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Determination of Multiresidue Pesticides in Botanical Dietary Supplements using Gas ChromatographyTriple Quadrupole Mass Spectrometry (GC-MS/MS) Yang Chen, Salvador Lopez, Douglas G. Hayward, Hoon Yong Park, Jon W Wong, Suyon S Kim, Jason Wan, Ravinder Reddy, Daniel Quinn, and David Steiniger J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00746 • Publication Date (Web): 21 Apr 2016 Downloaded from http://pubs.acs.org on April 23, 2016

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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To be submitted to:

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Determination of Multiresidue Pesticides in Botanical Dietary Supplements using Gas Chromatography-Triple Quadrupole Mass Spectrometry (GC-MS/MS)

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Yang Chen1, Salvador Lopez2, Douglas G. Hayward3, Hoon Yong Park3, Jon W. Wong3,

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Suyon S. Kim3, Jason Wan2, Ravinder M. Reddy1, Daniel J. Quinn4, David Steiniger4

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U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 6502 S. Archer Road, Bedford Park, IL 60501 2

Institute for Food Safety and Health, Illinois Institute of Technology, 6502 S. Archer Road, Bedford Park, IL 60501 3

U.S.Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Pkwy, College Park, MD 20740-3835 4

Thermo Fisher Scientific, 2215 Grand Avenue Pkwy, Austin, TX 78728

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Corresponding Authors: Yang Chen, Tel: (708)-924-0604; email: [email protected], Jon W. Wong, Tel: (240)-402-2172, email: [email protected]; Douglas G. Hayward, Tel: (240)-402-1654; email: [email protected] Keywords: multi-pesticide residue, botanicals, Carbon x/PSA, Carbon x/PSA/C18 SPE, dual phase SPE, triple-phase SPE GC-MS/MS, Running title: Multi-pesticide residue analysis in botanicals using GC-MS/MS

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ABSTRACT

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A simplified sample preparation method in combination with gas chromatography-triple

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quadrupole mass spectrometry (GC-MS/MS) analysis was developed and validated for the

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simultaneous determination of 227 pesticides in green tea, ginseng, gingko leaves, saw

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palmetto, spearmint, and black pepper samples. The botanical samples were hydrated with

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water and extracted with acetonitrile, magnesium sulfate, and sodium chloride. The

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acetonitrile extract was cleaned up using solid phase extraction with carbon coated

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alumina/primary-secondary amine with or without C18 . Recovery studies using matrix

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blanks fortified with pesticides at concentrations of 10, 25, 100 and 500 µg/kg resulted in

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average recoveries of 70-99% and relative standard deviation of 5-13% for all tested

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botanicals except for black pepper where lower recoveries of fortified pesticides were

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observed. Matrix-matched standard calibration curves revealed good linearity (r2 > 0.99)

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across a wide concentration range (1-1000 µg/L). Nine commercially-available tea and

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twenty three ginseng samples were analyzed using this method. Results revealed 36

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pesticides were detected in 9 tea samples at concentrations of 2-3500 µg/kg, and 61 pesticides

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were detected in 23 ginseng samples at concentrations of 1-12500 µg/kg.

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INTRODUCTION

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Botanical dietary supplements are plant-based materials valued for their possible

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medical or therapeutic properties such as aphrodisiacs, stress relievers, memory enhancers,

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and treatment for inflammation and infection. Such botanicals are widely regarded as

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valuable agricultural crops, and to prevent economic losses, pesticides may be used against

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mold, insects, and other relevant pests that may cause commodity damage. Despite the

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usefulness of pesticides in agricultural practices, there are concerns about their proper use and

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residue levels in plant commodities. Pesticides can remain, persist, or accumulate from

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application during the growing stages of the plant or from post-harvest treatment. Previous

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studies have shown that many organochlorine and organophosphorus pesticides have been

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detected in botanical products (1-3).

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A number of methods that are based on procedures for fresh plant-derived foods have

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been used for pesticide screening in dried botanical dietary supplements, spices, medicinal

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plants, herbals, teas and phytomedicines (4-8). These methods involve organic solvent

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extraction and clean-up procedures to remove co-extracted interfering components from the

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matrix. While these methods work well with fresh produce, there are serious challenges with

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dried botanicals due to the many varieties and complexities of botanical matrices and

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concentrated levels of natural products present in these low-moisture products. The

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complexity and variety of dried botanicals, along with the low concentration of many

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pesticides and small sample sizes, complicate the detection, identification, and quantitation of

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pesticide residues.

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One of these pesticide methods, QuEChERS (Quick, Easy, Cheap, Effective Rugged

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and Safe), has been widely used since its introduction over ten years ago (9). It involves

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salting-out acetonitrile extraction followed by cleanup using dispersive solid-phase extraction

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(d-SPE). The method has been modified to improve cleanup efficiency for more complex



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matrices such as botanicals by utilizing a combination of sorbent materials, such as

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graphitized carbon black (GCB), primary secondary amine (PSA), and octadecyl-linked silica

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(C18) for additional d-SPE cleanup. This modified QuEChERS procedure has been validated

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previously in our laboratory for the extraction of pesticides from botanicals prior to liquid

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chromatography-triple quadrupole mass spectrometry in tandem MS mode (LC-MS/MS)

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analysis (10). Hayward et al. (11) used a similar method for sample extraction and cleanup

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using column solid-phase extraction (SPE) for gas chromatography-triple quadrupole mass

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spectrometry in MS/MS mode (GC-MS/MS) analysis of 310 pesticides in 24 botanicals.

