Multiresidue Method for Quantification of Sulfonamides and Trimethoprim in Tilapia Fillet by Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry Using QuEChERS for Sample Preparation

A multiresidue method for detecting and quantifying sulfonamides (sulfapyridine, sulfamerazine, sulfathiazole, sulfamethazine, sulfadimethoxine, sulfamethoxazole, and sulfamethoxypyridazine) and trimethoprim in tilapia fillet (Oreochromis niloticus) using liquid chromatography coupled to mass spectrometry was developed and validated. The sample preparation was optimized using the QuEChERS approach. The chromatographic separation was performed using a C18 column and 0.1% formic acid in water and acetonitrile as the mobile phase in the isocratic elution mode. Method validation was performed based on the Commission Decision 2002/657/EC and Brazilian guideline. The validation parameters evaluated were linearity (r ≥ 0.99); limits of detection (LOD) and quantification (LOQ), 1 ng·g−1 and 5 ng·g−1, respectively; intraday and interdays precision (CV lower than 19.4%). The decision limit (CCα 102.6–120.0 ng·g−1 and 70 ng·g−1 for sulfonamides and trimethoprim, respectively) and detection capability (CCβ 111.7–140.1 ng·g−1 and 89.9 ng·g−1 for sulfonamides and trimethoprim, respectively) were determined. Analyses of tilapia fillet samples from fish exposed to sulfamethazine through feed (incurred samples) were conducted in order to evaluate the method. This new method was demonstrated to be fast, sensitive, and suitable for monitoring sulfonamides and trimethoprim in tilapia fillet in health surveillance programs, as well as to be used in pharmacokinetics and residue depletion studies.


Introduction
Brazil is one of the ve largest veterinary markets in the world, and aquaculture, in particular sh farming, is the fastest growing sector of animal food production in the country [1,2]. In sh farming, antimicrobials, including sulfonamides, are used for the treatment of bacterial diseases. Sulfonamides ( Figure 1) belong to an important group of synthetic antimicrobial agents that have been used in human and veterinary medicine for over 60 years. Recently, these drugs have been extensively employed in animals intended to produce food for human consumption since it is practically impossible to keep the production environment free of pathogenic organisms. Sulfonamides have become a useful tool for achieving high levels of productivity, thereby contributing to further growth, feed e ciency, and reduced mortality and morbidity [3]. However, sulfonamide residues are a major concern because of their potential risk to human health by development of bacterial resistance and adverse e ects, such as allergic reactions, in hypersensitive people [4].
Trimethoprim ( Figure 1) is a diaminopyrimidine antimicrobial agent, which is active against a wide range of Gram-positive and Gram-negative microorganisms including Escherichia coli and some Klebsiella, Proteus, and Staphylococcus species. In veterinary medicine, it is often used in combination with a sulfonamide to increase the antimicrobial activity of the sulfonamides but is excreted faster. Consequently, if no residues of sulfonamide are detectable, no residues of trimethoprim would be expected. Trimethoprim is of low acute mammalian toxicity, and there is no evidence for the potentiation of acute toxicity when it is administered in combination with a sulfonamide [5].
At its 40th session, the Codex Alimentarius Commission reported a maximum residue limit (MRL) value for sulfadimidine (sulfamethazine) of 100 µg·kg −1 in muscle, for species not speci ed [6]. According to the European Commission Regulation (EU) No. 37/2010 [7], for the muscle of n sh, the MRL value for individual sulfonamides, or the combined total residues of all substances belonging to the sulfonamide group, is 100 µg·kg −1 . In relation to trimethoprim, the MRL value is 50 µg·kg −1 . e MRL value relates to the muscle and skin in natural proportions. In Brazil, the use of sulfonamides in farm-raised sh is not permitted (it does not appear in the legislative framework) and, therefore, its use is considered out of label (prohibited substance). However, for monitoring purposes (and taking actions), the Brazilian National Plan for Control of Residues and Contaminants (PNCRC/Fish) establishes a reference limit of 100 μg·kg −1 for the residue of the individual sulfonamides (sulfachlorpyridazine, sulfadoxine, sulfamerazine, sulfadiazine, sulfamethoxazole, sulfathiazole, sulfamethazine, sulfaquinoxaline, and sulfadimethoxine) or the sum of them. Trimethoprim is not considered under the PNCRC/Fish sampling plan [8].
Studies on the determination of antimicrobial residues in foods of animal origin began in Belgium, the Netherlands, and Luxembourg in the late 1960s and early 1970s. In most European countries, research on residues and their application in inspection of slaughtered animals started later [9]. In relation to the sample preparation step, strategies such as salting out liquid-liquid extraction [10], solid-liquid extraction [11], and microscale matrix solid-phase dispersion [  have been employed to perform the extraction and cleanup of sulfonamides from sh and other biological matrices. More recently, Ziarrusta et al. [13] used focused ultrasound solid-liquid extraction (FUSLE) for extraction of uoroquinolones from sh tissues. e FUSLE method improves the extraction yield of target analytes (organic compounds), quantitatively, from biota samples. Regarding the systems of separation and detection, the high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is an analytical technique that has been used in the determination of veterinary drug residues. In this regard, a few sulfonamide multiresidue methods in food matrices have been described in the literature by this technique [14,15]. For instance, Abdallah et al. [16] determined sulfonamide residues in sheep, pork, beef, chicken, and dromedary, Nebot et al. [17] in bovine milk, Tsai et al. [18] in di erent sh species, and Jansomboon et al. [19] in Pangasius cat sh. Alternatively, a time-of-ight (TOF) mass spectrometer provides high sensitivity and accurate mass measurements (0.005 Da), enabling the detection of low concentrations (ng·g −1 ) of residues and contaminants in highly complex food matrices [15,20]. Nevertheless, to our knowledge, there is no reported multiresidue method for the combined quanti cation of sulfonamides and trimethoprim in tilapia llet using liquid chromatography coupled to quadrupole time-of-ight mass spectrometry (LC-QTOF/MS). e aim of this study was to develop and validate a rapid, simple (without the need of solid-phase extraction (SPE) cartridges or similar materials), and reliable multiresidue method for the identi cation and quanti cation of sulfonamides and trimethoprim in tilapia llets (Oreochromis niloticus) by LC-QTOF/MS, to be suitable for application in monitoring programmes as well as in pharmacokinetic and residue depletion studies. e sample preparation involved the QuEChERS (Quick, Easy, Cheap, E ective, Rugged, and Safe) approach as described by Lehotay et al. [21]. e validation was conducted in-house based on the Commission Decision 2002/657/EC [22] and Brazilian guideline [23]. To evaluate the precision of the method, analysis of tilapia llet samples from sh exposed to sulfamethazine through feed (incurred samples) was also conducted.

