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New Analytical Methods

Rapid Trace Detection and Isomer Quantitation of Pesticide Residues via Matrix-assisted Laser Desorption/ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Xinzhou Wu, Weifeng Li, Peng-Ran Guo, Zhi-xiang Zhang, and Han-Hong Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00427 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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Journal of Agricultural and Food Chemistry

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Rapid Trace Detection and Isomer Quantitation of

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Pesticide

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Desorption/ionization

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Cyclotron Resonance Mass Spectrometry

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Xinzhou Wu a, Weifeng Li b, Pengran Guo b, Zhixiang Zhang a,*, and Hanhong Xu a,*

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a

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bioresources, Key Laboratory of Natural Pesticide and Chemical Biology of the

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Ministry of Education, South China Agricultural University, Guangzhou, 510642,

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China.

Residues

via

Matrix-assisted

Fourier

Transform

Laser Ion

State Key Laboratory for Conservation and Utilization of Subtropical Agro-

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b

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Chemicals, Guangdong Engineering and Technology Research Center for Ambient

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Mass Spectrometry, Guangdong Institute of Analysis, Guangzhou, 510070, China

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*E-mail: [email protected]; [email protected]

Guangdong Provincial Key Laboratory of Emergency Test for Dangerous

ACS Paragon Plus Environment

1


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ABSTRACT

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Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron

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resonance mass spectrometry (MALDI-FTICR-MS) has been applied for rapid,

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sensitive, undisputed, and quantitative detection of pesticide residues on fresh leaves

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with little sample pretreatment. Various pesticides (insecticides, bactericides,

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herbicides, and acaricides) are detected directly in the complex matrix with excellent

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limits of detection down to 4 µg/L. FTICR-MS could unambiguously identify

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pesticides with tiny mass differences (~ 0.01775 Da), thereby avoiding false-positive

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results. Remarkably, pesticide isomers can be totally discriminated using diagnostic

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fragments, and quantitative analysis of pesticide isomers are demonstrated. The

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present results expand the horizons of the MALDI-FTICR-MS platform in the reliable

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determination of pesticides, with integrated advantages of ultra-high mass resolution

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and accuracy. This method provides growing evidence for the resultant detrimental

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effects of pesticides, expediting the identification and evaluation of innovative

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pesticides.

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KEYWORDS: Pesticide residues, isomer quantitation, limit of detection, MALDI,

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high-resolution FTICR-MS


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INTRODUCTION

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Gaining insight into the environmental behaviors of pesticides is key to

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understanding their conversion processes and resultant detrimental effects in the

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natural environment, especially in decreasing the foraging success and survival of

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honey bees worldwide.1-3 It provides scientific evidence for the discovery, rational use,

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and potential hazard evaluation of novel pesticides, facilitating the reduction of

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environmental pollution and increase of bee populations.4, 5 A series of environmental

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behaviors will be initiated once pesticides are sprayed onto the surface of foodstuffs,

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such as absorption, photolysis, and volatilization; therefore, scientific assessment of

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the effect of pesticides on the environmental safety is highlighted.

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Trace detection of detrimental pesticide residues in foodstuffs, such as fruits and

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vegetables, has received a significant amount of attention due to the toxicity or

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carcinogenicity of pesticides with regard to human health. A reasonable use of

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pesticides is beneficial for enhancing crop longevity and yield, whereas overuse will

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initiate safety problems.6-9 In spite of the potential for adverse effects, a wide array of

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pesticides have been adopted in the agricultural field, e.g., insecticides, bactericides,

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herbicides, acaricides.10-14 To ensure food safety and human health, many stringent

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directives have been proposed for establishing maximum residue limits (MRLs) for

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agrochemicals used in foodstuff.15 MRLs normally range from tens or hundreds of

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µg/kg up to several mg/kg on the basis of the biological activity of individual

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compounds. Rapid screening of pesticides is an important yet challenging issue which

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requires both high-sensitivity detection and reliable identification of known

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agrochemicals or unknown degradation products with potential health hazards. Hence,

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an appropriate strategy for direct and rapid pesticide residues detection with no or

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little sample pretreatment has long been needed.15-17


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Conventional separation analysis methods, such as gas chromatography (GC) and

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high-performance liquid chromatography (HPLC), have been widely applied in

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quantifying pesticides and food additives/extracts.18-22 However, these methods

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require pesticides on the surface of foodstuff to be dissolved, extracted, concentrated,

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and sometimes purified before analysis. Solvent-based sample preparation might

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generate contaminants, limiting high throughput mass spectrometry analysis of

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pesticides. A time-consuming gradient elution is also inevitable, and in return, solvent

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contamination and waste is self-evident.23, 24 Another disadvantage of this approach is

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that a severe matrix effect can compromise reliable analysis.