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Recently, Hayward et al. (12) studied the efficiencies of four SPE cleanup procedures for 170

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pesticides from green, black and oolong teas used for GC-MS/MS analysis. One of the SPE

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columns evaluated in this study was graphitized carbon coated on alumina/PSA (CCA/PSA)

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and eluted using acetone or ethyl acetate, instead of using a mixture of acetone/toluene (3:1)

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typically used for conventional graphitized carbon black/PSA dual layer SPE cartridges.

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Hayward et al. (12) concluded that the cleanup efficiency of using the dual layer CCA/PSA

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SPE, followed by elution using acetone or ethyl acetate were comparable to those using the

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GCB/PSA SPE and acetone/toluene elution, possibly as a result of the lighter carbon load in

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the CCA particles. The avoidance of using toluene during the SPE cleanup step is

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advantageous because there is less leaching of the matrix from the SPE cartridge into the GC

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final extract, which can result in lower temperatures and shorter times required for sample

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concentration, cleaner GC extracts, and faster sample preparation throughput.

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The objective of this study was to expand, evaluate and validate the use of acetonitrile

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salt-out extraction and CCA/PSA/C18 SPE cleanup for GC-MS/MS analysis of 227

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pesticides for a wider range of botanicals including green teas, ginseng, gingko, saw

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palmetto, spearmint, and black pepper.

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MATERIALS AND METHODS Chemicals and Botanical Matrices Twelve customer-made analytical standard mixtures, each containing 14 to 27 pesticides, at

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a concentration of 100 µg/mL in acetone, were purchased from AccuStandard, Inc. (New Haven,

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CT). Additional pesticides were obtained through the U.S. Environmental Protection Agency,

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National Pesticide Standard Repository (Ft. Meade, MD). The 12 standard mixtures were combined

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to provide a pesticide standard with a total of 227 pesticides. The internal standard, tris-(1,3-

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dichloroisopropyl) phosphate, was purchased from TCI America (Portland, OR). Pesticide-grade

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acetonitrile and toluene, HPLC-grade water and anhydrous sodium sulfate were purchased from

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Thermo Fisher Scientific (Pittsburgh, PA). The QuEChERS extraction kits containing 4 g of

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anhydrous magnesium sulfate and 1 g of sodium chloride packets were purchased from Agilent

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Technologies (Wilmington, DE). Solid-phase extraction (SPE) cartridges, carbon coated alumina

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(Carbon X)/primary-secondary amine (PSA) (250/500 mg) and C18 (500 mg) were purchased

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from United Science Corp (Center City, MN) and United Chemical Technologies, (Bristol, PA),

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respectively. Botanical dietary supplements used for this study (green tea, ginseng, gingko biloba

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leaves, saw palmetto, spearmint and black pepper) were obtained from retail commercial outlets,

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field studies or other “organic” retail sources. The botanicals were ground, homogenized and sieved

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through 18 mesh sieve prior to being used for the validation and recovery studies.

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Preparation of Analytical Standards

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An intermediate standard containing 5,000 ng/mL of each pesticide was prepared by diluting

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the twelve pesticide standard mixtures equally with toluene in a 20 mL volumetric flask and was

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used for preparing working standards and spike studies. Three other spiking solutions at

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concentrations of 100, 250, 1000 ng/mL were prepared by diluting the intermediate standard

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solution with toluene. Internal standard stock solution of 1,000 µg/mL was prepared by dissolving

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tris-(1,3-dichloroisopropyl) phosphate in acetonitrile. The working standards were obtained by



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diluting the intermediate standard solution mixture with matrix blanks to achieve working

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concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 ng/mL, each containing 150 ng/mL of

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internal standard tris-(1,3-dichloroisopropyl) phosphate. The matrix blanks were prepared by

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extracting blank matrices, following the same sample preparation protocol as the fortification

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studies.

