An In Situ Formation of Ionic Liquid for Enrichment of Triazole Fungicides in Food Applications Followed by HPLC Determination

An in situ formation of ionic liquid was used for preconcentration of four triazole fungicides in food samples. The microextraction method was used for the first time in the literature for preconcentration of triazole fungicides. In the developed method, tributylhexadecylphosphonium bromide ([P44412]Br) and potassium hexafluorophosphate (KPF6) were used for the formation of hydrophobic ionic liquid. After centrifugation, the fine microdroplets were produced in one step, providing the extraction step in a quick and environmentally friendly manner. The functional group of the hydrophobic ionic liquid was investigated using FT-IR. Various extraction parameters were studied and optimized. In the extraction method, 0.01 g of [P44412]Br and 0.01 g of KPF6, centrifugation at 4500 rpm for 10 min were used. The optimized technique provided a good linear range (90–1000 μg L−1) and high extraction recovery, with a low limit of detection (30–50 μg L−1). Methods for the proposed in situ formation of ionic liquid were successfully applied to honey, fruit juice, and egg matrices. The recoveries were obtained in a satisfactory range of 62–112%. The results confirmed the suitability of the proposed microextraction method for selective extraction and quantification of triazole fungicides.


Introduction
Triazole fungicides (TFs) are a class of extremely efficient systemic fungicides that contain a hydroxyl group (ketone group), a substituted phenyl group, and a 1,2,4-triazole group in the main chain [1]. The TFs are promoted with broad-spectrum, internal absorption characteristics, providing good stability for a long time [2]. They are commonly applied in the cultivation and postharvest storage of crops to protect them from fungal infections [3,4]. However, they may disturb endocrine activities, and have induced developmental toxicity in animals and human health [5]. For this reason, a reliable and effective method is still needed for the determination of triazole fungicides in various matrices.

Characterization of In Situ Ionic Liquid
The halide anion of tributylhexadecylphosphonium bromide ([P 44412 ]Br) and potassium hexafluorophosphate (KPF 6 ) was the main force for the formation of the hydrophobic ionic liquid. FT-IR spectra were used to confirm the formation of hydrogen bonding, as shown in Figure 1. In the FT-IR spectra, the characteristic peaks presented at 2850, 2920, and 2960 cm −1 were assigned to the C-H stretching or CH 3 stretching, while those at 1410 and 1465 cm −1 were attributed to the C-H bending and CH 3 bending vibrations of pure [P 44412 ]Br [26]. Moreover, the FT-IR spectra of the characteristic peaks showed the halogen compound vibrations at 577 and 795 cm −1 . This may have been due to a transfer of the bromide ion cloud electron to hydrogen bonding, and consequently, a decrease in the force constant [27]. Thus, the shift of the hexafluorophosphate counter-ion vibrations suggested the existence of hydrogen bonding between [P 44412 ]Br and KPF 6 when the hydrophobic ionic liquid was formed.
Molecules 2022, 27, 3416 3 bromide ion cloud electron to hydrogen bonding, and consequently, a decrease in force constant [27]. Thus, the shift of the hexafluorophosphate counter-ion vibrations gested the existence of hydrogen bonding between [P44412]Br and KPF6 when the hy phobic ionic liquid was formed.

Optimization of In Situ Metathesis-Reaction-Generated Ionic Liquid Combined with Liq Liquid Microextraction
Due to their low concentrations and matrix interferences in real samples, it is diff to directly analyze triazole fungicides. Therefore, a sample-preparation method is n sary before analysis. In this work, an in situ metathesis reaction that generated the liquid was combined with a liquid-liquid microextraction in the triazole fungicide an sis. In order to obtain a high extraction efficiency, various parameters had to be inv gated. The optimization was carried out using the aqueous solution (10 mL) contai 100 μg L −1 of each triazole fungicides. All experiments were performed at least in tripli In order to form the ionic liquid ([P44412][PF6]), [P44412]Br and KPF6 were selected cause the starting extraction solvent could be completely dissolved in the aqueous s tion, which promoted the analyte extraction in the absence of a dispersion solvent melting of this IL ([P44412][PF6]) was between 10 and 30 °C, and it could solidify at temperatures [24]. Moreover, [P44412][PF6] is denser than water, therefore it was eas collect as the bottom layer after centrifugation. The amount of [P44412]Br was studied in range of 0.01-0.10 g. The results are shown in Figure 2. The results showed that the m imum peak area was obtained at 0.01 g of [P44412]Br. Therefore, 0.01 g of [P44412]Br was sen.
The amount of KPF6 varied in a range of 0.01-0.10 g. The results are shown in Fi 3. The results showed that the maximum peak area was obtained at 0.01 g of KPF6. T the signal decreased at a higher amount of KPF6. Thus, 0.01 g of KPF6 was selected.

