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Fast screening of trace multiresidue pesticides on fruit and vegetable surfaces using ambient ionization tandem mass spectrometry
Journal Pre-proof Fast Screening of Trace Multiresidue Pesticides on Fruit and Vegetable Surfaces Using Ambient Ionization Tandem Mass Spectrometry Sy-Chyi Cheng, Ruei-Hao Lee, Jing-Yueh Jeng, Chi-Wei Lee, Jentaie Shiea PII:
S0003-2670(19)31494-1
DOI:
https://doi.org/10.1016/j.aca.2019.12.038
Reference:
ACA 237321
To appear in:
Analytica Chimica Acta
Received Date: 20 September 2019 Revised Date:
12 December 2019
Accepted Date: 15 December 2019
Please cite this article as: S.-C. Cheng, R.-H. Lee, J.-Y. Jeng, C.-W. Lee, J. Shiea, Fast Screening of Trace Multiresidue Pesticides on Fruit and Vegetable Surfaces Using Ambient Ionization Tandem Mass Spectrometry, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.038. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
ACA-19-2649 Rev-Clear
Fast Screening of Trace Multiresidue Pesticides on Fruit and Vegetable Surfaces Using Ambient Ionization Tandem Mass Spectrometry
Sy-Chyi Cheng1, Ruei-Hao Lee1, Jing-Yueh Jeng2, Chi-Wei Lee3,4, and Jentaie Shiea1,5,6,*
1
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan Department of Biotechnology, Chia Nan University of Pharmacy & Science, Tainan, Taiwan 3 Institute of Medical Science and Technology, National Sun Yat-Sen University, Taiwan 4 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 5 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2
*Corresponding author: Dr. Jentaie Shiea Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected]
1
ABSTRACT
An ambient ionization tandem mass spectrometric approach was developed to rapidly screen multiresidue pesticides on fruits and vegetables without sample preparation and chromatographic separation. The residual pesticides on fruits and vegetables were collected by sweeping a metallic probe across the sample surface for 2 cm. The analytes collected on the probe were desorbed and ionized in a thermal desorption electrospray ionization (TD-ESI) source, after which analyte ions were detected by a triple quadruple mass analyzer (QqQ) operated in multiple reaction monitoring (MRM) mode. With this TD-ESI/MS/MS approach, 308 pesticides were monitored, where a mixture containing 15 pesticide standards was successfully identified to demonstrate the capability of this approach to screen trace multiresidue pesticides. The approach had reasonable detection limits (< 50 ppb) and reproducibility (RSD: 8.43 %, n = 9) from the analysis of a benthiazole standard solution. Real samples including a tomato and bell pepper were analyzed using this TD-ESI/MS/MS approach. After TD-ESI/MS/MS analysis, the organic solvent extracts from the same samples were subjected to TD-ESI/MS/MS, gas chromatography mass spectrometry (GC-MS), and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for validation.
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INTRODUCTION
Pesticides are widely used to control weeds and insects in fields to increase crop yields. Due to misuse or overuse, pesticides can accumulate in or on crop products at harmful levels. People who are chronically exposed to pesticides may have an increased risk of contracting diseases such as cancer, Alzheimer’s disease, Parkinson’s disease, diabetes, and learning and developmental disorders [1-6]. Therefore, the levels of residual pesticides in agricultural products are strictly administrated and monitored to protect the health of the general public. Public health administration organizations such as the United States Environmental Protection Agency and European Community have restricted the maximum residue levels of pesticides that are permitted in agricultural products intended for human consumption. Since multiple pesticides are commonly applied on crop fields, analytical methods based on gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) have been developed to characterize multiresidue pesticides in agro products [7-11]. Pesticides are identified based on their chromatographic retention times, presence of molecular ions, and fragmentation profiles. To ensure high sensitivity for the detection of trace pesticides, tandem mass spectrometry instrumentation operated in multiple reaction monitoring (MRM) mode is commonly used to simultaneously monitor the pesticide’s parent and fragment ion pairs. Simple sample preparation methods such as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) have been developed to extract pesticides from sample matrix prior to GC-MS and LC-MS analysis [12-14]; however, these sample preparation processes are still laborious and time-consuming. In addition, the lengthy analytical time required for GC and LC is disadvantage for high-throughput analysis of a large number of samples. On the other hand, ambient ionization mass spectrometry (AMS) allows for rapid chemical analysis without sample preparation and chromatographic separation [15-18], so that analysis of specific chemical compounds on solid or in liquid samples can be completed within minutes or even seconds [19-26]. This makes AMS fulfil the demand for a method that can rapidly detect residual 3
pesticides in or on crop products. Actually, several AMS techniques such as direct analysis in real time (DART) [27-29], desorption electrospray ionization (DESI) [30], atmospheric pressure glow discharge ionization (APGDI) [31], and paper spray ionization (PSI) [32] have been utilized to characterize pesticides on fruits and in drinks. Although the sensitivities of using these AMS techniques for detecting residual pesticides was found to be good, the analysis was restricted to a limited number of samples due to limitations in slow sampling speed and sample switching and time required for sample pretreatment. An AMS approach capable to efficiently sample and characterize multiresidue pesticides on agro products is then needed for high-throughput sample analysis. Thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS) is an AMS technique that uses a metallic probe to collect trace amounts of analytes from solid or liquid samples [33]. The probe with the collected analytes is then inserted in a TD unit to thermally desorb the said analytes. The analytes are subsequently delivered into an ESI plume by a nitrogen flow, where they interact with charged solvent species through ion-molecule reactions (IMRs) to form analyte ions, which are then detected by the mass analyzer attached to the TD-ESI source. TD-ESI/MS has been used to rapidly characterize trace chemical compounds including pesticides, explosives, drugs, and plasticizers on various sample surfaces [33-37]. In addition, pesticides in the oral fluids or gastric juices of self-poisoning patients have been rapidly characterized via probe sampling followed by TD-ESI/MS analysis of patient biofluids to provide important toxicological and temporal information for timely decision-making during critical emergency room cases [38]. Although TD-ESI/MS has been successfully used to characterize chemical compounds on different sample surfaces, it is still difficult for high-throughput screening of trace multiresidue pesticides on fruit and vegetable surfaces because only a limited number of pesticides were monitored in a single MS analysis. Typically, a sample required four TD-ESI/MS analysis and more than 1.5 min to screening 100 pesticides the surface. To meet the demand of high-throughput screening of multiresidue pesticides on agro products, this study developed an analytical approach that combined multi-probe sampling, TD-ESI and tandem mass spectrometry (MS/MS) to monitor 4
more than 300 trace pesticides on fruit and vegetable surfaces within 30 seconds. To increase the detection sensitivity for residual pesticides, the mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Two MRM transitions of each pesticide were used to facilitate analyte detection. In addition to pesticide standard solutions, a locally purchased tomato and bell pepper were subjected to TD-ESI/MS/MS analysis to demonstrate the capability of the developed approach for high-throughput screening of trace multi-residual pesticides on real samples. For validation, the same tomato and bell pepper that were analyzed using direct TD-ESI/MS/MS were further treated with QuEChERS, after which the extracts were analyzed using GC-MS, LC-MS/MS, and TD-ESI/MS/MS.
