Determination of Three Typical Metabolites of Pyrethroid Pesticides in Tea Using a Modified QuEChERS Sample Preparation by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry

Pyrethroid pesticides are widely used on tea plants, and their residues of high frequency and concentration have received great attention. Until recently, the residues of typical metabolites of pyrethroid pesticides in tea were unknown. Herein, a modified “quick, easy, cheap, effective, rugged and safe” (QuEChERS) method for the determination of three typical metabolites of pyrethroid pesticides in tea, using ultra performance liquid chromatography tandem mass spectrometry, was developed. The mixture of florisil, octadecylsilane, and graphite carbon black was employed as modified QuEChERS adsorbents. A Kinetex C18 column achieved good separation and chromatographic peaks of all analytes. The calibration curves of 3-phenoxybenzoic acid (3-PBA) and 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) were linear in the range of 0.1–50 ng mL−1 (determination coefficient R2 higher than 0.999), and that of cis-3-(2-chloro-3,3,3-trifluoroprop-1-en-1-yl)-2,2-dimethylcyclopropanecarboxylic acid (TFA) was in the range of 1–100 ng mL−1 (R2 higher than 0.998). The method was validated and recoveries ranged from 83.0% to 117.3%. Intra- and inter-day precisions were lower than or equal to 13.2%. The limits of quantification of 3-PBA, 4-F-3-PBA, and TFA were 5, 2, and 10 μg kg−1, respectively. A total of 22 tea samples were monitored using this method, and 3-PBA and TFA were found in two green tea samples.


Introduction
It is estimated that 2.4 million tons of active pesticide ingredients are used annually, mainly in agriculture [1]. A more significant estimate indicates that pesticides are generally persistent; about half of the detected substances have already been eliminated, and another 10% to 20% are stable transformation products [2]. The issue of pesticide transformation products is important because they leave the active moiety intact, generate toxicologically more potent structures, and have smaller molecular mass and more polarity than their parent compounds, which increases their solubility and perdurability. The transformation products and/or metabolites of pesticides in crops may be due to impurities in formula products applied in the field, or the results of abiotic transformation and plant metabolism. Pesticides with their transformation products and metabolites enter the food chain and then are exposed to human beings, resulting in chronic and acute poisoning. though several approaches for the analysis of pyrethroid metabolites have been developed using UHPLC-MS/MS, these analytical protocols focused on environment water [17], soil [20], human urine, and plasma [21,22]. There are few methods for the determination of pyrethroid metabolites in agricultural crops and foods. For some simple matrices, such as human plasma and water [17], sample preparation did not involve clean-up [22]. However, for complex samples, such as soil [20], urine [21], wastewater [23], vegetables and fruits [24], and other substrates, it is necessary to carefully optimize the adsorbents and their dosage of dispersive solid-phase extraction (d-SPE) or SPE cartridges to obtain satisfactory purification effect. Therefore, the development of the UHPLC-MS/MS method for the complicated matrix is different from other current methods due to the optimization of sample preparation techniques. 21, 10, x FOR PEER REVIEW 3 of 12 derived with bis(trimethylsilyl) trifluoroacetamide (BSTFA) prior to GC-MS/MS analysis [19]. The UHPLC-MS/MS technique provided clear advantages for the analysis of transformation products and metabolites attributing to high selectivity, sensitivity, and accuracy. Although several approaches for the analysis of pyrethroid metabolites have been developed using UHPLC-MS/MS, these analytical protocols focused on environment water [17], soil [20], human urine, and plasma [21,22]. There are few methods for the determination of pyrethroid metabolites in agricultural crops and foods. For some simple matrices, such as human plasma and water [17], sample preparation did not involve cleanup [22]. However, for complex samples, such as soil [20], urine [21], wastewater [23], vegetables and fruits [24], and other substrates, it is necessary to carefully optimize the adsorbents and their dosage of dispersive solid-phase extraction (d-SPE) or SPE cartridges to obtain satisfactory purification effect. Therefore, the development of the UHPLC-MS/MS method for the complicated matrix is different from other current methods due to the optimization of sample preparation techniques. Tea is a complex matrix, rich in tea polyphenols, caffeine, polysaccharides, and other polar substances, which greatly interferes with the analysis of polar compounds, like pyrethroid pesticide metabolites. In this study, we developed a sensitive method based on UHPLC-MS/MS for the determination of typical pyrethroid metabolites in tea samples. UHPLC-MS/MS parameters, such as chromatographic column and mobile phase, were optimized, and three modified "quick, easy, cheap, effective, rugged and safe" (QuEChERS) techniques, i.e., original QuEChERS [25], Official CEN 15662 [26], AOAC 2007.1 [27], were evaluated, and the adsorbents were optimized step by step.