Optimization of In Situ Metathesis-Reaction-Generated Ionic Liquid Combined with Liquid-Liquid Microextraction
Due to their low concentrations and matrix interferences in real samples, it is difficult to directly analyze triazole fungicides. Therefore, a sample-preparation method is necessary before analysis. In this work, an in situ metathesis reaction that generated the ionic liquid was combined with a liquid-liquid microextraction in the triazole fungicide analysis. In order to obtain a high extraction efficiency, various parameters had to be investigated. The optimization was carried out using the aqueous solution (10 mL) containing 100 µg L −1 of each triazole fungicides. All experiments were performed at least in triplicate.
In order to form the ionic liquid ([P 44412 ][PF 6 ]), [P 44412 ]Br and KPF 6 were selected because the starting extraction solvent could be completely dissolved in the aqueous solution, which promoted the analyte extraction in the absence of a dispersion solvent; the melting of this IL ([P 44412 ][PF 6 ]) was between 10 and 30 • C, and it could solidify at low temperatures [24]. Moreover, [P 44412 ][PF 6 ] is denser than water, therefore it was easy to collect as the bottom layer after centrifugation. The amount of [P 44412 ]Br was studied in the range of 0.01-0.10 g. The results are shown in Figure 2. The results showed that the maximum peak area was obtained at 0.01 g of [P 44412 ]Br. Therefore, 0.01 g of [P 44412 ]Br was chosen. molar ratio of [P44412]Br to KPF6 was selected as 1:2.5 because the excess KPF6 ensured 100% completion of the in situ metathesis reaction [24].  Because the process of mass transfer and completely phase separation in an extraction procedure should be time-dependent, the effects of extraction speed and time on the peak area was studied [22]. The effect of centrifugation speed was evaluated in the range of 1500-5000 rpm (see Figure 4). It was found that the peak areas of all the analytes increased up to a 4500 rpm centrifugation speed, above which the peak areas slightly decreased due to disintegration of the phase of target analytes. Therefore, a centrifugation speed of 4500 rpm was selected. The extraction times studied ranged from 3 to 15 min, The amount of KPF 6 varied in a range of 0.01-0.10 g. The results are shown in Figure 3. The results showed that the maximum peak area was obtained at 0.01 g of KPF 6 . Then, the signal decreased at a higher amount of KPF 6. Thus, 0.01 g of KPF 6 was selected. The molar ratio of [P 44412 ]Br to KPF 6 was selected as 1:2.5 because the excess KPF 6 ensured 100% completion of the in situ metathesis reaction [24].  Because the process of mass transfer and completely phase separation in an extraction procedure should be time-dependent, the effects of extraction speed and time on the peak area was studied [22]. The effect of centrifugation speed was evaluated in the range of 1500-5000 rpm (see Figure 4). It was found that the peak areas of all the analytes increased up to a 4500 rpm centrifugation speed, above which the peak areas slightly decreased due to disintegration of the phase of target analytes. Therefore, a centrifugation speed of 4500 rpm was selected. The extraction times studied ranged from 3 to 15 min, Because the process of mass transfer and completely phase separation in an extraction procedure should be time-dependent, the effects of extraction speed and time on the peak area was studied [22]. The effect of centrifugation speed was evaluated in the range of 1500-5000 rpm (see Figure 4). It was found that the peak areas of all the analytes increased up to a 4500 rpm centrifugation speed, above which the peak areas slightly decreased due to disintegration of the phase of target analytes. Therefore, a centrifugation speed of 4500 rpm was selected. The extraction times studied ranged from 3 to 15 min, while other experimental conditions were kept constant (data not shown). The peak areas of most of the triazoles increased with increases in the extraction time, and reached the highest at 10 min. Therefore, 10 min was chosen to ensure an efficient extraction.
while other experimental conditions were kept constant (data not shown). The peak areas of most of the triazoles increased with increases in the extraction time, and reached the highest at 10 min. Therefore, 10 min was chosen to ensure an efficient extraction. Before HPLC analysis, acetonitrile (ACN) was added to dissolve the RTIL-rich phase due to its solubility property in the [P44412]PF6 phase and the compatibility with the mobile phase being used. The ACN volume was investigated in the range of 30-250 μL (as shown in Figure 5). It was found that ACN 200 μL provided the highest peak areas of all analytes. After that, the peak area decreased due to the dilution effect. When using ACN at less than 30 μL, the phase could not be completely dissolved. Thus, 200 μL of ACN was chosen.  Before HPLC analysis, acetonitrile (ACN) was added to dissolve the RTIL-rich phase due to its solubility property in the [P 44412 ]PF 6 phase and the compatibility with the mobile phase being used. The ACN volume was investigated in the range of 30-250 µL (as shown in Figure 5). It was found that ACN 200 µL provided the highest peak areas of all analytes. After that, the peak area decreased due to the dilution effect. When using ACN at less than 30 µL, the phase could not be completely dissolved. Thus, 200 µL of ACN was chosen. while other experimental conditions were kept constant (data not shown). The peak areas of most of the triazoles increased with increases in the extraction time, and reached the highest at 10 min. Therefore, 10 min was chosen to ensure an efficient extraction. Before HPLC analysis, acetonitrile (ACN) was added to dissolve the RTIL-rich phase due to its solubility property in the [P44412]PF6 phase and the compatibility with the mobile phase being used. The ACN volume was investigated in the range of 30-250 μL (as shown in Figure 5). It was found that ACN 200 μL provided the highest peak areas of all analytes. After that, the peak area decreased due to the dilution effect. When using ACN at less than 30 μL, the phase could not be completely dissolved. Thus, 200 μL of ACN was chosen.