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EXPERIMENTAL SECTION Chemicals and reagents Methanol (MeOH) and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany); acetic acid (AA), hexane, and ammonium acetate (NH4OAc) were purchased from J.T. Baker (Phillipsburg, NJ, U.S.A.). QuEChERS extraction kits were obtained from Agilent (Santa Clara, CA, U.S.A.). Pesticide standards were purchased from Agricultural Chemicals and Toxic Substances Research Institute (Taichung, Taiwan) and Sigma-Aldrich (St. Louis, U.S.A.). A Milli−Q Plus apparatus (Millipore, Molsheim, France) was used to produce distilled deionized water for sample preparation. TD-ESI/MS/MS analysis Figure 1a shows the analytical processes for using TD-ESI/MS/MS to characterize multiresidue pesticide on agro products. A stainless steel inoculating loop (60 mm long and 2.5 mm in diameter, Ming-Yuh Scientific Instruments Co., Taiwan) was used as the sampling probe to sweep across the surface of fruits and vegetables for a distance of 2 cm to collect trace pesticides. The probe was inserted into a TD-ESI source to thermally desorb the analytes in a preheated oven (280°C). A hot nitrogen gas stream (2.3 L/min) delivered the desorbed analytes from the oven down into an ESI plume. The analytes were ionized through ion-molecule reactions with the charged solvent species in the ESI plume. For liquid sample analysis, the sampling probe was dipped and removed from the sample solution to collect approximately 2 µL of liquid within the inoculating loop prior to desorption and ionization in the TD-ESI source. A triple quadrupole mass analyzer (Ultivo, Agilent, U.S.A.) connected to the TD-ESI source was operated in MRM mode to detect the characteristic ion pairs for pesticides. The flow rate of the drying gas was set at 5 L/min, the pressure of nebulizing gas was set at 4 psi, and the voltage of MS inlet was set at +4.5 kV for positive ion mode and −4 kV for negative ion mode, respectively. The ESI solution (5 mM NH4OAc in 40% MeOH) was delivered through a fused silica capillary at a flow rate of 160 µL/hr. A small butane torch was used to clean the sampling probe by burning it for ca. 5 seconds after each sample analysis to remove any residues on 6
the probe. The probe was then dipped in MeOH for immediately cooling. After that, the probe was ready to be used for next sampling. Analytical data obtained from TD-ESI/MS/MS analysis was processed using the MassHunter qualitative analysis software (Agilent, U.S.A.) to list the candidate pesticides detected on the sample. Optimization of MRM operational parameters Since the miniaturized triple quadrupole mass analyzer (Ultivo, Agilent) used in this study is configured differently compared to traditional triple quadrupole mass analyzers (especially with respect to the sizes of its lens and quadrupoles), the operational MRM parameters for detecting characteristic pesticide ion pairs must first be optimized. Pesticide standards were analyzed using TD-ESI/MS to determine the most abundant parent ion signals such as [M−H]-, [M+H]+, [M+NH4]+, [M+Na]+, or the main decomposition product ions of thermally labile pesticides. The standard pesticide solutions were then analyzed using direct-infusion ESI-MS/MS to discover the two most abundant fragment ions from the most abundant parent pesticide ions that were detected using TD-ESI/MS. The MassHunter Optimizer software was utilized to optimize the fragmentor voltage for each pesticide, after which the two most abundant product ions and their respectively optimized collision energies were determined. The fragmentor voltage was between 80 and 160 V, whereas the collision energy was between 2 and 60 eV. Two precursor-fragment ion transitions for each pesticide were monitored to assure a high accuracy of pesticide detection and identification. The MRM parameters for 616 transitions for 308 pesticides were established (Tables S1 and S2). Since the sensitivity of MRM analysis decreases when the number of transition channels exceed 300, the 308 pesticides were divided into two groups — the first group including the first 308 MRM transitions for the first 154 pesticides, and the second group including the next 308 MRM transitions for the next 154 pesticides — and analyzed using TD-ESI/MS/MS. Each sample analysis takes less than 30 seconds, so that it took less than 1 min to complete the two assays to cover all 616 MRM transitions for 308 pesticides. Unless stated otherwise, the dwell time was set at 1 ms to obtain at least 12 data points across the peak. Typically, 7
the scan time was 0.791 s for 1 ms dwell time and 15 data points could be obtained in 12 s. After optimization, a tomato and bell pepper purchased from local market were then subjected for TD-ESI/MS/MS to examine the capability of the developed AMS approach for fast screening trace multiresidue pesticides on agro products. Pesticide extraction from real samples The validation of TD-ESI/MS/MS for pesticide screening was done by comparing the results of direct TD-ESI/MS/MS analysis with those of LC-MS/MS and GC-MS analysis used in the conventional residue pesticide testing laboratory. So that, the bell pepper and tomato samples previously analyzed by TD-ESI/MS/MS were further processed and treated with QuEChERS and the extracts were analyzed by TD-ESI/MS/MS, LC-MS/MS, and GC-MS, respectively (Figure 1b). The sample was homogenized with a blender and 10 g of homogenized sample was weighed in a 50 mL PTFE centrifuge tube. A solution of 10 mL ACN with AA (1% v/v), NaOAc (1 g), and MgSO4 (4 g) were added in the tube. The solution in the tube was vigorously vortexed for 2 min and centrifuged at 3,000 × g for 1 min. An aliquot of 6 mL supernatant was transferred into a graduated centrifuge tube (containing 300 mg PSA and 900 mg MgSO4) and vortexed for 2 min. After centrifugation at 3,000 × g for 1 min, the supernatant was transferred into a glass vial and two test tubes (where 1 mL of supernatant was transferred into each test tube). The supernatant in the glass vial was analyzed using TD-ESI/MS/MS without further pretreatment. A rotary evaporator was used to evaporate the supernatant in the test tubes, after which the residue in one test tube was reconstituted in 1 mL mobile phase A (10% aqueous methanol with 5 mM NH4OAc) for LC-MS/MS analysis, whereas the residue in another test tube was dissolved in 1 mL acetone-hexane solution (1:1, v/v) for GC-MS analysis. Before LC-MS/MS and GC-MS analysis, the extracts were filtered through a disposable 0.45 µm PTFE membrane filter (UniRegion Bio-Tech, U.K.) to remove any particles and precipitate in the solution. Liquid Chromatography Mass Spectrometry and Gas Chromatography Mass Spectrometry A HPLC system (Shimadzu, Japan) with a binary pump (LC 20 ADXR), vacuum degasser 8
(DGU-20A3R), autosampler (SIL-20ACXR), column oven (CTO-20A), and Shim-pack GIST C18 column (3 µm, 4.6 x 150 mm, Shimadzu) was utilized to separate the pesticides in an aliquot of 3 µL QuEChERS extract solution. The mobile phase consisted of (A) 10% aqueous methanol with 5 mM NH4OAc and (B) 90% aqueous methanol with 5 mM NH4OAc, and was set at a flow rate of 1 mL/min for elution. The temperature of the column oven was set at 40°C during chromatographic separation. The gradient program of the mobile phase was started at 0% B, held for 0.2 min, increased to 40% B over 0.8 min, ramped to 95% B over 19.5 min and held for 4 min, and decreased to 0% B in 3 min. A triple quadrupole mass analyzer (LC-MS 8040, Shimadzu) equipped with an ESI source was used to detect pesticides under MRM mode. The operational parameters of the triple quadrupole mass analyzer were set as follows: interface voltage, +4.5 kV; nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation line (DL) temperature, 250°C; and heat block temperature, 450°C. A GC-MS system (GCMS-QP2010, Shimadzu) with an Rtx-5MS column (30 m × 0.25 mm × 0.25 µm) and Quick-DB GC-MS residual pesticides database (Shimadzu) was used to monitor multiresidue pesticides in the extracted solution obtained via QuEChERS. A helium stream was used as the carrier gas (1.2 mL/min) for GC-MS analysis. The injector temperature was set at 250°C and an aliquot of 1 µL extract solution was injected under splitless mode. The program of oven temperature was started at 60°C and held for 1 min, ramped to 160°C at a rate of 25°C/min, ramped to 240°C at a rate of 4°C/min, ramped to 290°C at a rate of 10°C/min, and held at 290°C for 11 min.