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Inspired by the pioneering efforts of R. G. Cooks’ group in 2004,25 multitudinous

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ambient mass spectrometric methods, such as desorption electrospray ionization

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(DESI),26, 27 direct analysis in real time (DART),15, 17 desorption atmospheric pressure

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chemical ionization (DAPCI),28 and atmospheric pressure glow discharge (APGD),29

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have been proposed. These methods are potential candidates for screening pesticides

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in their native condition with the integrated advantages of no or little sample

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pretreatment, in situ, high throughput, and high sensitivity. Moreover, ambient

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ionization can be coupled to a handheld mass spectrometer for in situ screening of

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pesticide residues on the fruit surface, facilitating the rapid quality monitoring of

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complex samples.27,

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convenient geometry and high resolving power.31 In practical applications, many

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pesticides, some detrimental and some not, might only differ by a tiny amount in mass

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or may exist as isomers. Therefore, many issues in pesticide analysis remain to be

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solved, and numerous possibilities for improvement still exist.

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However, there are some tradeoffs between achieving a

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Thanks to the unique features of ultra-high resolving power, mass accuracy and

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versatile fragmentation techniques, matrix-assisted laser desorption/ionization Fourier


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transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) has long

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been acknowledged as an excellent method for direct qualitative and quantitative

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analysis of mixtures with complex matrices.32,

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been reported for the analysis of pesticides by other high-resolution mass

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spectrometry, e.g., Orbitrap, the capability of MALDI-FTICR-MS in the rapid and

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direct solid analysis of pesticide residues without any chromatographic separation are

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still in infancy.34-37 Compared with the combination of MALDI and time-of-flight

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mass spectrometer, the spectral interference derived from MALDI matrices can be

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well resolved by virtue of the ultra-high resolving power of FTICR-MS, facilitating

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maximization of the analytical performance of MALDI for rapid screening of

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pesticide residues. Recently, a resolving power exceeding 1,000,000 has been

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reported for FTICR-MS; therefore, it promises the undisputed discrimination of

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pesticides with tiny mass differences, even smaller than 1.1 mDa (elemental

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compositions differing by SH313C versus 12C4).32, 33 Otherwise, these two peaks could

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be mistaken for the same compound, leading to wrong conclusions. In addition, the

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huge potential of high-resolution tandem MS techniques for the quantitative analysis

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of pesticide isomers has seldom been explored.

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Although numerous studies have

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The aim of the present study was to explore the potential of the MALDI-FTICR-MS

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technique for rapid, qualitative, and quantitative detection of pesticide residues on

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fresh leaves. This approach shows great promise for the simultaneous determination

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of pesticide mixtures undisputedly, even for pesticides with tiny mass variations

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arisen from elemental compositions differing by O2 versus S. LODs down to low

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µg/L for pure pesticides on leaves, and LODs in low to tens of µg/L range for ten

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pesticide mixtures on leaves can also be achieved through MALDI-FTICR-MS. The

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measured LODs for most pesticides are below the established MRLs, indicating its


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potential for precise discrimination and use as a universal trace detection method.

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Aside from these traits, direct quantitation of pesticide isomers was achieved for both

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detrimental agrochemicals and harmless pharmaceutical intermediates, showing the

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feasibility of the method for isomer discrimination and quantitative measurement by

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the relative intensity of diagnostic fragments versus the concentration ratio. The

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results indicate that the MALDI-FTICR-MS platform expands the ability of MS to

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integrate high-sensitivity detection with the reliable identification of pesticide

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mixtures with tiny mass differences, including the direct quantitative analysis of

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pesticide isomers in complex matrices.

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MATERIALS AND METHODS

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Chemicals and Materials. Dimethomorph, dimethoate, difenoconazole, tebuconazole,

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thiabendazole, spirotetramat, rotenone, flonicamid, epoxiconazole, oxasulfuron,

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simazine, diafenthiuron, cletoquine, and [(1S,3S,4S)-4-amino-3-hydroxy-5-phenyl-1-

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(phenylmethyl)pentyl]carbamic acid 1,1-dimethyl ethyl ester were purchased from

120

J&K Scientific Ltd. (Beijing, China). The matrix 2,5-dihydroxybenzoic acid (DHB)

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was purchased from Bruker Daltonics (Bremen, Germany), and HPLC-grade

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acetonitrile and formic acid were purchased from Tedia (Fairfield, USA) and Fluka

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Analytical (Sigma-Aldrich, Switzerland), respectively. Detailed information on the

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molecular classification, formula, average mass, and structure of pesticides used in the

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experiments is listed in Table S1 (Supporting Information).

