matrices. Solid-phase extraction reduces matrix interferences, improving accuracy, but reliance on specialized equipment and expertise may limit its use. The solid-phase enzyme immunoassay (EIA) method utilizes diflubenzuron-specific antibodies. After extraction, diflubenzuron reacts with antibodies, and its amount is measured via spectrophotometry. Wang et al.[17] developed an icELISA method for diclazuril determination in chicken samples, offering specificity, sensitivity, and accuracy comparable to instrumental methods but with faster results.

There are different ways to find out how much diflubenzuron is in pesticide mixtures. Each has its own level of sensitivity, accuracy, and difficulty, and the one we choose will depend on our analysis needs, such as the sample matrix, detection limit, and availability of equipment. However, HPLC stands out as a unique and crucial analytical technique for this purpose, offering sensitive, accurate, and reliable measurements of the active ingredient’s concentration in diverse pesticide formulations. Because it is so selective and sensitive, HPLC is one of a kind because it can effectively separate, identify, and quantify the target analyte from complex matrices like pesticide formulations. Accurately measuring the concentration of diflubenzuron is important for finding the right doses, making sure the product works to kill pests, and lowering the risks of exposure to non-target organisms and the environment. HPLC’s sensitivity allows for trace-level detection, which makes it easier to keep an eye on product stability and shelf life as well as quality control in the production of pesticides that contain diflubenzuron. This shows how important it is to make sure that these pesticides are safe, effective, and of high quality.[18,19]

CONCLUSIONS

The use of HPLC to determine the mass concentration of diflubenzuron in pesticide formulations provides accurate and reliable information to ensure effective pest control while minimizing potential damage to non-target organisms and the environment. The use of HPLC helps to detect any adulteration or contamination of the pesticide formulation, which is critical to ensure product safety and efficacy. By analyzing various pesticide formulations containing diflubenzuron, this study demonstrated the effectiveness of the HPLC method to accurately determine the concentration of diflubenzuron in preparative forms of the pesticide. This information is needed to determine the appropriate dosage for effective pest control, prevention of pesticide resistance and contamination, and environmental safety.

The results of this study highlight the importance of accurate and reliable methods for determining pesticide concentrations to achieve effective pest control while minimizing harm to non-target organisms and the environment. The use of HPLC to analyze the concentration of diflubenzuron in pesticide formulations can help in the development of safe and effective pesticide products, which are critical to the sustainability of agriculture and horticulture. Regulatory organizations can use the technique to enforce pesticide usage safety standards, and manufacturers can use it to regulate the caliber of their goods. In addition, the method can be used by researchers in environmental monitoring and pesticide residue analysis to study the behavior of diflubenzuron in different environmental matrices and to assess the potential risks associated with its use.

This method is crucial in ensuring that the concentration of diflubenzuron in pesticide formulations is within the acceptable range, resulting in an effective pesticide that can control target pests. The HPLC method for determining the mass concentration of diflubenzuron in pesticide formulations is a valuable tool for researchers, manufacturers, and regulatory agencies in the development, application, and monitoring of pesticide products. By continuing to improve and refine these methods, the safety and sustainability of agriculture and horticulture can be better ensured while meeting the increasing demand for food production worldwide. Further research could be conducted to validate the method on different matrices and to explore its applicability to other pesticides commonly used in agriculture.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.