Article pubs.acs.org/JAFC
Enantioselective Dissipation of Acephate and Its Metabolite, Methamidophos, during Tea Cultivation, Manufacturing, and Infusion Rong Pan,†,§ Hongping Chen,*,†,# Chen Wang,†,# Qinghua Wang,†,# Ying Jiang,†,# and Xin Liu*,†,# †
Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China # Key Laboratory of Tea Quality and Safety & Risk Assessment, Ministry of Agriculture, Hangzhou 310008, China §
S Supporting Information *
ABSTRACT: The enantioselective dissipation of acephate and its metabolite, methamidophos, was investigated during tea cultivation, manufacturing, and infusion, using QuEChERS sample preparation technique and gas chromatography coupled with a BGB-176 chiral column. Results showed that (+)-acephate and (−)-acephate dissipated following first-order kinetics in fresh tea leaves with half-lives of 1.8 and 1.9 days, respectively. Acephate was degraded into a more toxic metabolite, methamidophos. Preferential dissipation and translocation of (+)-acephate may exist in tea shoots, and (−)-methamidophos was degraded more rapidly than (+)-methamidophos. During tea manufacturing, drying and spreading (or withering) played important roles in the dissipation of acephate enantiomers. The enantiometic fractions of acephate changed from 0.495−0.496 to 0.479−0.486 (P ≤ 0.0081), whereas those of methamidophos changed from 0.576−0.630 to 0.568−0.645 (P ≤ 0.0366 except for green tea manufacturing on day 1), from fresh tea leaves to made tea. In addition, high transfer rates (>80%) and significant enantioselectivity (P ≤ 0.0042) of both acephate and its metabolite occurred during tea brewing. KEYWORDS: acephate, enantioselectivity, metabolite, tea, manufacturing, infusion
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INTRODUCTION Tea is one of the most popular beverages worldwide, with multiple biological activities, such as anticancer,1 antioxidant,2 and relaxation.3 However, various pests and diseases seriously reduce tea yield and economic benefits in tea cultivation countries, such as China and India. Acephate, which is a registered pesticide in Chinese tea plantations,4 is widely used to control pests and ensure tea production with a maximum residue limit (MRL) of 0.1 mg/kg. Much stricter MRLs have been formulated at 0.05 and 0.02 mg/kg in the European Union (EU) and the United States, respectively. Similar to most of the chiral organophosphorus pesticides, acephate (Figure 1) has a phosphorus chiral center, resulting in two enantiomers. Acephate can be degraded into a more toxic chiral metabolite, methamidophos (Figure 1), in rice,5 mango,6 and other crops. However, there is a zero tolerance level for methamidophos, cited in China and Japan, and the relationship between acephate and methamidophos enantiomers in tea is still unknown. Theoretically, enantiomers of chiral pesticides present diverse bioactivities,7,8 toxicities,9,10 and environmental fates.11−13 The (+)-forms of acephate and methamidophos were 5 and 6.3 times more toxic to houseflies, respectively, whereas those of (−)-form showed approximately 2.5 times more potency to German cockroach in 6 h.14 Additionally, (+)-methamidophos has been reported to be 7 times more toxic to Daphnia magna than (−)-methamidophos in 48 h. By contrast, (−)-methamidophos was 8.0−12.4 times more potent to acetylcholinesterases of bovine erythrocytes than its antipode.15 Wang et al.16 observed that enantioselective degradation of acephate and © 2015 American Chemical Society
Figure 1. Structures of acephate and methamidophos enantiomers.