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Preparation of Pesticide Mixtures and Pesticide-sprayed Broad Bean Leaves.

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Individual stock solutions of all the pesticides listed in Table S1 were first dissolved

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in acetonitrile at a concentration of 1,000 mg/L. Subsequently, the pesticide solutions

129

were diluted with deionized water into a series of concentrations ranging from 0.005

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to 10 mg/L for further investigation. And pesticide mixtures contain ten different


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species,

including

spirotetramat,

dimethomorph,

rotenone,

difenoconazole,

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tebuconazole, epoxiconazole, thiabendazole, oxasulfuron, simazine, diafenthiuron.

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Unless otherwise stated, the concentrations of all the pesticide residues referred to in

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this manuscript indicate the mass concentration (mg/L or µg/L) in the solution before

135

drying.

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Prior to the preparation of pesticide-sprayed broad bean leaves, broad bean seeds

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were cultivated in vermiculite after immersion in water for one day. A plant growth

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chamber with the temperature and humidity of 22±1 °C and 80±5%, respectively, was

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used for 14 h of daytime light exposure. The broad bean seeds sprouted after 6 days of

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cultivation in vermiculite and were fed with chemical fertilizer every week. Then,

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uniformly growing broad bean leaves after four weeks were chosen as the test plants,

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and realistic simulations of pesticide spraying were executed. The pesticide solutions

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of different concentrations were sprayed to the broad bean leaves. They were

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naturally air dried for 3 h and then used for the subsequent MALDI-FTICR-MS or

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HPLC experiments. Specifically, leaves were cut into numerous small leaves pieces

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with size of about 6 mm×6 mm. All the leaves pieces were fixed on a commercial

147

MALDI plate by conductive graphite adhesive. Five pieces of each concentration

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were used for parallel experiments, and each piece occupied a specific position of

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MALDI plate, facilitating the observation and positioning of sampling leaves pieces

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with different pesticide species and concentrations using the built-in CCD system. In

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the MALDI-FTICR-MS analysis, 1 µL droplet of DHB matrix in a water-acetonitrile-

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formic acid mixture (50/50/0.1, v/v/v) was deposited on the surface of each pesticide-

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sprayed leaves pieces and air-dried.

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MALDI-FTICR-MS Analysis. All high-resolution mass analysis was performed

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on a FTICR mass spectrometer equipped with a 7T actively shielded magnet (SolariX


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XR 7T, Bruker Daltonic GmbH, Bremen, Germany) in positive ion mode.

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Electrospray ionization (ESI) and MALDI sources can be switched in FTICR-MS, but

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MALDI source was used in the whole experiments. All experiments were conducted

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using a smartbeam laser which was set to the medium spot size with a repetition rate

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of 100 Hz and with 100 laser shots. To achieve the desired signal intensity and

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guarantee homogeneous sampling, laser irradiance of a “random” walk movement

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was employed. Eight transients were accumulated per final spectrum from m/z 150 to

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1,000 using 1 M data sets unless otherwise specified. For each transient, an ion

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accumulation time of 1 ms and a time-of-flight of 1 ms were adopted. The FTICR

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mass spectrum was externally calibrated with respect to a sample containing cesium

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iodide. All data were processed using Bruker DataAnalysis 4.4 software and

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interpreted manually. Tandem MS (MS/MS) analyses were acquired via collision-

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induced dissociation (CID). Prior to executing MS/MS analysis, the ions of interest

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were chosen by quadrupole and isolated in a hexapole cell for subsequent CID

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experiments. The isolation window was set at 5 and the collision energy was adjusted

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ranging from 3.5 to 20 eV. To avoid confusion, m/z values involved in the mass

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spectrum of this article correspond to the mono-isotope of the mentioned ion specie

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with the most abundance unless otherwise noted.

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High Performance Liquid Chromatography (HPLC) Analysis. Separation and

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analysis of the extracted solutions was conducted on an Agilent 1290 HPLC system

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using a C18 reversed-phase column (1.8 µm; 100 mm × 2.1 mm i.d.; Agilent) with a

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flow rate of 0.1 mL/min at 30 °C. The eluent was made of water and acetonitrile in an

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isocratic elution and detected by UV absorption at different wavelength. The detailed

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conditions of gradient elution and UV absorption wavelengths of four types of

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pesticides and pesticide isomers are listed in Table S2 of the Supporting Information.


