Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of environmental chemistry and pesticide residue analysis, and in specific, relates to a method for extraction and determination of validamycin residues in aqueous environments by utilizing a cloud point extraction (CPE) technique that employs surfactant-based micellar systems to achieve an eco-friendly, efficient, and sensitive extraction of validamycin from water samples, such as paddy fields.
Background of the invention:
[0002] Validamycin is a water-soluble aminoglycoside antibiotic and fungicide, primarily used in agriculture to combat fungal pathogens. Chemically known as validamycin A, it is a secondary metabolite produced by Streptomyces hygroscopicus. Its broad-spectrum antifungal activity makes it highly effective against diseases in staple crops such as rice, wheat, sugarcane, and vegetables. Validamycin functions by inhibiting trehalase enzymes in fungi, thereby halting energy metabolism and mycelial growth. Due to its targeted efficacy, it is extensively applied in paddy fields, where rice cultivation is widespread. However, improper application and overuse have resulted in residual build-up in soil and water bodies adjacent to agricultural fields, raising serious environmental and health concerns. Its persistence in aqueous environments necessitates sensitive monitoring techniques to track trace-level contamination and understand the dynamics of pesticide runoff from treated areas.

[0003] Sheath blight is one of the most prevalent and destructive fungal diseases affecting rice and other cereal crops, caused by Rhizoctonia solani. It manifests as water-soaked lesions on the leaf sheath near the waterline, which progress rapidly under high humidity and warm temperatures. If left uncontrolled, sheath blight might lead to reduced tillering, poor grain filling, and lodging, ultimately resulting in significant yield losses and diminished crop quality. In severe outbreaks, yield reduction might range between 20 and 50 percent. Due to the economic impact of this disease, farmers are heavily reliant on chemical fungicides such as validamycin for disease management. The intensive and repeated use of such fungicides has not only led to the accumulation of chemical residues in soil and water but has also raised concerns over potential resistance development in pathogenic fungi, underscoring the need for responsible monitoring and mitigation strategies in integrated crop management systems.

[0004] While validamycin is effective in controlling fungal infestations, its residual presence in crops and water bodies poses a potential threat to both environmental and human health. Pesticide residues might accumulate in soil and surface water near treated fields, subsequently entering the food chain through crop uptake or irrigation systems. From a human health perspective, ingestion of contaminated produce or water can lead to long-term toxic effects, especially affecting vulnerable populations such as children, pregnant women, and the elderly. Prolonged consumption of food products laced with pesticide residues is linked to carcinogenic, mutagenic, and endocrine-disrupting effects. These risks highlight the necessity for regular monitoring of fungicide residues in agricultural environments and call for the development of safer, more sustainable detection and remediation technologies.

[0005] Conventional methods for removing validamycin and similar pesticide residues, such as soil washing, crop rotation, and microbial bioremediation, often require long remediation times and extensive field trials and are sensitive to environmental conditions. These approaches are not always scalable or effective, particularly in removing low-concentration residues from water bodies like irrigation canals. Physical methods like activated carbon filtration and membrane separation are cost-prohibitive and inefficient for field use. Consequently, these limitations highlight the need for alternative, rapid, sensitive, and eco-friendly methods for validamycin residue detection and removal that are both effective and environmentally sustainable.

[0006] In existing technology, a method for detecting validamycin in environmental and food samples includes high-performance liquid chromatography (HPLC), gas chromatography (GC) with flame ionization or mass spectrometry detection (GC-FID, GC-MS), and liquid chromatography-mass spectrometry (LC-MS). While these methods offer high sensitivity and specificity, they have significant drawbacks. They require sophisticated instruments, extensive sample preparation, and skilled operators, making them unsuitable for on-site or routine field monitoring. Furthermore, they often use large volumes of toxic organic solvents, conflicting with green chemistry principles. High operational costs and infrastructure needs also limit their use in resource-constrained settings. Therefore, there is a need for simpler, cost-effective, and environmentally friendly detection methods such as cloud point extraction (CPE) to support sustainable agricultural practices.

