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

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- aminosulphonic 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, 1.01 μgmL−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 effective. In this article, the method has been validated by applying it to samples from different environmental conditions.

1. Introduction

Pyraclostrobin (Methyl N-[2-(1-(4-chlorophenyl)-1Hpyrazol-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 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 are very persistent in the environment [3]. Pyraclostrobin is a lipophilic fungicide that is harmful to aquatic creatures, particularly to the fish. The distribution of pyraclostrobin residue in fish tissues under chronic toxicity has received a 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].

Figure 1.

Molecular formula & 3D Structure of Pyraclostrobin.

Several techniques has been applied for the determination of pyraclostrobin like Scanning electron microscope [9], rapid resolution liquid chromatography tandem 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 in implication for the routine analysis of different matrices. This sheds light to the need of alternative simple and sensitive methods for pyraclostrobin analysis. The 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 to determining pyraclostrobin in diverse environmental samples.

Common name	Pyraclostrobin
IUPAC name	(Methyl N-[2-(1-(4-chlorophenyl)1Hpyrazol-3-yloxymethyl) phenyl] (N-methoxy) Carbamate)
Trade name	Merivon
Appearance	Viscous Liquid
Molecular formula	C19H18ClN3O4
Weight	387.82 gmol^−1
Solubility (in water)	1.9 mg/L at 20 ºC

2. Experimental section

2.1 Chemicals, reagents and solution preparations

Pyraclostrobin (purify 98%), and sodium nitrite (NaNO₂), 4-Amino sulphonic acid were procured from Sigma-Aldrich (ACS reagent, ≥99%, MA, USA). Hydrochloric acid (HCl) were purchased from Hi-Media (AR reagent, 99% Mumbai, India). All the solutions were prepared in ultrapure water and the same was 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. 1% NaNO₂ were 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. 1% solution of para-amino sulphonic 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, a 3 mL of HCl and 3 mL of NaNO₂ was mixed with 25 mL of solution containing 4-Amino sulphonic 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 the 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 from agricultural area. The liquid samples were stored at 3°C for spectrophotometric analysis.

2.2.2 Solid samples

The solid samples potato, soyabean, peanut, wheat grain, pea, banana, mango, papaya and environmental soil were collected using clean polyethylene bags and wash several times with distilled water. The samples were cut small pieces and dried in hot-air-oven around 40 min. The samples were crushed with a pestle and homogenized. After this, 5.0 g vegetables and soil sample were added in 50 mL water and kept overnight at room temp. The next day, the sample was gently mixed with 50 mL ethanol and then centrifuged at 12,000 rpm for 15 minutes. The supernatant part of the vegetable sample were 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 for the quantitative determination of organochlorine (pyraclostrobin) using para-amino sulphonic 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, a 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 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 turn to light red color indicating the formation of color complex between pyraclostrobin and PAS as coupling reagents. The absorbance of the pyraclostrobin: PAS complex was measured in spectrophotometry at λ_max = 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 λ_max = 600 nm for pyraclostrobin complex was selected for quantitative determination. The calibration curve was drawn between the different concentrations of pyraclostrobin (3.0–12 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 λ_max value revealed a good correlation.

Figure 2.

The schematic representation for determination of pyraclostrobin.

2.3.1 Apparatus

The UV-Vis absorption spectrums were measured on a double beam spectrophotometer make Cary-60 UV-Vis spectrophotometer (Agilent technologies) along with 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 12,000 rpm for this coupling reaction. All glassware’s 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 reagents 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 pH of 4.0 (acidic medium). The coupling reagent of pesticide (pyraclostrobin) showed relatively a 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 pesticide 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 the suitable qualitative and quantitative analysis of pyraclostrobin as model compounds in an organic medium. For this, an aliquot 1.0 mL from test solution containing 3.0–12 μg mL⁻¹ of pyraclostrobin in 25 mL volumetric flask and then 1.0 mL reagent such PAS was introduced into the sample solution, resulting the formation of pyraclostrobin: PAS complex. The ultrapure water was used to record blank UV-Vis absorption spectrum which is used as a reference 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 3.0–12 μg mL⁻¹in the final solution volume at 25 mL. Here, the Beer’s-Lambert law explains the use of the terms absorbance, molar absorptivity and Sandell’s sensitivity relating to UV-visible absorption spectrometry was 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 a six replicate analyses of a solution containing 4.0 μg mL⁻¹.

Figure 3.

The schematic mechanism of complex formation.