metabolism to methamidophos varied with soils caused by microorganism. Therefore, it is insufficient to evaluate the risk of chiral acephate on the basis of its racemate. Studies of enantioselective dissipation of chiral pesticides may provide a more accurate understanding of their fate during tea cultivation, manufacturing, and infusion. Received: Revised: Accepted: Published: 1300
October 14, 2014 January 13, 2015 January 13, 2015 January 13, 2015 DOI: 10.1021/jf504916b J. Agric. Food Chem. 2015, 63, 1300−1308
Article
Journal of Agricultural and Food Chemistry
mL of acetonitrile by homogenization at 12000 rpm for 2 min. Afterward, 5 g of NaCl and 1 g of MgSO4 were added in the centrifuge tube. The sample was mixed on a vortex mixer immediately for 30 s, followed by a centrifugation at 5000 rpm for 10 min. An aliquot of 2 mL was transferred from the supernatant to a 5 mL centrifuge tube, which contains 200 mg of PSA, 200 mg of C18, 50 mg of GCB, and 100 mg of MgSO4. Then the whole centrifuge tube was vortexed for 30 s before centrifugation (4000 rpm, 10 min). An aliquot of 1 mL was transferred and evaporated to dryness using a nitrogen flow controller. Residues were dissolved in 1 mL of acetone and filtered through a 0.22 μm membrane for final determination by GC-FPD. For the fresh tea leaves collected within 7 days after acephate application, preparation was similar to the made tea samples mentioned above in addition to presoaking. To reduce the limits of quantification (LOQs) and meet the demand of detection, an additional concentration step was used prior to QuEChERS cleanup for the fresh tea leaves collected on days 10−35. The fresh tea leaves (4 g) were homogenized with 20 mL of acetonitrile in a centrifuge tube at 12000 rpm for 2 min. Then 5 g of NaCl and 2 g of MgSO4 were added. Vortexing was performed for 30 s followed by centrifugation for 10 min at 4000 rpm. An aliquot of 10 mL was transferred and evaporated to 2 mL for further cleanup. Subsequent cleanup steps were similar to those for made tea samples, except the dissolved volume used was 0.5 mL of acetone. For tea soup, liquid−liquid extraction was used to extract target pesticides from 50 mL of tea soup (dissolved with 10% NaCl) with 50 mL of dichloromethane. The organic phase was evaporated to dryness and dissolved in 2 mL of acetone for final determination. For spent tea leaves, residues after tea brewing were sheared into pieces and transferred into a centrifuge tube, before the addition of acetonitrile (10 mL). The subsequent steps were similar to those for made tea sample. Instrumentation. Acephate and methamidophos enantiomers were determined by gas chromatography (Agilent 7890 A, Wilmington, DE, USA) coupled with FPD. Enantiomeric separation was performed by a BGB-176 capillary column (30 m × 0.25 mm × 0.25 μm, BGB, Adiswil, Switzerland), having 20% 2,3-dimethyl-6-tertbutyldimethylsilyl-β-cyclodextrin dissolved in BGB-15 (15% phenyl-, 85% methylpolysiloxane). Carrier gas was high-purity nitrogen with a constant flow of 1.2 mL/min, and injection volume was 1 μL. The temperatures of the injector and detector were 230 and 245 °C, respectively. Oven temperature was maintained at 80 °C for 1 min, then increased to 220 °C at 10 °C/min, and held for 5 min. Data Analysis. To evaluate chromatographic peaks of chiral acephate and methamidophos, the resolution (R) was calculated on the basis of the following equation: R = 1.18 × [tR(2) − tR(1)]/[W1/2(1) + W1/2(2)]. tR(1) and tR(2) here stand for the retention times of the first and second enantiomers eluted from the column, respectively. W1/2(1) and W1/2(2) represent the half-peak width of the first and second enantiomers eluted from the column. Generally, the dissipation of chiral pesticides hypothetically follows first-order kinetics.21 In this study, this kinetic was calculated by Sigmaplot 12.5. Enantiomeric fractions (EF), as given by Harner et al.,22 were used to express the differences between enantiomers, that is, EF = E1/(E1 + E2), where E1 and E2 represent the concentrations of the first and second enantiomers eluted from the column, respectively. In this study, E1 stands for (+)-acephate or (+)-methamidophos. The values of EF ranged from 0.000 to 1.000, wherein EF = 0.500 referred to the racemic mixture. Transfer rates of acephate or methamidophos enantiomers from made tea to infusion were calculated using eq 1
The behavior of pesticides in green tea shoots and the effects of manufacturing on residues in made tea, as well as their transfer rates during tea brewing, have been extensively reported.17−19 However, few studies have reported on the enantioselective dissipation behavior of chiral pesticides in tea matrix, except for indoxacarb and cis-epoxiconazole.20 In this study, a rapid method has been developed for the separation and determination of acephate and methamidophos enantiomers using gas chromatography with a QuEChERS sample preparation technique. Enantioseletive dissipation of acephate and methamidophos in tea was investigated during cultivation, manufacturing, and infusion.