Analytical Performance of the Proposed Method
Under the chosen extraction condition, a series of experiment were conducted to study the enrichment factors (EFs), extraction recoveries (ERs), linear ranges, limits of detection (LODs), limits of quantitation (LOQs), and precisions (intraday and interday). The results  Table 1. The calibration graphs were linear over concentration ranges of 90-1000 µg L −1 for myclobutanil, triadimefon, and tebuconazole, and 150-1000 µg L −1 for hexaconazole, with a coefficient of determination in the range of 0.9988-0.9991. The LODs were evaluated as the concentrations giving a signal-to-noise ratio of 3 (S/N = 3), and were in the range of 30-50 µg L −1 . The LOQs (S/N = 10) were in the range of 90-150 µg L −1 . The relative standard deviation (RSD) was determined using five solutions of 150 µg L −1 of each triazole fungicide. The RSD values of the retention times and peak areas were in the ranges of 0.40-0.80% and 4.84-6.23%, respectively. The EFs were in the range of 8.53-12.26. The ERs were in the range of 59.71-85.82. The chromatograms obtained from the proposed preconcentration method and direct analysis (without preconcentration) are shown in Figure 6. It was found that the developed method showed high chromatographic signals when compared to the direct analysis.

Analysis of Real Samples
To study the applicability of the proposed method, an in situ formation and preconcentration method using the ionic liquid was applied in the determination of triazole fungicides in honey, fruit juice, and egg samples. A standard addition method was applied to study the matrix effect (ME). ME(%) is expressed as the ratio of the slopes obtained from calibration curves of each analyte spiked into the samples to those obtained after extraction using the proposed method, according to Equation (1) [28]:

