Applications of Colorimetric Analysis in Agriculture Enviornmental Medical and Pharmaceutical Industries

#### **Chapter 6**

## Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid Coupling Reagent in Agricultural Soil/Environmental Samples by Spectrophotometric Analysis

*Chhaya Bhatt, Manish Kumar Rai and Joyce Rai*

#### **Abstract**

A simple and sensitive method for the determination of pyraclostrobin, a widely used fungicide, is described here, which is based on diazotization and coupling with 4-aminosulfonic acid in alkaline medium. The reaction mechanism is based on the pre-equilibrium formation of amine and diazonium salt followed by a rate-limiting attack of the diazonium ion at an N-atom (N-coupling) to appear the corresponding red-colored azo complex. The λmax, molar absorptivity and Sandell's sensitivity related to the UV-visible absorption spectrometry were found λmax = 600 nm, 2.7 × 104 L mol−1 cm−1 and 1.01 × 10−5 μg cm−2, respectively. Some of the important parameters like linearity range, limit of detection (LOD), limit of quantification (LOQ ), correlation coefficient (R2 ) and recovery% were calculated 3 to 12 μgmL−1, 1.01μgmL−1, 3.08 μgmL−1, 0.984 and 93.5–99.3%, respectively, for the determination of organochlorine like pyraclostrobin using coupling reagent. The advantages of the present method are its simplicity, high selectivity and cost-effectiveness. In this article, the method has been validated by applying it to samples from different environmental conditions.

**Keywords:** spectrophotometry, pyraclostrobin, coupling reaction, recovery percentage, agricultural and environmental samples

#### **1. Introduction**

Pyraclostrobin (methyl N-[2-(1-(4-chlorophenyl)-1 h-pyrazol-3-yloxymethyl) phenyl](N-methoxy) Carbamate) (**Figure 1**) is a new and widely used fungicide of the strobilurin group [1]. Pyraclostrobin acts through inhibition of mitochondrial

**Figure 1.** *Molecular formula and 3D structure of Pyraclostrobin.*

respiration by blocking electron transfer within the respiratory chain that results incessation of fungal growth [2]. Despite their beneficial agricultural applications, agrochemicals in general. It can be extremely toxic to humans and animals and is very persistent in the environment [3]. Pyraclostrobin is a lipophilic fungicide that is harmful to aquatic creatures, particularly to fish. The distribution of pyraclostrobin residue in fish tissues under chronic toxicity has received significant attention in recent years, but little is known about its distribution in fish tissues under acute toxicity conditions [4]. Pyraclostrobin is a broad-spectrum fungicide that protects against powdery mildew, rust, web blotch, downy mildew and rice blast, which are all caused by oomycetes, ascomycetes, basidiomycetes, and asexual fungal species [5]. Currently, the pyraclostrobin is the most widely used fungicide group in the world, employed extensively in agricultural production [6]. The unreasonable usage of this fungicide has led to excess contaminants flowing into the aquatic environment by farming irrigation and surface runoff, which has caused fungicide accumulation in the soil and water ecosystem [7]. This contamination affects the aquatic organisms, food safety and human health in a negative way. As a result, pyraclostrobin determination research has become a prominent field of research [8].

Several techniques have been applied for the determination of pyraclostrobin, like Scanning electron microscope [9], rapid resolution liquid chromatographytandem mass spectrometry [10], matrix solid-phase dispersion (MSPD) and gas chromatography, gas chromatography-mass spectrometry in selected ion monitoring mode (GC–MS, SIM) [11], QuEChERS and ultra-high-performance liquid chromatography-tandem mass spectrometry [12, 13]. However, these methods are sophisticated, expensive, time-consuming, and difficult to implement for the routine analysis of different matrices. This sheds light on the need for alternative simple and sensitive methods for pyraclostrobin analysis. Spectrophotometry is considered the most convenient analytical technique because of its inherent simplicity, low cost and wide availability in most quality control laboratories. Currently, a spectrophotometer method for determining pyraclostrobin in diverse environmental samples has been developed. In this article, we have proposed a spectrophotometer-based method for determining pyraclostrobin in diverse environmental samples.