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Instrumentation

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The analytical instrument comprised of a Trace GC 1310 gas chromatograph coupled

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to a TSQ 8000 triple quadrupole mass spectrometer and a TriPlus RSH liquid autosampler

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with a programed temperature vaporization injector (PTV) (Thermo Fisher Scientific, San

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Jose, CA). The PTV was operated in splitless mode with an open baffled fused silica

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deactivated glass liner (2 mm i.d. × 120 mm). The injector temperature was programed at

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75°C for 0.1 min, ramped to 300°C at 2.5oC/sec, held for 3 min, followed by ramping to 330

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°C at 14.5 °C/sec, held for 20 min. The injection volume was 2.0 µL, with split vent flow of

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50 mL/min and splitless time of 1.0 min. Separation was performed on a TG-5SilMS column

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(30 m × 0.25 mm × 0.25 µm, Thermo Scientific, San Jose CA) using helium as the carrier gas

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at a constant flow rate of 1.2 mL/min. The column temperature was programmed to start at

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40oC for 1.5 min, increased to 90°C at 25°C/min, held for 1.5 min, ramped to 180°C at

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25°C/min, to 280°C at 5°C/min to a final temperature of 300°C at 10°C/min, and held for 6

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min, with a total run time of 35 min. The transfer line and ion source temperatures were 280

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and 300°C, respectively. The mass spectrometer was operated in electron impact (EI)

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ionization mode using timed selected reaction monitoring (t-SRM) using retention time

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windows of 0.9 min. Three MS/MS transitions were selected for each pesticide and optimized

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for sensitivity and selectivity were determined by collision energy experiments. The MS/MS

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transitions and their respective collision energies, chromatographic retention times, as well as


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the molecular and chemical information of each pesticide are provided in Supporting Table

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S1.

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The pesticides were identified based on retention times, the presence of the precursor-

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product transition ions, and their respective ion ratios using the built-in TraceFinder pesticide

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identification software of the TSQ 8000 system. The software consists of a master method

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containing retention times and transition parameters for 600 pesticides. Quantitation was

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performed using the peak area ratio responses of the analyte in the matrix-matched standards

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to that of the internal standard using linear or quadratic curve produced using the built-in

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TraceFinder software.

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For the pesticides that have high abundance and meet sensitivity requirement, the

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transitions were directly exported to a new method. For those pesticides that were not in the

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master method or had low abundances, the retention times and transitions were created and

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optimized using the build-in auto selected reaction monitoring (AutoSRM) program. The

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AutoSRM created a full-scan method for precursor ion selection and selected ion monitoring

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(SIM) methods with collision energy (CE) setting at 10, 20 and 30 eV for product ion

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selections, followed by further CE optimization for the selected product ions. The retention

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time window of 0.9 min was used for the t-SRM considering the diversity of matrices

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evaluated in the study. The t-SRM data for a particular pesticide was acquired at a constant

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cycle time at its expected retention time, therefore increasing dwell time to ensure optimum

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number of data points for each peak and constant sensitivity.

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Sample Preparation and Recovery Studies

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The recovery studies were conducted by fortifying 100 µL of 100, 250, 1000 and 5000

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ng/mL spiking standard solutions to 50 mL polypropylene centrifuge tubes containing 1.0 ± 0.05 g

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blank botanical matrices to achieve the fortification levels of 10, 25, 100, and 500 µg/kg,

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respectively. The spiked samples were thoroughly vortexed and allowed to stand for 10 min to



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allow absorption of pesticides in the matrices. The fortification step was eliminated for the blank

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matrix and incurred samples. The dried plant material was hydrated with HPLC-grade water (10

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mL) for 15 min and acetonitrile (10 mL) containing internal standard (60 µg/L of tris-(1,3-

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dichloroisopropyl)-phosphate) followed by the addition of 4 g anhydrous magnesium sulfate and 1 g

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sodium chloride for salting-out acetonitrile extraction. Samples were shaken vigorously at 1000

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strokes/min for 1 min using GenoGrinder (SPEX Sample Prep, Metuchen, NY) followed by

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centrifugation at 4200 × g for 5 min. The resulting supernatants were further processed using solid

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phase extraction (SPE)

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The acetonitrile supernatant (1.25 mL) was loaded on the Carbon-X/PSA or C18/Carbon-

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X/PSA SPE column (topped with approximately 100-200 mg anhydrous sodium sulfate and

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preconditioned using two column volumes ~12 mL of acetone), and subsequently the SPE column

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was eluted with approximately 12 mL acetone. The acetonitrile extracts from ginseng, tea, and

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gingko biloba were cleaned up using 500 mg Carbon-X/500 mg PSA, whereas the spearmint and

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saw palmetto were cleaned up using a second C18 SPE cartridge attached on top of the Carbon

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X/PSA dual layer cartridge. The QuEChERS acetonitrile extract (approximately 10 mL) from the

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black pepper was carefully transferred to a clean glass test tube with a cap and gently rinsed with 5

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mL acetonitrile-saturated hexane. The hexane was removed and the process was repeated again and

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the hexane-rinsed acetonitrile extract was loaded onto the two cartridge setup for C18/Carbon-

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X/PSA cleanup. The eluting acetone extract including the loading acetonitrile supernatant were

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collected in a 15 mL glass centrifuge tube and the cleaned extract was concentrated to

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approximately 100 µL at 50oC under a gentle stream of nitrogen using a nitrogen evaporator (N-

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Evap, Organomation, Associates, Berlin, MA). Special attention was made to avoid the evaporation

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of the extracts to go to complete dryness to prevent losses of the pesticide analytes. To the reduced

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extract, 500 µL toluene, 50 µL QC standards, and 25 mg anhydrous magnesium sulfate were added.