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RESULTS AND DISCUSSION

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LODs of MALDI-FTICR-MS for Four Types of Pesticides. Prior to MALDI-MS

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analysis of the pesticide mixtures, four illustrative pesticides, including rotenone

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(insecticide), tebuconazole (bactericide), oxasulfuron (herbicide), and diafenthiuron

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(acaricide), were chosen for the evaluation of methodological LODs. In the initial

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experiments, many different MALDI matrices (e.g., DHB, α-cyano-4-hydroxy-

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cinamic acid, sinapic acid) have been tested, and DHB was chosen for its higher

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detection sensitivity. First, the MALDI-FTICR mass spectrum of pure broad bean

189

leaves with DHB matrix added was investigated. As shown in Figure 1a, only DHB

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matrix as well as endogenous compounds of broad bean leaves, such as C18 caffeate

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(C27H44O4), kaempferol glucoside (C21H20O11), and C20 ferulate (C30H50O4), were

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detected.38-40 However, an unambiguous FTICR mass spectrum of rotenone, which

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was sprayed onto the broad bean leaves at a concentration of 1 mg/L, was also

194

obtained (Figure 1b). In addition to the matrix background and endogenous

195

compounds of leaves, the molecular ion of rotenone with a matched theoretical

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isotope pattern was clearly observed. Rapid detection of the other three pesticide

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molecules (tebuconazole, oxasulfuron, and diafenthiuron) can also be achieved with

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high signal-to-noise ratio (SNR) (see Figure S1 of the Supporting Information). In

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addition, HPLC results of these four types of pesticides were also obtained for

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comparison as shown in Figure S2 of the Supporting Information. The compositions

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of extracted solutions from pesticide-sprayed leaves are very complex, and

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interference peaks derived from many endogenous compounds and the solvent

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background are unavoidable. Thus, four pure pesticides at the concentration of 1 mg/L

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without leaf matrix can be clearly detected, whereas only ambiguous information on

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whether pesticide residues remain on the leaf surface can be acquired when extracted


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solutions from pesticide-sprayed leaves were introduced. It would take a lot of time to

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optimize the elution conditions and purify the pesticides of interest for precise

208

discrimination via this method. Compared to HPLC analysis, MALDI-FTICR-MS is

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of great utility for the rapid screening and detection of pesticide residues in the

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complex matrix. Incremental concentrations from 0.01 to 10 mg/L were used to

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calculate methodological LODs of four types of pesticides, which were sprayed on

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leaves directly. LODs were calculated using at least five replicates at each

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concentration level of the calibration curve by definition.41, 42 As shown in Figure 2,

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good linearity of calibration curves and excellent correlation coefficients (R2) better

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than 0.98 can be acquired. The LODs for rotenone, tebuconazole, oxasulfuron, and

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diafenthiuron were calculated to be 4 µg/L, 9 µg/L, 5 µg/L, and 7 µg/L, respectively.

217

This method offers excellent LODs of one or two orders of magnitude lower than the

218

MRLs for pesticide residues. Compared to conventional separation analysis methods,

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these results demonstrate that MALDI-FTICR-MS, with the advantage of direct solid

220

analysis and little sample pretreatment, is a very sensitive method for the detection of

221

pesticide residues in practical applications.

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LODs of MALDI-FTICR-MS for Ten Pesticide Residue Mixtures. To further

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elucidate the feasibility and universality of this method in practical pesticide residue

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detection, a batch of pesticide residue mixtures containing ten pesticides was sprayed

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onto broad bean leaves and investigated. The mixtures included four different

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classifications of insecticides, bactericides, herbicides, and acaricides, and the detailed

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information is listed in Table S3 of the Supporting Information. The corresponding

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mass spectrum at a mixture concentration of 500 µg/L is shown in Figure 3. Some

229

peaks are saturated for showing the trace signals. All the pesticide molecules at such

230

low concentration could be detected unambiguously due to the ultra-high resolving


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power and mass accuracy of FTICR-MS. In an effort to identify the known pesticides

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and elucidate their molecular structures in the mixture system, tandem mass

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spectrometry (MS/MS) with multiple dissociation techniques is indispensable,

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whereas the high resolving power and mass accuracy of FTICR-MS make it a method

235

of choice for rapid screening without additional effort. The theoretical and

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experimental m/z values of ten pesticide mixtures, as well as mass errors in the low

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ppm range are listed in Table S3 of the Supporting Information. These results indicate

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that the method facilitates the rapid identification of trace pesticide residues with

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proof-positive. In theory, better mass accuracy can be obtained by optimizing

240

instrumental parameters, but there is a trade-off between implementing desirable

241

sensitivity and mass accuracy. Even so, mass accuracy down to low ppm is sufficient

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for the differentiation of pesticide mixtures even in the intricate matrices. On closer

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inspection of Figure 3, it can be inferred that the signal intensities for different kinds

244

of pesticides are not uniform, despite the same spray concentration being applied.