9
RESULTS AND DISCUSSION To examine the capability of TD-ESI/MS/MS for rapidly screening trace pesticide, solutions of benthiazole at different concentrations ranging from 1 ppb to 1 ppm were prepared and analyzed. Since two main fragment ions (m/z 136 and 180) were obtained from protonated benthiazole ion ([M+H]+, m/z 239) by infusion ESI-MS/MS analysis, the transitions of m/z 239→180 and m/z 239→136 were used to monitor benthiazole in the sample solutions during MRM analysis. For sampling, a probe was dipped into and quickly removed from the benthiazole solution to collect ca. 2 µL of sample solution for TD-ESI/MS/MS analysis. Figure 2a displays the MRM chromatogram for benthiazole, where the detection limit of benthiazole in solution was found to be between 10 to 50 ppb. To perform rapid screening of multiple pesticides in a sample solution, 308 MRM transitions for 154 pesticides were monitored per scan under 1 ms dwell time. This dwell time was shorter than that used in conventional LC-MS/MS analysis (i.e., 5−100 ms). This makes the sensitivity of benthiazole detected using TD-ESI/MS/MS be lower than that of conventional LC-MS/MS. It was found that increasing the dwell time to 100 ms decreased the detection limit of benthiazole from 50 ppb to <10 ppb (data not shown). To maintain the capability of high-throughput analysis, 308 MRM transitions and a dwell time of l ms were used throughout the study. Figure 2b shows the MRM chromatogram for nine consecutive analyses of the benthiazole standard solution (500 ppb). The analysis was completed in 6.2 minutes and the relative standard deviations (RSDs) of the analyses of two MRM transitions for benthiazole (m/z 239→180 and m/z 239→136) were calculated to be 8.4% and 10.5%, respectively. The average time to complete a TD-ESI/MS/MS analysis with 308 MRM transitions was approximately 40 seconds. The experimental results suggested the high-throughput capabilities of TD-ESI/MS/MS for the rapid screening of trace pesticide. The applicability of TD-ESI/MS/MS for screening multiresidue pesticides was examined by analyzing a solution containing fifteen pesticide standards (500 ppb each) including atrazine, benalaxyl, clomazone, dimethenamid, dimethoate, dimethomorph, etirimol, fenamiphos, fenthion, flazasulfuron, flusilazole, mepanipyrim, metolcarb, metribuzin, and spirotetramat. Conventionally, 10
these pesticides could be detected using LC-MS/MS through a method (MOHWP0055.03) published by the Taiwan Food and Drug Administration (TFDA) [39]. The raw data acquired by TD-ESI/MS/MS was further processed by the MassHunter qualitative analysis software, which listed the candidate pesticides detected in the sample. To reduce the possibility of false positive or false negative results, two ion pair transitions for monitoring each pesticide were set in the MRM analysis. To confirm the presence of a pesticide in the sample solution, the duration and intensity ratio of the ion signal of two MRM transitions for the pesticide must well match with each other. For example, the duration of two MRM transitions of the atrazine ion (m/z 216→174, solid line; m/z 216→68, dashed line) shown in Figure 3a was exactly the same (1-1.2 min), and the ratios of (m/z 216→174)/(m/z 216→68) for two analysis (2.52 and 2.69) were similar to those obtained via LC-MS/MS analysis (2.49), indicating that the transitions of (m/z 216→174) and (m/z 216→68) resulted from fragmentation of the same molecular ion. The same phenomenon was found in all 15 pesticide standards (Figures 3a-o). Since the volatility between individual pesticides can vary, the shape of the MRM signals for atrazine (a higher volatility pesticide) was sharper than that of benalaxyl (a lower volatility pesticide) (comparing Figure 3a with 3b). The time required to complete a replicate analysis (including sampling, desorption/ionization, and detection), data processing, and pesticide identification was less than 4 min. Figures 3p-t show the MRM chromatograms of five randomly selected pesticides (bendiocarb, bromacil, demeton-S-methyl, ethion, and vamidothion) which were not presented in the standard solution. Although the signals of two MRM transitions for bendiocarb (m/z 224→109 and m/z 224→81) seemed to be positive (Figure 3p), the duration of both MRM signals did not well match, suggesting both signals were from different ion pairs. Bendiocarb was therefore concluded to be absent in the sample solution. Similar results were obtained for demeton-S-methyl (m/z 231→89 and m/z 231→61), where two MRM transitions were detected in the first analysis (Figure 3r), but the resultant peak of the transition m/z 231→61 did not match that of m/z 231→89. For bromacil, only one transition pair (m/z 261→205) was detected on the MRM chromatogram (Figures 3q). No 11
obvious ion-pair transition was found in the MRM chromatograms of ethion and vamidothion (Figures 3s & t). These five pesticides were then concluded to be absent in the sample solution. To examine the capability of TD-ESI/MS/MS for fast screening of multiresidue pesticides on real samples, a bell pepper and tomato purchased from a local market were analyzed. Without any sample preparation, a sampling probe was swept across the surface of the sample for 2 cm. The probe was then immediately inserted into the TD-ESI source for further analysis. The process repeated five times for each sample. Figure 4 displays the MRM chromatograms for quintuplicate TD-ESI/MS/MS analysis of the sample collected from the bell pepper, where samples were collected at different positions on the bell pepper. The intact ion signals for three fungicides -propamocarb, pyraclostrobin, and fluopicolide (Figures 4a-c), one miticide -spirotetramat (Figure 4d), and two insecticides -chlorantraniliprole and dinotefuran (Figures 4e and g) were detected on the bell pepper surface. The intensities of the pesticide ion signals between each of the five replicates were unequal, indicating that the pesticides were not uniformly distributed across the bell pepper surface, so that the intensities of pesticide ions could vary based on the sampling position. The presence of these pesticides on the bell pepper was determined based on the following criteria: (1) detection of two MRM transitions of each pesticide molecular ion, (2) well match between the duration of two MRM transitions of the molecular pesticide ion and fragment ions, (3) the peak area ratio of two MRM transitions of the analyte must match that of pesticide standard, and (4) detection of the two MRM transitions at intensities higher than 102 counts. In addition, even though the molecular ion of the thermally labile kresoxim-methyl (m/z 314) was not detected using TD-ESI/MS/MS, the presence of kresoxim-methyl in the sample was still confirmed via the detection of two transitions of a thermally decomposed product ion (m/z 282) from kresoxim-methyl (m/z 282→222 and m/z 282→254) (Figure 4f). This conclusion was confirmed by TD-ESI/MS/MS analysis of a kresoxim-methyl standard (data not shown). After examining the bell pepper surface using TD-ESI/MS/MS, the sample was homogenized and extracted using the QuEChERS method, after which aliquots of the extracts were analyzed using 12
TD-ESI/MS/MS, GC-MS, and LC-MS/MS, respectively. The total time required for an experienced technician to complete these analysis was about 2 hours. Table 1 shows the pesticides detected on the bell pepper using TD-ESI/MS/MS and in its QuEChERS extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS. Comparing the TD-ESI/MS/MS results of bell pepper surface and its QuEChERS extracts, only four pesticides (propamocarb, pyraclostrobin, fluopicolide, and spirotetramat) were detected in the QuEChERS extracts, whereas seven pesticides were directly detected on the sample surface
(propamocarb,
pyraclostrobin,
fluopicolide,
spirotetramat,
chlorantraniliprole,
kresoxim-methyl, and dinotefuran). This difference in the number of detected pesticides between both approaches may owing to: (1) the pesticides on the sample surface were diluted or lost during QuEChERS extraction, which did not occur during direct surface analysis using TD-ESI/MS/MS; and (2) the interference from the matrix via surface sampling was much less than that via QuEChERS extraction, even though both samples were analyzed using TD-ESI/MS/MS. Five pesticides (propamocarb, pyraclostrobin, chlorantraniliprole, kresoxim-methyl, and λ-cyhalothrin) were detected in the QuEChERS extract using GC-MS (Figure S1), whereas seven pesticides (propamocarb, pyraclostrobin, fluopicolide, spirotetramat, chlorantraniliprole, acetamiprid, and fenobucarb) were detected using LC-MS/MS (Figure S2). Dinotefuran — detected via direct surface analysis using TD-ESI/MS/MS — was not detected during analysis of the QuEChERS extracts. This indicates that dinotefuran was lost during QuEChERS extraction. On the other hand, kresoxim-methyl was detected via direct surface analysis using TD-ESI/MS/MS and QuEChERS extraction followed by GC-MS analysis, but was not detected via QuEChERS extraction followed by LC-MS/MS analysis, as the residual pesticide method for LC-MS/MS did not include this pesticide. The nonpolar λ–cyhalothrin was more suited for GC-MS analysis, whereas acetamiprid and fenobucarb were more suited for LC-MS/MS analysis. In summary, seven pesticides were detected from the bell pepper via direct surface analysis using TD-ESI/MS/MS. After the sample was extracted by QuEChERS, four pesticides were detected using TD-ESI/MS/MS, five pesticides were detected using GC-MS, and seven pesticides were detected using LC-MS/MS (Table 1). 13