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The mixture was vortexed for 30 sec, centrifuged at 1258 × g for 5 min. The resulting supernatant


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was transferred into an amber glass autosampler vial and stored at - 20 oC prior to GC-MS/MS

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analysis.

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The working standards were obtained by diluting the intermediate standard solution mixture

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with matrix blanks (containing the internal standard, tris-(1,3-dichloroisopropyl) phosphate) to

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achieve working concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 ng/mL. The matrix blank

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samples used for preparation of the matrix-matched standard were pre-screened for presence of any

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target pesticide residue prior to use for the matrix-matched standard preparation. The matrix blanks

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were prepared by extracting blank matrices following the same sample preparation protocol as the

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fortification study.

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Determination of Matrix-Dependent Instrument Quantitation Limit (MDIQL)

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Matrix-matched standards at concentrations of 2, 5, 10, 20 and 50 ng/mL, each with

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eight replicates were analyzed and the results were used for the determination of the matrix

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dependent-instrument quantitation limit (MDIQL). The peak area ratio response of a pesticide

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to that of the internal standard was plotted against the corresponding matrix-matched standard

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curve and the concentration was calculated. The lowest level of pesticide in the standard that

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produces results within ± 25% of the expected accuracy for the qualify ion with minimum of

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one confirmation ion was used for MDIQL determination of that pesticide. The MDIQL were

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calculated according to the U.S. Environmental Protection Agency (EPA) procedure used to

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determine the method limit of quantitation (13). The standard deviations (SD) of the

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responses were calculated from the eight injections for each pesticide and the MDIQL was

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calculated at 3 × SD× 3 × dilution factor. [i.e., 2.998 × SD (critical t 0.010 = 2.998 at degree of

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freedom (df) = 7) × 3 × dilution factor] (13). Means and standard deviations of the recoveries

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in the fortified samples, matrix dependent instrument quantitation limit (MDIQL) were

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determined using Microsoft Excel 2010.

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Analysis of Pesticide Residues in Commercially Available Botanical Samples



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Commercially available tea samples (green tea, black tea and oolong tea) and ginseng

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samples were analyzed for pesticide residues using the sample preparation method in

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combination with GC-MS/MS analysis developed in the current study.

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RESULTS AND DISCUSSION

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Instrument Performance and Method Optimization A standard mixture containing the 227 pesticides in toluene (5,000 ng/mL) was

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analysed using the GC-MS/MS and the chromatograms and precursor-to-product ion

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transitions of each pesticide were examined using the TraceFinder software. The 227

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pesticides evaluated in this study along with their retention times, transitions and collision

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energy are provided in Supporting Table S1. The quantitative calibration and linearity

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evaluation for each pesticide were performed using matrix-matched standards in the

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concentration range of 1-1000 ng/mL (r2 > 0.99). The linearity ranges for the majority of

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pesticides in all matrices except for the black pepper were in the range of 1 to 1000 ng/mL

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with regression coefficients greater than 0.99, except for allethrin, captan, dicofol,

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dioxathion, ethoxyquin, isoproturon, lufenuron, metalaxyl, and trichlorfon. Of the 227

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pesticides fortified in the black pepper extract, the chromatographic peaks of 50 pesticides

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were not present and could not be identified even at concentration up to 1000 µg/L. This is

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probably contributed to the high concentrations of chemical components still present in the

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black pepper extract that are overwhelming the presence and interfering the chromatographic

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separation of the less concentrated pesticide analytes.

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Recovery of Pesticides

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Recovery studies were conducted by fortifying the 227 pesticides into blank matrices

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of green tea, ginseng, gingko leaves, saw palmetto, spearmint and black pepper, to

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concentrations of 10, 25, 100 and 500 µg/kg. The number of pesticides with recoveries of 1-

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49%, 50-69%, 70-120%, and >121%, as well as the average recovery and relative standard

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deviation at each fortification level for each matrix are presented in Table 1. Individual

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pesticide recoveries in each matrix are provided in Supporting Table S2. For green tea,

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ginseng, gingko leaves, saw palmetto and spearmint, a majority (> 90%) of the fortified



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pesticides showed an average recovery of 76 - 99%, with standard deviations of 4 - 12%.

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The number of pesticides with acceptable recoveries within the 70 - 120% range at the 10 and

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500 µg/kg fortification levels were 170, 181, 202, 165, 154 and 170, 197, 209, 196, 199 for

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green tea, ginseng, gingko biloba, saw palmetto, and spearmint, respectively. These average

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recovery levels for the fortified pesticides are comparable with those reported previously by

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Hayward et al. (11, 12). The number of non-detected pesticides decreased with increasing

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fortification levels. At the 10 µg/kg fortification, 16, 12, 13, 29 and 29 pesticides were not

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detected in green tea, ginseng, gingko, saw palmetto and spearmint, respectively, whereas 0,

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1, 3, 11, 9 pesticides were not detected at the 500 µg/kg for the respective matrices. The non-

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detects of allethrin, captan, dioxathion, ethoxyquin, isoproturon, lufenuron, metalaxyl and

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trichorfon in some matrices at the low fortification levels were due to low instrument