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This effect is rationalized by the discrepant ionization efficiency based on the proton

246

transfer mechanism.43 Hence, competition ionization phenomenon will occur in the

247

classes of pesticides with different physicochemical properties. The comprehensive

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treatment of foodstuff using different kinds of pesticides for integrated collaboration,

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such as using insecticide and bactericide, is inevitable in practical applications. The

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calibration curves of ten pesticide mixtures were obtained for incremental

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concentrations from 0.01 to 10 mg/L to calculate LOD values. Good linearity of

252

calibration curves and excellent R2 (> 0.95) can be obtained for all the molecules in

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the pesticide mixtures, and the results are depicted in Figure S3 of the Supporting

254

Information, indicating this method has the potential for quantitative analysis of

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diverse complex pesticides in practical systems. Listed in Table 1 are categories, R2,


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and LODs for the ten pesticide mixtures. Excellent LODs of low to tens of µg/L for

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the ten residue pesticide mixtures on the leaf surface could be acquired. Compared

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with detection of a single pesticide residue, obtaining higher LODs for the pesticide

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mixtures is reasonable due to competitive inhibition of ionization processes.

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The Effect of High Mass Resolution on Pesticide Discrimination. Many studies

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have been conducted using a MALDI source coupled with time-of-flight mass

262

spectrometer for the detection of pesticide residues and vegetal endogenous

263

compounds.44, 45 However, its limited mass resolution always produces a dilemma in

264

the analysis of practical mixture systems when their m/z values are very close to each

265

other. Mass resolution of FTICR-MS is relatively insensitive to high kinetic energy

266

dispersal of given m/z ions when high laser energy is applied, whereas the

267

performance of time-of-flight mass spectrometer (e.g., mass resolution and accuracy)

268

is significantly dropped as laser energy increases, especially in complex matrix.46 The

269

performance worsens when the pesticide residues of interest encounter endogenous

270

peak interferences from leaves and other harmless pesticides with adjacent mass,

271

thereby a wrong conclusion might be reached. To this end, FTICR-MS is becoming

272

an indispensable method for the direct discrimination of molecules with tiny mass

273

differences in complex environments due to its ultra-high mass resolution and mass

274

accuracy. Therefore, three groups of pesticides with minuscule mass differences have

275

been used to investigate the effect of high mass resolution on pesticide discrimination.

276

The information on mass differences is listed in Table S4 of the Supporting

277

Information. As shown in Figure 4, a tiny mass difference such as 0.0467 Da and

278

0.0420 Da can be well resolved due to the increased mass resolution and mass

279

accuracy of FTICR-MS. Otherwise, these two peaks would be mistaken for being

280

produced from the same compound. Resolving power above 200,000 can be easily


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achieved for thiabendazole via FTICR-MS; therefore, the method is sufficient for the

282

undisputed discrimination of pesticide analogues with mass differences within 2 mDa

283

for a pesticide with m/z 400.

284

In practice, another important fact to note is whether or not pesticide molecules

285

contain sulfur atoms. This poses a challenge for conventional low-resolution mass

286

spectrometry. Unlike chlorine or bromine atoms with easy-to-diagnose isotope

287

patterns, accurate identification of sulfur-containing compounds normally requires the

288

high resolving power of MS because one striking feature of spectra derived from

289

sulfur-containing compounds is the split peaks at the second isotopic peak (A+2),

290

which can only be observed provided that the resolving power is higher than 100,000,

291

as shown in the bottom right of Figure 5c (marked in blue). To further validate the

292

feasibility of the MALDI-FTICR-MS method, another pair of a pesticide and a

293

medical intermediate with a smaller mass difference, only differing in two oxygen

294

atoms (31.98982 Da for mono-isotope mass) versus one sulfur atom (31.97207 Da for

295

mono-isotope mass), was examined. As depicted in the inset of Figure 5a, the tiny

296

mass difference of 0.01775 Da between diafenthiuron (C23H32N2OS) and [(1S,3S,4S)-

297

4-amino-3-hydroxy-5-phenyl-1-(phenylmethyl)pentyl]carbamic

298

ethyl ester (C23H32N2O3) could also be clearly distinguished. It is of paramount

299

importance to differentiate harmless compounds from detrimental pesticides in

300

practical applications. Moreover, the split peaks at the (A+2) isotope pattern (m/z

301

range from 387.15 to 387.25) of diafenthiuron (C23H32N2OS) were observed distinctly

302

as shown in Figure 5c, facilitating the undoubted identification of pesticides

303

containing sulfur atoms. The theoretical spectra for C23H32N2O3 and C23H32N2OS are

304

marked in purple and blue, respectively, further supporting that both molecules, of

305

which one contains a sulfur atom, can be unambiguously discriminated. Compared


acid

1,1-dimethyl

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with other MS techniques, the ultra-high resolution power of FTICR-MS and the

307

isotopic fine structure peak shows great promise in discriminating pesticides with tiny

308

mass differences directly in complex matrices, as well as whether they contain

309

heteroatoms, such as sulfur. Under most circumstances, higher resolving power is

310

indispensable to resolve closely spaced mass doublets in complex systems with

311

increasing numbers of atoms. This method also affords further, yet unverified,

312

possibilities.

313

Quantitation of Pesticide Isomers via High Resolution Tandem FTICR-MS. A

314

lingering notion of mass spectrometry for isomer discrimination has been the problem

315

of indistinguishable peaks for identical masses, not to mention achieving a

316

quantitative analysis. It is worth mentioning that some isomers of pesticides of

317

interest is hard to distinguish when no extra chromatographic separation step or ion-

318

mobility spectrometry are employed. However, solvent-based sample preparation

319

process is unavoidable, impeding the rapid and direct pesticide isomer residues

320

analysis.22, 47-49 To circumvent this issue, an attempt at the discrimination of pesticide

321

isomers sprayed on leaves was conducted by tandem FTICR-MS using CID

322

fragmentation mode. As shown in Figure 6a, the fragment-rich spectra of pesticide

323

isomers for tebuconazole (bactericide) and cletoquine (medical intermediate, see

324

Table S1 of the Supporting Information) were acquired. The spectra provide the same

325

protonated molecular ion peaks in the parent mass spectrum (data not shown), but

326

dramatic differences are observed in the tandem mass spectrum for these two isobaric

327

molecules (Figure 6a), which is an essential prerequisite for the comprehensive

328

identification of isomers.

329

corresponding to m/z 165.04659 and m/z 151.03090 can be attributed to fragments

330

resulting from the loss of C7H6Cl and C8H8Cl, respectively, the conjugated fragments

For tebuconazole,

the primary fragment peaks


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331

of which can be observed at m/z 125.01527 and m/z 139.03093. Additionally, the

332

characteristic fragment peaks of cletoquine at m/z 247.09972 and m/z 179.03710 can

333

be attributed to fragments originating from the loss of C2H6NO and C7H16NO. The

334

fragment corresponding to m/z 130.12266 is the conjugated fragment of [M+H-

335

C7H16NO]+. To make our discussion more concrete, a mixture of tebuconazole and

336

cletoquine was prepared and sprayed onto fresh leaves. All of the characteristic

337

fragments from both isomers can be visualized and highlighted in Figure 6b. This

338

paves the way for the rapid discrimination of pesticide isomers by their diagnostic

339

product ions without the need for other separation methods. In addition, the

340

comparative results from the HPLC spectra of tebuconazole only, cletoquine only,

341

and tebuconazole/cletoquine mixtures are depicted in Figure S4 of the Supporting

342

Information. However, it is hard to distinguish between these isobaric pesticides in the

343

HPLC spectra unless the time-consuming gradient elution is adopted.

344

In addition, MALDI-FTICR-MS opens up the possibility of direct quantifying

345

pesticide isomers based on distinguishing fragments. To further test this possibility, a

346

quantitative correlation between the relative intensities of two characteristic fragments

347

from two isomers and their concentration ratio was investigated. To provide insight

348

into the diagnostic fragments of tebuconazole and cletoquine, such as m/z 151.03090

349

and m/z 125.01527 for tebuconazole and m/z 247.09972, m/z 179.03710, and m/z

350

130.12266 for cletoquine, calibration curves of I179.03710/I125.01527 (marked in pink) and

351

I247.09972/I125.01527 (marked in blue) were acquired as shown in Figure 7. Of particular

352

interest is that good linearity of calibration curves and excellent R2 better than 0.99

353

can be acquired. Similar results can be obtained with respect to I130.12266/I125.01527,

354

I130.12266/I151.03090, I179.03710/I151.03090, and I247.09972/I151.03090, as shown in Figure S5 of the


15


Page 16 of 32

355

Supporting Information, indicating the potential of tandem FTICR-MS for the

356

quantitative analysis of pesticide isomers.

357

In this work, the figures of merit of the analytical methodology for the rapid trace

358

screening of pesticide residues on fresh leaves via MALDI-FTICR-MS have been

359

demonstrated. Compared to HPLC analysis, the results herein suggest that this

360

technique is of great potential for rapid, high-throughput, sensitive determination of

361

trace pesticide residues in a complex matrix. LODs down to 4 µg/L for pure pesticides,

362

as well as LODs of low to tens of µg/L for pesticide mixtures were obtained. More

363

importantly, reliable identification of agrochemicals poses a challenge for

364

conventional low-resolution mass spectrometry. Undisputed identification of

365

pesticides with adjacent mass, even for the mass difference between

366

Da) and

367

this method opens new possibilities for the discrimination of pesticide isomers by

368

their diagnostic product ions. Good qualitative linearity of calibration curves can be

369

obtained, further supporting the notion that this method is of great benefit for the

370

direct quantitative analysis of pesticide isomers. Every year, a considerable number of

371

new pesticides are discovered or synthetized with adjacent masses and mutual isomers,

372

which further emphasizes the MALDI-FTICR-MS instrument as a versatile platform

373

for expediting the identification and determination of new pesticides.

374

ASSOCIATED CONTENT

375

Supporting Information

376

This material is available free of charge via the Internet at http://pubs.acs.org.

377

Information on the pesticide compounds used in the experiments, gradient elution and

378

UV absorption wavelength conditions for HPLC detection, MALDI-FTICR-MS and

379

HPLC spectra of rotenone, tebuconazole, oxasulfuron, and diafenthiuron residues, the

32

16

O2 (31.98982

S (31.97207 Da), was directly achieved using FTICR-MS. Furthermore,


16

Page 17 of 32


380

theoretical and experimental m/z values as well as mass errors of ten pesticide

381

mixtures, calibration curves of ten pesticide mixtures, calibration curves of pesticide

382

isomers with respect to m/z 130.12266/125.01527, m/z 130.12266/151.03090, m/z

383

179.03710/151.03090, and m/z 247.09972/151.03090 versus concentration ratio of

384

cletoquine and tebuconazole can be found in the Supporting Information.

385

AUTHOR INFORMATION

386

Corresponding Author

387

*E-mail:

388

[email protected];

389

[email protected].

390

ORCID

391

Hanhong Xu: 0000-0001-7841-2396

392

Xinzhou Wu: 0000-0001-5079-4994

393

Funding

394

We gratefully acknowledge the financial support provided by the National Natural

395

Science Foundation of China (grant 31672044), the Natural Science Foundation of

396

Guangdong Province (grant 2014A030311044 and 2016A030313387), and the

397

Science and Technology Program of Guangzhou (grant 201707020013).

398

Notes

399

The authors declare no competing financial interest.

400

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401 402 403 404

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560


21


Page 22 of 32

561

Figure captions

562

Figure 1. MALDI-FTICR mass spectrum of a) only DHB matrix on broad bean

563

leaves and b) rotenone residues sprayed onto broad bean leaves with DHB matrix at

564

the concentration of 1 mg/L.

565

Figure 2. Calibration curves of a) rotenone, b) tebuconazole, c) oxasulfuron, and d)

566

diafenthiuron range from 10 µg/L to 10,000 µg/L. Error bars represent the standard

567

deviation from five measurements.

568

Figure 3. Typical MALDI-FTICR mass spectrum of ten pesticide mixtures at the

569

concentration of 500 µg/L. Molecular ion peaks of ten pesticides are marked in blue,

570

MALDI matrix peaks are marked in brown, and some endogenous peaks derived from

571

broad bean leaf are marked in green.

572

Figure 4. High-resolution MALDI-FTICR mass spectrum of a) dimethoate

573

(C9H6F3N3O) and flonicamid (C5H12NO3PS2) and b) thiabendazole (C10H7N3S) and

574

simazine (C7H12ClN5). The concentration of all three mixtures were 1 mg/L. Some

575

interference peaks from leaves are not marked to avoid overcrowding in the mass

576

spectrum.

577

Figure 5. High-resolution MALDI-FTICR mass spectrum of diafenthiuron

578

(C23H32N2OS)

579

(phenylmethyl)pentyl]carbamic acid 1,1-dimethyl ethyl ester (C23H32N2O3), which

580

only differ by

581

from 240 to 420 and expanded views at b) m/z 385.0-387.5 and c) m/z 387.15-387.35.

582

The experimental spectrum and isotope pattern are marked in black, and the

583

theoretical spectra for C23H32N2O3 and C23H32N2OS are marked in purple and blue,

584

respectively. The concentration of the residue mixture was 1 mg/L.

585

Figure 6. a) CID tandem mass spectrum of two pure isomers (C16H22ClN3O, MW =

586

307.83172), tebuconazole (marked in blue) and cletoquine (marked in pink), acquired

587

with a dissociation energy of 20 eV. The pesticide isomers at a concentration of 5

and

16

O2 and

32

[(1S,3S,4S)-4-amino-3-hydroxy-5-phenyl-1-

S atoms. a) The corresponding spectrum in the m/z range


22

Page 23 of 32


588

mg/L were sprayed onto fresh leaves. b) CID tandem mass spectrum of isomer

589

mixtures of tebuconazole and cletoquine was acquired with a dissociation energy of

590

20 eV. The concentration ratio of tebuconazole (5 mg/L) and cletoquine (0.25 mg/L)

591

was 20:1 to obtain a relatively uniform signal intensity of fragments.

