Development and validation of stability-indicating RP-HPLC method for the estimation of azoxystrobin in its formulations

Introduction

Azoxystrobin is a broad-spectrum fungicide with pyrimidine rings that is used in agriculture to protect crops against fungal infections. It was initially released in 1998 as a new fungicide with a unique biochemical method of action. It is used on grape vines, cereals, potatoes, apples, bananas, citrus, tomatoes, and other crops to prevent spore germination. Rusts, Downey and powdery mildew, rice blast, and apple scab are among the diseases it combats. The Azoxystrobin pesticide is less toxic to humans, other mammals, birds, insects, and earthworms, but it has the ability to penetrate soil and control fungal growth very effectively. The azoles class included the Epoxiconazole chemical. This chemical regulates the metabolism of fungal cells, which in turn regulates fungal growth. The combo product was used to reduce fungus development on crops all over the world. Because the molecules are chemically distinct, their functions are likewise distinct. The action of regulating the fungus in a different way resulted in the control of a wide spectrum of fungus. In the field of plant culture, this combination product has proven to be effective. For a better understanding, the full pesticide molecule must be examined for purity, stability, and other raw material, in-process, and solvent impurities. During the analysis, any analytical methods must be simple, repeatable, and cost-effective.HPLC is a simple and widely used analytical device that is used for qualitative and quantitative analysis efficiently in terms of cost, time, and simplicity. Furthermore, this process is repeatable and may be applied to quality control as well as research and development.

WU Ying-xuan et al.1 used High Performance Liquid Chromatography and Electrospray Ionization Tandem Mass Spectrometry to concurrently identify Azoxystrobin residues in legumes. At four spiking concentration levels of 0.05, 0.1, 0.2, and 0.5 mg/kg, the devised technique was verified. The linear ranges were 2.5 to 50 g/L, with average recoveries ranging from 89 to 99 percent and relative standard deviations ranging from 2.2 to 8.5 percent. Ehab M.H. Abdelraheemet al.2 used HPLC-UV to validate a technique for extracting and quantifying Azoxystrobin residues in green beans and peas, and the results were verified by GC–MS. For green beans and peas, mean recoveries varied from 83.69 % to 91.58 % and 81.99 % to 107.85 %, respectively, in HPLC-UV analysis. In GC–MS analysis, mean recoveries varied from 76.29 % to 94.56 % and 80.77 % to 100.91 %, respectively. The approach has been shown to be effective for extracting and determining Azoxystrobin residues in green beans and peas based on these findings. P.Marczewska et al.3 used high performance liquid chromatography with diode array detector (HPLC-DAD) in suspension concentrate pesticide formulations to create a technique for the simultaneous qualitative and quantitative measurement of Azoxystrobin and its related impurity (Z)-azoxystrobin. Individual recovery rates for azoxystrobin and (Z)-azoxystrobin were 97–103 % and 90–110 %, respectively. The impurity ((Z)-azoxystrobin) had a limit of quantification (LOQ) of 0.3 µg mL⁻¹, which was acceptable because it was less than the maximum permissible level under the regulations.Monica et al.4 described a unique and sensitive technique for extracting, preconcentrating, and determining azoxystrobin and chlorothalonil, two extensively used fungicides. Solid-phase extraction (SPE) using a polymeric substance functionalized with gold nanoparticles (AuNPs) as sorbent is followed by high-performance liquid chromatography (HPLC) with a diode array detector in the proposed process (DAD). When applied to drinking and ambient water samples, the suggested approach enabled for the identification of fungicides as low as 0.05 µg L⁻¹ and provided good recoveries (75–95%). For the detection of isopyrazam (IZM) and azoxystrobin (AZT) in cucumbers, Dan Hu et al.5 suggested a quick and sensitive analytical approach based on high-performance liquid chromatography–tandem mass spectrometry. At fortification doses of 1, 20, and 500 μg kg⁻¹(n = 3), the suggested technique resulted in excellent recovery of IZM and AZT (91.48 to 114.62 %) and relative standard deviations of less than 13.1 %.IZM and AZT quantification limits were 0.498 and 0.499 μg kg⁻¹, respectively, substantially below the maximum residue level (0.5 mg kg⁻¹) specified for this kind of material.SHI Feng et al.6 established a technique for determining azoxystrobin residue in Citrus Shatangju. The following were the HPLC conditions:The mobile phase is V(acetonitrile)/V(H2O)=70/30, with a flow rate of 1.0 mL/min, an injection volume of 10 mL, and a detection wavelength of 257 nm. The average recovery of azoxystrobin was 85.23 %～92.04 %, and the lowest detection concentration was 0.01 g/g, respectively, which might be in line with pesticide residue standards. G. P. Balayiannis and colleagues7 devised and validated a technique for determining the active ingredients (a.s.) azoxystrobin, topramezone, acetamiprid, fluometuron, and folpet in commercially available formulations. All individual chemicals were recovered in the range of 97.8%–100.9 %.