Analysis of Real Samples
To study the applicability of the proposed method, an in situ formation and preconcentration method using the ionic liquid was applied in the determination of triazole fungicides in honey, fruit juice, and egg samples. A standard addition method was applied to study the matrix effect (ME). ME(%) is expressed as the ratio of the slopes obtained from calibration curves of each analyte spiked into the samples to those obtained after extraction using the proposed method, according to Equation (1) [28]: ME (%) = slope of spiked real sample slope of standard solution × 100 In the absence of the ME, the slope of the calibration curves from both the standard solution and spiked real samples should be similar (ME (%) ≈ 100%). However, in the presence of ME, the signal intensity for the analytes can decrease or increase. Generally, ME values between 80 and 120% indicate no ME, ME values between 50 and 80% or 120 and 150% indicate minor MEs, and ME values less than 50% or greater than 150% indicate major MEs [29]. It was found that the MEs (%) were in the range of 76.9-113.9%.
In order to confirm the accuracy of the proposed method, the relative recoveries (RRs) were investigated using an analysis of three real samples spiked with five triazole fungicides at a concentration of 150 µg·L −1 within one day. As shown in Table 2, acceptable recoveries (62-112%) with relative standard deviations (RSDs) of less than 7.9% were obtained. The obtained results showed that no triazole fungicide residues were detected in all samples. These results confirmed that the proposed microextraction method could successfully be utilized to estimate triazole fungicide residues at trace levels in real samples with high accuracy and validity. The chromatograms of blank and spiked samples are shown in Figure 7. In the absence of the ME, the slope of the calibration curves from both the standard solution and spiked real samples should be similar (ME (%) ≈ 100%). However, in the presence of ME, the signal intensity for the analytes can decrease or increase. Generally, ME values between 80 and 120% indicate no ME, ME values between 50 and 80% or 120 and 150% indicate minor MEs, and ME values less than 50% or greater than 150% indicate major MEs [29]. It was found that the MEs (%) were in the range of 76.9-113.9%.
In order to confirm the accuracy of the proposed method, the relative recoveries (RRs) were investigated using an analysis of three real samples spiked with five triazole fungicides at a concentration of 150 μg·L −1 within one day. As shown in Table 2, acceptable recoveries (62-112%) with relative standard deviations (RSDs) of less than 7.9% were obtained. The obtained results showed that no triazole fungicide residues were detected in all samples. These results confirmed that the proposed microextraction method could successfully be utilized to estimate triazole fungicide residues at trace levels in real samples with high accuracy and validity. The chromatograms of blank and spiked samples are shown in Figure 7.     Table 3 shows a comparison of the developed microextraction in this work with other published methods [1,4,29,30]. The ionic liquid could be prepared under facile conditions, which simplified the experimental operation, and no complicated instrument was required. Compared to the other sample-preparation methods, the proposed method provided a wide linear calibration range (150-1000 µg L −1 ), a high precision (less than 5), and acceptable recoveries (61-112) within shorter extraction times for simultaneous extraction determinations for various samples. In conclusion, the proposed microextraction method was demonstrated as a simple, fast, effective, and environmentally friendly technique.

Instrumentations
The HPLC analysis was conducted on a Waters 1525 Binary HPLC pump (Water, Massachusetts, USA) equipped with a diode array detector (DAD). A Rheodyne injector with a 20 µL injection loop was used. Empower 3 software was used to acquire and analyze the chromatographic data. A Purospher ® STAR RP-18 endcapped (4.6 × 150 mm, 5 µm) column (Merck, Darmstadt, Germany) with an isocratic elution of acetonitrile and water at a ratio of 50:50 (%v/v) was used for the separations. The mobile phase flow rate was 1 mL min −1 . The detection wavelength was set at 220 nm.
The Fourier-transformed infrared spectra (FTIR) of the ionic liquid phase were measured using a Bruker Invenio-S FT-IR (Bruker Corp., Massachusetts, USA). Diamond-lensattenuated total reflectance (ATR) was used. A centrifuge (Centurion, Aberdeen, England) also was used.

Honey Samples
Honey samples were purchased from a supermarket in Maha Sarakham province. A total of 5 g of the sample was weighed into a 50 mL volumetric flask and diluted to the marker. The solution was filtered through a Whatman (no. 1) filter paper. After that, the filtrate was passed through a 0.45 µm nylon membrane filter before extraction using the proposed method.

Fruit Juice Samples
Passion fruit juice and pomegranate juice (commercial juice samples) were bought from the supermarket in Maha Sarakham province. An aliquot of fruit juice (30.0 mL) was centrifuged at 4000 rpm for 10 min and then filtered through a Whatman (no. 1) filter paper. The solution was then passed through a 0.45 µm nylon membrane filter before extraction using the proposed method.