592

Figure 7. Quantitative calibration curves of cletoquine and tebuconazole in mixture

593

form. The calibration curves were obtained by the relative intensities of characteristic

594

fragments from cletoquine and tebuconazole, such as I179.03710/I125.01527 (marked in

595

pink) and I247.09972/I125.01527 (marked in blue), versus the concentration ratio of

596

cletoquine and tebuconazole, which ranged from 0.05 to 20. The concentration of

597

molecules for higher ratios was kept at 5 mg/L.


23


Page 24 of 32

Table 1. Correlation coefficients (R2) of calibration curves and LODs for ten pesticides acquired via MALDI-FTICR-MS pesticides

category

R2

LOD (µg/L)

spirotetramat

insecticide

0.9788

21

dimethomorph

insecticide

0.9942

13

rotenone

insecticide

0.9987

42

difenoconazole

bactericide

0.9701

8

tebuconazole

bactericide

0.9891

48

epoxiconazole

bactericide

0.9831

26

thiabendazole

bactericide

0.9890

5

oxasulfuron

herbicide

0.9968

6

simazine

herbicide

0.9799

96

diafenthiuron

acaricide

0.9837

22


24

Page 25 of 32


Figure graphics

Figure 1. MALDI-FTICR mass spectrum of a) only DHB matrix on broad bean leaves and b) rotenone residues sprayed onto broad bean leaves with DHB matrix at the concentration of 1 mg/L.


25


Page 26 of 32

Figure 2. Calibration curves of a) rotenone, b) tebuconazole, c) oxasulfuron, and d) diafenthiuron range from 10 µg/L to 10,000 µg/L. Error bars represent the standard deviation from five measurements.


26

Page 27 of 32


Figure 3. Typical MALDI-FTICR mass spectrum of ten pesticide mixtures at the concentration of 500 µg/L. Molecular ion peaks of ten pesticides are marked in blue, MALDI matrix peaks are marked in brown, and some endogenous peaks derived from broad bean leaf are marked in green.


27


Page 28 of 32

Figure 4. High-resolution MALDI-FTICR mass spectrum of a) dimethoate (C9H6F3N3O) and flonicamid (C5H12NO3PS2) and b) thiabendazole (C10H7N3S) and simazine (C7H12ClN5). The concentration of all three mixtures were 1 mg/L. Some interference peaks from leaves are not marked to avoid overcrowding in the mass spectrum.


28

Page 29 of 32


Figure 5. High-resolution MALDI-FTICR mass spectrum of diafenthiuron (C23H32N2OS)

and

[(1S,3S,4S)-4-amino-3-hydroxy-5-phenyl-1-

(phenylmethyl)pentyl]carbamic acid 1,1-dimethyl ethyl ester (C23H32N2O3), which only differ by

16

O2 and

32

S atoms. a) The corresponding spectrum in the m/z range

from 240 to 420 and expanded views at b) m/z 385.0-387.5 and c) m/z 387.15-387.35. The experimental spectrum and isotope pattern are marked in black, and the theoretical spectra for C23H32N2O3 and C23H32N2OS are marked in purple and blue, respectively. The concentration of the residue mixture was 1 mg/L.


29


Page 30 of 32

Figure 6. a) CID tandem mass spectrum of two pure isomers (C16H22ClN3O, MW = 307.83172), tebuconazole (marked in blue) and cletoquine (marked in pink), acquired with a dissociation energy of 20 eV. The pesticide isomers at a concentration of 5 mg/L were sprayed onto fresh leaves. b) CID tandem mass spectrum of isomer mixtures of tebuconazole and cletoquine was acquired with a dissociation energy of 20 eV. The concentration ratio of tebuconazole (5 mg/L) and cletoquine (0.25 mg/L) was 20:1 to obtain a relatively uniform signal intensity of fragments.


30

Page 31 of 32


Figure 7. Quantitative calibration curves of cletoquine and tebuconazole in mixture form. The calibration curves were obtained by the relative intensities of characteristic fragments from cletoquine and tebuconazole, such as I179.03710/I125.01527 (marked in pink) and I247.09972/I125.01527 (marked in blue), versus the concentration ratio of cletoquine and tebuconazole, which ranged from 0.05 to 20. The concentration of molecules for higher ratios was kept at 5 mg/L.


31


Page 32 of 32

Graphic for table of contents


32

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