Sample Preparation
A 2.0 g portion of previously grounded tea was weighed into a 50 mL centrifuged tube. An internal standard, named 2-PBA, was added, and its concentration was 50 µg kg −1 by adding 100 µL of 2-PBA standard solution at 1 µg mL −1 . The internal standard fortified in tea samples was used to calibrate the loss of target compound during sample preparation. For the matrix-matched standard solutions, the internal standard 2-PBA was added to the concentration at 10 µg L −1 . Then, 2 mL of water was added and the tea sample was mixed on a vortex for 1 min. After that, 10 mL acetonitrile containing 1% acetic acid was used to extract the target compounds using an oscillator for 10 min. Then, 1.0 g of dehydrate citrate sodium and 0.5 g of disodium monohydrogen citrate were added to the mixture. The mixture was homogenized on a vortex for 1 min and then centrifuged at 10,000 rpm for 10 min. Following this, 2 mL of supernatant was transferred into a 5 mL centrifuge tube containing 200 mg florisil, 200 mg C18, and 100 mg GCB. The mixture was mixed on a vortex for 1 min and then centrifuged at 10,000 rpm for 10 min. The supernatant was filtered through a 0.22-µm membrane into the LC vial for UHPLC-MS/MS analysis.

Validation Procedure
The method was validated in accordance with the SANTE/11813/2017 standard, in terms of linearity, matrix effect, sensitivity, accuracy, and precision [28]. Linearity was evaluated using a matrix-matched standard solution at seven levels in the range of 0.1-200 µg L −1 in triplicates. Matrix matched standard solutions were prepared using the same type of analyzed tea samples, and internal standard 2-PBA was added to the concentration at 10 µg mL −1 . Matrix effect (ME) was evaluated comparing the slope of the calibration curve obtained from the solvent and matrix-matched standard solution in the same levels, according to the following equation: ME (%) = (slope of matrix-matched calibration curve/slope of solvent calibration curve-1) × 100. The limits of detection (LOD) and quantification (LOQ) were estimated from the UHPLC-MS/MS signal through the lowest spiked level. LOD was defined as the lowest spiked concentration that provided a signal to noise ratio of 3, and LOQ was defined as the lowest spiked concentration with recovery in the range of 70 -130% with relative standard deviations (RSDs) below 20%. Recoveries were calculated by comparing the measured values and spiked concentrations. Accuracy was expressed as recovery and assessed by the detecting concentrations in blank tea samples spiked with 10, 50, and 100 µg kg −1 . Precision was estimated as intraday and interday variability by recovery experiment of three replicates in three days.

UHPLC-MS/MS Optimization
The MRM parameters were optimized by direct injection of individual standard solution at 0.5 µg mL −1 through a syringe pump at the flow rate of 10 µL min −1 . Regarding analytes as acid compounds, ESI in negative mode was employed, and their deprotonated molecular ions [M − H] − were acquired by full scan in the mass range of m/z 50-300. Each deprotonated molecular ion of three target compounds and internal standard could achieve two transitions. The ion transition with a higher signal was used for quantification, and another transition was used for confirmation. The isomers of 3-PBA and 2-PBA had the same transitions. The declustering potential and collision energy were optimized to improve sensitivity. Table 1 shows MRM parameters for each compound.
The mobile phase was also investigated. Compared with methanol, acetonitrile had high elution ability, and better chromatographic peaks for all compounds were observed. Acid water with 0.1% formic acid resulted in no retaining on Kinetex column, poor separation of 3-PBA and 2-PBA, and mass spectrometric signal decreasing eight times. Therefore, a Kinetex C18 column with mobile phase using water and acetonitrile was employed for the separation of target compounds.

Modified QuEChERS Development
Three extraction methods, namely, original QuEChERS [25], Official CEN 15662 [26], and AOAC 2007.1 [27], were evaluated. As shown in Figure 3, the highest recoveries of all analytes for CEN 15662 treatment were obtained among three extraction methods. Acidified acetonitrile was generally used for the optimal extraction of acid compounds to achieve high extraction efficiency [31,32]. In this study, the analytical compounds and internal standard 2-PBA were acid compounds with log P less than 4.0. Therefore, the citrate-buffering salts employed for the Official CEN 15662 method helped to improve the recoveries of target compounds when compared with the original QuEChERS and AOAC 2007.1 method, without using acidified acetonitrile. The mobile phase was also investigated. Compared with methanol, acetonitrile had high elution ability, and better chromatographic peaks for all compounds were observed. Acid water with 0.1% formic acid resulted in no retaining on Kinetex column, poor separation of 3-PBA and 2-PBA, and mass spectrometric signal decreasing eight times. Therefore, a Kinetex C18 column with mobile phase using water and acetonitrile was employed for the separation of target compounds.

Modified QuEChERS Development
Three extraction methods, namely, original QuEChERS [25], Official CEN 15662 [26], and AOAC 2007.1 [27], were evaluated. As shown in Figure 3, the highest recoveries of all analytes for CEN 15662 treatment were obtained among three extraction methods. Acidified acetonitrile was generally used for the optimal extraction of acid compounds to achieve high extraction efficiency [31,32]. In this study, the analytical compounds and internal standard 2-PBA were acid compounds with log P less than 4.0. Therefore, the citrate-buffering salts employed for the Official CEN 15662 method helped to improve the recoveries of target compounds when compared with the original QuEChERS and AOAC 2007.1 method, without using acidified acetonitrile.
The selection of QuEChERS adsorbents is crucial for the recovery of analytes and the removal of the tea matrix. Several adsorbents, such as PSA, C18, GCB, florisil, and MWCNTs, have been employed for cleaning up the tea matrix [33][34][35]. In this study, seven adsorbents, e.g., NH3-NTs, MTCNTs, GCB, C18, PSA, florisil, and ZrO, were investigated. Two milliliters of tea extracts spiked with three target compounds and internal standard at 100 μg L −1 was added in a 5 mL centrifuged tube individually containing NH3-NTs  Table 2. Satisfied recoveries of all target compounds were achieved in the range of 92.5-108.3% for five adsorbents, except PSA and PVPP, in which recoveries ranged from 55.7% to 111.9%, and from 86.4-97.9%, respec-   were cleaned up by three carbon materials, such as NH3-NTs, MWCNTs, and GCB, because these adsorbents were particularly effective for tea co-extracted pigments [36]. Tea extracts were dark and opaque before and after clean up by ZrO, indicating that a few tea matrices were removed.   The selection of QuEChERS adsorbents is crucial for the recovery of analytes and the removal of the tea matrix. Several adsorbents, such as PSA, C18, GCB, florisil, and MWCNTs, have been employed for cleaning up the tea matrix [33][34][35]. In this study, seven adsorbents, e.g., NH 3 -NTs, MTCNTs, GCB, C18, PSA, florisil, and ZrO, were investigated. Two milliliters of tea extracts spiked with three target compounds and internal standard at 100 µg L −1 was added in a 5 mL centrifuged tube individually containing NH 3 Table 2. Satisfied recoveries of all target compounds were achieved in the range of 92.5-108.3% for five adsorbents, except PSA and PVPP, in which recoveries ranged from 55.7% to 111.9%, and from 86.4-97.9%, respectively. As shown in Figure 4, tea extracts became light yellow and transparent after they were cleaned up by three carbon materials, such as NH 3 -NTs, MWCNTs, and GCB, because these adsorbents were particularly effective for tea co-extracted pigments [36]. Tea extracts were dark and opaque before and after clean up by ZrO, indicating that a few tea matrices were removed. Table 2. Recoveries of target compounds after clean-up by amino carbon nanotubes (NH 3 -NTs), multi-walled carbon nanotubes (MWCNTS), graphite carbon black (GCB), octadecylsilane (C18), primary secondary (PSA), polyvinylpyrrolidone (PVPP), florisil, and zirconia (ZrO).

