High Performance Liquid Chromatography – Ultraviolet Method for the Determination of Fludioxonil Fungicide Residues: Application on Rice Grains Cultivated in Pakistan

An analytical method was developed and validated for the determination of udioxonil in rice samples. Rice samples for the study were collected from different regions of Pakistan. The method was based on safe and cost-effective extraction of udioxonil from rice grains using acetone and methanol (1:1), ecient clean-up through homogenous mixture of acidic aluminum (12 g) and activated charcoal (1 g) followed by liquid chromatographic determination with UV detection. Quantication was performed on Prospher Star C 18 (5 µm, 25 x 0.46 cm) column maintaining the temperature 40ºC and detector wavelength 212 nm using mobile phase 50:50 v/v methanol-water (pH 3.3) employing ow rate 1.0 mL.min -1 and 20 µL injection volume. The method showed linearity with correlation coecient greater than 0.998. The proposed method was precisely validated for rice sample of all regions, showing recoveries higher than 98%. Rice samples collected from Badin, Multan, Hyderabad, Lahore, Jahania and Sarghoda was found to have udioxonil residues 0.046, 0.045, 0,043, 0.040, 0.024 and 0.016 mg.kg -1 respectively, all below the maximum residual limit (MRL) level i.e. 0.05 mg.kg -1 whereas samples collected from Khanewal and Gularchi showed udioxonil residue above MRL i.e 0.065 and 0.058 mg.kg -1 respectively. However, udioxonil residues was not detected in rice sample collected from city Makhdumpur.


Introduction
Rice is the important staple sustenance and leading food commodity of developing world, which ranks third on the basis of its consumption. However rice crop is subjected to a number of diseases affecting its growth as well as quality and reducing the crop yield worldwide. Pakistan's chief summer crop is rice, which is cultivated over almost 11% area of total agricultural land distinguishing Pakistan as a top producer of rice commodity. Pakistani rice is famous for its taste and aroma and it is a major source of foreign export earnings. Punjab and Sindh are major rice producing provinces in Pakistan, and accounts for about 88% of total rice production (U.S. Department of Agriculture 2017).
Rice commodity is majorly affected by fungi e.g. fusarium species spoiled by penicillium and Aspergillus (Park et al. 2005). Fludioxonil, chemically 4-(2, 2-di uoro-1,3-benzodioxol-4-yl)-1H-pyrrole-3-carbonitril ( Fig. 1) is non-systematic, colorless and odorless phenylpyrrole fungicide. It is strongly effective against fungal pathogens like stem-base browning, snow mold and seeding blight and can be used to inhibit The determination of pesticide residues in food commodities is of major importance in relation to public health. Regular monitoring of pesticide residue is one of the necessary steps to achieve adequate level of consumer protection. Asia is on top in the world with highest average pesticide usage i.e. 6.5-60 kg.ha − 1 (Carvalho 2017). Pakistan is the second largest pesticide consumer country in south Asia (Waheed et al. 2017). Various analytical methods have been reported for the quantitative determination of udioxonil in variety of fruits, vegetables and food commodities. Residue of udioxonil was quantitatively analyzed in blueberries (Munitz, Resnik, and Montti 2013) and in grape and lettuce (Marín et al. 2003) by GC-NPD.  (Mercader et al. 2014). However, in spite of importance of rice as one of the most common staple foods, very few studies have been conducted on determination of udioxonil residues in rice e.g. udioxonil residues were quantitatively analyzed in brown rice and rice straw by gas chromatographic method (Ko, Ah-Young et al. 2015). To the best of our knowledge, no liquid chromatographic methods-ultraviolet have been reported for udioxonil residues in rice samples.
Present study reports development and validation of simple and cost effective liquid-chromatographic method with UV-detection for the determination of udioxonil residues in rice grains. After appropriate pretreatment method for its extraction and puri cation, liquid chromatography with UV-detection was used as analytical technique followed by validation employing FDA guidelines (FDA 2015). Rice samples were collected from nine different regions of two major rice-producing provinces of Pakistan namely Punjab (Khanewal, Multan, Lahore, Jahania, Sarghoda and Makhdumpur) and Sindh (Gularchi, Badin Hyderabad).

Chromatographic conditions
Separation was achieved on Purospher Star, C 18 (5 µm, 25 x 0.46 cm) column (Merck, Germany) maintaining the column temperature at 40ºC with optimized parameters including mobile phase methanol: water 50:50 (v/v) with pH adjusted at 3.3 using o-phosphoric acid (85%), observing detector response at 212 nm. Fludioxonil was eluted isocratically maintaining ow rate 1.0 mL.min − 1 . Prior to introducing into the system, all the solutions were ltered through 0.45 µm millipore lter followed by degassing on ultrasonic bath (Elma LC-30H model Singen, Germany).