Egg Yolk Sample
Chicken eggs were purchased from local markets in Maha Sarakham province. The yolk was separated from the white to reduce interference, since in the analysis of egg collected from animals treated with anthelmintics, it is known that the interferences are greater in the yolk [31,32]. Fortification of the sample was performed directly in the yolk, and a period of about 12 h was allowed to elapse before continuing with any of the extraction processes in order to improve the interaction between the analytes and the matrix compounds [33]. A total of 10.00 g of egg yolk was mixed well with 0.2 g of anhydrous Na 2 SO 4 . After that, 1% (v/v) acetic acid in acetonitrile (2.00 mL) was added and shaken vigorously by hand for 1 min, and the homogenized eggs were centrifuged at 3500 rpm for 5 min for complete fat and protein precipitation. The supernatants were collected using a microsyringe. The solutions were diluted with deionized water to 10.00 mL, 100 µL of acetic acid was added, and the solutions were centrifuged to ensure the complete precipitation of fat and proteins [32]. The samples were spiked with the triazole fungicides at different concentrations before fat and protein precipitation. The obtained clear solutions were then extracted using the proposed microextraction method.

In Situ Metathesis-Reaction-Generated Ionic Liquid Combined with Liquid-Liquid Microextraction
A schematic diagram of the microextraction procedure is shown in Figure 8. The mixed solution included the standard or sample solution (10.00 mL), 0.01 g of [P 44412 ]Br and 0.01 g of KPF 6 , which were added into a 15 mL conical centrifuge tube. Then, the tube was shaken to dissolve the [P 44412 ]Br and KPF 6 and to complete the in situ metathesis reaction. The solution was then centrifuged at 4500 rpm for 10 min. After that, the centrifuge tube was cooled in an ice bath until the [P 44412 ]PF 6 was generated. The [P 44412 ]PF 6 phase (upper phase) was separated and then diluted with acetonitrile (200 µL) to decrease the viscosity before being injected into the HPLC system.

Evaluation of Enrichment Factor (EF), Extraction Recovery (ER), Relative Recovery (RR), and Matrix Effect (ME)
To study the effects of experimental conditions on the extraction efficiency, the EF was calculated between the analyte concentration in the final phase (Cfinal) and the initial concentration in the analyte in aqueous sample solution (C0) according to the following equations: The %ER was expressed as the total percentage amount of the target analytes extracted into the sediment phase using the proposed microextraction method: where Vsed and V0 are the volume of sediment phase and the sample solution, respectively. The %RR was defined as the % amount of analyte recovered from the matrix (real samples) with reference to the extracted standard (standard spiked into the same matrix). RR(%) = C found -C real C added × 100 (4) where Cfound is the concentration of analyte after adding a known amount of working standard to the real sample, Creal is the analyte concentration in the real sample, and Cadded represents the concentration of a known amount of working standard that was spiked into the real samples. To study the effects of experimental conditions on the extraction efficiency, the EF was calculated between the analyte concentration in the final phase (C final ) and the initial concentration in the analyte in aqueous sample solution (C 0 ) according to the following equations: EF = C final /C 0 The %ER was expressed as the total percentage amount of the target analytes extracted into the sediment phase using the proposed microextraction method: where V sed and V 0 are the volume of sediment phase and the sample solution, respectively. The %RR was defined as the % amount of analyte recovered from the matrix (real samples) with reference to the extracted standard (standard spiked into the same matrix). RR (%) = C found − C real C added × 100 (4) where C found is the concentration of analyte after adding a known amount of working standard to the real sample, C real is the analyte concentration in the real sample, and C added represents the concentration of a known amount of working standard that was spiked into the real samples.

Conclusions
In the present study, an in situ extraction and preconcentration method using an ionic liquid for separation and preconcentration of triazole fungicides in honey, fruit juice, and egg samples was performed prior to high-performance liquid chromatographic analysis. The halide anion of tributylhexadecylphosphonium bromide ([P 44412 ]Br) and potassium hexafluorophosphate (KPF 6 ) was used for the formation of the hydrophobic ionic liquid. In the proposed microextraction method, forming the immiscible IL extraction phase and the transfer of analytes occurred simultaneously. The metathesis reaction and extraction were accomplished in single step, making the transfer of the analytes into the extracting phase very quick and efficient. The proposed method provided good repeatability, a wide linearity range, a high enrichment factor, and an acceptable extraction recovery for each compound, and matrix effects did not interfere with the quantification process. Therefore, the proposed method is recommended as a fast, simple, sensitive, and environmentally friendly sample-preparation technique.