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

#### **2. Experimental section**

#### **2.1 Chemicals, reagents and solution preparations**

Pyraclostrobin (purify 98%), sodium nitrite (NaNO2) and 4-amino sulfonic acid were procured from Sigma-Aldrich (ACS reagent, ≥99%, MA, USA). Hydrochloric acid (HCl) was purchased from Hi-Media (AR reagent, 99% Mumbai, India). All the solutions were prepared in ultrapure water, and they were used throughout the studies. A stock standard solution of pyraclostrobin (1000 μgmL−1) was prepared in an appropriate amount of analyte by dissolving in ultrapure water. Appropriate dilutions were made to obtain solutions containing pyraclostrobin in the concentration ranges from 3.0 to 12 μgmL−1. About 1% NaNO2 was prepared by dissolving 1.0 g of each targeted compound in 100 mL of ultrapure water. In addition, 1 N HCl solution was prepared by dissolving 8.3 mL of concentrated HCl in 100 mL of ultrapure water. A 1% solution of para-amino sulfonic acid (PAS) was prepared by taking 0.05 g of substance in 5.0 mL ethanol containing 10 mL of volumetric flask. To prepare a coupling reagent, 3 mL of HCl and 3 mL of NaNO2 were mixed with 25 mL of solution containing 4-amino sulfonic acid reagent. All sample solutions were stored at 5°C until the analysis.

#### **2.2 Sample collections and preparations**

#### *2.2.1 Liquid samples*

Water samples were collected in bottles with special care avoiding air trapping, followed by sealing with Teflon-lined screw caps, kept in ice and transported to the laboratory before 24 h. The liquid sample is collected near the agricultural area. The liquid samples were stored at 3°C for spectrophotometric analysis.

#### *2.2.2 Solid samples*

The solid samples of potato, soybean, peanut, wheat grain, pea, banana, mango, papaya and environmental soil were collected using clean polyethylene bags and washed several times with distilled water. The samples were cut into small pieces and dried in hot-air oven for around 40 min. The samples were crushed with a pestle and homogenized. After this, 5.0 g of vegetables and soil samples were added to 50 mL water and kept overnight at room temperature. The next day, the sample was gently mixed with 50 mL ethanol and then centrifuged at 12000 rpm for 15 minutes. The supernatant part of the vegetable sample was filtered through a 0.45 mm Whatman filter paper and stored at 3°C for 24 hours. Finally, the prepared agricultural and environmental soil samples were used to quantitatively determine organochlorine (pyraclostrobin) using para-amino sulfonic acid (PAS) as a coupling reagent in spectrophotometric analysis.

#### **2.3 Spectrophotometric determination of pyraclostrobin using coupling reagent**

The schematic procedure for the determination of organochlorine using PAS as a coupling reagent in agricultural and environmental samples is depicted in **Figure 2**. For this, 1.0 mL of pyraclostrobin (4.0 μg mL−1) was taken into 25 mL of clean and dry volumetric flask, and then 1.0 mL of a coupling reagent (PAS) was introduced

#### **Figure 2.**

*The schematic representation for determination of pyraclostrobin.*

into the sample under the 3–5°C temperatures. The pH of the sample solution was adjusted at 4.0 with the help of 1 N HCl and 1% NaOH solutions. The sample solution was turned to light red, indicating the formation of a color complex between pyraclostrobin and PAS as coupling reagents. The absorbance of the pyraclostrobin:PAS complex was measured in spectrophotometry at λmax = 600 nm for the quantitative determination of pyraclostrobin. Subsequently, standard calibration curves of different concentrations of pyraclostrobin were prepared by performing different sets of experiments in triplicate sets of analyses by spectrophotometry. The blank sample was prepared under a similar condition. The λmax = 600 nm for pyraclostrobin complex was selected for quantitative determination. The calibration curve was drawn between the different concentrations of pyraclostrobin (3.0 to12 μg mL−1) and their respective absorbance or value of color intensity using linear least square (LLS) equation, i.e., y = mx + c. Regression analysis of Beer's law plots at their respective λmax value revealed a good correlation.