Calibration curve
Accurately weighted 0.01 g udioxonil standard was dissolved in 25 mL methanol to obtain 400 mg.L − 1 stock standard solutions. It was prepared once and stored at 4°C protected from light. Six calibration standards of udioxonil within the linearity range 0.25-16 µg.mL − 1 were prepared in 25 mL volumetric ask. Calibration standard were prepared fresh daily and ltered through 0.45 µm lter before injecting in to the system.

Method validation
Validation of method was performed according to Food and Drug Administration (FDA) guidelines in term of accuracy, linearity, precision, speci city, system suitability, limit of detection (LOD) and limit of quanti cation (LOQ). E ciency of column performance was evaluated in term of tailing factor, number of theoretical plates and capacity factor. Six different concentration levels were used for calibration study. Linearity and regression characteristics were evaluated by using intercept, slope, correlation coe cient, standard error and standard error estimate. Percent recovery was calculated to determine accuracy of the method. LOD and LOQ were also calculated. For robustness study, minor deliberate changes were introduced in order to check the persistence of method. A robust assay for analysis of udioxonil was performed and validated. The chromatographic parameters were deliberately varied including mobile phase composition methanol: water 50:50 ± 2mL, pH 2.6-3.6, wavelength 212 nm ± 2, ow rate in range of 0.7-1.2 mL.min − 1 and chromatographic response was monitored.  Figure 2. presents distribution of rice sampling regions and strategy to obtain representative rice sample for the study. Purposeful sampling was carried out to get the representative samples. Rice eld was equally divided into nine parts by sketching imaginary lines. Approximating 50 g of rice samples were randomly collected from each part, homogenously mixed and considered as one composite sample. All the nine composite samples were collected following the same strategy.

Forti cation
Accurately weighed 100 g of all nine rice samples were separately soaked in 50 mL acetone followed by addition of 100 µL of 10 ppm udioxonil working standard solution and homogenized. The contents were allowed to penetrate in rice grains by keeping in the dark for 24 hours. After the grains have been well dried, all the samples were separately pulverized with a mechanical hand grinder to increase the surface area and to ensure better extraction of udioxonil from rice grain sample.

Extraction
The extraction was accomplished following the procedure described by Uddin et.al (Uddin et al. 2011).
Along with forti ed samples, a blank was also concurrently processed for extractions. Into a centrifuging tube, accurately weighed 4 g of already forti ed and pulverized rice sample was transferred followed by addition of 75 mL mixture of acetone and methanol (1:1) in two times. The contents were vigorously stirred and centrifuged at 2500 rpm for three minutes, supernatant was collected into the conical ask passing through Whatman lter paper supported by lter funnel. Into a separating funnel already containing 200 mL of 2.5% sodium sulfate solution, the ltered rice extract was transferred followed by addition of 25 mL of dichloromethane by rinsing the respective conical ask. The contents were vigorously stirred and then allowed for layer separation. The lower layer containing udioxonil extract was collected in a conical ask; the procedure was repeated with addition of 25 mL portion of dichloromethane twice in order to get maximum extraction of udioxonil. Furthermore, the contents were passed through the glass column containing about 25 g of anhydrous sodium sulfate (Na 2 SO 4 ) supported by glass wool. Finally, a 10 mL portion of dichloromethane was further passed to sweep the contents from the column completely. Analysis was performed in triplicate for each sample.

Clean-up
The extract (moisture-free) was concentrated up to 1-2 mL by rotary evaporator. The concentrated extract was carefully shifted to a glass column containing homogenous mixture of acidic aluminum (12 g) and activated charcoal (1 g) between the layers of anhydrous Na 2 SO 4 , followed by addition of 120 mL dichloromethane through the column. Prior to column preparation, acidic aluminum, charcoal and sodium sulfate were activated at 110°C for 6 hours.

Preparation of sample for analysis
The eluted extract was then placed on rotary evaporator for complete evaporation of solvent. The dried ask contains the expected residues of udioxonil which was then dissolved in 2 mL methanol for its quantitative determination. The samples in 2 mL methanol were mostly observed to be opaque. The clear and transparent samples obtained by ltration through 0.45 µm millipore lter paper was injected to the system for chromatographic analysis. Steps for extraction and clean-up were repeated for all the other samples before preparation for chromatographic analysis.

Method Optimization
In order to establish the optimum reliable analytical condition and to obtain the maximum sensitivity for identi cation and quanti cation of udioxonil, numerous parameters were varied to set the best chromatographic condition. Firstly, the maximum wavelength of udioxonil was measured on Shimadzu-1800 double beam UV-vis spectrophotometer. Figure 3 represent the spectra of udioxonil showing maximum wavelength at 212 nm. Further instrumental parameters including ow rate, composition and ratio of mobile phase and its pH were separately studied by injecting 10 µg.mL − 1 udioxonil standard solution prepared in methanol into the chromatograph. Furthermore, in order to get the improved selectivity in reversed-phase high performance liquid chromatography (HPLC), different combination of organic solvents were tried such as acetonitrile:water and methanol:water. It was observed that increasing the percentage of organic modi er causes a reduction in retention i.e. acetonitrile showed less retention time of analyte as compare to methanol. However, because of being carcinogenic nature of acetonitrile, methanol was preferred. Methanol:water in the ratio 80:20, 70:30, 60:40 and 50:50 with pH in the range 2.0-4.0 were tried in order to avoid retention and selectivity changes. The ow rate of mobile phase was varied between 0.7 mL.min − 1 and 1.2 mL.min − 1 . The best results in terms of short retention time, high resolution and good peak symmetry were obtained with mobile phase ratio 50:50 v/v methanol: water adjusting eluent pH 3.3 at detector wavelength 212 nm and ow rate 1.0 mL.min − 1 . The representative chromatogram of standard udioxonil is shown in Fig. 4.