3.3 Mechanism for the determination of pyraclostrobin using PAS coupling reagent

In this method, para-amino sulphonic 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 giving a mixture of products. The remaining arene and azo group reduced the color intensity of 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 sulphonic 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 a 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), breaking N–O (pi). This is based on more stability of aromatic di azonium salts as compared to aliphatic diazonium salts, because the electron-rich benzene ring stabilizes the N ≡ N group or delocalised 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 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 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 give the red color of pyraclostrobin:PAS azo complex is following. The diazonium salts act as an electrophilic nature and attacking 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 N=N double bond. Azo compounds are stable so the dyes do not fade.

UV-Vis spectrophotometry was used to reduce interference and avoid of the overlapped absorption bands during the determination of pyraclostrobin in an aqueous medium in the visible regions. This theory is based on energy absorbed causes changes in electronic energy there by the electrons are promoted from a ground state to an excited state [17]. The chromophoric functional groups are responsible to give 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 reagent or dyes and are also responsible for intensifying the color and give UV-Vis spectra at 600 nm (λ_max). Thus, the use of the color pyraclostrobin: PAS complex was successfully demonstrated for analysis of OCl e.g., 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 (λ_max) 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 pesticide 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, 1.0 mL optimum volume of PAS reagent 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).

Figure 4.

Effect of volume of reagent.

Figure 5.

Effect of concentration of reagent.

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 ranged of 2.0–6.5 during the extraction and pre-concentration of pyraclostrobin from real samples (Figure 6). The color intensity of sample solution was changed with the increases in pH upto 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 condition. Therefore, the concentration at pH 4.0 was selected for the quantitative analysis through UV-Vis spectrophotometry.

Figure 6.

Effect of pH.

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 group 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⁻¹of pyraclostrobin prior to coupling reaction and analyzed by the spectrophotometric method at 600 wavelengths. The tolerance limits of different foreign species ions along with other class of pesticides are given in Table 1 and Figure 7. The peak intensity or maximum λ_max 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 any color change with PAS coupling reagent or not any 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 spectrophtometric method was found free from interference of diverse substances and other pesticides, which are commonly associated with the determination of OCl like pyraclostrobin from environmental and agricultural samples.

Foreign species	Tolerance limit (mg/L)	Foreign ions	Tolerance limit (mg/L)
Fenvalrate	250	Na^+	600
Ethion	300	K^+	450
Carbendazim	450	Ca^2+	220
Acetampride	300	Zn^2+	450
Triazophos	150	Ba^2+	400
Kitazin	600	Al^3+	500
Glyphosate	450	SO4^2−	750
Metribuzin	250	Ni^2+	600
Permethrin	500	Cl^−	550

Table 1.

Effect of foreign species and ions.

Figure 7.

Effect of various pollutants and pesticides.

3.6 Analytical validation (determination) of pyraclostrobin using spectrophotometry

Some of the essence parameters such as linearity range, molar adsorptivity and sandell’s sensitivity, accuracy and precision, robustness and ruggedness, limit of detection (LOD), limit of quantification (LOQ), correlation coefficient (R²), 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 adsorptivity 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 absorptivities of the resulting colored complex and RSD % of response factors for 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 3.0–12 μg/1.0 mL of Pyraclostrobin solution. We have also calculated absorptivity and Sandell’s sensitivity (Table 2).

Figure 8.

Calibration curve for the different concentration of pyraclostrobin (3–12 μg mL⁻¹).

Figure 9.

UV-Visible absorption spectrum of red colored dye complex.

Parameters	Values for the reaction
λ max (nm)	600
Beer’s law limit (μg mL^−1)	3–12 μg/10 mL
Detection limits (μg mL^−1)	0.077
Quantification limits (μg mL^−1)	0.235
Molar absorptivity (L mol^−1 cm^−1)	2.7 × 10^4
Sandell’s sensitivity (μg cm^−2)	1.0 × 10^−5
Regression equation (y = bx + a)	y = 0.026x + 3.089
Intercept (a)	3.089
Slope (b)	0.026
Standard deviation	0.008
Relative standard deviation (%)	1.11
Correlation coefficient (r^2)	0.984

Table 2.

Optical characteristics and statistical data for the reaction.

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 of method was calculated by spiking definite concentrations in real environmental and agricultural samples. A good recovery % obtained with coupling reagent coupled with spectrophotometry was calculated in the range of 84.75–108.5% for pyraclostrobin in agricultural and environmental samples, displays in Table 3. The precision is another parameter that should be validated to know 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 the Beer’s range and finding out the absorbance. The different level of pesticide concentration (6 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 shows the precision, accuracy, and the selectivity for the determination of OCL like pyraclostrobin in agricultural and environmental samples.