[0007] Therefore, there is a need for a method that develops a simple and efficient approach for the extraction and quantification of validamycin residues in water samples using a mixed micellar cloud point extraction (CPE) technique. There is also a need for a method that contributes to environmental and human health safety by enabling effective monitoring of pesticide contamination in water bodies near paddy and wheat fields. Furthermore, there is also a need for a method that offers a cost-effective, accessible, and scalable solution that might be applied in laboratories and on-site testing in rural agricultural settings.
Objectives of the invention:
[0008] The primary objective of the present invention is to provide a method for extraction and determination of validamycin residues in aqueous environments by utilizing a cloud point extraction (CPE) technique that employs surfactant-based micellar systems to achieve an eco-friendly, efficient, and sensitive extraction of validamycin from water samples, such as paddy fields.

[0009] Another objective of the present invention is to provide a method that develops a simple and efficient approach for the extraction and quantification of validamycin residues in water samples using a mixed micellar cloud point extraction (CPE) technique.

[0010] Another objective of the present invention is to provide a method that utilizes eco-friendly surfactants, sodium dodecyl sulfate (SDS) and Triton X-114 (TX-114), thereby avoiding the use of harmful organic solvents common in conventional analytical techniques.

[0011] Another objective of the present invention is to provide a method that optimizes key extraction parameters such as potential of hydrogen (pH), surfactant concentration, salting-out agent (NaCl), equilibrium temperature, and incubation time to achieve maximum recovery of validamycin.

[0012] Another objective of the present invention is to provide a method that improves analytical sensitivity and detection limits, making the method suitable for trace-level detection of validamycin residues in agricultural runoff and field water.

[0013] Yet another objective of the present invention is to provide a method that offers a cost-effective, accessible, and scalable solution that might be applied in laboratories and on-site testing in rural agricultural settings.

[0014] Further objective of the present invention is to provide a method that contributes to environmental and human health safety by enabling effective monitoring of pesticide contamination in water bodies near paddy and wheat fields.
Summary of the invention:
[0015] The present disclosure proposes a method for extraction and determination of validamycin in water using a cloud point extraction. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0016] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for extraction and determination of validamycin residues in aqueous environments by utilizing a cloud point extraction (CPE) technique that employs surfactant-based micellar systems to achieve an eco-friendly, efficient, and sensitive extraction of validamycin from water samples, such as paddy fields.

[0017] According to one aspect, the invention provides a method for detecting and determining validamycin in a water sample using cloud point extraction. At one step, the non-ionic surfactant Triton X-114 in a concentration of about 2.0% w/v and the anionic surfactant Sodium Dodecyl Sulfate (SDS) in a concentration of about 0.14% w/v are added to a water sample, thereby forming a surfactant-containing mixture. At another step, the sodium chloride (NaCl) in a concentration of about 3.0% w/v is added to the surfactant-containing mixture to promote micellar phase formation. At another step, the adjusts the potential of hydrogen (pH) of the resulting mixture to at least 4.0 using an acetate buffer, thereby forming a pre-extraction solution.

[0018] At another step, the pre-extraction solution is heated to an equilibrium temperature of at least 50 °C, maintaining the temperature for a time period of at least 15 min to induce cloud point extraction and enable phase separation of a surfactant-rich phase and an aqueous phase. At another step, the heated mixture is centrifuged and cooled to complete the phase separation process, and decants the aqueous phase and collects the surfactant-rich phase containing concentrated validamycin. Further, at another step, the absorbance of the surfactant-rich phase is measured at a wavelength of 640 nm using UV-visible spectrophotometry to determine the concentration of the validamycin in the water sample.

[0019] In one embodiment herein, the water sample containing validamycin. The water sample is collected from agricultural runoff, paddy fields, irrigation canals, and surface water near treated crop zones. In one embodiment herein, the pH of the resulting mixture is adjusted using the sodium acetate-acetic buffer to maintain the pH at 4.0 ± 0.2. In one embodiment herein, the incubation temperature ranges from 45 °C to 55 °C, and the incubation time period ranges from 10 min to 20 min.

[0020] Additionally, the centrifugation is performed at a speed that varies between 3000 rpm and 5000 rpm for a time period that ranges from 5 min to 10 min to facilitate phase separation. The cooling of the heated mixture at temperature ranges from 4 °C to 10 °C to enhance the separation of the surfactant-rich phase. In one embodiment herein, the UV-visible spectrophotometric measurement is performed at a wavelength range of 630 nm to 650 nm, with 640 nm being the optimal wavelength.

[0021] In one embodiment herein, the Triton X-114 and SDS are added to the water sample through a controlled distillation process, thereby ensuring homogenous dispersion and enhanced micelle formation. In one embodiment herein, the method is configured to achieve a recovery efficiency of at least 95 percent for validamycin in the tested water sample. Additionally, the method is configured to provide a limit of detection (LOD) for validamycin of 1.32 ng/mL and lower under optimized conditions.