#### *2.3.1 Apparatus*

The UV-vis absorption spectra were measured on a double beam spectrophotometer made of Cary-60 UV-vis spectrophotometer (Agilent Technologies) along with

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

99.99% accuracy of quartz glass. The absorption spectrum of the pyraclostrobin:PAS complex was recorded at 600 nm using a quartz cuvette with a path length of 1 cm. pH measurements were done using pH 700 EUTECH instrument. The centrifuge REMI R-4C was used at 12000 rpm for this coupling reaction. All glasswares were cleaned prior to use with an ultrasonic cleaning bath, PCI Analytics Pvt. Ltd., India, Model 3.5 L100H/DIC using mild detergents and rinsed with distilled water. The ultrapure water for solution preparation was obtained from a thermo fisher scientific Barnstead 234 Smart2Pure water system (conductivity 18.2 Ω 235−1).

#### **3. Results and discussion**

#### **3.1 Coupling reagent of pyraclostrobin**

In the present method, the organic reagent dye (i.e., PAS) combines with a pesticide (pyraclostrobin) to form a pyraclostrobin:PAS complex by the coupled reaction through the diazonium ion (-N〓N-). The color change of the sample solution from colorless to red indicated the formation of pyraclostrobin:PAS complex at a pH of 4.0 (acidic medium). The coupling reagent of pesticide (pyraclostrobin) showed a relatively narrow range of PAS reagent concentration that is suitable for quantitative analysis. Thereafter, UV-vis spectrophotometric with coupling reagent is a single-step extraction and pre-concentration process, recycled and the waste generated was easy to manage for the determination of pesticides like pyraclostrobin in different environmental and agricultural samples.

#### **3.2 Spectral characteristic and quantitative screening of OCl (pyraclostrobin:PAS) complex**

The UV-visible spectrophotometer was successfully applied for resolving complex mixtures for determination of pyraclostrobin in varieties of environmental and vegetable samples; recently, we have exploited the use of UV-vis spectrophotometry for the quantitative analysis of pesticide in environmental, vegetables and food samples based on the measurement of selected characteristic absorption bands of analyte complex with organic reagents in the UV-vis spectra [14]. In the present work, UV-visible spectrophotometer was used to acquire a suitable qualitative and quantitative analysis of pyraclostrobin as model compounds in an organic medium. For this, an aliquot of 1.0 mL from a test solution containing 3.0 to 12 μg mL−1 of pyraclostrobin in a 25 mL volumetric flask, and then 1.0 mL reagent such as PAS was introduced into the sample solution, resulting in the formation of pyraclostrobin:PAS complex. The ultrapure water was used to record the blank UV-vis absorption spectrum, which is used as a reference for the other spectral measurements. The absorption spectrum of the azo (pyraclostrobin:PAS) complex is depicted in **Figure 3**. The red color pyraclostrobin:PAS complex formed in the proposed reaction showed maximum absorbance at 600 nm. The color system obeyed Beer's law in the range of 3.0 to 12 μg mL−1in the final solution volume of 25 mL. Here, the Beers–Lambert law explains the use of the terms absorbance, molar absorptivity, and Sandell's sensitivity relating to UV-visible absorption spectrometry, which were calculated and validated. The molar absorptivity and Sandell's sensitivity of red color pyraclostrobin:PAS complex were calculated to be 2.7 × 104 L mol−1 cm−1 and 1.0 × 10−5 μg cm−2, respectively. The reproducibility of the method was assessed by carrying out six replicate analyses of a solution containing 4.0 μg mL−1 (**Table 1**).

#### **Figure 3.**

*The schematic mechanism of complex formation.*


#### **Table 1.**

*Reproducibility of the method.*

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

#### **3.3 Mechanism for the determination of pyraclostrobin using PAS coupling reagent**

In this method, para-amino sulfonic acid (PAS) is used as a new reagent for the formation of a color azo complex with target analytes, which is acts as a strong electrophilic in nature. PAS, a coupling reagent, reacts with OCl (pyraclostrobin) during the consumption of 4.0 mM of PAS dye to give a mixture of products. The remaining arene and azo groups reduced the color intensity of the reagent through disruption of the conjugation system in the PAS dye [15].

The schematic of the mechanism for selective detection of pyraclostrobin with PAS dye for coupling reaction is completed following step, and reactions are shown in **Figure 3**. The first step is diazotization process where the aromatic amine (p-amino sulfonic acid) is treated with nitrous acid or sodium nitrite, which is converted into nitrous acid in the presence of a strong acid like HCl at below 5°C temperature. This results in the loss of H2O and the formation of new triple-bonded nitrogen atoms. This is followed by two proton transfers from nitrogen to oxygen accompanied by reorganization of the pi bonding framework such as forming N∙N (pi) and breaking N∙O (pi). This is based on the greater stability of aromatic diazonium salts than aliphatic diazonium salts because the electron-rich benzene ring stabilizes the N≡N group or delocalized system [16]. If the temperature rises above 5°C, the benzene diazonium chloride decomposes to form phenol and nitrogen gas is given off (**Figure 3**). The second step is the coupling reaction, where a benzene diazonium chloride reacts with a benzene ring having pyraclostrobin as a pesticide to produced red color of azo compound. The mechanism involves an initial attack of a coupling agent (phenols or anilines) on an electrophilic diazonium ion, followed by the loss of a proton. This process is known as the coupling reaction (**Figure 3**). The reason for PAS reagent couples with pyraclostrobin in an acidic medium and gives the red color of pyraclostrobin:PAS azo complex is following. The diazonium salts act as an electrophilic nature and attack the nucleophilic site during the reaction. A colored precipitate of azo compound is formed immediately on reaction of diazonium salt with amines or phenols. Azo dyes are compounds that contain two aromatic fragments connected by an N〓N double bond. Azo compounds are stable, so the dyes do not fade.

UV-vis spectrophotometry was used to reduce interference and avoid overlapped absorption bands during the determination of pyraclostrobin in an aqueous medium in the visible regions. This theory is based on energy absorbed, which causes changes in electronic energy when the electrons are promoted from a ground state to an excited state [17]. The chromophoric functional groups are responsible for giving color due to the electrons excitation of the outer bonding electrons or lone pairs to these dyes. The auxochromes are the functional groups that are an integral part of PAS reagents or dyes and are also responsible for intensifying the color and giving UV-vis spectra at 600 nm (λmax). Thus, the use of the color pyraclostrobin:PAS complex was successfully demonstrated for analysis of Ocl, for example, pyraclostrobin in UV-vis spectrophotometry.

#### **3.4 Optimization and analytical parameters**

The coupling reagent of pyraclostrobin from sample solution was monitored by considering the absorption band obtained at 600 (λmax) in UV-vis spectrophotometry. The optimum conditions for analytical parameters such as reagent concentrations,

volume of reagent, volume of sample, effect of pH, extraction time and stirring rate were investigated for obtaining the efficient extraction of pesticides like pyraclostrobin from environmental and agricultural samples. The influences of each of the following variables on the reaction were tested.

#### *3.4.1 Effect of PAS reagent concentration and volume*

The effect of concentration and volume of PAS reagent were investigated for optimum coupling reaction as well as formation of stable pyraclostrobin:PAS complex by varying the concentration of reagent from 1.0 to 5.5 mM and volume of reagent from 0.2 to 2.0 mL. The absorbance of reaction solution increases with the reagent concentration, and the highest absorption intensity was attained at 5.5 mM. Similarly, a 1.0 mL optimum volume of PAS reagent is used for the production of maximum and reproducible color complex. Therefore, the 5.5 mM reagent concentration with 1.0 mL volume was used throughout the work (**Figures 4** and **5**).