Sample	Pyraclostrobin in original found (X)	Pyraclostrobin added in μg (Y)	Total found (Z)	Difference (μg) (Z-X)	Recovery (%) (Z-X)/Yx100	RSD
Water**	0.26	10	9.72	9.45	94.5	0.395
Soil***	0.21	10	9.78	9.56	95.6	0.170
Potato***	0.52	10	10.38	9.86	98.6	0.396
Soyabean***	0.31	10	10.24	9.93	99.3	0.408
Peanut***	0.38	10	10.20	9.82	98.2	0.764
Wheatgrain***	0.24	10	10.15	9.91	99.1	0.510
Pea***	0.25	10	9.83	9.58	95.8	0.396
Banana***	0.43	10	10.30	9.87	98.7	0.663
Mango***	0.27	10	9.72	9.44	94.4	0.172
Papaya***	0.32	10	9.67	9.35	93.5	0.174

Table 3.

Recovery of pyraclostrobin in environmental samples.

^*

Mean of three replicate Analysis Amount of sample 50 mL.

^**

Amount of sample 5 gm.

^***

Amount of sample 5 gm.

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 2). For this wavelength, negligible absorbance was observed for blank reagent. We had not observe any color dye azo formation in the presence of other classes of pesticide. In the study area, maximum absorption was obtained at 600 nm. The main reasons are less spectral interference and having 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 the 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 robustness the different parameters like pH, reagent concentration, wavelength range and time were remains unaffected by small deliberate variations. Method ruggedness was expressed as RSD % by six analysts with both the spectrophotometric on different days. The results showed no statistical differences between instrument suggesting that the developed methods were robust and rugged (Table 2).

3.6.5 LOD and LOQ

LOD is defined as a minimum quantity of substance that the instrument can respond to with signal-to-noise ratio to be three (S/N = 3) (or 3σ/k); LOQ is minimum quantity of substance when S/N = 10 (or 10σ/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 μgmL⁻¹and 3.08 μgmL⁻¹, respectively (Table 2).

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 to choose this region is that the numbers 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 spectrophotometric method. The absorbance of the red color of sample containing pyraclostrobin: PAS complex was measured in spectrophotometry at λ_max = 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 spectrophotometric method. 10 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 towards 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 standard concentrations 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 maximum wavelength (λ_max) at 600 nm, indicating the absence of the analytes. The concentration of pyraclostrobin was obtained using the linear regression equation model (Figure 8).

No. of days	Absorbance
1	0.709
2	0.715
3	0.719
4	0.707
5	0.722
6	0.727
7	0.730
Mean	0.718

Table 4.

Reproducibility of the method.

Concentration of pyraclostrobin = 6 μg/10 mL.

Table 5 provides the analytical data for determination of pyraclostrobin using coupling reagent 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) level as compared to other instrumental methods such as solid-phase micro extraction (SPME) GC-MS, ultra-performance liquid chromatography tandem mass spectrometry UPLC-MS/MS, HPLC-UV (Table 5). The results are indicating 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 and sensitive and cost-effective as compared to different spectroscopic and chromatographic methods [22, 24, 25, 26].

Methods	Linear range (μgmL^−1)	LOD (μgmL^−1)	LOQ (μgmL^−1)	correlation coefficient (R^2)	Samples	Ref.
solid-phase micro extraction (SPME) GC-MS	1.00–100.0	0.30	1.00	0.993	Environment	[22]
ultra-performance liquid chromatography tandem mass spectrometry UPLC-MS/MS	0.0001–0.1	0.70	2.23	0.999	aquatic ecological	[4]
LC-MS/MS	0.2–200	0.06	0.2	0.998	Environment	[23]
UV-visible spectrophotometer	3–12	0.077	0.235	0.984	Environment and agricultural	Present method

Table 5.

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

4. Conclusion

The present investigations were carried out with a view to examination the potential of para-amino sulphonic 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 determination of pyraclostrobin at lowest concentration and it can be easily detect by naked eyes due to prominent color variation of the sample solution with and without 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 quantity chemical and reagent, and involve time-consuming sample preparation processes. Our strategic endeavor was initiated with a coupling reagent followed by the analysis of pyraclostrobin on a convenient and simple spectrophotometric method without any requirement of specific cleanup and samples preparation steps. The current approach is simple, cost effective, and does not required specific sample preparation for better extraction, leading to enhanced extraction and recovery % in UV-visible region during the analysis. The method has shown a good sensitivity, reliability and easy preparation of the reagent compared with other existing extractive spectrophotometric determination methods.