[0022] In one embodiment herein, the method is a novel, eco-friendly, and efficient for the detection and quantification of validamycin in water samples, thereby originating from paddy fields and agricultural runoff, by employing a mixed micellar cloud point extraction (CPE) technique combined with UV-Visible spectrophotometry. The method utilizes a dual-surfactant system consisting of Triton X-114, a non-ionic surfactant, and Sodium Dodecyl Sulfate (SDS), an anionic surfactant, to facilitate the extraction of the validamycin into a surfactant-rich phase under optimized physicochemical conditions.

[0023] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0025] FIG. 1 illustrates a flowchart of a method for detecting and determining validamycin in a water sample using cloud point extraction, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2 illustrates a flow diagram of a systematic process for extracting and detecting the validamycin from water samples using the mixed micellar cloud point extraction method, in accordance to an exemplary embodiment of the invention.

[0027] FIG. 3 illustrates a graphical representation of an effect of pH on the recovery of the validamycin in a cloud point extraction method, in accordance to an exemplary embodiment of the invention.

[0028] FIG. 4 illustrates a graphical representation of an effect of the concentration of the surfactant Triton X-114, in accordance to an exemplary embodiment of the invention.

[0029] FIG. 5 illustrates a graphical representation of an effect of the concentration of the sodium chloride, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0030] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0031] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for extraction and determination of validamycin residues in aqueous environments by utilizing a cloud point extraction (CPE) technique that employs surfactant-based micellar systems to achieve an eco-friendly, efficient, and sensitive extraction of validamycin from water samples, such as paddy fields.

[0032] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for detecting and determining validamycin in a water sample using cloud point extraction. The method develops a simple and efficient approach for the extraction and quantification of validamycin residues in water samples using a mixed micellar cloud point extraction (CPE) technique. The method optimizes key extraction parameters such as potential of hydrogen (pH), surfactant concentration, salting-out agent (NaCl), equilibrium temperature, and incubation time to achieve maximum recovery of validamycin.

[0033] At step 102, the non-ionic surfactant Triton X-114 in a concentration of about 2.0% w/v and the anionic surfactant sodium dodecyl sulfate (SDS) in a concentration of about 0.14% w/v are added to a water sample, thereby forming a surfactant-containing mixture. At step 104, the sodium chloride (NaCl) in a concentration of about 3.0% w/v is added to the surfactant-containing mixture to promote micellar phase formation. At step 106, the potential of hydrogen (pH) of the resulting mixture is adjusted to at least 4.0 using an acetate buffer, thereby forming a pre-extraction solution.

[0034] At step 108, the pre-extraction solution is heated to an equilibrium temperature of at least 50 °C, maintaining the temperature for a time period of at least 15 min to induce cloud point extraction and enable phase separation of a surfactant-rich phase and an aqueous phase. At step 110, the heated mixture is centrifuged and cooled to complete the phase separation process, and decants the aqueous phase and collects the surfactant-rich phase containing concentrated validamycin. Further, at step 112, the absorbance of the surfactant-rich phase is measured at a wavelength of 640 nm using UV-visible spectrophotometry to determine the concentration of the validamycin in the water sample.

[0035] In one embodiment herein, the water sample includes validamycin. The water sample is collected from agricultural runoff, paddy fields, irrigation canals, and surface water near treated crop zones. In one embodiment herein, the pH of the resulting mixture is adjusted using the sodium acetate-acetic buffer to maintain the pH at 4.0 ± 0.2. In one embodiment herein, the incubation temperature ranges from 45 °C to 55 °C, and the incubation time period ranges from 10 min to 20 min.

[0036] Additionally, the centrifugation is performed at a speed that varies between 3000 rpm and 5000 rpm for a time period that ranges from 5 min to 10 min to facilitate phase separation. The cooling of the heated mixture at the temperature ranges from 4 °C to 10 °C to enhance the separation of the surfactant-rich phase. In one embodiment herein, the UV-visible spectrophotometric measurement is performed at a wavelength range of 630 nm to 650 nm, with 640 nm being the optimal wavelength.

[0037] In one embodiment herein, the Triton X-114 and SDS are added to the water sample through a controlled distillation process, thereby ensuring homogenous dispersion and enhanced micelle formation. In one embodiment herein, the method is configured to achieve a recovery efficiency of at least 95 percent for validamycin in the tested water sample. Additionally, the method is configured to provide a limit of detection (LOD) for validamycin of 1.32 ng/mL and lower under optimized conditions.