#### *3.4.2 Influence of variable pH as an extracting medium*

The reactivity of pyraclostrobin was investigated by the effect of different pH values in the range of 2.0 to 6.5 during the extraction and pre-concentration of pyraclostrobin from real samples (**Figure 6**). The color intensity of the sample solution was changed with the increases in pH up to 4.0 to 6.0 and beyond the suddenly decreased due to the repulsive force of ions. In addition, the maximum absorption band was obtained when maintaining the pH 4.0 at 3°C temperature, indicating the formation of stable pyraclostrobin:PAS complex at acidic conditions. Therefore, the concentration at pH 4.0 was selected for the quantitative analysis through UV-vis spectrophotometry.

**Figure 4.** *Effect of volume of reagent.*

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

**Figure 5.** *Effect of concentration of reagent.*

#### **3.5 Effect of interfering ions and cross-contaminants**

Under the optimized experimental conditions, we investigated the interference study that was carried out for selective determination of pyraclostrobin as OCl in sample solution using PAS as coupling reagent. Selectivity describes whether methodology could discriminate interference of similar groups of compounds in the presence of analytes, which is determined by relative absorbance value by spectrophotometric method [18]. To assess the validity of the proposed method, the effects of various common foreign species and other pesticides were added to the standard solution containing 4.0 μg mL−1 of pyraclostrobin prior to the coupling reaction and analyzed by the spectrophotometric method at 600 wavelengths. The tolerance limits of different foreign species ions, along with other classes of pesticides, are


#### **Table 2.**

*Optical characteristics and statistical data for the reaction.*

#### **Figure 7.**

*Effect of various pollutants and pesticides.*

given in **Table 2** and **Figure 7**. The peak intensity or maximum λmax of pyraclostrobin remained unchanged in the presence of tested organic and inorganic chemical species at the optimized conditions of the proposed method. In addition, there was no color change with the PAS coupling reagent, or no coupled reaction was found in the presence of other classes of pesticides under the optimized conditions, at the same time as only pyraclostrobin displayed the color change from colorless to red color during the coupling reaction (**Figure 2**). Therefore, the spectrophotometric method was found free from interference of diverse substances and other pesticides commonly associated with determining OCl like pyraclostrobin from environmental and agricultural samples.

#### **3.6 Analytical validation (determination) of pyraclostrobin using spectrophotometry**

Some of the essence parameters such as linearity range, molar adsorptive and Sandell's sensitivity, accuracy and precision, robustness and ruggedness, limit of

#### *Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

detection (LOD), limit of quantification (LOQ ), correlation coefficient (R<sup>2</sup> ) and correlation estimation (R) for the determination of pyraclostrobin were estimated to determine the plausibility of using coupling reagent.

#### *3.6.1 Linearity range, molar adsorptive and Sandell's sensitivity*

The linearity range was obtained by constructing the calibration curve using the absorbance value versus different concentrations of pyraclostrobin, shown in **Figure 8**. The statistical parameters were given in the regression equation (y = mx + c) calculated from the calibration graphs, along with the standard deviation (SD) of the slope (m) and the intercept (c) on the ordinate and the SD residuals (SDy/x). The linearity of calibration graphs was proved by high values of the correlation coefficient (r) and small values of the y-intercepts of the regression equations. The absorption spectrum of the red color pyraclostrobin:PAS complex is shown in **Figure 9**, with maximum absorption at 600 nm. The apparent molar absorptivity of the resulting colored complex and RSD % of response factors for the spectrophotometric method were also calculated and validated (**Table 2**). Beer's law was obeyed, and the linearity graph is shown in **Figure 9** in the concentration range of 3.0 to 12 μg/1.0 mL of Pyraclostrobin solution. We have also calculated absorptivity and Sandell's sensitivity (**Table 3**).

#### *3.6.2 Accuracy and precision*

The accuracy of the method was determined by calculating the recovery percentage (%) of pyraclostrobin and by comparing the results with standard reference method (ICH Q2 R1 standard guideline) [19, 20]. The recovery % for pyraclostrobin method was calculated by spiking definite concentrations in real environmental and agricultural samples. A good recovery % obtained with coupling reagent

**Figure 8.** *Calibration curve for the different concentrations of pyraclostrobin (3–12 μg mL−1).*

**Figure 9.** *UV-visible absorption spectrum of red-colored dye complex.*


#### **Table 3.**

*Effect of foreign species and ions.*

coupled with spectrophotometry was calculated in the range of 84.75–108.5% for pyraclostrobin in agricultural and environmental samples, as displayed in **Table 4**. The precision is another parameter that should be validated to determine the reproducibility of the results (ICH Q2 R1 standard guideline). The precision of the proposed method was ascertained by actual determination of fixed concentration of the pesticide within Beer's range and finding the absorbance. The different levels of pesticide concentration (six times) are prepared three different times in a day and studied for intra-day variation and three different days to study inter-day variation. The percentage relative standard deviation (% RSD) of the predicted concentration from the regression equation is taken as precision. Accordingly, the RSD % was found to be 1.9% for pyraclostrobin. These results show the precision, accuracy and selectivity for the determination of OCL-like pyraclostrobin in agricultural and environmental samples.

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*


*\* Mean of three replicate Analysis Amount of sample 50 mL.*

*\*\*Amount of sample 5 gm.*

*\*\*\*Amount of sample 5 gm.*

#### **Table 4.**

*Recovery of pyraclostrobin in environmental samples.*

#### *3.6.3 Reproducibility*

The solution containing 4.0 μg mL−1 of pyraclostrobin was evaluated for reproducibility by performing three replicate analyses for 7 days, and then standard deviation (±SD) and relative standard deviation (RSD%) were calculated at ±0.008 and 1.11%, respectively (**Table 3**). For this wavelength, negligible absorbance was observed for blank reagents. We did not observe any color dye azo formation in the presence of other classes of pesticides. In the study area, maximum absorption was obtained at 600 nm. The main reasons are less spectral interference and high absorption of pyraclostrobin in an aqueous medium. The measurement repeatability expresses the closeness of the results obtained with the same sample using the reagent coupled with spectrophotometric analysis in a short period of time to give the smallest possible variation in all experimental results.

#### *3.6.4 Robustness and ruggedness*

The robustness and ruggedness were evaluated for the coupling reagent coupled with spectrophotometric determination of pyraclostrobin in agricultural and environmental samples. For evaluation of the method's robustness, the different parameters like pH, reagent concentration, wavelength range and time remain unaffected by small, deliberate variations. Method ruggedness was expressed as RSD % by six analysts using both spectrophotometric methods on different days. The results showed no statistical differences between instruments, suggesting that the developed methods were robust and rugged (**Table 3**).

#### *3.6.5 LOD and LOQ*

LOD is defined as a minimum quantity of substance that the instrument can respond to with a signal-to-noise ratio of three (S/N = 3) (or 3σ/k); LOQ is the

minimum quantity of substance when S/N = 10(or10σ/k) with measured precision during routine laboratory operating conditions [21]. Where σ is the standard deviation of replicate analysis values under the same conditions as for the sample analysis in the absence of the analyte, and k is the sensitivity, namely the slope of the calibration graph. The value of LOD and LOQ in the present work was calculated to be 1.01 and 3.08 μgmL−1, respectively (**Table 3**).

