On the use of in-source fragmentation in ultra high

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New Analytical Methods

On the use of in-source fragmentation in ultra highperformance liquid chromatography-electrospray ionizationhigh resolution mass spectrometry for pesticide residue analysis Zeying He, Yaping Xu, Yanwei Zhang, Bingjie Liu, and Xiaowei Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04583 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Journal of Agricultural and Food Chemistry

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On the use of in-source fragmentation in ultra high-performance liquid chromatography-

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electrospray ionization-high resolution mass spectrometry for pesticide residue analysis

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Zeying Hea, Yaping Xua, Yanwei Zhanga, Bingjie Liub, Xiaowei Liu a *

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aKey

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Agriculture, Agro-Environmental Protection Institute, Ministry of Agriculture and Rural Affairs,

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Tianjin 300191, P.R. China

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bSCIEX,

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*Corresponding author:

Laboratory for Environmental Factors Control of Agro-product Quality Safety, Ministry of

Analytical Instrument Trading Co., Ltd, Beijing 100015, China

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Xiaowei Liu, Key Laboratory for Environmental Factors Control of Agro-product Quality Safety,

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Ministry of Agriculture, Agro-Environmental Protection Institute, Ministry of Agriculture and Rural

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Affairs, Tianjin 300191, P.R. China;

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Tel.: +86 022-23611006; Fax: +86 022-23611006;

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E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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In this work, a highly efficient pesticide residue screening and quantification method was

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established using UPLC-QTOF based on in-source fragmentation. Over 400 pesticides were tested,

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among which, 96 pesticides displayed in-source fragmentation. A novel concept of in-source

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fragment fraction (i-SFF) was proposed to evaluate the extent of in-source fragmentation, which

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was found to be chemical structure and source parameter dependent. A high-resolution MS/MS

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library containing 403 pesticides and 126 fragments was created and was applied for library

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searching of pesticide residues in vegetables and fruits. The introduction of in-source fragments

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effectively circumvented misannotation and occurrence of false negatives. The quantification

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ability for the fragments was validated in terms of recovery, linearity, and LOQ and its superiority

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to the parent pesticides was established. Finally, the proposed method was applied for analysis of

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real samples and proficiency test samples and false negative results were successfully avoid in the

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analysis.

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Key words: in-source fragmentation, in-source fragments, pesticide residue, UPLC-ESI-QTOF,

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MS/MS library

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INTRODUCTION

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Pesticides are widely used in agricultural practice to control pests, plant disease and weed in

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order to promote crop yield and quality. In China alone, 689 different pesticides are commercially

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available, and as many as 41282 formulations of these products haven been registered until May

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2019.1 Furthermore, close to 1400 pesticides are used world-wide, which highlights widespread

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use in modern agriculture. 2

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While pesticides confer distinct advantages, they also lead to several challenges due to the

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presence of pesticide residues in agricultural products, even they are produced under good

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agricultural practices. To ensure food safety, strict maximum residue levels (MRLs) of pesticides in

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food products have been specified by various nations and international organizations such as the

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Codex Alimentarius Commission (CAC)3 and European Union (EU).4

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Considering the high diversity of the pesticide physiochemical characteristics and MRLs in

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various matrices, the development of highly effective methods for residual screening and analysis

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of broad spectrum of pesticides is high critical. Ultra high performance liquid chromatography or

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gas chromatography (UPLC/GC) tandem mass spectrometry analysis using instruments such as

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triple quadrupole (QQQ), quadrupole-linear ion trap (QTRAP), and quadrupole-high resolution

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accurate mass spectrometry are the most commonly used approaches for pesticide residue

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analysis and high throughput screen.5 UPLC-quadrupole tandem high-resolution mass

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spectrometry, including quadrupole-time of flight (Q-TOF) and quadrupole-Orbitrap (Q-Orbitrap),

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have shown to be effective tools and are widely employed for pesticide residue screen and