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MATERIALS AND METHODS
Field Trial. The field trial was conducted in a tea plantation (variety, Longjing 43; area, 666.7 m2; location, Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, China) from June 9 to July 14, 2014. The plantation was divided equally into six plots for treatment or control, leaving one row of bushes to separate each plot. After being diluted by a factor of 1:400, acephate (30% EC) was sprayed on tea leaf surfaces in five replications with a handoperated knapsack sprayer. The control was sprayed with a similar amount of water. Before application, fresh tea leaves collected in this plantation were analyzed, and no acephate or methamidophos was found. Sampling and Sample Preparation. The fresh tea leaves (one bud with two leaves) were collected randomly from tea trees in each plot on 0 (2 h), 1, 3, 5, 7, 10, 14, 21, 28, and 35 days after application to investigate the enantioselective dissipation of acephate and its metabolite during cultivation. Meanwhile, to determine the effects of tea manufacturing on the enantioselective dissipation of acephate and its metabolite, fresh tea leaves were picked from both treatment and control on days 1 and 3 and then made into green and black tea immediately. Samples collected at each stage of manufacturing were analyzed on the day of collection prior to storage. The tea manufacturing steps were as follows. For green tea manufacturing: (a) spreading, that is, the plucked leaves were spread on a bamboo mat for 5 h at room temperature (23−25 °C) with good ventilation; (b) fixing, that is, the spread leaves were fried in a caldrontype water-removing machine at 220−230 °C to inactivate various enzymes; and (c) drying, that is, fixed leaves were dried by hot air at 110 °C to a final moisture concentration of approximately 3%. For black tea manufacturing: (a) withering, that is, the plucked leaves were spread on withering troughs for approximately 15 h at room temperature (23−25 °C); (b) rolling, that is, withered leaves were rolled by hand for 30−40 min at different pressures; (c) fermentation, that is, rolled leaves were stacked together and covered with a wet nylon net to maintain high relative humidity (about 95%) for 5−6 h to facilitate enzymatic oxidation; and (d) drying, a process similar to that in green tea manufacturing. A total of 3 g of made tea was brewed with 150 mL of boiling water for 5 min. The infusion was then filtered out, and spent leaves were left. Both infusion and spent leaves were cooled for further analysis in the laboratory. Chemicals and Reagents. Racemate standards of acephate and methamidophos were obtained from Agro-Environment Protection Institute (1000 mg/L, Tianjin, China). Organic solvents were of analytical grade and purchased from Merk (Darmstadt, Germany). The sorbents of primary−secondary amine (PSA), graphitized carbon black (GCB), and octadecylsilane (C18) were purchased from Agela (Tianjin, China). Analytical grade sodium chloride (NaCl) and anhydrous magnesium sulfate (MgSO4) (Zhejiang, China) were baked for 3 h at 650 °C prior to use. Ultrapure water was made from a Milli-Q water purification system (Bedford, MA, USA) in the laboratory. Sample Pretreatment. For made tea, samples (2 g) were weighed into a 50 mL centrifuge tube and presoaked with 1 mL of boiled ultrapure water for 15 min. Target pesticides were extracted with 10
transfer (%) = (m1 − m2)/m1 × 100
(1)
where m1 and m2 stand for the concentrations of the target compounds (mg/kg) in made tea and spent tea leaves, respectively. Validation Study. This method was validated by analyzing its performance characteristics, such as linearity, recovery, limit of detection (LOD), LOQ, and matrix effect (ME). For sensitivity evaluation, the linearity was determined using mixed standard solutions at five concentration levels, ranging from 0.08 to 1.6 1301
DOI: 10.1021/jf504916b J. Agric. Food Chem. 2015, 63, 1300−1308
Article
Journal of Agricultural and Food Chemistry
Figure 2. Typical chromatograms of acephate and methamidophos enantiomers in matrix standard solution (fresh tea leaves, 0.8 mg/kg) (A), fresh tea leaves on day 3 (B), fresh tea leaves on day 14 (C), green tea on day 3 (D), spent leaves of green tea on day 3 (E), black tea on day 3 (F), and spent leaves of black tea on day 3 (G). Peaks: 1, (+)-methamidophos; 2, (−)-methamidophos; 3, (+)-acephate; 4, (−)-acephate.