Figure 1

Chemical structure of Azoxystrobin

Chemicals Used

Here, we used Azoxystrobin working Standard, Amistar Fungicide, Methanol- AR, Sodium Hydroxide — AR, Hydrochloric Acid — AR, Acetonitrile and Millipore Water.

Preparation of Azoxystrobin Standard Solution

Weigh accurately about 50 mg of Azoxystrobinworking Standard and transfer to a 50 ml volumetric flask. Add 20 ml of diluent and sonicate to dissolve. Dilute to volume with diluent and mix. Transfer 1.0 ml of solution into a 10 ml of volumetric flask and dilute to volume with the diluent and mix.

(Dilution scheme: 50mg +50.0 ml + 1 ml /10.0 ml)

Forced degradation

The forced degradation experiments are carried out to determine the stability indicating nature of assay method and to look for any deteriorated compounds. Azoxystrobin WS and the sample (AMISTAR FUNGICIDE) are exposed to 5N HCl, 5N NaOH, thermal degradation, and UV degradation. All of the aforesaid solutions were chromatographed and the chromatograms were recorded. For degradation, the following stress conditions are used.

Table 3

System suitability – forced degradation

Sr. No.	Area of azoxystrobin
1	2012.30
2	2001.38
3	2008.47
4	2003.08
5	2023.58
Mean	2009.76
Standard Deviation (±)	8.86
(%) Relative Standard Deviation	0.44

Table 4

Conditions — forced degradation

Sample stress condition	Description of stress condition
Acid degradation	5N HCl heated at about 60oC for 10 min on a water bath.
Alkali degradation	5N NaOH heated at about 60oC for 10 min on a water bath.
Thermal degradation	105ºC for 12 hours
UV degradation	expose to UV-radiation for 7 days

Figure 2

Chromatogram of Azoxystrobin sample in Acid degradation

Figure 3

Chromatogram of Azoxystrobin sample in Base degradation

Figure 4

Chromatogram of Azoxystrobin sample in Thermal degradation

Figure 5

Chromatogram of Azoxystrobin sample in UVdegradation

Table 5

% of degradation by applying different conditions

Acid Stress	% Degradation
Standard	0.020
Sample	0.015
Alkali Stress	% Degradation
Standard	0.008
Sample	0.008
Thermal Stress	% Degradation
Standard	0.625
Sample	0.020
UV Stress	% Degradation
Standard	0.429
Sample	0.037

Acceptance criteria

Correlation coefficient should be greater than or equal to 0.999. According to the analytical procedure, the system suitability requirements fulfilled the pre-established acceptance criteria. (Refer to Table 5 for system suitability results).

Table 6

System suitability - Linearity of sample

Sr. No.	Area of Azoxystrobin
1	S 2120.54
2	2113.57
3	2111.26
4	2125.79
5	2121.20
Mean	2118.47
Standard Deviation (±)	5.94
(%) Relative Standard Deviation	0.28

The average peak area of Azoxystrobin peak was measured at each concentration level and linearity graph was plotted against the sample concentration in percentage. The results of linearity study are as given in Table 6.

Table 7

Results of linearity of sample

Linearity Level	Sample Concentration (in %)	Sample Concentration (inppm)	Peak Area	Correlation Coefficient
Level – 1	50	50	1149.52	0.999
Level – 2	75	75	1636.57
Level – 3	100	100	2130.60
Level – 4	125	125	2651.12
Level – 5	150	150	3173.48

Figure 6

inearity graph of Azoxystrobin sample

Figure 7

Chromatogram of Azoxystrobin sample

Summary and Conclusion

The validation data presented in this study reveals that the analytical method of assaying Azoxystrobin in AMISTAR FUNGICIDE by HPLC is determined to be suitable, selective, specific, precise, linear, accurate, and robust. At room temperature, the analytical solution is determined to be stable for up to 48 hours. Hence, it is concluded that the analytical method has been validated and may be used for regular analysis and stability testing.

Source of Funding

None.

Conflict of Interest

None.

Acknowledgments

The author thanks Analog labs Hyderabad, India and Department of Chemistry, Sri Venkateswara University, Tirupati, India for providing laboratory facilities.