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quantification.6-8 3



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The typical pesticide residue analysis under these approaches involves the collision-induced

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dissociation (CID) of the pesticide pseudomolecular ions (protonated, deprotonated, or

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ammoniated) , followed by which, the dissociated product ions are detected by high resolution

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MS/MS scans in and UPLC-ESI-QTOF instrument. While these techniques are widely applicable,

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some pesticides with particular chemical structures are challenging to analyze using this approach

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due to their ionization problems in the ion source. The process for such dissociation is called in-

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source collision-induced dissociation or in-source fragmentation, wherein, fragments of the

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molecular or pseudomolecular ion are generated between the atmospheric pressure source and

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the high-vacuum region of the mass analyzer.9 Due to the difference in the voltage which exist

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between the orifice (transfer capillary) and Qjet (skimmer), the ions reach sufficiently high

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velocities to collide with remaining solvent and dry gas molecules, which result in in-source

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fragmentation. Therefore, the higher the decluster potential (DP)/ fragmentor voltage, the more

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intensive is the in-source fragmentation. Besides, source temperature could also affect the extent

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of in-source fragmentation according to our study.

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In-source fragmentation is particularly important when using instruments with a single-stage

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mass analyzer.10,

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fragmentation can result in undesirable problems, such as misannotation of non-target

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compounds,12 reduced detection sensitivity and false negatives or positives for target

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compounds.13 On the other hand, it can be used for chemical structure interpretation14 and

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analytical method development of certain kinds of compounds.15-17 The extent of in-source

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fragmentation depends on the types and parameters of the ion source, and the chemical

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structures of analytes. Although electrospray ionization (ESI) is an atmospheric ionization

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However, it should be avoided for tandem mass analysis, since in-source

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technique which is widely considered the “softest ionization”, in-source fragmentation is still

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inevitable for some compound classes, particularly with lactams and lactones. Under this scenario,

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we should take advantage in-source fragmentation and develop specific analytical methods for

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these analytes.

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While pesticide residue analysis by in-source fragmentation with an HPLC-ESI-QTOF has been

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reported in several studies,13,

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relevance and value for pesticide residue analysis has not been well explored. In this study, a high-

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resolution screening and quantification method based on in-source fragmentation was developed

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and applied for real sample analysis. Furthermore, the effects of source parameters including DP

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and temperature on the extent of in-source fragmentation were also investigated. Notably, a high-

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resolution MS/MS library consisting of a wide scope of pesticides and their in-source fragments

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was created. The proposed method was demonstrated to be a powerful technique for efficient

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pesticide screening and quantification and was found to be particularly useful for avoiding false

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negative results.

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in-depth analysis of in-source fragmentation effects and its

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EXPERIMENTAL

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Regents and materials

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Methanol and acetonitrile of HPLC grade were obtained from Fisher Scientific (Fair Lawn, NJ).

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Formic acid and ammonium formate were purchased from Sigma Aldrich (Darmstadt Germany).

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Water was purified by a Milli-Q system (Millipore, Billerica, MA, USA). The pesticide standards

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were obtained from Dr. Ehrenstorfer (Ausberg, Germany). The standard stock solutions were

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prepared in acetonitrile (20 mg/L) and were stored at -20 °C until use. 5



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For sample preparation, the required QuEChERS extraction salt packets (Bond Elut QuEChERS

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P/N 5982-5650: anhydrous MgSO4, 4 g; sodium citrate, 1 g; sodium hydrogencitrate sesquihydrate,

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0.5 g; sodium chloride, 1 g), and dispersive solid phase extraction tubes (Bond Elut QuEChERS P/N

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5982-5056 containing 150 mg of PSA and 900 mg of anhydrous MgSO4; Bond Elut QuEChERS P/N

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5982-5256 containing 150 mg of PSA, 15 mg of GCB and 885 mg of Anhydrous MgSO4) were

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obtained from Agilent Technologies, Lake forest, CA.