Instrumentation.
e identi cation and quantitation of sulfonamides and trimethoprim was carried out using an UPLC-Q-TOF system comprising an Acquity UPLC system coupled to a hybrid quadrupole orthogonal time-of-ight (Q-TOF) mass spectrometer (SYNAPT HDMS Q-TOF mass spectrometer) with electrospray source ionization (ESI) in positive mode. e software of acquisition control and data treatment was the MassLynx

Blank and Incurred Fish Samples.
e blank samples of tilapia (Oreochromis niloticus) with no detectable analyte concentration used for the development and validation of the analytical method were provided by a local producer (Rio Doce sh farm, São João da Boa Vista, SP) with a guarantee that the sh were not exposed to the compounds that were the analytical focus of this work. Nonetheless, to ensure the viability of the blank samples, they were analysed, and the chromatograms did not show the presence of any interference at the retention time corresponding to the studied analytes. For validation of the analytical method, blank samples and incurred samples (truly contaminated samples) were used, that is, samples of sh exposed to SMZ through feed, obtained from an experiment conducted at Embrapa Environment, Jaguariuna, SP, Brazil, where tilapia were given SMZ at a dose level of 422 mg·kg −1 body weight, for 11 consecutive days. e incurred samples used in this study were from sh slaughtered by thermal shock and immersion Journal of Analytical Methods in Chemistry in an ice bath, 12 h after stopping medication. All samples were stored in a freezer (−20°C) until analysis [24]. e experiment with sh to obtain the incurred samples was approved by the Ethics Committee on Animal Experiments of Embrapa Environment (Protocol No. 001/2013) [25].