[0038] In one embodiment herein, the method is a novel, eco-friendly, and efficient for the detection and quantification of validamycin in water samples, thereby originating from paddy fields and agricultural runoff by employing a mixed micellar cloud point extraction (CPE) technique combined with UV-Visible spectrophotometry. The method utilizes a dual-surfactant system consisting of Triton X-114, a non-ionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant, to facilitate the extraction of the validamycin into a surfactant-rich phase under optimized physicochemical conditions.

[0039] In another embodiment herein, the method begins by preparing the water sample suspected to contain trace levels of validamycin. To this sample, Triton X-114 is added at a concentration of 2.0% w/v, and sodium dodecyl sulfate (SDS) is added at a concentration of 0.14% w/v. In one variant of the method, both surfactants might be introduced into the water sample via a controlled distillation process, thereby allowing for homogeneous dispersion and improved micelle formation in the aqueous medium.

[0040] Subsequently, the sodium chloride (NaCl) is added to the mixture in a concentration of 3.0% w/v to act as a salting-out agent. The presence of NaCl decreases the cloud point temperature and promotes micellar aggregation, thereby enhancing the partitioning of the validamycin into the micellar phase. The pH of the resulting solution is then adjusted to approximately 4.0 using an acetate buffer, which has been shown to optimize the recovery of the validamycin during extraction.

[0041] The resulting pre-extraction solution is then subjected to heating at a temperature of at least 50 °C, which is maintained for an incubation period of at least 15 min. This thermal treatment initiates the cloud point phenomenon, during which the solution separates into two distinct phases, such as an upper aqueous phase and a lower surfactant-rich phase, the latter containing the concentrated validamycin. After incubation, the solution is centrifuged at a suitable speed (e.g., 4000 rpm) for the time period of at least 10 min to accelerate phase separation. The mixture is then cooled, typically to a temperature range of 4 °C to 10 °C, to stabilize the surfactant-rich phase. The aqueous supernatant is carefully decanted, and the surfactant-rich phase is isolated for subsequent analysis.

[0042] The final step involves the quantitative determination of the validamycin in the surfactant-rich phase using UV-Visible spectrophotometry, where the absorbance is measured at a wavelength of 640 nm. A calibration curve generated from known concentrations of the validamycin is used to determine the analyte concentration in the original water sample. Under the optimized conditions herein, the method achieves the limit of detection (LOD) of 1.32 ng/mL, a linear detection range between 0.153 µg/mL and 3.074 µg/mL, and a recovery efficiency of up to 98 percent. The method demonstrates excellent reproducibility, with a standard deviation of 0.005041 across multiple trials.

[0043] In one embodiment herein, the method provides a cost-effective, green chemistry-based alternative to traditional analytical techniques such as HPLC, GC-MS, and LC-MS, which are often expensive, complex, and environmentally hazardous due to the use of organic solvents. Additionally, the method might be performed with relatively simple laboratory equipment, making it suitable for implementation in resource-limited settings and field-based environmental monitoring applications. The effect of equilibrium temperature and incubation time was varied in the range of 10 °C -70 °C and the time period of at least 5 min to 60 min. The maximum absorbance was obtained at 50 °C for time period of at least 15 min.

[0044] According to another exemplary embodiment of the invention, FIG. 2 refers to a flow diagram 200 of a systematic process for extracting and detecting the validamycin from water samples using the mixed micellar cloud point extraction method. At step 202, the Triton X-114, a non-ionic surfactant, is added at a concentration of 2.0% w/v, and the Sodium Dodecyl Sulfate (SDS), an anionic surfactant, is added at the concentration of 0.14% w/v, thereby preparing the mixture. These surfactants facilitate the formation of micelles in solution. The unique combination of a non-ionic and anionic surfactant improves the extraction efficiency by enhancing hydrophobic interactions with the validamycin, which becomes entrapped within the micellar core.

[0045] Additionally, the addition of the non-ionic surfactant Triton X-114 and the anionic surfactant Sodium Dodecyl Sulfate (SDS) to the water sample is performed through a distillation-based delivery system. This ensures controlled vapor-phase transfer and uniform dispersion of the surfactants into the aqueous medium, enhancing the formation of mixed micelles and improving extraction efficiency.