Similarly, five samples were collected from different surface locations on a tomato for direct TD-ESI/MS/MS analysis. Figure 5 displays the MRM chromatograms for direct surface analysis of the residual pesticides on the tomato. Two fungicides (azoxystrobin and pyraclostrobin) and three insecticides (dinotefuran, methomyl, and chlorantraniliprole) were detected using direct TD-ESI/MS/MS (Figures 5a-e), where the time required to complete a quintuplicate analysis was less than 4 min. The ion pair transition of the decomposition product of thiodicarb (m/z 106) was also detected (m/z 106→88 and m/z 106→58) (Figure 5f). After surface analysis using TD-ESI/MS/MS, the tomato was subjected to QuEChERS extraction followed by TD-ESI/MS/MS, GC-MS, and LC-MS/MS analysis. Table 1 shows that with the exception of chlorantraniliprole and thiodicarb, four pesticides detected on tomato surface using direct TD-ESI/MS/MS were also detected in the QuEChERS extracts that were analyzed using TD-ESI/MS/MS. However, only two volatile fungicides (azoxystrobin and pyraclostrobin) were detected using GC-MS (Figure S3), whereas two fungicides (azoxystrobin and pyraclostrobin) and six insecticides (dinotefuran, methomyl, chlorantraniliprole, thiodicarb, thiamethoxam, and clothianidin,) were detected using LC-MS/MS (Figure S4). The results indicated that direct analysis of the sample surface using TD-ESI/MS/MS could detect not only volatile pesticides (assigned via GC-MS analysis of the extracts) but also semi-volatile pesticides (assigned via LC-MS/MS analysis of the extracts). Structures of the pesticides detected in real samples are shown in Figure S5. The Venn diagrams showed in Figure 6 display the number of unique and shared pesticides detected using TD-ESI/MS/MS, GC-MS, and LC-MS/MS. For the bell pepper, 86% (6 of 7) of the pesticides detected on the surface using TD-ESI/MS/MS were also detected in the extracts using GC-MS and LC-MS/MS. The pesticides detected on the tomato surface using TD-ESI/MS/MS were all detected in the extracts using GC-MS and LC-MS/MS. These results indicate that TD-ESI/MS/MS is able to efficiently screen trace multi-residual pesticides on the surfaces of agricultural products such as bell pepper and tomato. Pesticide signal intensities that varied based on sampling location reflected the non-homogenous distribution of these pesticides across the sample 14
surface even though this was not benefit for quantitative analysis. CONCLUSION TD-ESI/MS/MS combined with probe sampling was utilized to rapidly screen trace multiresidue pesticides on agricultural products and in their extracts. A metallic probe was swept across sample surfaces to collect trace pesticides, regardless of the sample size and shape. Since sample pretreatment and chromatographic separation are unnecessary, the analytical time required to screen residual pesticides on sample surfaces was less than 1 min. Over 300 concurrent MRM transitions were monitored per analysis so that we could simultaneously detect a large number of pesticides. The analysis of tomato and bell pepper showed that 86–100% of the pesticides detected on sample surfaces using TD-ESI/MS/MS were also detected in their QuEChERS extracts using GC-MS and LC-MS/MS. The analytical time and labor required for direct surface TD-ESI/MS/MS analysis were obviously much less than that of conventional QuEChERS extraction followed by GC-MS and LC-MS/MS analysis. It should be noted that surface analysis using TD-ESI/MS/MS is not yet suited for quantitative analysis because the residual pesticides were non-homogeneously distributed on agricultural products. Since TD-ESI/MS/MS was combined with sample preparation methods such as liquid extraction and solid-phase microextraction for quantification of drugs in blood and urine. The combination of QuEChERS extraction and TD-ESI/MS/MS is therefore potential to quantify residue pesticides in agro products. In addition, pesticides with very low volatilities and polarities demonstrated poor desorption and ionization efficiencies in the TD-ESI source, and were instead more suited for analysis using LC-MS/MS or GC-MS.
Acknowledgement This work was partially supported by the Research Center for Environmental Medicine at Kaohsiung Medical University, Kaohsiung, Taiwan as part of the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. 15
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Table 1. Residual pesticides identified using TD-ESI/MS/MS, GC-MS, and LC-MS/MS (the term “nd” stands for “not detected”). TD-ESI/MS/MS Sample
Bell Pepper
Tomato
a b
Pesticide Propamocarb Pyraclostrobin Fluopicolide Spirotetramat Chlorantraniliprole Kresoxim-methyl Dinotefuran Acetamiprid Fenobucarb λ-cyhalothrin Azoxystrobin Pyraclostrobin Dinotefuran Methomyl Chlorantraniliprole Thiodicarb Thiamethoxam Clothianidin
Surface
LC-MS/MS
GC-MS
QuEChERS extract
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
nd
✓
✓
✓
nd
✓
nd
✓
✓
✓b
nd
nd
✓
✓
nd
nd
nd
nd
nd
✓
nd
nd
nd
✓
nd
nd
nd
nd
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
nd
✓
✓
✓
nd
✓
nd
✓
nd
✓b
nd
✓
nd
nd
nd
✓
nd
nd
nd
✓
nd
a
Successful analyte detection when the injection volume was increased to 5 µL Decomposition product
19
Figure legends Figure 1. (a) Schematic illustration of the TD-ESI/MS/MS method for rapidly screening multiresidue pesticides on vegetable surfaces. (b) A flow chart showing the analytical procedure for fruit and vegetable samples in this work. Figure 2. TD-ESI/MS/MS MRM chromatogram for (a) benthiazole standard solutions at concentrations ranging from 5 ppb to 1 ppm, and (b) nine consecutive analysis of benthiazole pesticide solution at a concentration of 500 ppb. Figure 3. TD-ESI/MS/MS MRM chromatograms for two consecutive analysis of a mixture solution containing 15 pesticides (500 ppb each). (a-o) Pesticides identified in the sample solution: (a) atrazine, (b) benalaxyl, (c) clomazone, (d) dimethenamid, (e) dimethoate, (f) dimethomorph, (g) ethirimol, (h) fenamiphos, (i) fenthion, (j) flazasulfuron (decomposition product), (k) flusilazole, (l) mepanipyrim, (m) metolcarb, (n) metribuzin, and (o) spirotetramat. (p-t) Five randomly selected pesticides which were absent in the sample solution: (p) bendiocarb, (q) bromacil, (r) demeton-S-methyl, (s) ethion, and (t) vamidothion. Figure 4. TD-ESI/MS/MS MRM chromatograms for (a) propamocarb, (b) pyraclostrobin, (c) fluopicolide, (d) spirotetramat, (e) chlorantraniliprole, (f) kresoxim-methyl (decomposition product), and (g) dinotefuran detected on a bell pepper. Figure 5. TD-ESI/MS/MS MRM chromatograms for (a) azoxystrobin, (b) pyraclostrobin, (c) dinotefuran, (d) methomyl, (e) chlorantraniliprole, and (f) thiodicarb (decomposition product) detected on a tomato. Figure 6. The Venn diagrams showing the number of unique and shared pesticides detected on a (a) bell pepper and (b) tomato and in their extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS.
20
Figure 1. (a) Schematic illustration of the TD-ESI/MS/MS method for rapidly screening multiresidue pesticides on vegetable surfaces. (b) A flow chart showing the analytical procedure for fruit and vegetable samples in this work.
21
Figure 2. TD-ESI/MS/MS MRM chromatogram for (a) benthiazole standard solutions at concentrations ranging from 5 ppb to 1 ppm, and (b) nine consecutive analysis of benthiazole pesticide solution at a concentration of 500 ppb.
22
Figure 3. TD-ESI/MS/MS MRM chromatograms for two consecutive analysis of a mixture solution containing 15 pesticides (500 ppb each). (a-o) Pesticides identified in the sample solution: (a) atrazine, (b) benalaxyl, (c) clomazone, (d) dimethenamid, (e) dimethoate, (f) dimethomorph, (g) ethirimol, (h) fenamiphos, (i) fenthion, (j) flazasulfuron (decomposition product), (k) flusilazole, (l) mepanipyrim, (m) metolcarb, (n) metribuzin, and (o) spirotetramat. (p-t) Five randomly selected pesticides which were absent in the sample solution: (p) bendiocarb, (q) bromacil, (r) demeton-S-methyl, (s) ethion, and (t) vamidothion.
23
Figure 4. TD-ESI/MS/MS MRM chromatograms for (a) propamocarb, (b) pyraclostrobin, (c) fluopicolide, (d) spirotetramat, (e) chlorantraniliprole, (f) kresoxim-methyl (decomposition product), and (g) dinotefuran detected on a bell pepper.
24
Figure 5. TD-ESI/MS/MS MRM chromatograms for (a) azoxystrobin, (b) pyraclostrobin, (c) dinotefuran, (d) methomyl, (e) chlorantraniliprole, and (f) thiodicarb (decomposition product) detected on a tomato.
25
Figure 6. The Venn diagrams showing the number of unique and shared pesticides detected on a (a) bell pepper and (b) tomato and in their extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS.
26
Highlights TD-ESI/MS/MS was developed to rapidly screen 308 residual pesticides on fruits and vegetables within 1 minute. The samples were directly characterized without sample preparation and chromatographic separation The identities of the pesticides detected on bell peppers and tomatoes using TDESI/MS/MS were confirmed by GC-MS and LC-MS/MS analysis of sample extracts.