Adsorbents
Recoveries (%) extracts were dark and opaque before and after clean up by ZrO, indicating that a few tea matrices were removed.    Although the decolorization effect of C18 and florisil was not obvious, considering the fact that C18 and florisil were useful and employed to remove some mid-polar and high lipid contents [37,38], the mixture of 200 mg C18 and 200 mg florisil combination with individual MWCNTs and GCB was further evaluated as modified QuEChERS. As shown in Table 3, the mixture

Method Performance
The linearity of the present method was evaluated using acetonitrile and matrixmatched calibration curves at seven different concentration levels in the range of 0.1 to 100 µg L −1 in triplicates at each level. 2-PBA at 10 µg L −1 was used as an internal standard for calibration of 3-PBA, 4-F-3-PBA, and TFA. The calibration curves of 3-PBA, 4-F-3-PBA, and TFA were obtained by plotting the peak area ratios against the concentrations. Satisfactory linearity of three analytes was obtained with determination coefficients (R 2 ) higher than 0.998 in both acetonitrile and tea matrix-matched calibration solutions (Table 4). Table 4. Linear range and equation with correlation coefficient (R 2 ), matrix effect (ME, %), the limit of detection (LOD, µg kg −1 ), and the limit of quantification (LOQ, µg kg −1 ). Matrix effects (MEs) were evaluated by comparison of the slope ratio between matrixmatched calibration curves and acetonitrile calibration curves. All target compounds showed a strong matrix suppression effect, ranging from −53.2% to −83.7%. The black tea matrix resulted in a more serious suppression effect than that of the green tea matrix. Strong Mes were also observed for the determination of pyrethroids degradation products in human urine samples [18]. Therefore, the matrix-matched calibrations were used for quantification purposes, employing blank green tea samples and black tea samples.

Compounds
Blank tea samples spiked at 1, 2, 5, and 10 µg kg −1 were determined for the evaluation of LODs and LOQs. As shown in Table 4, LODs of 3-PBA, 4-F-3-PBA and TFA were 1.9-2.2, 0.5 and 5.2-6.1 µg kg −1 , respectively, while LOQs were 5, 2, and 10 µg kg −1 for 3-PBA, 4-F-3-PBA and TFA, respectively, in green tea and black tea samples. Mortimer et al. developed a sensitive gas chromatography electronic capture detector (GC-ECD)-based method for the determination of pyrethroid pesticides metabolites, where the LODs of 3-PBA and TFA were 5.0 and 1.4 µg L −1 [12]. Although the sensitivity of the previous study reported by Mortimer et al. was similar to this developed method, its complicated and time-consuming derivatization step was a shortage. Meanwhile, the LODs depended on the coextractive interferences because of the poor selectivity of GC-ECD. Table 5 shows the intraday and interday accuracy and precision estimated through recovery trials, spiking blank samples at 10, 50, and 100 µg kg −1 . Satisfactory recoveries were obtained in the range of 83.0-108.6% for 3-PBA, 82.9-92.3% for 4-F-3-PBA, and 107.8-117.3% for TFA in green tea and black tea. The precisions expressed as RSDs of 3-PBA, 4-F-3-PBA and TFA ranged from 2.4% to 13.2%, 1.8% to 9.2%, and 1.2% to 9.5% for intraday precision, while the interday RSDs of 3-PBA, 4-F-3-PBA and TFA ranged from 3.1% to 11.0%, 4.0% to 11.1%, and 2.2% to 8.0%, respectively. Typical UHPLC-MS/MS chromatograms of three target compounds and the internal standard are shown in Figure 5. Table 5. Recovery, relative standard deviations (RSDs), LOD, and LOQ were obtained for the target compounds in green tea (GT) and black tea (BT).  PBA and TFA were 5.0 and 1.4 μg L −1 [12]. Although the sensitivity of the previous study reported by Mortimer et al. was similar to this developed method, its complicated and time-consuming derivatization step was a shortage. Meanwhile, the LODs depended on the coextractive interferences because of the poor selectivity of GC-ECD. Table 5 shows the intraday and interday accuracy and precision estimated through recovery trials, spiking blank samples at 10, 50, and 100 μg kg −1 . Satisfactory recoveries were obtained in the range of 83.0-108.6% for 3-PBA, 82.9-92.3% for 4-F-3-PBA, and 107.8-117.3% for TFA in green tea and black tea. The precisions expressed as RSDs of 3-PBA, 4-F-3-PBA and TFA ranged from 2.4% to 13.2%, 1.8% to 9.2%, and 1.2% to 9.5% for intraday precision, while the interday RSDs of 3-PBA, 4-F-3-PBA and TFA ranged from 3.1% to 11.0%, 4.0% to 11.1%, and 2.2% to 8.0%, respectively. Typical UHPLC-MS/MS chromatograms of three target compounds and the internal standard are shown in Figure 5.

Analysis of Real Sample
The proposed method was applied to analyze 12 green tea samples and 10 black tea samples. Only two green tea samples were found to be contaminated with 3-PBA at 28.7 µg kg −1 and TFA at 12.9 µg kg −1 for one sample, and 3-PBA at 9.5 µg kg −1 for another sample. All black tea samples did not contain 3-PBA, 4-F-3-PBA, and TFA. To illustrate the formation of 3-PAB and TFA, we detected pyrethroid pesticides in both positive green tea samples referring to the previous method [39]. The results show that the positive sample with 3-PBA and TFA contained cypermethrin (649 µg kg −1 ), bifenthrin (1021 µg kg −1 ), and cyhalothrin (406 µg kg −1 ), while another positive green tea sample with 3-PBA only contained cypermethrin (31 µg kg −1 ). The results indicate that 3-PBA could be transformed from cypermethrin and cyhalothrin, while TFA was converted from bifenthrin and cyhalothrin.
Although pyrethroid pesticides were not found in the present black tea samples, 3-PBA and other metabolites could be produced during tea fermentation when pyrethroid pesticides were applied in the tea plant. Hu et al. found a novel golden flower fungus from fu brick tea, named Eurotium cristatum ET1 strain, which could efficiently degrade both cypermethrin and 3-PBA [40]. They also found that 3-PBA could be formed when ET1 degraded cypermethrin in brick tea, which was one of the heavy fermentation teas. Therefore, metabolites of pyrethroid pesticides could appear in both unfermented and fermented tea samples, and, thus, both pesticides and their metabolites should be monitored.

Conclusions
A modified QuEChERS method was developed for the determination of three typical pyrethroid metabolites, namely 3-PBA, 4-F-3-PBA, and TFA in tea using UHPLC-MS/MS. The UHPLC-MS/MS parameters were optimized and three versions of QuEChERS technique were compared. Various types of adsorbents were evaluated for obtaining high recoveries of target compounds and removal of tea matrices. Method validation was employed, and high recoveries and precisions were obtained, indicating the developed method fulfilled with the analysis of 3-PBA, 4-F-3-PBA, and TFA in tea.