[0046] At step 204, the process begins with the preparation of the aqueous solution containing the target analyte, validamycin. This water sample is collected from a water source suspected of contamination, such as runoff from agricultural fields where the validamycin is commonly used as a fungicide. The water sample is kept in a controlled environment, thereby ensuring that no degradation or contamination occurs prior to analysis. At step 206, the sodium chloride (NaCl) is introduced into the mixture at a concentration of 3.0% w/v. This acts as a salting-out agent, which further promotes micellar phase separation by decreasing the solubility of the surfactants in water. The ionic strength of the solution is increased, leading to the aggregation of micelles into a distinct surfactant-rich phase.

[0047] At step 208, the resulting solution is subjected to heating at the temperature of at least 50 °C for the incubation period of at least 15 min. This heating step induces the cloud point phenomenon, where the micelles become sufficiently aggregated due to thermal energy and phase separation begins. The thermal environment is carefully maintained to achieve optimal separation without degradation of the validamycin. At step 210, the heating process is completed, the solution is centrifuged to assist in physically separating the dense surfactant-rich phase from the lighter aqueous phase. The sample is then cooled to stabilize the formed phases. Cooling enhances the density difference between the two layers, making the boundary more distinct and the separation more reliable.

[0048] At step 212, after centrifugation and cooling, the aqueous supernatant (the top layer) is carefully decanted, leaving behind the bottom surfactant-rich phase. This phase includes the extracted Validamycin that has been concentrated due to its partitioning into the micelles formed by the surfactants. At step 214, the surfactant-rich phase is then collected with precision, thereby ensuring that no contamination from the aqueous phase interferes with the analysis. This phase includes the analyte in a form suitable for direct quantification.

[0049] At step 216, the collected surfactant-rich phase is analysed using UV-Visible spectrophotometry. The absorbance of the solution is measured at a wavelength of 640 nm, which corresponds to the maximum absorption of the coloured complex formed with the validamycin. This measurement provides a quantitative determination of the validamycin concentration in the original water sample.

[0050] According to another exemplary embodiment of the invention, FIG. 3 refers to a graphical representation 300 of an effect of pH on the recovery (%) of the validamycin in a cloud point extraction method. In one embodiment herein, the pH levels affect the recovery efficiency of the validamycin during the cloud point extraction (CPE) process. With pH values ranging from 1 to 13 on the x-axis and percentage recovery on the y-axis, the experiment used specific buffer systems at each pH to maintain chemical consistency. The x-axis represents the pH of the solution, ranging from 3.0 to 5.8, with increments of 0.2. The y-axis represents the recovery (%), ranging from 0 percent to 100 percent, with increments of 20 percent. The recovery reaches its peak at a pH of around 4.0, where the recovery is approximately 88 percent. This indicates that a pH of 4.0 provides the most favourable conditions for the extraction of Validamycin using this specific CPE method and surfactant.

[0051] When the pH increases beyond the optimal value of 4.0, the recovery of Validamycin starts to decline. At pH 4.2, the recovery is still high (around 85 percent), but it gradually decreases to approximately 80 percent at pH 4.6, around 72 percent at pH 5.0, and further down to approximately 52 percent at pH 5.8. Overall, the trend follows a bell-shaped curve, confirming that CPE performance is highly pH-sensitive, with pH 4 identified as the optimal condition. These findings highlight the importance of pH control through acetate buffering for improving extraction efficiency in the validamycin detection.

[0052] According to another exemplary embodiment of the invention, FIG. 4 refers to a graphical representation 400 of an effect of the concentration of the surfactant Triton X-114. In one embodiment herein, the x-axis represents the concentration of TX-114 (% w/v), ranging from 0.0 to 2.8% w/v, with increments of 0.4% w/v. The y-axis represents the recovery (%), ranging from 0 to 100%, with increments of 20 percent. The line plot shows the trend of the recovery percentage as the concentration of TX-114 changes. Starting from a low concentration of TX-114 (around 0.6% w/v), the recovery is relatively low, approximately 46%. As the concentration of TX-114 increases, the recovery shows a significant upward trend.