Sample CRediT author statement Sy-Chyi Cheng: Writing - Original Draft, Writing - Review & Editing Ruei-Hao Lee: Methodology, Verification Jing-Yueh Jeng: Resources Chi-Wei Lee: Investigation Jentaie Shiea: Conceptualization, Supervision
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0003-2670(19)31494-1
DOI:
https://doi.org/10.1016/j.aca.2019.12.038
Reference:
ACA 237321
To appear in:
Analytica Chimica Acta
Received Date: 20 September 2019 Revised Date:
12 December 2019
Accepted Date: 15 December 2019
Please cite this article as: S.-C. Cheng, R.-H. Lee, J.-Y. Jeng, C.-W. Lee, J. Shiea, Fast Screening of Trace Multiresidue Pesticides on Fruit and Vegetable Surfaces Using Ambient Ionization Tandem Mass Spectrometry, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.038. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Graphical Abstract
ACA-19-2649 Rev-Clear
Fast Screening of Trace Multiresidue Pesticides on Fruit and Vegetable Surfaces Using Ambient Ionization Tandem Mass Spectrometry
Sy-Chyi Cheng1, Ruei-Hao Lee1, Jing-Yueh Jeng2, Chi-Wei Lee3,4, and Jentaie Shiea1,5,6,*
1
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan Department of Biotechnology, Chia Nan University of Pharmacy & Science, Tainan, Taiwan 3 Institute of Medical Science and Technology, National Sun Yat-Sen University, Taiwan 4 Department of Emergency Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan 5 Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, Taiwan 6 Research Center for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 2
*Corresponding author: Dr. Jentaie Shiea Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung, 80424 Taiwan Tel/Fax: +88675253933 Email: [email protected]
1
ABSTRACT
An ambient ionization tandem mass spectrometric approach was developed to rapidly screen multiresidue pesticides on fruits and vegetables without sample preparation and chromatographic separation. The residual pesticides on fruits and vegetables were collected by sweeping a metallic probe across the sample surface for 2 cm. The analytes collected on the probe were desorbed and ionized in a thermal desorption electrospray ionization (TD-ESI) source, after which analyte ions were detected by a triple quadruple mass analyzer (QqQ) operated in multiple reaction monitoring (MRM) mode. With this TD-ESI/MS/MS approach, 308 pesticides were monitored, where a mixture containing 15 pesticide standards was successfully identified to demonstrate the capability of this approach to screen trace multiresidue pesticides. The approach had reasonable detection limits (< 50 ppb) and reproducibility (RSD: 8.43 %, n = 9) from the analysis of a benthiazole standard solution. Real samples including a tomato and bell pepper were analyzed using this TD-ESI/MS/MS approach. After TD-ESI/MS/MS analysis, the organic solvent extracts from the same samples were subjected to TD-ESI/MS/MS, gas chromatography mass spectrometry (GC-MS), and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for validation.
2
INTRODUCTION
Pesticides are widely used to control weeds and insects in fields to increase crop yields. Due to misuse or overuse, pesticides can accumulate in or on crop products at harmful levels. People who are chronically exposed to pesticides may have an increased risk of contracting diseases such as cancer, Alzheimer’s disease, Parkinson’s disease, diabetes, and learning and developmental disorders [1-6]. Therefore, the levels of residual pesticides in agricultural products are strictly administrated and monitored to protect the health of the general public. Public health administration organizations such as the United States Environmental Protection Agency and European Community have restricted the maximum residue levels of pesticides that are permitted in agricultural products intended for human consumption. Since multiple pesticides are commonly applied on crop fields, analytical methods based on gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) have been developed to characterize multiresidue pesticides in agro products [7-11]. Pesticides are identified based on their chromatographic retention times, presence of molecular ions, and fragmentation profiles. To ensure high sensitivity for the detection of trace pesticides, tandem mass spectrometry instrumentation operated in multiple reaction monitoring (MRM) mode is commonly used to simultaneously monitor the pesticide’s parent and fragment ion pairs. Simple sample preparation methods such as QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) have been developed to extract pesticides from sample matrix prior to GC-MS and LC-MS analysis [12-14]; however, these sample preparation processes are still laborious and time-consuming. In addition, the lengthy analytical time required for GC and LC is disadvantage for high-throughput analysis of a large number of samples. On the other hand, ambient ionization mass spectrometry (AMS) allows for rapid chemical analysis without sample preparation and chromatographic separation [15-18], so that analysis of specific chemical compounds on solid or in liquid samples can be completed within minutes or even seconds [19-26]. This makes AMS fulfil the demand for a method that can rapidly detect residual 3
pesticides in or on crop products. Actually, several AMS techniques such as direct analysis in real time (DART) [27-29], desorption electrospray ionization (DESI) [30], atmospheric pressure glow discharge ionization (APGDI) [31], and paper spray ionization (PSI) [32] have been utilized to characterize pesticides on fruits and in drinks. Although the sensitivities of using these AMS techniques for detecting residual pesticides was found to be good, the analysis was restricted to a limited number of samples due to limitations in slow sampling speed and sample switching and time required for sample pretreatment. An AMS approach capable to efficiently sample and characterize multiresidue pesticides on agro products is then needed for high-throughput sample analysis. Thermal desorption electrospray ionization mass spectrometry (TD-ESI/MS) is an AMS technique that uses a metallic probe to collect trace amounts of analytes from solid or liquid samples [33]. The probe with the collected analytes is then inserted in a TD unit to thermally desorb the said analytes. The analytes are subsequently delivered into an ESI plume by a nitrogen flow, where they interact with charged solvent species through ion-molecule reactions (IMRs) to form analyte ions, which are then detected by the mass analyzer attached to the TD-ESI source. TD-ESI/MS has been used to rapidly characterize trace chemical compounds including pesticides, explosives, drugs, and plasticizers on various sample surfaces [33-37]. In addition, pesticides in the oral fluids or gastric juices of self-poisoning patients have been rapidly characterized via probe sampling followed by TD-ESI/MS analysis of patient biofluids to provide important toxicological and temporal information for timely decision-making during critical emergency room cases [38]. Although TD-ESI/MS has been successfully used to characterize chemical compounds on different sample surfaces, it is still difficult for high-throughput screening of trace multiresidue pesticides on fruit and vegetable surfaces because only a limited number of pesticides were monitored in a single MS analysis. Typically, a sample required four TD-ESI/MS analysis and more than 1.5 min to screening 100 pesticides the surface. To meet the demand of high-throughput screening of multiresidue pesticides on agro products, this study developed an analytical approach that combined multi-probe sampling, TD-ESI and tandem mass spectrometry (MS/MS) to monitor 4
more than 300 trace pesticides on fruit and vegetable surfaces within 30 seconds. To increase the detection sensitivity for residual pesticides, the mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Two MRM transitions of each pesticide were used to facilitate analyte detection. In addition to pesticide standard solutions, a locally purchased tomato and bell pepper were subjected to TD-ESI/MS/MS analysis to demonstrate the capability of the developed approach for high-throughput screening of trace multi-residual pesticides on real samples. For validation, the same tomato and bell pepper that were analyzed using direct TD-ESI/MS/MS were further treated with QuEChERS, after which the extracts were analyzed using GC-MS, LC-MS/MS, and TD-ESI/MS/MS.