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responses of analytes in the matrices. The low recoveries were likely due to poor extraction

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efficiency during acetonitrile extraction, adsorption to the sorbent during SPE clean-up,

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chemical instability or decomposition, and matrix interference during chromatographic

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analysis. These pesticides include acephate, anthraquinone, cyprodinil, chlorothalonil

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ethoxyquin, fludioxinil, folpet, thiabendazole and phenmedipham, fluridone, prochloraz,

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pyrimethanil, pentachloroaniline and pyraclostrobin. (10, 14-16). The high recoveries (145 -

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2000%) of dicofol in green tea and non-detection or low recoveries in the other matrices may

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be attributed to its response change during the batch analysis. This was observed by

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monitoring the pesticide responses in the 20 ng/mL matrix-matched standard at the early,

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middle and end stages of the sequence as peak area responses tended to increase for green tea

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and ginseng and decrease for gingko biloba, saw palmetto, and spearmint matrices.

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QuEChERS typically uses acetonitrile as the extraction solvent followed by dispersive

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SPE with PSA and magnesium sulfate (9). The use of PSA helps remove organic acids, polar

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pigments, some sugars, and fatty acids prior to GC-MS/MS analysis of pesticides. This


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method works well for high moisture vegetal-based matrices but does not work well for food

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matrices containing high levels of pigments and fats, which are prevalent in many botanical

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products. Therefore cleanup is essential for satisfactory method performance as well as

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general maintenance of the GC-MS/MS, particularly the liner, column, and ion source. The

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effective use of graphitized carbon black (GCB) for SPE was reported by Fillion et al. (21) to

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remove non-polar pigments such as carotenoids and chlorophyll (22). Pang et al. (23) applied

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a similar approach using an acetonitrile salt-out extraction step, followed by SPE cleanup

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with GCB/PSA whereas Lozano et al. (24) applied a modified QuEChERS (Quick, Easy,

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Cheap, Rugged and Safe) procedure for the analysis of pesticides in tea samples. GCB has

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non-specific retention not only to chlorophyll, but also to planar pesticides containing

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aromatic moieties, resulting in lower recoveries. Typically, the eluting solvent contains

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toluene as a competing planar agent to desorb the pesticides from the GCB sorbent although

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some of the matrix components may also be eluted as well (25). Wong et al. (15) applied

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dispersive SPE with C8 to dried ginseng sample extracts followed by dual layer SPE with

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GCB and PSA to remove these matrix components that may interfere with the analysis of

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pesticides. The use of GCB for the sample clean-up of plant-based materials for pesticide

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analysis has been well established but newer carbon-based materials such as silica-dispersed

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carbon (26) and alumina (27) and multiwalled carbon nanotubes (28), have been recently

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introduced as chromatographic media for separation processes or as sorbents in removing

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chemical co-extractives from the sample matrix.

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cleanup efficiency of using carbon-coated alumina (Carbon X) followed by elution using

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acetone or ethyl acetate without the use as toluene was comparable to those using the

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GCB/PSA acetone/toluene SPE procedures in black and green teas. The SPE approach using

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a combination of Carbon X/PSA or C18/Carbon X/PSA seems to work well with ginseng,

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Gingko biloba, saw palmetto, spearmint, and green tea in this current study.

Hayward et al. (12) reported that the



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However, when this promising approach was applied to black pepper, less than 50%

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of the pesticides fortified in black pepper showed a 70 - 120% recovery range, specifically

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with an average recovery range of 66 - 79% at the 10 and 25 µg/kg spike levels. Additional

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clean-up involving the rinsing of the acetonitrile extracts with acetonitrile-saturated hexane to

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further eliminate lipophilic co-extracts of the black pepper matrix was also implemented as

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described by Przybylski and Segard (29). Significant signal loss was observed for a large

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number of target pesticides and chromatographic signals could not be observed even at the

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1000 ng/mL level for 50 pesticides. This may be due to the low ionization efficiency in the

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mass analyzer caused by the overwhelming presence of interferring co-extractives from the

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black pepper matrix. Black pepper, similar to other botanicals, contains high levels of

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unsaturated amides, flavonoids, fatty esters, steroids, propenlyphenols and alkaloids (17).

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The combined use of the hexane rinses and SPE cleanup with the C18/Carbon X/PSA

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sorbents ought to be capable of removing high levels of co-matrix chemical components such

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as fatty acid esters, sterols, polyphenolic, pigments, and non-polar compounds in the black

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pepper extract. However, black pepper extracts consist of essential oils containing primarily

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of terpenes and terpenoids compounds, such as β-caryophylline, limonene, β-pinene, and

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piperine (18, 19). Since many synthetic pyrethroid and chlorinated pesticides have been

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derived from terpene- and terpenoid-based (as well as other classes of) natural products (20),

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the chemical analysis of pesticides in botanicals containing essential oils and oleoresins is

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further complicated since the chemical and physical properties of both natural and synthetic

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constituents are similar and difficult to separate from one another using conventional

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chromatographic techniques. From a previous study (11), these botanicals, especially spices,

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whose desirable features are due to the presence of these essential oils, are also posing

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problems for pesticide analysis. Effective schemes need to be investigated to address and

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resolve these problems for a majority of these botanicals that consist of these essential oils.


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Matrix Dependent Instrument Quantitation Limit (MDIQL) The MDIQLs were established using the lowest concentration of matrix-matched

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standards that produced within 25% of the expected levels with presence of at least one of the

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confirmation ions. The results obtained are shown in Supporting Table S1 and summarized

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in Table 1. More than 90% of analytes in all matrices (except black pepper) have MDIQL of

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20 µg/kg) in all matrices possibly due to a lack of stable

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product ions or thermal instability of the pesticide. The high MDIQLs of dicofol were due to

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its response changes throughout the analysis. The MDIQL of thermally labile lufenuron

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varied between matrices, from 10 µg/kg in ginseng and gingko to >80 µg/kg in saw palmetto

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and spearmint. Sharp chromatographic peak shape of lufenuron in sensing and gingko was

339

observed compared to broaden peaks in saw palmetto and spearmint, possibly due to

340

chemical interaction between analyte and the matrix.

341

The average MDIQL for the spiked pesticides in green tea, ginseng, gingko biloba,

342

saw palmetto and spearmint was between 6 and 9 µg/kg and 29 µg/kg for black pepper.

343

Higher Limits of Quantitation (LOQs) for pesticides in black pepper were also reported by

344

Lacina et al. (30). They compared two extraction methods (aqueous acetonitrile extraction

345

followed by a partition step and aqueous acetonitrile extraction) for 288 pesticides and 38

346

mycotoxins in wheat, sunflower seeds, paprika, black pepper, and apple baby food using LC-

347

MS. They found the majority of the LOQ in the two spices were in the 11-50 ppb range

348

compared to < 10 ppb for the other matrices. Signal suppression of 30-70% was observed for

349

50% of the analytes in diluted (factor of 24) black pepper extract and there was difficulty to

350

identify 18 analytes due to the interference attributed to the presence of the matrix co-

351

extractives. Signal suppression of other chemicals beside pesticides, particularly mycotoxins

352

in black pepper have also been observed by Yogendrarajah et al. (31) and Ferrer et al. (32).



353

Ferrer et al (32) revealed 67% of the compounds in black pepper showed strong signal

354

suppression (>50%), along with curcuma (42%), nutmeg (36%), ginger (28%), curry (28%),

355

paprika (19%) and chilli powder (7%).

356

Pesticide Detection in Incurred Samples.

357

Nine commercially available tea samples (green tea, black tea and oolong tea), also

358

measured by Hayward et al. (12) and twenty-three ginseng samples were analyzed for

359

pesticide residues using the improved method in combination with GC-MS/MS analysis

360

developed in the current study. Thirty-six pesticides were detected in the nine tea samples as

361

listed in Table 2, including twenty-eight insecticides, six fungicides (azoxystrobin,

362

difenconazole, diphenylamine, fenbuconazole, propargite and tebuconazole), one herbicide

363

(clomazone) and one bird repellant (anthraquinone). Among the detected insecticides, there

364

were six pyrethroids (bifenthrin, cyhalothrin, cypermethin, deltamethrin, fenvalerate and

365

permethrin), six organophosphates (chlorpyrifos, ethion, methidation, monocrotophos,

366

pirimiphos methyl and triazophos), eleven organochlorines (alpha-, beta-, delta- and gamma-

367

BHC, p,p’-DDE, p,p’- DDD, and p,p’-DDT, alpha- and beta-endosulfan and heptachlor) and

368

five other compounds (acetamiprid, buprofezin, o-phenylphenol, pyridaben, and

369

tebufenpyrad). Nine out of the thirty-six detected pesticides are listed in the Codex MRL

370

(http://www.codexalimentarius.net/pestres/data/index.html).

371

In the nine incurred tea samples tested, seven detected acetamiprid (33-382 µg/kg),

372

six detected bifenthrin (3.2 -3,542 µg/kg) and six detected cypermethrin (56-343 µg/kg).

373

Similar findings were previously reported by Hayward et al. (12) for bifenthrin and

374

cypermethrin in the same nine teas. Chen et al. (14) reported eleven of the eighteen tea

375

samples containing acetamiprid. The detection of acetamiprid in tea has also been reported by

376

Huang et al (33) who analyzed 3042 tea samples and in addition, found the following

377

pesticides: fenvalerate (0.05-0.25 mg/kg), cypermethrin (0.01-0.05 mg/kg), fenpropathrin


Page 16 of 29

Page 17 of 29


378

(0.03-0.3 mg/kg), buprofezin (0.06-0.25 mg/kg) and triazophos (0.02-0.2 mg/kg), with 74.3%

379

of the samples having fenvalerate levels exceeding the EU MRL. The frequency of detection

380

of fenpropathrin was 73.4 %, 16.4 % and 22.7% for oolong tea, green tea and black tea,

381

respectively.

382

The concentrations of the pesticide residues detected in the tea samples ranged from 1

383

to 3500 µg/kg, with the highest concentration pesticide being bifenthrin and the most

384

frequently detected pesticide being acetamiprid. The pesticides found in these tea samples

385

were those also present in the MRL lists established by European Union and Japan for tea.

386

The pesticide concentrations in the incurred tea samples were below the Japanese MRL, with

387

the exception of clomazone (61 µg/kg), exceeding the MRL tolerance of 20 µg/kg. The EU

388

has established MRL for eight of the detected pesticides (bifenthrin, cyhalothrin,

389

cypermethrin, deltamethin, endosulphan, ethion, propargite and tebufenyrad), while the MRL

390

for other pesticides were based on the limit of analytical determination of 10 – 100 µg/kg.

391

Ten detected pesticides the incurred tea samples with concentration over the EU MRL were

392

acetamiprid (3 out of 7), alpha, beta and delta BHC, (1 out of 1), buprofenzin (2 out of 4),

393

clomazone (1 out of 1), diphenylamine (1 out of 4), monocrotophos (1 out of 2), pyridaben (1

394

out of 1) and triazophos (1 out of 3).

395

Table 3 provides the results of sixty-one pesticide residues detected in twenty-three

396

incurred ginseng samples, ranging from three in sample G22, to twenty in sample G11. The

397

most frequently detected pesticides were anthraquinone (detected in all twenty-three ginseng

398

samples, 20-40 µg/kg) and the fungicides quintozene (seventeen samples, 14-2800 µg/kg),

399

pentachlorobenzene (fifteen, 3-157 µg/kg), hexachlorobenzene (twelve, 2-174 µg/kg),

400

iprodione (twelve, 5-457 µg/kg), tecnazene (twelve, 3-69 µg/kg ), pentachloroaniline (eleven,

401

80-557 µg/kg), pentachlorothioanisole (eleven, 7-203 µg/kg), and tetrachloroaniline (ten, 1-

402

15 µg/kg). The least frequent pesticide was chlorothalonil (G2, 64.8 µg/kg). Other pesticides



403

detected were chlorpyrifos (G8, 81.6 µg/kg), fenpropathrin (G15, 198.1 µg/kg ), fenvalerate

404

(G17, 19.4 µg/kg), heptachlor (G2, 3.7 µg/kg), nonachlor cis & trans (G4, 18.6 & 4.3 µg/kg),

405

oxychlordane (G4, 8.8 µg/kg), parathion (G15, 110.7 µg/kg), phorate and phorate sulfoxide

406

(G23, 143 & 369 µg/kg), pyraclostrobin (G2, 9.3 µg/kg), tetrahydrophthalimide (G19, 19

407

µg/kg), tetramethrin (G15, 232.9 µg/kg) and tricyclazole (G17, 73.3 µg/kg).

408

The sample preparation method based on acetonitrile extraction and solid phase

409

extraction using carbon coated alumina/primary-secondary amine with or without C18

410

evaluated in the current study was sufficient for extraction and cleanup of pesticides from dry

411

botanical samples for GC-MS/MS analysis of up to 227 multi-residues. This method was also

412

successfully used to determine pesticide residues in commercially-available tea and ginseng

413

samples.

414


Page 18 of 29

Page 19 of 29


415 416

ACKNOWLEDGMENTS

417

This project was supported in part by a Research Program appointment at the Center for Food

418

Safety and Applied Nutrition administrated by the Oak Ridge Institute for Science and

419

Education via an interagency agreement between the US Department of Energy and the US

420

Food and Drug Administration. We also thank the National Institutes of Health, Office of

421

Dietary Supplements for partial support of this work and the U.S. Environmental Protection

422

Agency, National Pesticide Standard Repository for additional pesticide standards.

423



424

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425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470

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Hayward, D.G.; Wong, J.W.; Park, H.Y. Determinations for pesticides on black, green, oolong, and white teas by gas chromatography triple quadrupole mass spectrometry. J. Agric. Food Chem. 2015, 63(37), 8116-8124.

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Wong, J.W.; Zhang, K.; Tech, K.; Hayward, D.G.; Krynitsky, A. J.; Cassias, I.; Schenck, F. J.; Banerjee, K.; Dasgupta, S.; Brown, D. Multiresidue pesticide analysis of ginseng powders using acetonitrile- or acetone-based extraction, solid-phase extraction cleanup, and gas chromatography-mass spectrometry/selective ion monitoring (GC-MS/SIM) or –tandem mass spectrometry (GC-MS/MS). J. Agric. Food Chem. 2010, 58(10), 5884-5896.

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587 588 589 590 591 592 593 594 595 596 597 598

SUPPORTING INFORMATION Supporting Table 1. GC-MS/MS parameters used to analyzed pesticides (compound name, CAS number, molecular formula, weight and structure) including retention times, precursor → product ion transitions and collision energies. Supporting Table S2. Recoveries (average and relative standard deviations), linear ranges, matrix-matched instrument quantitation levels, linearities and regression coefficients (R2) for individual pesticides in each botanical commodity.

599


Page 24 of 29

Page 25 of 29

600 601 602 603 604 605 606 607 608 609 610 611 612 613 614


TABLE CAPTIONS

Table 1. Recoveries of pesticides spiked at 10, 25, 100 and 500 µg/kg in green tea, ginseng, gingko biloba leaves, saw palmetto, spearmint and black pepper; and matrix-dependent instrument quantitation Limit (MDIQL).

Table 2. Pesticides detected and their concentrations (average ± standard deviation, µg/kg, n = 5) in the nine incurred tea samples analyzed using the GC-MS/MS method.

Table 3. Pesticides detected and their concentrations (average ± standard deviation, µg/kg, n = 5) in the twenty three incurred ginseng samples analyzed using the GC-MS/MS method.



615 616 617

Page 26 of 29

Table 1. Recoveries of pesticides spiked at 10, 25, 100 and 500 µg/kg in green tea, ginseng, gingko biloba leaves, saw palmetto, spearmint and black pepper; and matrix-dependent instrument quantitation Limit (MDIQL).

Botanicals

Green Tea

Ginseng

Gingko Leaves

Saw Palmetto

Spearmint

Black Pepper

Spiking levels (µg/kg) 10 25 100 500 10 25 100 500 10 25 100 500 10 25 100 500 10 25 100 500 10 25 100 500

ND 16 8 4 0 12 5 2 1 13 8 5 3 29 20 16 11 29 20 10 9 92 73 63 53

Number of pesticides with recoveries of 120% 5 10 15 8 16 18 17 15 3 3 4 3 10 11 8 12 10 14 16 15 15 22 11 14

25 28 51 46 10 17 7 13 6 6 15 11 22 17 3 7 32 11 10 5 27 73 36 46

170 173 157 170 181 184 198 197 202 209 201 209 165 177 199 196 154 181 191 199 89 58 117 114

11 8 0 3 8 3 3 1 3 1 2 1 1 2 1 1 3 2 3 1 4 1 0 0

618 619 620


Average recovery ± RSD (%) 88 ± 7 84 ± 8 76 ± 8 83 ± 6 91 ± 9 82 ± 6 87 ± 5 84 ± 4 99 ± 9 97 ± 6 83 ± 10 90 ± 5 78 ± 10 81 ± 7 88 ± 7 89 ± 5 81 ± 10 85 ± 8 84 ± 8 88 ± 8 79 ± 12 66 ± 7 75 ± 6 72 ± 5

Average MDIQL (µg/kg) 6

7

7

7

9

29

Page 27 of 29

621 622


Table 2. Pesticides detected and their concentrations (average ± standard deviation, µg/kg, n = 5) in the nine incurred tea samples analyzed using the GC-MS/MS method No. Pesticides

Detec.

Acetamiprid Anthraquinone

7 6

Azoxystrobin BHC, alpha BHC, Beta BHC, delta BHC, gamma (lindane) Bifenthrin Buprofezin Chlorpyrifos Clomazone Cyhalothrin (lamda) Cypermethrin DDD, pp DDE, pp DDT, pp Deltamethrin Difenoconazole Diphenylamine Endosulphan, alpha Endosulphan, beta Endosulphan, sulphate Ethion Fenbuconazole Fenvalerate Heptachlor Methidathion Monocrotophos Permethrin Phenylphenol, oPirimiphos-methyl Propargite Pyridaben Tebuconazole Tebufenpyrad Triazophos

1 1 1 1 1 6 4 5

Sample IDs (Number of pesticides detected) T1

T2

T3

T4

T5

T6

T7

T8

T9

(7) 72 ± 4

(12) 58 ± 1.5

(1)

(14) 186 ± 15 22 ± 15

(14) 382 ± 35 5 ± 0.2

(13) 52 ± 13 5.9 ± 0.9

(4) 33 ± 3

(10)

(14) 865 ± 25 11.7 ± 0.8

9.3

a

56.8 ± 5.7 42.7 ± 4.0 34.0 ± 4.2 16.8 ± 1.5 18 ± 1.2 319 ± 4 28 ± 0.5

96 ± 5.1 37 ± 3 6.6 ± 0.4

1 5 6 2 2 2 3 3 4

10.8 ± 0.8 56 ± 5 5.2 ± 0.6 1.5 ± 0.5 18 ± 1.2 12 ± 0.4

60 ± 3 100 ± 12

127 ± 14 75 ± 4 21 ± 2.5 61 ± 8 95 ± 3 136 ± 4

49 ± 3.5

3.2 ± 0.3

3542 ± 134 142 ± 8 2 ± 0.2

3.0 ± 0.2 21 ± 0.8 115 ± 2

44 ± 1 343 ± 14

49 ± 3 8.6 ± 1.3 6.0 ± 1.2 35 ± 2.0

3.3 ± 0.1 236 ± 19

2 1 2 2 1 2 1 1 2 1 2 1 2 3 1 1 3

4.8 ± 0.8

24 ± 0.8

22 ± 0.8

2.2 ± 0.2 23 ± 4 14 ± 2

MS - ACS Publications - American Chemical Society

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