[0053] The recovery continues to increase with increasing TX-114 concentration, reaching a peak or plateau region. The highest recovery is observed at a TX-114 concentration of around 2.0% w/v, where the recovery is approximately 88 percent. Beyond the optimal concentration, further increases in the TX-114 concentration appear to have a minimal positive or even a slightly negative impact on the recovery. The recovery at 2.4% w/v is around 82%, and at 2.6% w/v, it is approximately 81 percent, thereby indicating a slight decrease after the peak. There is an optimal concentration of the non-ionic surfactant TX-114 (around 2.0% w/v) for achieving the maximum recovery of the target analyte using this cloud point extraction method.

[0054] According to another exemplary embodiment of the invention, FIG. 5 refers to a graphical representation 500 of an effect of the concentration of the sodium chloride. In one embodiment herein, the x-axis represents the concentration of SDS (% w/v), ranging from 0.0 to 0.3% w/v, with increments of 0.05% w/v. The y-axis represents the recovery (%), ranging from 30 percent to 100 percent, with increments of 10 percent. At a low concentration of SDS (0.05% w/v), the recovery is approximately 53 percent. As the concentration of SDS increases to 0.1% w/v, the recovery shows a noticeable increase to around 64 percent. Further increase to 0.12% w/v leads to a more significant jump in recovery, reaching approximately 75 percent.

[0055] The highest recovery is achieved at an SDS concentration of 0.14% w/v, where the recovery peaks at approximately 88 percent. This suggests that this concentration of SDS provides the most favourable conditions for the analyte's extraction in the presence of the other components of the cloud point extraction system (like Triton X-114). Beyond the optimal concentration, increasing the SDS concentration leads to a decrease in the recovery percentage. At 0.16% w/v SDS, the recovery drops to around 81 percent, further decreasing to approximately 79 percent at 0.2% w/v and around 75% at 0.3% w/v. There is an optimal concentration of the anionic surfactant SDS (around 0.14% w/v) that maximizes the recovery of the target analyte in this mixed micellar cloud point extraction method. Concentrations of SDS lower than this optimum may not provide sufficient interaction or synergistic effect with the non-ionic surfactant (TX-114) for efficient extraction.

[0056] Table. 1
Validamycin
TX-114 SDS NaCl
Concentration
%w/v Recovery (%) Concentration
%w/v Recovery (%) Concentration
%w/v Recovery (%)
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.5 45.8
60.2
67.6
73.5
76.9
75
80.8
87.5
82.3
80.8 0.05
0.1
0.12
0.14
0.16
0.2
0.22
0.25 53.2
64.4
75.1
88.2
81.0
79.4
75.1
74.0 0.4
0.6
0.8
1.0
1.2
1.5
2.0 77.7
78.8
88.5
81.3
85.7
86.2
86.9

[0057] In one embodiment herein, the concentrations of the non-ionic surfactant TX-114 affect the recovery of the validamycin. The TX-114 concentration ranges from 0.6% w/v to 2.5% w/v. The corresponding recovery percentages at a lower TX-114 concentration of 0.6% w/v, the recovery is at least 45.8 percent. The TX-114 concentration increases, the recovery generally improves, reaching a peak of at least 87.5 percent at a concentration of 2.0% w/v. This suggests that 2.0% w/v is the optimal TX-114 concentration within this tested range for efficient validamycin extraction. The optimal concentration increases in TX-114 concentration (to 2.2% w/v and 2.5% w/v) lead to a slight decrease in recovery range from 82.3 percent and 80.8 percent, respectively.

[0058] In another embodiment herein, the impact of the anionic surfactant SDS concentration on Validamycin recovery. The SDS concentration ranges from 0.05% w/v to 0.25% w/v. The recovery percentages demonstrate at the recovery of at least 53.2 percent at the lowest tested SDS concentration (0.05% w/v). The consistent increase in recovery as the SDS concentration rises, reaching a maximum of 88.2 percent at a concentration of 0.14% w/v. This indicates that 0.14% w/v is the optimal SDS concentration for the validamycin recovery under the conditions tested. The optimal SDS concentration, the recovery starts to decline, with values of at least 81.0 percent at 0.16% w/v and further decreasing to at least 74.0 percent at 0.25% w/v.

[0059] In another embodiment herein, the influence of the salting-out agent NaCl concentration on the recovery of Validamycin. The NaCl concentration ranges from 0.4% w/v to 2.0% w/v. The recovery percentages show an initial recovery of at least 77.7 percent at the NaCl concentration of 0.4% w/v. The recovery increases with increasing NaCl concentration, reaching a peak of 88.5 percent at a concentration of 0.8% w/v. This suggests that 0.8% w/v is the optimal NaCl concentration for maximizing the validamycin recovery in this CPE system.