described above. According to the previous study by Wang,23 the (+)-form would be eluted from the column first, followed by the (−)-form for both acephate and methamidophos (Figure 2A). Addition of water to the tea matrix can significantly improve the extraction efficiency.24,25 In this study, the effects of adding different levels of water (0, 1, 5 mL) on extraction efficiency were investigated in both spiked made tea and positive made tea. The average recoveries of presoaking with 0, 1, and 5 mL of water were 61.3, 52.5, and 38.2% for acephate and 68.6, 60.0, and 42.5% for methamidophos, respectively. The results indicated that addition of water would reduce the recoveries of both pesticides, which is explained by the pesticides going into the water due to their high polarity.26 However, extraction yield of acephate in positive samples by presoaking was 11.3− 13.9 times that without presoaking. Methamidophos was determined with 0.8−1.0 mg/kg by presoaking, whereas it was not detected without presoaking (Supporting Information Table 4). This indicated that presoaking was necessary to facilitate polar pesticide separation from the matrix. However, it is hard to overcome the great loss of acephate and
mg/kg for each enantiomer in multiple matrices. The LODs and LOQs for each enantiomer were confirmed on the basis of signal-tonoise (S/N) ratios of 3 and 10, respectively. The matrix effect was calculated by the ratio of the slopes for corresponding matrix-matched and solvent standards. Matrix-matched standard solutions were prepared with blank samples and used for quantification by external standard method. To evaluate the accuracy and precision of this method, recovery was evaluated by spiking blank materials at two different levels (0.04 and 0.4 mg/kg for fresh tea leaves, whereas 0.4 and 1.6 mg/kg for other matrices).
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RESULTS AND DISCUSSION Method Optimization and Validation. A commercial BGB-176 column was chosen for chiral separation and determination of acephate and methemidophos enantiomers by GC-FPD. Gas chromatographic conditions, such as oven temperature program and flow rate of carrier gas, were optimized step by step for quicker analysis (Supporting Information Tables 1−3). Ultimately, satisfactory capacity (Racephate = 1.37 and Rmethamidophos = 1.97) was obtained for the whole analysis of each enantiomer within 25 min as 1302
DOI: 10.1021/jf504916b J. Agric. Food Chem. 2015, 63, 1300−1308
Article
Journal of Agricultural and Food Chemistry
Table 1. Linear Regression Equations, Correlation Coefficients (r), Recoveries, Matrix Effect (ME), Limits of Detection (LOD), and Limits of Quantitation (LOQ) for Acephate and Methamidophos Enantiomers in Different Tea Matrices
(+)-acephate
(−)-acephate
(+)-methamidophos
(−)-methamidophos
recoveryb (%)
LOD (mg/kg)
LOQ (mg/kg)
51.6 57.3 50.9 54.5 52.0 54.1 51.2 53.7 52.6 55.3
± ± ± ± ± ± ± ± ± ±
3.5 1.8 3.3 2.7 3.4 2.7 3.1 3.0 2.9 2.5
0.005
0.015
0.1
0.3
0.03
0.1
0.1
0.3
0.03
0.1
0.04 0.40 0.4 1.6 0.4 1.6 0.4 1.6 0.4 1.6
51.8 57.5 50.8 54.7 52.3 55.3 51.9 55.1 53.1 55.6
± ± ± ± ± ± ± ± ± ±
3.4 2.0 3.3 2.7 3.5 2.8 3.6 2.9 3.4 2.7
0.005
0.015
0.1
0.3
0.03
0.1
0.1
0.3
0.03
0.1
0.04 0.40 0.4 1.6 0.4 1.6 0.4 1.6 0.4 1.6
58.4 65.0 58.7 62.9 59.8 63.8 58.5 62.8 59.9 64.1
± ± ± ± ± ± ± ± ± ±
3.4 2.0 3.6 2.9 3.5 2.7 3.8 3.0 3.6 2.9
0.003
0.01
0.03
0.1
0.03
0.1
0.03
0.1
0.03
0.1
0.04 0.40 0.4 1.6 0.4 1.6 0.4 1.6 0.4 1.6