#### **3.7 Applications to agricultural and environmental samples**

The recommended method was successfully applied to the determination of OCl-like pyraclostrobin in various agricultural and environmental samples from rural residential sites of Raipur city. The reason for choosing this region is that a large number of crops are produced frequently. The details for the sample preparation procedure of agricultural and environmental samples are discussed in the experimental section and **Figure 2**. Finally, the prepared vegetables, soil, and environmental water samples were used for the quantitative determination of OCl (pyraclostrobin) using coupling reagent in the spectrophotometric method. The absorbance of the red color of the sample containing pyraclostrobin:PAS complex was measured in spectrophotometry at λmax = 600 nm for quantitative determination of pyraclostrobin in vegetable and environmental samples at a very trace level under optimum conditions, which is displayed in **Figure 9** followed by the spectrophotometric method. Ten agricultural and environmental samples were tested, which were obtained from rural farm sites of Raipur city, Chhattisgarh. Five agricultural and two environmental samples were found to be positive toward the presence of pyraclostrobin in significant concentrations (**Table 4**). The concentrations of pyraclostrobin were determined in pure real samples, and in the same samples after spiking, the standard concentration is 4.0 μgmL−1. Blank samples, those containing undetectable quantities of pesticide, were analyzed to evaluate the selectivity of the proposed coupling reagent coupled with spectrophotometric method. In addition, the blank samples did not show


#### **Table 5.**

*The comparison of present UV-Visible method with other reported methods for the determination of pyraclostrobin in varieties of the samples.*

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

maximum wavelength (λmax) at 600 nm, indicating the absence of the analytes. The concentration of pyraclostrobin was obtained using the linear regression equation model (**Figure 8**).

**Table 5** provides the analytical data for determination of pyraclostrobin using coupling reagents from agricultural and environmental samples in spectrophotometry and compared with the results of other reported methods in terms of linearity range, LOD and sample matrices. In the present work, coupling reagent was performed without using toxic solvents in spectrophotometry for the determination of pyraclostrobin in different agricultural and environmental samples at very microgram (μg) levels as compared to other instrumental methods such as solid-phase microextraction (SPME) GC-MS, ultra-performance liquid chromatography-tandem mass spectrometry UPLC-MS/MS and HPLC-UV (**Table 5**). The results indicate that a many-fold enhancement in the recovery % and extraction efficiency will be acquired using PAS coupling reagent-assisted microextraction of pyraclostrobin as compared to the other methods. The present method based on PAS:pyraclostrobin complex is found to be simple, eco-friendly, sustainable, selective, sensitive and cost-effective as compared to different spectroscopic and chromatographic methods [22, 24–26].

#### **4. Conclusion**

The present investigations were carried out with a view to examining the potential of para-amino sulfonic acid (PAS) as a coupling reagent for OCl-like pyraclostrobin present in various agricultural and environmental samples and its subsequent determination by coupling reagent coupled with spectrophotometric method. The pyraclostrobin:PAS complex is very sensitive for the determination of pyraclostrobin at the lowest concentration, and it can be easily detected by the naked eye due to the prominent color variation of the sample solution with and without the addition of analytes. The routine analysis of OCl is normally performed using sophisticated instruments such as GC-MS, LC/ESI-MS, HPLC, RS-TLC and UHPLC/QqTOF-MS, which are generally large in size, require rigorous training to operate, require large quantities of chemicals and reagent, and involve time-consuming sample preparation processes. Our strategic endeavor was initiated with a coupling reagent followed by the analysis of pyraclostrobin using a convenient and simple spectrophotometric method without any requirement for specific cleanup and sample preparation steps. The current approach is simple, cost-effective and does not require specific sample preparation for better extraction, leading to enhanced extraction and recovery % in UV-visible regions during the analysis. The method has shown good sensitivity, reliability and ease of preparation of the reagent compared with other existing extractive spectrophotometric determination methods.

*Advances in Colorimetry*

#### **Author details**

Chhaya Bhatt1 \*, Manish Kumar Rai1 \* and Joyce Rai<sup>2</sup>

1 School of Studies in Chemistry, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

2 Chhattisgarh Council of Science and Technology, Raipur, Chhattisgarh, India

\*Address all correspondence to: chhaya05bhatt@gmail.com and mkjkchem@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Colorimetric Determination of Pyraclostrobin Fungicide Using P-Amino-sulphonic Acid… DOI: http://dx.doi.org/10.5772/intechopen.111833*

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Section 6