[0060] The NaCl concentration increases the optimum (to 1.0% w/v, 1.2% w/v, 1.5% w/v, and 2.0% w/v) result in a gradual decrease in the recovery percentage, although the recovery remains relatively high (above 80 percent). These optimal concentrations probable represent the conditions that provide the most favourable micellar environment and phase separation for the efficient partitioning and extraction of the validamycin from the aqueous matrix. The decrease in recovery beyond these optimal concentrations suggests that higher concentrations of these components might lead to counteracting effects, such as changes in micelle structure, increased viscosity, and reduced analyte partitioning into the surfactant-rich phase.

[0061] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, the method is disclosed. The proposed method develops a simple and efficient approach for the extraction and quantification of validamycin residues in water samples using a mixed micellar cloud point extraction (CPE) technique. The proposed method utilizes eco-friendly surfactants, Sodium Dodecyl Sulfate (SDS) and Triton X-114 (TX-114), thereby avoiding the use of harmful organic solvents common in conventional analytical techniques. The proposed method contributes to environmental and human health safety by enabling effective monitoring of pesticide contamination in water bodies near paddy and wheat fields.

[0062] The proposed method optimizes key extraction parameters such as potential of hydrogen (pH), surfactant concentration, salting-out agent (NaCl), equilibrium temperature, and incubation time to achieve maximum recovery of validamycin. The proposed method improves analytical sensitivity and detection limits, making the method suitable for trace-level detection of validamycin residues in agricultural runoff and field water. The proposed method offers a cost-effective, accessible, and scalable solution that might be applied in laboratories and on-site testing in rural agricultural settings.

[0063] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application. 
, Claims:CLAIMS:
I/We Claim:
1. A method for detecting and determining validamycin in a water sample using cloud point extraction, comprising:
adding a non-ionic surfactant Triton X-114 in a concentration of about 2.0% w/v and an anionic surfactant Sodium Dodecyl Sulfate (SDS) in a concentration of about 0.14% w/v to a water sample to form a surfactant-containing mixture;
adding sodium chloride (NaCl) in a concentration of about 3.0% w/v to the surfactant-containing mixture to promote micellar phase formation;
adjusting potential of hydrogen (pH) of the resulting mixture to at least 4.0 using an acetate buffer, thereby forming a pre-extraction solution;
heating the pre-extraction solution to an equilibrium temperature of at least 50 °C and maintaining the temperature for a time period of at least 15 min to induce cloud point extraction and enable phase separation of a surfactant-rich phase and an aqueous phase;
centrifuging and cooling the heated mixture to complete the phase separation process, and decanting the aqueous phase and collecting the surfactant-rich phase containing concentrated validamycin; and
measuring the absorbance of the surfactant-rich phase at a wavelength of 640 nm using UV-visible spectrophotometry to determine the concentration of the validamycin in the water sample.
2. The method as claimed in claim 1, wherein the water sample includes validamycin, wherein the water sample is collected from agricultural runoff, paddy fields, irrigation canals, and surface water near treated crop zones.
3. The method as claimed in claim 1, wherein the pH value of the resulting mixture is adjusted using the sodium acetate-acetic buffer to maintain the pH at 4.0 ± 0.2.
4. The method as claimed in claim 1, wherein the incubation temperature varies between 45 °C and 55 °C, and the incubation time period of at least 10 min to 20 min.
5. The method as claimed in claim 1, wherein the centrifugation is performed at a speed that varies between 3000 rpm and 5000 rpm for a time period of at least 5 min to 10 min to facilitate phase separation.
6. The method as claimed in claim 1, wherein the cooling of the heated mixture at the temperature varies between 4 °C and 10 °C to enhance the separation of the surfactant-rich phase.
7. The method as claimed in claim 1, wherein the UV-Visible spectrophotometric measurement is performed at a wavelength varies between 630 nm and 650 nm, with 640 nm being the optimal wavelength.
8. The method as claimed in claim 1, wherein the Triton X-114 and SDS are added to the water sample through a controlled distillation process, thereby ensuring homogenous dispersion and enhanced micelle formation.
9. The method as claimed in claim 1, wherein the method is configured to achieve a recovery efficiency of at least 95 percent for validamycin in the tested water sample.
10. The method as claimed in claim 1, wherein the method is configured to provide a limit of detection (LOD) for validamycin of 1.32 ng/mL and lower under optimized conditions.