58.7 65.3 58.9 63.2 59.8 64.6 58.8 64.1 60.0 63.9
± ± ± ± ± ± ± ± ± ±
3.3 2.1 3.5 2.9 3.6 2.9 3.4 3.0 2.9 3.2
0.003
0.01
0.03
0.1
0.03
0.1
0.03
0.1
0.03
0.1
matrix
regression eq
r
MEa
spike level (mg/kg)
acetone fresh tea leaves
y = 887x − 12 y = 1241x + 3
0.9974 0.9993
1.40
green tea
y = 925x − 5
0.9974
1.04
black tea
y = 1505x + 2
0.9983
1.70
spent leaves of green tea
y = 973x − 6
0.9968
1.10
spent leaves of black tea
y = 1749x + 2
0.9994
1.97
0.04 0.40 0.4 1.6 0.4 1.6 0.4 1.6 0.4 1.6
acetone fresh tea leaves
y = 955x − 7 y = 1384x + 2
0.9994 0.9992
1.45
green tea
y = 1015x − 7
0.9970
1.06
black tea
y = 1585x + 5
0.9979
1.66
spent leaves of green tea
y = 1108x − 10
0.9971
1.16
spent leaves of black tea
y = 1891x + 3
0.9990
1.98
acetone fresh tea leaves
y = 1725x − 25 y = 1702x + 4
0.9992 0.9995
0.99
green tea
y = 1742x + 5
0.9974
1.01
black tea
y = 2284x + 1
0.9989
1.32
spent leaves of green tea
y = 1798x + 4
0.9977
1.04
spent leaves of black tea
y = 2295x + 2
0.9988
1.33
acetone fresh tea leaves
y = 1841x − 9 y = 1877x + 3
0.9993 0.9997
1.02
green tea
y = 1901x + 2
0.9985
1.03
black tea
y = 2416x − 1
0.9991
1.31
spent leaves of green tea
y = 2038x + 2
0.9979
1.11
spent leaves of black tea
y = 2502x − 2
0.9994
1.36
enantiomer
a ME. matrix effect. It was calculated by the ratio of slope of matrix and solvent, where matrix suppression takes place with ME 1.00. bValues are presented as the mean ± SD (n = 5).
selected to be the addition volume before extraction by acetonitrile. For other tea matrices with certain amounts of water, there is no need of presoaking, and recoveries are similar to made tea samples. To overcome the interference of coextraction, a mixture of adsorbents (200 mg of PSA, 200 mg of C18, 50 mg of GCB, and 100 mg of MgSO4) was used as a QuEChERS technique to clean up the tea matrix. We investigated the effect of the
methamidophos that occurred during extraction from the dried matrix by water presoaking.27,28 We also investigated ethyl acetate, dichloromethane, and acetone as extraction solvent, and recoveries of acephate and methamidophos enantiomers ranged from 36.1 to 58.9% (Supporting Information Table 5). This result indicated that acetonitrile was the best choice for sample extraction among these solvents, which was in agreement with Kanrar et al.29 Thus, 1 mL of water was 1303
DOI: 10.1021/jf504916b J. Agric. Food Chem. 2015, 63, 1300−1308
Article
Journal of Agricultural and Food Chemistry
Figure 3. Dissipation of rac-acephate in fresh tea leaves (A) and EF value versus time curves of acephate and its metabolite (B). Residues have been corrected with the mean recovery (54% for acephate enantiomers and 62% for methamidophos enantiomers).
Enantioselective Dissipation of Residues in Fresh Tea Leaves. Enantioselective dissipation of acephate and its metabolite, methamidophos, in the tea plantation was investigated, and the results are shown in Figure 3. Dissipation kinetics of (+)-acephate and (−)-acephate were fitted in a first-order kinetic model with correlation coefficients of 0.9880 and 0.9899, respectively. Half-lives of (+)-acephate and (−)-acephate were about 1.8 and 1.9 days, respectively. The initial deposits of (+)-acephate and (−)-acephate were 36.14 and 36.49 mg/kg, respectively. Then these deposits decreased to 12.50 and 12.76 mg/kg correspondingly 3 days after their field application, which showed a great reduction of approximately 65%. The concentrations of each acephate enantiomer were