5
EXPERIMENTAL SECTION Chemicals and reagents Methanol (MeOH) and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany); acetic acid (AA), hexane, and ammonium acetate (NH4OAc) were purchased from J.T. Baker (Phillipsburg, NJ, U.S.A.). QuEChERS extraction kits were obtained from Agilent (Santa Clara, CA, U.S.A.). Pesticide standards were purchased from Agricultural Chemicals and Toxic Substances Research Institute (Taichung, Taiwan) and Sigma-Aldrich (St. Louis, U.S.A.). A Milli−Q Plus apparatus (Millipore, Molsheim, France) was used to produce distilled deionized water for sample preparation. TD-ESI/MS/MS analysis Figure 1a shows the analytical processes for using TD-ESI/MS/MS to characterize multiresidue pesticide on agro products. A stainless steel inoculating loop (60 mm long and 2.5 mm in diameter, Ming-Yuh Scientific Instruments Co., Taiwan) was used as the sampling probe to sweep across the surface of fruits and vegetables for a distance of 2 cm to collect trace pesticides. The probe was inserted into a TD-ESI source to thermally desorb the analytes in a preheated oven (280°C). A hot nitrogen gas stream (2.3 L/min) delivered the desorbed analytes from the oven down into an ESI plume. The analytes were ionized through ion-molecule reactions with the charged solvent species in the ESI plume. For liquid sample analysis, the sampling probe was dipped and removed from the sample solution to collect approximately 2 µL of liquid within the inoculating loop prior to desorption and ionization in the TD-ESI source. A triple quadrupole mass analyzer (Ultivo, Agilent, U.S.A.) connected to the TD-ESI source was operated in MRM mode to detect the characteristic ion pairs for pesticides. The flow rate of the drying gas was set at 5 L/min, the pressure of nebulizing gas was set at 4 psi, and the voltage of MS inlet was set at +4.5 kV for positive ion mode and −4 kV for negative ion mode, respectively. The ESI solution (5 mM NH4OAc in 40% MeOH) was delivered through a fused silica capillary at a flow rate of 160 µL/hr. A small butane torch was used to clean the sampling probe by burning it for ca. 5 seconds after each sample analysis to remove any residues on 6
the probe. The probe was then dipped in MeOH for immediately cooling. After that, the probe was ready to be used for next sampling. Analytical data obtained from TD-ESI/MS/MS analysis was processed using the MassHunter qualitative analysis software (Agilent, U.S.A.) to list the candidate pesticides detected on the sample. Optimization of MRM operational parameters Since the miniaturized triple quadrupole mass analyzer (Ultivo, Agilent) used in this study is configured differently compared to traditional triple quadrupole mass analyzers (especially with respect to the sizes of its lens and quadrupoles), the operational MRM parameters for detecting characteristic pesticide ion pairs must first be optimized. Pesticide standards were analyzed using TD-ESI/MS to determine the most abundant parent ion signals such as [M−H]-, [M+H]+, [M+NH4]+, [M+Na]+, or the main decomposition product ions of thermally labile pesticides. The standard pesticide solutions were then analyzed using direct-infusion ESI-MS/MS to discover the two most abundant fragment ions from the most abundant parent pesticide ions that were detected using TD-ESI/MS. The MassHunter Optimizer software was utilized to optimize the fragmentor voltage for each pesticide, after which the two most abundant product ions and their respectively optimized collision energies were determined. The fragmentor voltage was between 80 and 160 V, whereas the collision energy was between 2 and 60 eV. Two precursor-fragment ion transitions for each pesticide were monitored to assure a high accuracy of pesticide detection and identification. The MRM parameters for 616 transitions for 308 pesticides were established (Tables S1 and S2). Since the sensitivity of MRM analysis decreases when the number of transition channels exceed 300, the 308 pesticides were divided into two groups — the first group including the first 308 MRM transitions for the first 154 pesticides, and the second group including the next 308 MRM transitions for the next 154 pesticides — and analyzed using TD-ESI/MS/MS. Each sample analysis takes less than 30 seconds, so that it took less than 1 min to complete the two assays to cover all 616 MRM transitions for 308 pesticides. Unless stated otherwise, the dwell time was set at 1 ms to obtain at least 12 data points across the peak. Typically, 7
the scan time was 0.791 s for 1 ms dwell time and 15 data points could be obtained in 12 s. After optimization, a tomato and bell pepper purchased from local market were then subjected for TD-ESI/MS/MS to examine the capability of the developed AMS approach for fast screening trace multiresidue pesticides on agro products. Pesticide extraction from real samples The validation of TD-ESI/MS/MS for pesticide screening was done by comparing the results of direct TD-ESI/MS/MS analysis with those of LC-MS/MS and GC-MS analysis used in the conventional residue pesticide testing laboratory. So that, the bell pepper and tomato samples previously analyzed by TD-ESI/MS/MS were further processed and treated with QuEChERS and the extracts were analyzed by TD-ESI/MS/MS, LC-MS/MS, and GC-MS, respectively (Figure 1b). The sample was homogenized with a blender and 10 g of homogenized sample was weighed in a 50 mL PTFE centrifuge tube. A solution of 10 mL ACN with AA (1% v/v), NaOAc (1 g), and MgSO4 (4 g) were added in the tube. The solution in the tube was vigorously vortexed for 2 min and centrifuged at 3,000 × g for 1 min. An aliquot of 6 mL supernatant was transferred into a graduated centrifuge tube (containing 300 mg PSA and 900 mg MgSO4) and vortexed for 2 min. After centrifugation at 3,000 × g for 1 min, the supernatant was transferred into a glass vial and two test tubes (where 1 mL of supernatant was transferred into each test tube). The supernatant in the glass vial was analyzed using TD-ESI/MS/MS without further pretreatment. A rotary evaporator was used to evaporate the supernatant in the test tubes, after which the residue in one test tube was reconstituted in 1 mL mobile phase A (10% aqueous methanol with 5 mM NH4OAc) for LC-MS/MS analysis, whereas the residue in another test tube was dissolved in 1 mL acetone-hexane solution (1:1, v/v) for GC-MS analysis. Before LC-MS/MS and GC-MS analysis, the extracts were filtered through a disposable 0.45 µm PTFE membrane filter (UniRegion Bio-Tech, U.K.) to remove any particles and precipitate in the solution. Liquid Chromatography Mass Spectrometry and Gas Chromatography Mass Spectrometry A HPLC system (Shimadzu, Japan) with a binary pump (LC 20 ADXR), vacuum degasser 8
(DGU-20A3R), autosampler (SIL-20ACXR), column oven (CTO-20A), and Shim-pack GIST C18 column (3 µm, 4.6 x 150 mm, Shimadzu) was utilized to separate the pesticides in an aliquot of 3 µL QuEChERS extract solution. The mobile phase consisted of (A) 10% aqueous methanol with 5 mM NH4OAc and (B) 90% aqueous methanol with 5 mM NH4OAc, and was set at a flow rate of 1 mL/min for elution. The temperature of the column oven was set at 40°C during chromatographic separation. The gradient program of the mobile phase was started at 0% B, held for 0.2 min, increased to 40% B over 0.8 min, ramped to 95% B over 19.5 min and held for 4 min, and decreased to 0% B in 3 min. A triple quadrupole mass analyzer (LC-MS 8040, Shimadzu) equipped with an ESI source was used to detect pesticides under MRM mode. The operational parameters of the triple quadrupole mass analyzer were set as follows: interface voltage, +4.5 kV; nebulizer gas flow, 3 L/min; drying gas flow, 15 L/min; desolvation line (DL) temperature, 250°C; and heat block temperature, 450°C. A GC-MS system (GCMS-QP2010, Shimadzu) with an Rtx-5MS column (30 m × 0.25 mm × 0.25 µm) and Quick-DB GC-MS residual pesticides database (Shimadzu) was used to monitor multiresidue pesticides in the extracted solution obtained via QuEChERS. A helium stream was used as the carrier gas (1.2 mL/min) for GC-MS analysis. The injector temperature was set at 250°C and an aliquot of 1 µL extract solution was injected under splitless mode. The program of oven temperature was started at 60°C and held for 1 min, ramped to 160°C at a rate of 25°C/min, ramped to 240°C at a rate of 4°C/min, ramped to 290°C at a rate of 10°C/min, and held at 290°C for 11 min.