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Instrument and software

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The high-resolution screen and quantitative analysis were performed on a Quadrupole Time-

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of-Flight mass spectrometry (TripleTOF 6600, SCIEX) coupled to an ExionLC UPLC system consisting

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of a binary pump, degasser, autosampler and column oven. Separation of pesticides were

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performed on a C18 column (HSS T3 2.1 x 100 mm, 1.7 µm, 100 Å, WATERS, Torrance CA USA). The

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column temperature was set at 40 °C, and the flow rate was 0.3 mL/min. The mobile phase A was

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water, and mobile phase B was methanol, both of which containing 2mM ammonium formate and

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0.1% formic acid. The gradient was programmed as follows: 0-1 min: 5 % elute B, 1-2 min: gradient

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increase to 20 % elute B, 2-3 min: gradient increase to 50 % elute B, 3-10 min gradient increase to

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95 % elute B, 10-12 min: hold 95 % elute B, 12-12.1 min: 5 % elute B; 12.1-15 min: hold 5 % elute

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B. The injection volume was 5 µL.

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The source parameters were: ISVF, 5500 V in positive mode; temperature, 550 °C; nebulizing

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gas (GS1), 50 psi; heater gas (GS2), 50 psi; curtain gas, 35 psi. The mass acquisition was performed

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using information-dependent acquisition (IDA) that consisted of survey scan and dependent

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product ion scan in a single run. The survey scan was performed in a full-scan TOF-MS between 6


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m/z 70-900 with DP and collision energy (CE) at 60 V and 10 eV, respectively. The IDA-MS/MS was

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performed under the following conditions: MS/MS threshold, 100 cps; ion tolerance, 50 mDa;

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collision energy (CE) was ramped over an interval by entering a CES value, and the CE and CES was

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set at 35 and 15 eV, respectively (i.e., 35±15 eV).

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The Analyst 1.7 software was used for data acquisition. High-resolution MS/MS library creation

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and data processing, including TOF-MS quantification, TOF-MS (mass accuracy of precursor ion

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and isotope ration) and MS/MS sepctra screen were carried out using Sciex OS 1.5 software. The

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software calculates XICs at an extraction window of 0.02 Da against an XIC table containing target

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pesticides and fragments to give retention time, isotope difference, and mass error information.

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Concurrently, the obtained MS/MS spectra were searched against the pre-created high-resolution

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MS/MS library for giving library search confidence information, i.e., the library score (purity score).

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Finally, a combined score calculated based on mass error, isotope difference, and library score will

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be given for each analyte to evaluate its performance. The QTOF MS was calibrated in high

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sensitivity mode and the automated calibration device system (CDS) was set to perform an

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external calibration every ﬁve samples using APCI calibrate solution.

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Sample preparation

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The samples were prepared by adopting the QuEChERS (quick, easy, cheap, effective, rugged,

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and safe) method developed in our previous study.19 A portion (10 g) of the homogenized samples

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were weighed in a 50 mL centrifuge tube Then 10 mL of acetonitrile was added and followed by a

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ceramic homogenizer, and the QuEChERS extraction salts (P/N 5982-5650). The tube was

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immediately sealed, and the contents were shaken manually for 1 min. The extract was then 7



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centrifuged at 5000 rpm for 5 min and 6-mL of the supernatant was transferred into the dispersive

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solid phase extraction tubes (P/N 5982-5056 for common samples, P/N 5982-5256 for samples

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with a high content of chlorophyll, e.g. lettuce and leek) for clean-up. The extracts in the tubes

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were vortexed for 1 min, followed by centrifugation for 5 min at 5000 rpm. The supernatant was

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filtered through a PTEE filter (0.22 μm) and was subjected to UPLC-QTOF analysis.

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Validation

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The analytical performance of selected pesticides and their in-source fragments were evaluated

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in terms of recovery, repeatability, limit of quantification (LOQ), linearity, and matrix effects. The

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validation was carried out with leek, kidney bean and apple, which were free of the selected

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pesticides. The accurate mass extracted ion chromatograms (XIC) of the quasi-molecular ions of

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each pesticide (M+H or +NH4) and their in-source fragments were used for data analysis.

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RESULTS AND DISCUSSION

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In-source fragmentation in pesticide residue analysis

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Several factors, such as matrix effects, matrix isobaric interferences, and in-source

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fragmentation, are known to affect the detectability of pesticides in high-resolution non-target

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screening13. However, the negative effects from matrices and the isobaric interferences can be

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alleviated by sample pretreatment measures such as improved clean-up, and sample dilution.

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Nevertheless, in-source fragmentation cannot be avoided during the mass spectrometry analysis

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for certain pesticides. In previous study, the high rate of false negative results for aldicarb was

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attribute to high ion suppression, which make the MS2 scan not triggered because of the low 8


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abundance of the precursor ion.20 However, in this study, we found this is actually not the case,

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and that the extensive in-source fragmentation is the main cause for the false negative results.

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In-source fragmentation in LC-MS/MS analysis occurs due to the vulnerability of chemical bonds

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in the target analytes. It has been reported that, pharmaceuticals or their phase I and II metabolites,

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containing lactones, lactams or disulfide functionalities undergo fragmentation in the electrospray

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ion source.21-23 Therefore, it is reasonable to assume that in-source fragmentation of pesticides

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containing these functionalities is possible.

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In this study, we tested over 400 pesticides, selected from the lists of Maximum Residue Limits

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for Pesticides in China and European Proficiency Tests in Fruit and Vegetables (EUPT-FV 21), to

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examine their propensity for in-source fragmentation. Different extents of in-source

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fragmentation were observed for 96 pesticides among the tested samples, and the detailed

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information from the analysis, including molecular formula, adduct, exact mass, retention time,

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and extent of in-source fragmentation of these pesticides and their corresponding fragments are

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given in table 1.

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Overall, 126 fragments were detected for these 96 pesticides, and two or more fragments were

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generated in parallel for about half of them. To evaluate the extent of in-source fragmentation

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under different source parameters, we introduce here, a novel concept of in-source fragment

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fraction (i-SFF) which can be calculated according to the following equation:

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𝐼𝑖 ― 𝑆

i ― SFF = 𝐼𝑖 ― 𝑆 + 𝐼𝑝

(1)

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Where Ip is the intensity of the protonated, or ammoniated pesticide, and Ii-S is the intensity of its

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in-source fragments. The i-SFF value ranges between 0 and 1, with 0 indicating no in-source

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fragmentation and 1 represents complete in-source fragmentation. 9



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We investigated the effects of DP and source temperature on i-SFF. For most of the pesticides,

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within the tested DP values between 0 and 150 V, the responses were almost constant when the

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DP was lower than 60 V, and decreased dramatically with further increase in the DP. Nevertheless,

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different pattern was observed for most of the fragments. After a relatively steady-state between

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0 and 60 V, a slow increase in the extent of fragmentation was observed with further increase in

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the DP. Finally, the i-SFF values for most of the pesticides reached a steady-state at DP values

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between 0 and 60 V and indicated an increase in the extent of in-source fragmentation within 60

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to 150 DP range.

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According to the i-SFF values at the 60 V DP, we categorized the analyzed pesticides into

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different classes based on the extent of in-source fragmentation, wherein pesticides with i-SFF≥

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0.7 are classified as severe in-source fragmentation pesticides, those with i-SFF 75. The mass errors of some fragments such as acetochlor fragment m/z 148 and terbufos

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fragment m/z 103 were found to be higher than 5 ppm leading to a lower combined score. Since

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a mass accuracy

On the use of in-source fragmentation in ultra high

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