Method validation
In order to establish the appropriateness of method for its future application, developed method was validated according to Food and Drug Administration (FDA) guidelines. Validated parameters include system suitability, linearity, precision, accuracy, limit of detection, limit of quanti cation and robustness (FDA 2015).

System suitability test
System suitability is an important step of method validation which represents the e ciency of column. It was evaluated by injecting the standard udioxonil solution into the system six times on each day of analysis. The data obtained for system suitability of the proposed method represented in Table 1 shows capacity factor (k') 2.177, theoretical plates (N) 5106, tailing factor (T) 1.16 and separation factor (α) 1.22. Number of theoretical plates above 2000 and tailing factor below 2 show good system suitability of the method.

Precision
Repeatability and precision of our method was con rmed by introducing the seven calibration standard of udioxonil three times within the same day (intra-day precision) and on two consecutive days (interday precision) of method validation. The % relative standard deviation (RSD) values within-day and in between two consecutive days was found to be in the range 0.63-1.71% and 0.09-1.33% respectively. Results expressed in Table 3 in terms of relative standard deviation (RSD) lie in the acceptance criteria.

Accuracy
Accuracy, one of the foremost parameter of method validation, was evaluated in the term of percentage recovery of udioxonil from rice sample. Recoveries were determined by adding known amount of udioxonil (0.10 µg.kg − 1 ) in rice grain samples of Punjab and Sindh. The recoveries were observed between 98.26-105.64% and 98.52-103.30% for two consecutive days of analysis (Table 3).

Limit of detection and quanti cation
Detection and quantitation limits of udioxonil were determined in relation to the chromatographic signal higher than three times and ten times to the baseline noise respectively. LOD and LOQ of udioxonil were found to be 0.0042 and 0.0126 µg.mL − 1 demonstrating the suitability of proposed method with respect to MRL for udioxonil in rice grains (Table 2).

Robustness
The robustness of the proposed method was assessed by evaluating the capability of method to withstand intended variation in the chromatographic parameters of developed analytical method. Parameters including mobile phase composition and pH, wavelength and ow rate were intentionally changed and compatibility of method was assessed. Theoretical plates and tailing factor represented in Table 4 con rms suitability of method for routine analysis.   Khanewal and Gularchi samples i.e 0.065 and 0.058 mg.kg − 1 respectively. It represents that untrained farmers had excessively sprayed the pesticide on rice eld without considering its potential dangerous effects. Fludioxonil was not detected in rice sample collected from city Makhdumpur. It may be due to human error or it is possible that its quantity is below the detection limit. One possible reason may be that udioxonil have not been sprayed in the eld. The results are represented in Table 5 and comparison of chromatographic response of un-spiked and spiked samples has been depicted in Fig. 5.

Conclusion
An inexpensive, simple and e cient LC-UV method for quantitative determination of udioxonil residues in rice commodity has been reported for the laboratories that don't have access to modern extraction techniques. Developed method has been successfully applied for analysis of rice samples that were collected from Khanewal, Gularchi, Badin, Multan, Hyderabad, Lahore, Jahania, Sarghoda and Makhdumpur region of Punjab and Sindh. Analysis involved extraction of udioxonil from rice with reduced amount of solvent and less matrix effect. Chromatographic analysis presented the sensitivity and speci city for the udioxonil determination in rice samples; the method was then validated according to the FDA guidelines 2015, proving suitability of method. Results demonstrated satisfactory recovery and reproducibility con rming excellent accuracy and precision of method. Results demonstrated detection of udioxonil in rice samples collected from Badin, Multan, Hyderabad, Lahore, Jahania and Sarghoda were found to be below its MRL level whereas its concentration was high in Khanewal and Gularchi samples. However, it was not detected in rice sample collected from city Makhdumpur. It is concluded that the proposed method can be applied in the laboratories those are not equipped with recent extraction techniques and modern instruments.

Declarations
Ethics approval and consent to participate Not applicable because the manuscript does not contain any individual's/person data.

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Availability of data and materials
All the data generated or analyzed during this study are included within the article.

Competing interests
The authors declare that they have no known competing nancial interest or personal relationship that cud have appear to in uence the work reported in this paper.