9
RESULTS AND DISCUSSION To examine the capability of TD-ESI/MS/MS for rapidly screening trace pesticide, solutions of benthiazole at different concentrations ranging from 1 ppb to 1 ppm were prepared and analyzed. Since two main fragment ions (m/z 136 and 180) were obtained from protonated benthiazole ion ([M+H]+, m/z 239) by infusion ESI-MS/MS analysis, the transitions of m/z 239→180 and m/z 239→136 were used to monitor benthiazole in the sample solutions during MRM analysis. For sampling, a probe was dipped into and quickly removed from the benthiazole solution to collect ca. 2 µL of sample solution for TD-ESI/MS/MS analysis. Figure 2a displays the MRM chromatogram for benthiazole, where the detection limit of benthiazole in solution was found to be between 10 to 50 ppb. To perform rapid screening of multiple pesticides in a sample solution, 308 MRM transitions for 154 pesticides were monitored per scan under 1 ms dwell time. This dwell time was shorter than that used in conventional LC-MS/MS analysis (i.e., 5−100 ms). This makes the sensitivity of benthiazole detected using TD-ESI/MS/MS be lower than that of conventional LC-MS/MS. It was found that increasing the dwell time to 100 ms decreased the detection limit of benthiazole from 50 ppb to <10 ppb (data not shown). To maintain the capability of high-throughput analysis, 308 MRM transitions and a dwell time of l ms were used throughout the study. Figure 2b shows the MRM chromatogram for nine consecutive analyses of the benthiazole standard solution (500 ppb). The analysis was completed in 6.2 minutes and the relative standard deviations (RSDs) of the analyses of two MRM transitions for benthiazole (m/z 239→180 and m/z 239→136) were calculated to be 8.4% and 10.5%, respectively. The average time to complete a TD-ESI/MS/MS analysis with 308 MRM transitions was approximately 40 seconds. The experimental results suggested the high-throughput capabilities of TD-ESI/MS/MS for the rapid screening of trace pesticide. The applicability of TD-ESI/MS/MS for screening multiresidue pesticides was examined by analyzing a solution containing fifteen pesticide standards (500 ppb each) including atrazine, benalaxyl, clomazone, dimethenamid, dimethoate, dimethomorph, etirimol, fenamiphos, fenthion, flazasulfuron, flusilazole, mepanipyrim, metolcarb, metribuzin, and spirotetramat. Conventionally, 10
these pesticides could be detected using LC-MS/MS through a method (MOHWP0055.03) published by the Taiwan Food and Drug Administration (TFDA) [39]. The raw data acquired by TD-ESI/MS/MS was further processed by the MassHunter qualitative analysis software, which listed the candidate pesticides detected in the sample. To reduce the possibility of false positive or false negative results, two ion pair transitions for monitoring each pesticide were set in the MRM analysis. To confirm the presence of a pesticide in the sample solution, the duration and intensity ratio of the ion signal of two MRM transitions for the pesticide must well match with each other. For example, the duration of two MRM transitions of the atrazine ion (m/z 216→174, solid line; m/z 216→68, dashed line) shown in Figure 3a was exactly the same (1-1.2 min), and the ratios of (m/z 216→174)/(m/z 216→68) for two analysis (2.52 and 2.69) were similar to those obtained via LC-MS/MS analysis (2.49), indicating that the transitions of (m/z 216→174) and (m/z 216→68) resulted from fragmentation of the same molecular ion. The same phenomenon was found in all 15 pesticide standards (Figures 3a-o). Since the volatility between individual pesticides can vary, the shape of the MRM signals for atrazine (a higher volatility pesticide) was sharper than that of benalaxyl (a lower volatility pesticide) (comparing Figure 3a with 3b). The time required to complete a replicate analysis (including sampling, desorption/ionization, and detection), data processing, and pesticide identification was less than 4 min. Figures 3p-t show the MRM chromatograms of five randomly selected pesticides (bendiocarb, bromacil, demeton-S-methyl, ethion, and vamidothion) which were not presented in the standard solution. Although the signals of two MRM transitions for bendiocarb (m/z 224→109 and m/z 224→81) seemed to be positive (Figure 3p), the duration of both MRM signals did not well match, suggesting both signals were from different ion pairs. Bendiocarb was therefore concluded to be absent in the sample solution. Similar results were obtained for demeton-S-methyl (m/z 231→89 and m/z 231→61), where two MRM transitions were detected in the first analysis (Figure 3r), but the resultant peak of the transition m/z 231→61 did not match that of m/z 231→89. For bromacil, only one transition pair (m/z 261→205) was detected on the MRM chromatogram (Figures 3q). No 11
obvious ion-pair transition was found in the MRM chromatograms of ethion and vamidothion (Figures 3s & t). These five pesticides were then concluded to be absent in the sample solution. To examine the capability of TD-ESI/MS/MS for fast screening of multiresidue pesticides on real samples, a bell pepper and tomato purchased from a local market were analyzed. Without any sample preparation, a sampling probe was swept across the surface of the sample for 2 cm. The probe was then immediately inserted into the TD-ESI source for further analysis. The process repeated five times for each sample. Figure 4 displays the MRM chromatograms for quintuplicate TD-ESI/MS/MS analysis of the sample collected from the bell pepper, where samples were collected at different positions on the bell pepper. The intact ion signals for three fungicides -propamocarb, pyraclostrobin, and fluopicolide (Figures 4a-c), one miticide -spirotetramat (Figure 4d), and two insecticides -chlorantraniliprole and dinotefuran (Figures 4e and g) were detected on the bell pepper surface. The intensities of the pesticide ion signals between each of the five replicates were unequal, indicating that the pesticides were not uniformly distributed across the bell pepper surface, so that the intensities of pesticide ions could vary based on the sampling position. The presence of these pesticides on the bell pepper was determined based on the following criteria: (1) detection of two MRM transitions of each pesticide molecular ion, (2) well match between the duration of two MRM transitions of the molecular pesticide ion and fragment ions, (3) the peak area ratio of two MRM transitions of the analyte must match that of pesticide standard, and (4) detection of the two MRM transitions at intensities higher than 102 counts. In addition, even though the molecular ion of the thermally labile kresoxim-methyl (m/z 314) was not detected using TD-ESI/MS/MS, the presence of kresoxim-methyl in the sample was still confirmed via the detection of two transitions of a thermally decomposed product ion (m/z 282) from kresoxim-methyl (m/z 282→222 and m/z 282→254) (Figure 4f). This conclusion was confirmed by TD-ESI/MS/MS analysis of a kresoxim-methyl standard (data not shown). After examining the bell pepper surface using TD-ESI/MS/MS, the sample was homogenized and extracted using the QuEChERS method, after which aliquots of the extracts were analyzed using 12
TD-ESI/MS/MS, GC-MS, and LC-MS/MS, respectively. The total time required for an experienced technician to complete these analysis was about 2 hours. Table 1 shows the pesticides detected on the bell pepper using TD-ESI/MS/MS and in its QuEChERS extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS. Comparing the TD-ESI/MS/MS results of bell pepper surface and its QuEChERS extracts, only four pesticides (propamocarb, pyraclostrobin, fluopicolide, and spirotetramat) were detected in the QuEChERS extracts, whereas seven pesticides were directly detected on the sample surface
(propamocarb,
pyraclostrobin,
fluopicolide,
spirotetramat,
chlorantraniliprole,
kresoxim-methyl, and dinotefuran). This difference in the number of detected pesticides between both approaches may owing to: (1) the pesticides on the sample surface were diluted or lost during QuEChERS extraction, which did not occur during direct surface analysis using TD-ESI/MS/MS; and (2) the interference from the matrix via surface sampling was much less than that via QuEChERS extraction, even though both samples were analyzed using TD-ESI/MS/MS. Five pesticides (propamocarb, pyraclostrobin, chlorantraniliprole, kresoxim-methyl, and λ-cyhalothrin) were detected in the QuEChERS extract using GC-MS (Figure S1), whereas seven pesticides (propamocarb, pyraclostrobin, fluopicolide, spirotetramat, chlorantraniliprole, acetamiprid, and fenobucarb) were detected using LC-MS/MS (Figure S2). Dinotefuran — detected via direct surface analysis using TD-ESI/MS/MS — was not detected during analysis of the QuEChERS extracts. This indicates that dinotefuran was lost during QuEChERS extraction. On the other hand, kresoxim-methyl was detected via direct surface analysis using TD-ESI/MS/MS and QuEChERS extraction followed by GC-MS analysis, but was not detected via QuEChERS extraction followed by LC-MS/MS analysis, as the residual pesticide method for LC-MS/MS did not include this pesticide. The nonpolar λ–cyhalothrin was more suited for GC-MS analysis, whereas acetamiprid and fenobucarb were more suited for LC-MS/MS analysis. In summary, seven pesticides were detected from the bell pepper via direct surface analysis using TD-ESI/MS/MS. After the sample was extracted by QuEChERS, four pesticides were detected using TD-ESI/MS/MS, five pesticides were detected using GC-MS, and seven pesticides were detected using LC-MS/MS (Table 1). 13