Sample Preparation by QuEChERS.
Tilapia llet samples were ground using a domestic food processor. Triturated samples (2.5 g) were weighed in a 50 mL polypropylene tube, and ACN (5 mL) was added and then homogenized using a Turrax for 30 seconds. e homogenized sample was then added of 5 mL ACN, the tubes were shaken vigorously by vortexing for 1 min and placed in an ultrasonic bath for 5 min. Next, 2.0 g of anhydrous magnesium sulfate and 0.75 g of sodium acetate were added to the homogenized samples and vortexed for 1 min and centrifuged at 17,500 × g for 10 min, at 5°C. For sample cleanup, an aliquot of 5.0 mL of supernatant was volumetrically pipetted to another tube containing 150 mg of PSA and 0.5 g of anhydrous magnesium sulfate. e tube was subsequently vortexed for 30 seconds and centrifuged at 17,500 × g again for 5 min, at 5°C. A 2.0 mL aliquot of the supernatant was pipetted and transferred to another tube, and the solvent was completely evaporated under nitrogen stream, in an ice bath, to avoid losses of the analytes. Next, the residue was suspended in 0.5 mL of the mobile phase (ACN : 0.1% aqueous formic acid, 95 : 5 v/v). To facilitate the dissolution of analytes, the tubes were placed in ultrasonic bath for 5 min. Finally, the resulting extracts were ltered through a cellulose lter unit (0.22 µm pore size) directly into the vial and injected in the LC-QTOF/MS system. A schematic representation of the sample preparation procedure is shown in Figure 2. e sulfonamide and trimethoprim identity was con rmed by obtaining the accurate mass of the protonated molecular ion, as well as by the consideration of fragment ions in order to obtain the identi cation points (IPs) according to Commission Decision 2002/657/EC [22] ( Table 1).

Validation Parameters.
e purpose of this step was to establish the performance parameters and the minimum requirements of acceptance that must be satis ed such that the analytical method presented in this study is considered validated. e recommendations of the European Community [22] and the Guide to Analytical Methods Validation of the Brazilian Ministry of Agriculture, Livestock, and Supply [23] were used as reference to perform the method validation.
After optimization of the preparation procedure (extraction and cleanup), the validation of the analytical method was performed. e following validation parameters were evaluated: selectivity; linearity, sensitivity, and matrix e ect; precision (intra-and interday); accuracy; and decision limit (CCα) and detection capability (CCβ). e limit of detection (LOD) and limit of quanti cation (LOQ) were also assessed to evaluate the potential use of the analytical method in pharmacokinetic and residue depletion studies where lower LOD and LOQ are required. Selectivity of the method was evaluated by comparing the chromatograms obtained from blank samples (n � 10) and the samples spiked with sulfonamides and trimethoprim standard solutions (n � 10). e chromatograms were evaluated for the presence of the analytical signal at the same retention time observed for the mass-to-charge ratio (m/z) of the analytes of interest. e results were analysed by the method of least squares , and the linearity was expressed through the coe cient of determination (R 2 ) which was adopted as R 2 ≥ 0.99, as recommended by the Brazilian validation guide [23]. e matrix e ect was evaluated by comparing three di erent concentrations (12.5, 50.0, and 100.0 ng·g −1 ) of sulfonamides and trimethoprim, prepared in solvent and forti ed extracts. e intermediate precision was expressed by CV% of the results of three di erent concentrations with ve replicates of each concentration on three di erent days by the same analyst. e calculation of the decision limit (CCα) and the detection capability (CCβ) was based on the Commission Decision 2002/657/EC [22]. e decision limit is de ned as the lowest concentration level at which the method can discriminate with a statistical certainty of 1−α if the analyte is present. For substances with an MRL, the value of α is considered to be 5%. e calculation was performed by analysing 20 blank samples forti ed with the analyte at the MRL level. e concentration of the MRL plus 1.64 times the standard deviation corresponds to the CCα (α � 5%). e detection capability (CCβ) is the lowest amount of the substance that can be detected, identi ed, and/or quanti ed in a sample with an acceptable error probability (β). For substances with an MRL, the determination of CCβ can be accomplished by the analysis of 20 blank samples forti ed with the analyte in the decision limit (CCα). e value of CCα plus 1, 64 times the standard deviation, corresponds to the CCβ (β � 5%).
For each sulfonamide and trimethoprim, the LOD and LOQ were established by analysing the forti ed matrix with standard solution of the analytes. LOD was determined based on signal-to-noise approach.
us, LOD was expressed as the lowest concentration with a signal equal to three times the signal-to-noise ratio. e LOQ was taken as the rst level of the analytical curve, which was measured with acceptable precision (CV ≤ 20%) [26].

Results and Discussion
e representative sulfonamide veterinary drugs were chosen based on a study of their use in sh farming around the world, those monitored by the Brazilian National Plan for Control of Residues and Contaminants (PNCRC/Fish) of the Brazilian Ministry of Agriculture, Livestock, and Food Supply and those used for other animal species that could potentially be illegally employed in sh farming. us, sulfamethazine, sulfathiazole, sulfadimethoxine, sulfamerazine, sulfamethoxazole (monitored by the PNCRC/Fish [8]), sulfapyridine, sulfamethoxypyridazine, and trimethoprim (regulated for veterinary use [7], although not regulated for use in sh farming in Brazil) were selected. e maximum residue limit (MRL) adopted for all the sulfonamides (individual or the combined total residues) was 100 μg·kg −1 , and 50 μg·kg −1 for trimethoprim [7].

Sample Preparation Based on QuEChERS.
Dispersive solid-phase extraction (d-SPE) technique and QuEChERS have been previously used for the determination of veterinary drug residues in animal uids and tissues [16,27,28], but not for the concomitant determination of sulfonamides and trimethoprim in sh llet. It is well known that the step of sample preparation (extraction of analytes and cleanup of the extract) is crucial.
is approach can in uence the magnitude of the matrix e ect, depending on the amount of endogenous substances from which it is coextracted. Acetonitrile has been widely used in the extraction of analytes from complex matrices as it extracts analytes with few interfering compounds (e.g., low amount of lipophilic coextractives from the sample) and further promotes the precipitation of proteins. is is necessary because the lower the quantity of interfering content present in the extract, the less matrix e ect is observed, which leads to a better quality analysis [29]. Kruve et al. [30] reported the minimizing matrix e ect in LC-ESI-MS analysis by using extrapolative dilution. It was demonstrated by several tests using QuEChERS sample preparation procedure that the use of a greater volume of acetonitrile for analyte extraction of complex matrices tends to reduce the matrix e ect, possibly eliminating the matrix e ect if a suitable dilution is achieved. It should be mentioned that although LC-ESI-QTOF/MS technique is very selective, possible interference caused by matrix substances can lead to suppression or an increase in the ionization of the analytes of interest [31]. us, this study explores the extraction of sulfonamides and trimethoprim by using QuEChERS procedure making use of acetonitrile as the extracting solvent and extrapolative dilution.
Preliminary studies have shown that for the quantication of sulfonamides and trimethoprim in tilapia llet using the proportion of acetonitrile : sample 4 : 1 (v/w) showed the best results with fewer coextracts, thus decreasing the presence of interfering compounds. It is noteworthy that although the amount of sample used in this study was four times lower than that used by Lehotay et al. [21], it was possible to achieve an LOQ of 5 ng·g −1 for all analytes, consequently to the LC-ESI-QTOF/MS system used. Literature data show that the LOQ for SDZ was 36 ng·g −1 , using 5 g of the sample [32]. Stubbings and Bigwood [33] showed an LOQ for SP, STZ, SMZ, SDMX, SMX, and SMR of 50 ng·g −1 , also using 5 g of the sample. e addition of salts to promote the salting out e ect has been shown to enhance the optimization of the analyte recovery percentages in multiresidue methods since it increases the solubility of these molecules in the organic phase [34,35]. In the QuEChERS approach reported by Lehotay et al. [21], 6 g of anhydrous magnesium sulfate and 2.5 g of sodium acetate trihydrate were used. In the present method for extracting sulfonamides and trimethoprim from tilapia llet, 2 g of anhydrous magnesium sulfate and 0.75 g of sodium acetate trihydrate were employed. At the cleanup step, PSA and/or C18 were used. Since no signi cant variation was observed between them in relation to recovery values, we opted for the use of PSA only. is nding may be observed because the fat content in tilapia llet is low. ere are studies in matrices that have considerably higher fat content in which the concomitant use of PSA and C18 is required for a better cleanup of the sample extract [33]. [22], the identity con rmation of a substance is performed by a system of identi cation points (IPs). e mass accuracy of a highresolution mass spectrometer acquires 2 IPs for the precursor ion and 2.5 for each transition product. e resolution of mass spectrometer used in this study (SYNAPT HDMS Q-TOF) is more than 10,000, which fall within the criteria established by the guide as a high-resolution MS. Under the conditions selected, the protonated molecule and one fragmented ion for each analyte could be monitored, thus reaching the requirements to con rm their identity in accordance with Commission Decision 2002/657/EC [22]. For the quantitative purpose, only the sulfonamides and trimethoprim molecular ions were monitored.

Analytical Method Validation.
e method selectivity was evaluated by analysing ten samples free of analytes (blank samples) and comparing them to the chromatograms obtained from samples spiked with the sulfonamides and trimethoprim. Peaks for interfering compounds with the same retention times as the analytes of interest with the same m/z were not observed. erefore, the method performed is satisfactorily selective. Figure 3 shows the chromatograms of each analyte studied.

Journal of Analytical Methods in Chemistry
To study the linearity, sensitivity, and matrix e ect, the analytical results at the following concentrations were compared: 5.0, 12.5, 25.0, 50.0, 75.0, 100.0, 125.0, and 250.0 ng·mL −1 . Measurements were carried out for the analytes dissolved in the solvent, in the forti ed extract, and in the forti ed blank matrix (matrix-matched). e matrix e ect, expressed as a percentage, was calculated from the division between the areas obtained for the analyte in solvent and in the forti ed extract, at the same concentration level [36]. e highest matrix e ect value observed was 18,98%, which is below the maximum acceptable value by the validation guides (20%) [22]. us, the matrix e ect was considered irrelevant for this method. However, when comparing the analytical curves in extract with the curves in the forti ed blank matrix, it was noted that the slope (angular coe cient) of the curve for the matrix-matched sample was much lower, indicating the loss of analytes (sulfonamides and trimethoprim) during sample preparation step (extraction and cleanup).
us, for the present method a matrix-matched analytical curve must be employed.
Accuracy was evaluated from recovery tests (%), as recommended by the Commission Decision 2002/657/EC when no certi ed reference material (CRM) is available [22].
e experiment was carried out through the recovery test of the spiked samples at 3 levels (10.0, 20.0, and 40.0 ng·g −1 ), evaluating each level using 5 independent replicates on 3 consecutive days. Analytes SP, SMR, and TMP had satisfactory recovery values (between 79.5 and 103.6%), SMZ and SMPD showed intermediate recoveries (between 64.6 and 80.0), and STZ, SDMX and SMX exhibit lower recovery values (between 38.4 and 52.9) ( Table 2). Low recovery values for sulfonamides have been reported. Won et al. [37] reached a recovery of 58.8% for SDMX after extraction of this molecule from marine products, such as common eel, blue crab, shrimp, and at sh, among others. Sulfonamides' low recoveries have also been reported in other matrices. Summa et al. [38] report recoveries for SMX and SDM, extracted from eggs, around 60% and 55%, respectively. A review dealing about the presence of sulfonamides in edible tissues reports recoveries of various sulfonamides ranging from 40 to 67% for honey, 45-85% for pork veal, and 57-63% for salmon muscle [39]. Although recovery values found for some sulfonamides were below the percentage established in the validation guide [22], the method has been shown to be precise (CV% found is within the value speci ed in the validation guide), and the required LOQ was achieved, which leads us to consider that the method is suitable for the intended purpose. Nevertheless, this corroborates the need to use matrix-matched analytical curves for the quantication of the analytes in samples of unknown origin. e precision of the method was determined through intraday precision (repeatability) and interdays (intermediate precision) at three spiked levels and was expressed as coe cient of variation (CV%). e intraday and interdays precision were evaluated in the concentration levels at 10.0, 20.0, and 40.0 ng·g −1 , with 5 replicates at each level. Working in this concentration range, we can ensure the precision and accuracy since in the most dispersive points, the CV is ≤20%. e repeatability (analysed on the same day and same equipment) and the interdays precision (intermediate precision) are shown in Table 3.
For compounds with concentration levels lower than 100 ng·g −1 , the Commission Decision 2002/657/EC [22] and Brazilian validation guideline [23] recommend a maximum acceptable CV ≤ 20%. As shown in Table 3, the validation parameters (intraday and interdays precision) meet the speci cations recommended by both guides since they recommend a CV ≤ 20%. e decision limit (CCα) is a parameter that takes into account the precision of the method for establishing a critical reference level, from which we can conclude that a sample is classi ed as nonconforming with a probability of error of 5%. An additional critical parameter, detection capability (CCβ), is calculated for use with nonconforming samples in order to con rm their concentration, and their identities are conrmed with an error probability of 5% (β � 5%). e decision limit (CCα) and detection capability (CCβ) values for each of the analyte studied are shown in Table 4. For sulfonamides, values varied from 102.6 to 120.0 µg·kg −1 and from 111.7 to 140.1 µg·kg −1 for CCα and CCβ, respectively. For trimethoprim, those values were 70.0 and 89.9 µg·kg −1 , respectively. us, considering the MRL values of 100 µg·kg −1 and 50 µg·kg −1 , respectively, for sulfonamides and trimethoprim, established by the regulatory framework of the European Union in n sh [7], we can conclude that the method reported here is suitable for application in surveillance programmes of residues of sulfonamides and trimethoprim in n sh muscle samples. e evaluation of LOD and LOQ for the determination of sulfonamides and trimethoprim residues in tilapia llet was performed using the matrix-matched analytical curve forti ed with the analytes. e LOD and LOQ of the method were 1.0 ng·g −1 and 5.0 ng·g −1 for all sulfonamides and trimethoprim, respectively. e LOQ was validated by analyse of 10 replicates that showed a CV ≤ 20% for all of the analytes. is indicates that due to the low value of LOQ obtained, the method can be used by restrictive regulatory agencies of countries such as Japan [40], which, for the multiresidue method intended for quanti cation of veterinary drug residues in animal and shery products, adopt for individual sulfonamides and trimethoprim a LOQ value of 10 ng·g −1 and 20 ng·g −1 , respectively.

Analysis of Incurred Samples.
To assess the method developed, analysis was performed on genuinely contaminated (incurred) sh samples obtained from an experiment in laboratory where the shes were exposed to SMZ through the feed. is study was related to the e ects of dietary exposure to SMZ on the haematological parameters and hepatic oxidative stress biomarkers in Nile tilapia [25]. e residue of SMZ in the muscle of 10 independent samples analysed in the same day was 1,062.9 ± 53.2 ng·g −1 (mean value ± standard deviation), and the precision (CV%) was 5.0%. Due to the high concentration levels, the extract of the samples was diluted prior to injection to adjust the concentration to t the range of the analytical curve. is corroborates the precision of the method and provides con dence that it is appropriate Journal of Analytical Methods in Chemistry for the intended purpose and can be used by regulatory agencies in health surveillance programs, as well as in pharmacokinetics and residue depletion studies.

Conclusions
A multiresidue method for determination of sulfonamides STZ, SMX, SMR, SMPD, SDMX, SP, and SMZ and trimethoprim (TMP) in tilapia llet was developed and validated. e analytes selected were those most frequently used worldwide in sh farming and those with the greatest potential for illegal use. QuEChERS approach with extrapolative dilution was shown to be a simple and inexpensive sample preparation process that can be easily used in routine analysis. Quantitation by liquid chromatographyquadrupole time-of-ight mass spectrometry (LC-ESI-QTOF/MS) showed to be a selective and low detectability method.
us, the method is suitable for application in    SP, sulfapyridine; STZ, sulfathiazole; SMZ, sulfamethazine; SDMX, sulfadimethoxine; SMX, sulfamethoxazole; SMPD, sulfamethoxypyridazine; SMR, sulfamerazine; TMP, trimethoprim. a e MRL value adopted for the calculation of CCα and CCβ for all sulfonamides was 100 ng·g −1 [6]. b e MRL value adopted for the calculation of CCα and CCβ for TMP was 50 ng·g −1 [6]. 8 Journal of Analytical Methods in Chemistry monitoring programmes of residues of sulfonamides and trimethoprim in tilapia llet, even by countries such as Japan that adopt low LOQ values for analytical methods to be used in food for determination of residues of substances such as veterinary drugs. Also, it was shown to be appropriate to be used in pharmacokinetic and residue depletion studies.

Conflicts of Interest
e authors declare that there are no con icts of interest regarding the publication of this paper.