Similarly, five samples were collected from different surface locations on a tomato for direct TD-ESI/MS/MS analysis. Figure 5 displays the MRM chromatograms for direct surface analysis of the residual pesticides on the tomato. Two fungicides (azoxystrobin and pyraclostrobin) and three insecticides (dinotefuran, methomyl, and chlorantraniliprole) were detected using direct TD-ESI/MS/MS (Figures 5a-e), where the time required to complete a quintuplicate analysis was less than 4 min. The ion pair transition of the decomposition product of thiodicarb (m/z 106) was also detected (m/z 106→88 and m/z 106→58) (Figure 5f). After surface analysis using TD-ESI/MS/MS, the tomato was subjected to QuEChERS extraction followed by TD-ESI/MS/MS, GC-MS, and LC-MS/MS analysis. Table 1 shows that with the exception of chlorantraniliprole and thiodicarb, four pesticides detected on tomato surface using direct TD-ESI/MS/MS were also detected in the QuEChERS extracts that were analyzed using TD-ESI/MS/MS. However, only two volatile fungicides (azoxystrobin and pyraclostrobin) were detected using GC-MS (Figure S3), whereas two fungicides (azoxystrobin and pyraclostrobin) and six insecticides (dinotefuran, methomyl, chlorantraniliprole, thiodicarb, thiamethoxam, and clothianidin,) were detected using LC-MS/MS (Figure S4). The results indicated that direct analysis of the sample surface using TD-ESI/MS/MS could detect not only volatile pesticides (assigned via GC-MS analysis of the extracts) but also semi-volatile pesticides (assigned via LC-MS/MS analysis of the extracts). Structures of the pesticides detected in real samples are shown in Figure S5. The Venn diagrams showed in Figure 6 display the number of unique and shared pesticides detected using TD-ESI/MS/MS, GC-MS, and LC-MS/MS. For the bell pepper, 86% (6 of 7) of the pesticides detected on the surface using TD-ESI/MS/MS were also detected in the extracts using GC-MS and LC-MS/MS. The pesticides detected on the tomato surface using TD-ESI/MS/MS were all detected in the extracts using GC-MS and LC-MS/MS. These results indicate that TD-ESI/MS/MS is able to efficiently screen trace multi-residual pesticides on the surfaces of agricultural products such as bell pepper and tomato. Pesticide signal intensities that varied based on sampling location reflected the non-homogenous distribution of these pesticides across the sample 14
surface even though this was not benefit for quantitative analysis. CONCLUSION TD-ESI/MS/MS combined with probe sampling was utilized to rapidly screen trace multiresidue pesticides on agricultural products and in their extracts. A metallic probe was swept across sample surfaces to collect trace pesticides, regardless of the sample size and shape. Since sample pretreatment and chromatographic separation are unnecessary, the analytical time required to screen residual pesticides on sample surfaces was less than 1 min. Over 300 concurrent MRM transitions were monitored per analysis so that we could simultaneously detect a large number of pesticides. The analysis of tomato and bell pepper showed that 86–100% of the pesticides detected on sample surfaces using TD-ESI/MS/MS were also detected in their QuEChERS extracts using GC-MS and LC-MS/MS. The analytical time and labor required for direct surface TD-ESI/MS/MS analysis were obviously much less than that of conventional QuEChERS extraction followed by GC-MS and LC-MS/MS analysis. It should be noted that surface analysis using TD-ESI/MS/MS is not yet suited for quantitative analysis because the residual pesticides were non-homogeneously distributed on agricultural products. Since TD-ESI/MS/MS was combined with sample preparation methods such as liquid extraction and solid-phase microextraction for quantification of drugs in blood and urine. The combination of QuEChERS extraction and TD-ESI/MS/MS is therefore potential to quantify residue pesticides in agro products. In addition, pesticides with very low volatilities and polarities demonstrated poor desorption and ionization efficiencies in the TD-ESI source, and were instead more suited for analysis using LC-MS/MS or GC-MS.
Acknowledgement This work was partially supported by the Research Center for Environmental Medicine at Kaohsiung Medical University, Kaohsiung, Taiwan as part of the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. 15
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Table 1. Residual pesticides identified using TD-ESI/MS/MS, GC-MS, and LC-MS/MS (the term “nd” stands for “not detected”). TD-ESI/MS/MS Sample
Bell Pepper
Tomato
a b
Pesticide Propamocarb Pyraclostrobin Fluopicolide Spirotetramat Chlorantraniliprole Kresoxim-methyl Dinotefuran Acetamiprid Fenobucarb λ-cyhalothrin Azoxystrobin Pyraclostrobin Dinotefuran Methomyl Chlorantraniliprole Thiodicarb Thiamethoxam Clothianidin
Surface
LC-MS/MS
GC-MS
QuEChERS extract
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
nd
✓
✓
✓
nd
✓
nd
✓
✓
✓b
nd
nd
✓
✓
nd
nd
nd
nd
nd
✓
nd
nd
nd
✓
nd
nd
nd
nd
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
✓
nd
✓
✓
✓
nd
✓
nd
✓
nd
✓b
nd
✓
nd
nd
nd
✓
nd
nd
nd
✓
nd
a
Successful analyte detection when the injection volume was increased to 5 µL Decomposition product
19
Figure legends Figure 1. (a) Schematic illustration of the TD-ESI/MS/MS method for rapidly screening multiresidue pesticides on vegetable surfaces. (b) A flow chart showing the analytical procedure for fruit and vegetable samples in this work. Figure 2. TD-ESI/MS/MS MRM chromatogram for (a) benthiazole standard solutions at concentrations ranging from 5 ppb to 1 ppm, and (b) nine consecutive analysis of benthiazole pesticide solution at a concentration of 500 ppb. Figure 3. TD-ESI/MS/MS MRM chromatograms for two consecutive analysis of a mixture solution containing 15 pesticides (500 ppb each). (a-o) Pesticides identified in the sample solution: (a) atrazine, (b) benalaxyl, (c) clomazone, (d) dimethenamid, (e) dimethoate, (f) dimethomorph, (g) ethirimol, (h) fenamiphos, (i) fenthion, (j) flazasulfuron (decomposition product), (k) flusilazole, (l) mepanipyrim, (m) metolcarb, (n) metribuzin, and (o) spirotetramat. (p-t) Five randomly selected pesticides which were absent in the sample solution: (p) bendiocarb, (q) bromacil, (r) demeton-S-methyl, (s) ethion, and (t) vamidothion. Figure 4. TD-ESI/MS/MS MRM chromatograms for (a) propamocarb, (b) pyraclostrobin, (c) fluopicolide, (d) spirotetramat, (e) chlorantraniliprole, (f) kresoxim-methyl (decomposition product), and (g) dinotefuran detected on a bell pepper. Figure 5. TD-ESI/MS/MS MRM chromatograms for (a) azoxystrobin, (b) pyraclostrobin, (c) dinotefuran, (d) methomyl, (e) chlorantraniliprole, and (f) thiodicarb (decomposition product) detected on a tomato. Figure 6. The Venn diagrams showing the number of unique and shared pesticides detected on a (a) bell pepper and (b) tomato and in their extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS.
20
Figure 1. (a) Schematic illustration of the TD-ESI/MS/MS method for rapidly screening multiresidue pesticides on vegetable surfaces. (b) A flow chart showing the analytical procedure for fruit and vegetable samples in this work.
21
Figure 2. TD-ESI/MS/MS MRM chromatogram for (a) benthiazole standard solutions at concentrations ranging from 5 ppb to 1 ppm, and (b) nine consecutive analysis of benthiazole pesticide solution at a concentration of 500 ppb.
22
Figure 3. TD-ESI/MS/MS MRM chromatograms for two consecutive analysis of a mixture solution containing 15 pesticides (500 ppb each). (a-o) Pesticides identified in the sample solution: (a) atrazine, (b) benalaxyl, (c) clomazone, (d) dimethenamid, (e) dimethoate, (f) dimethomorph, (g) ethirimol, (h) fenamiphos, (i) fenthion, (j) flazasulfuron (decomposition product), (k) flusilazole, (l) mepanipyrim, (m) metolcarb, (n) metribuzin, and (o) spirotetramat. (p-t) Five randomly selected pesticides which were absent in the sample solution: (p) bendiocarb, (q) bromacil, (r) demeton-S-methyl, (s) ethion, and (t) vamidothion.
23
Figure 4. TD-ESI/MS/MS MRM chromatograms for (a) propamocarb, (b) pyraclostrobin, (c) fluopicolide, (d) spirotetramat, (e) chlorantraniliprole, (f) kresoxim-methyl (decomposition product), and (g) dinotefuran detected on a bell pepper.
24
Figure 5. TD-ESI/MS/MS MRM chromatograms for (a) azoxystrobin, (b) pyraclostrobin, (c) dinotefuran, (d) methomyl, (e) chlorantraniliprole, and (f) thiodicarb (decomposition product) detected on a tomato.
25
Figure 6. The Venn diagrams showing the number of unique and shared pesticides detected on a (a) bell pepper and (b) tomato and in their extracts using TD-ESI/MS/MS, GC-MS, and LC-MS/MS.
26
Highlights TD-ESI/MS/MS was developed to rapidly screen 308 residual pesticides on fruits and vegetables within 1 minute. The samples were directly characterized without sample preparation and chromatographic separation The identities of the pesticides detected on bell peppers and tomatoes using TDESI/MS/MS were confirmed by GC-MS and LC-MS/MS analysis of sample extracts.
Sample CRediT author statement Sy-Chyi Cheng: Writing - Original Draft, Writing - Review & Editing Ruei-Hao Lee: Methodology, Verification Jing-Yueh Jeng: Resources Chi-Wei Lee: Investigation Jentaie Shiea: Conceptualization, Supervision
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: