Subscriber access provided by UOW Library
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
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.
Page 1 of 29
Journal of Agricultural and Food Chemistry
1
To be submitted to:
2
Journal of Agricultural and Food Chemistry
3 4 5 6
Determination of Multiresidue Pesticides in Botanical Dietary Supplements using Gas Chromatography-Triple Quadrupole Mass Spectrometry (GC-MS/MS)
7
Yang Chen1, Salvador Lopez2, Douglas G. Hayward3, Hoon Yong Park3, Jon W. Wong3,
8
Suyon S. Kim3, Jason Wan2, Ravinder M. Reddy1, Daniel J. Quinn4, David Steiniger4
9 10 11 12 13 14 15 16 17 18 19
1
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
20 21 22 23 24 25 26 27 28 29 30 31
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
1 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
32
ABSTRACT
33 34
A simplified sample preparation method in combination with gas chromatography-triple
35
quadrupole mass spectrometry (GC-MS/MS) analysis was developed and validated for the
36
simultaneous determination of 227 pesticides in green tea, ginseng, gingko leaves, saw
37
palmetto, spearmint, and black pepper samples. The botanical samples were hydrated with
38
water and extracted with acetonitrile, magnesium sulfate, and sodium chloride. The
39
acetonitrile extract was cleaned up using solid phase extraction with carbon coated
40
alumina/primary-secondary amine with or without C18 . Recovery studies using matrix
41
blanks fortified with pesticides at concentrations of 10, 25, 100 and 500 µg/kg resulted in
42
average recoveries of 70-99% and relative standard deviation of 5-13% for all tested
43
botanicals except for black pepper where lower recoveries of fortified pesticides were
44
observed. Matrix-matched standard calibration curves revealed good linearity (r2 > 0.99)
45
across a wide concentration range (1-1000 µg/L). Nine commercially-available tea and
46
twenty three ginseng samples were analyzed using this method. Results revealed 36
47
pesticides were detected in 9 tea samples at concentrations of 2-3500 µg/kg, and 61 pesticides
48
were detected in 23 ginseng samples at concentrations of 1-12500 µg/kg.
49
2 ACS Paragon Plus Environment
Page 2 of 29
Page 3 of 29
Journal of Agricultural and Food Chemistry
INTRODUCTION
50 51
Botanical dietary supplements are plant-based materials valued for their possible
52
medical or therapeutic properties such as aphrodisiacs, stress relievers, memory enhancers,
53
and treatment for inflammation and infection. Such botanicals are widely regarded as
54
valuable agricultural crops, and to prevent economic losses, pesticides may be used against
55
mold, insects, and other relevant pests that may cause commodity damage. Despite the
56
usefulness of pesticides in agricultural practices, there are concerns about their proper use and
57
residue levels in plant commodities. Pesticides can remain, persist, or accumulate from
58
application during the growing stages of the plant or from post-harvest treatment. Previous
59
studies have shown that many organochlorine and organophosphorus pesticides have been
60
detected in botanical products (1-3).
61
A number of methods that are based on procedures for fresh plant-derived foods have
62
been used for pesticide screening in dried botanical dietary supplements, spices, medicinal
63
plants, herbals, teas and phytomedicines (4-8). These methods involve organic solvent
64
extraction and clean-up procedures to remove co-extracted interfering components from the
65
matrix. While these methods work well with fresh produce, there are serious challenges with
66
dried botanicals due to the many varieties and complexities of botanical matrices and
67
concentrated levels of natural products present in these low-moisture products. The
68
complexity and variety of dried botanicals, along with the low concentration of many
69
pesticides and small sample sizes, complicate the detection, identification, and quantitation of
70
pesticide residues.
71
One of these pesticide methods, QuEChERS (Quick, Easy, Cheap, Effective Rugged
72
and Safe), has been widely used since its introduction over ten years ago (9). It involves
73
salting-out acetonitrile extraction followed by cleanup using dispersive solid-phase extraction
74
(d-SPE). The method has been modified to improve cleanup efficiency for more complex
3 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
75
matrices such as botanicals by utilizing a combination of sorbent materials, such as
76
graphitized carbon black (GCB), primary secondary amine (PSA), and octadecyl-linked silica
77
(C18) for additional d-SPE cleanup. This modified QuEChERS procedure has been validated
78
previously in our laboratory for the extraction of pesticides from botanicals prior to liquid
79
chromatography-triple quadrupole mass spectrometry in tandem MS mode (LC-MS/MS)
80
analysis (10). Hayward et al. (11) used a similar method for sample extraction and cleanup
81
using column solid-phase extraction (SPE) for gas chromatography-triple quadrupole mass
82
spectrometry in MS/MS mode (GC-MS/MS) analysis of 310 pesticides in 24 botanicals.
83
Recently, Hayward et al. (12) studied the efficiencies of four SPE cleanup procedures for 170
84
pesticides from green, black and oolong teas used for GC-MS/MS analysis. One of the SPE
85
columns evaluated in this study was graphitized carbon coated on alumina/PSA (CCA/PSA)
86
and eluted using acetone or ethyl acetate, instead of using a mixture of acetone/toluene (3:1)
87
typically used for conventional graphitized carbon black/PSA dual layer SPE cartridges.
88
Hayward et al. (12) concluded that the cleanup efficiency of using the dual layer CCA/PSA
89
SPE, followed by elution using acetone or ethyl acetate were comparable to those using the
90
GCB/PSA SPE and acetone/toluene elution, possibly as a result of the lighter carbon load in
91
the CCA particles. The avoidance of using toluene during the SPE cleanup step is
92
advantageous because there is less leaching of the matrix from the SPE cartridge into the GC
93
final extract, which can result in lower temperatures and shorter times required for sample
94
concentration, cleaner GC extracts, and faster sample preparation throughput.
95
The objective of this study was to expand, evaluate and validate the use of acetonitrile
96
salt-out extraction and CCA/PSA/C18 SPE cleanup for GC-MS/MS analysis of 227
97
pesticides for a wider range of botanicals including green teas, ginseng, gingko, saw
98
palmetto, spearmint, and black pepper.
99
4 ACS Paragon Plus Environment
Page 4 of 29
Page 5 of 29
100 101 102
Journal of Agricultural and Food Chemistry
MATERIALS AND METHODS Chemicals and Botanical Matrices Twelve customer-made analytical standard mixtures, each containing 14 to 27 pesticides, at
103
a concentration of 100 µg/mL in acetone, were purchased from AccuStandard, Inc. (New Haven,
104
CT). Additional pesticides were obtained through the U.S. Environmental Protection Agency,
105
National Pesticide Standard Repository (Ft. Meade, MD). The 12 standard mixtures were combined
106
to provide a pesticide standard with a total of 227 pesticides. The internal standard, tris-(1,3-
107
dichloroisopropyl) phosphate, was purchased from TCI America (Portland, OR). Pesticide-grade
108
acetonitrile and toluene, HPLC-grade water and anhydrous sodium sulfate were purchased from
109
Thermo Fisher Scientific (Pittsburgh, PA). The QuEChERS extraction kits containing 4 g of
110
anhydrous magnesium sulfate and 1 g of sodium chloride packets were purchased from Agilent
111
Technologies (Wilmington, DE). Solid-phase extraction (SPE) cartridges, carbon coated alumina
112
(Carbon X)/primary-secondary amine (PSA) (250/500 mg) and C18 (500 mg) were purchased
113
from United Science Corp (Center City, MN) and United Chemical Technologies, (Bristol, PA),
114
respectively. Botanical dietary supplements used for this study (green tea, ginseng, gingko biloba
115
leaves, saw palmetto, spearmint and black pepper) were obtained from retail commercial outlets,
116
field studies or other “organic” retail sources. The botanicals were ground, homogenized and sieved
117
through 18 mesh sieve prior to being used for the validation and recovery studies.
118
Preparation of Analytical Standards
119
An intermediate standard containing 5,000 ng/mL of each pesticide was prepared by diluting
120
the twelve pesticide standard mixtures equally with toluene in a 20 mL volumetric flask and was
121
used for preparing working standards and spike studies. Three other spiking solutions at
122
concentrations of 100, 250, 1000 ng/mL were prepared by diluting the intermediate standard
123
solution with toluene. Internal standard stock solution of 1,000 µg/mL was prepared by dissolving
124
tris-(1,3-dichloroisopropyl) phosphate in acetonitrile. The working standards were obtained by
5 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
125
diluting the intermediate standard solution mixture with matrix blanks to achieve working
126
concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 ng/mL, each containing 150 ng/mL of
127
internal standard tris-(1,3-dichloroisopropyl) phosphate. The matrix blanks were prepared by
128
extracting blank matrices, following the same sample preparation protocol as the fortification
129
studies.
130
Instrumentation
131
The analytical instrument comprised of a Trace GC 1310 gas chromatograph coupled
132
to a TSQ 8000 triple quadrupole mass spectrometer and a TriPlus RSH liquid autosampler
133
with a programed temperature vaporization injector (PTV) (Thermo Fisher Scientific, San
134
Jose, CA). The PTV was operated in splitless mode with an open baffled fused silica
135
deactivated glass liner (2 mm i.d. × 120 mm). The injector temperature was programed at
136
75°C for 0.1 min, ramped to 300°C at 2.5oC/sec, held for 3 min, followed by ramping to 330
137
°C at 14.5 °C/sec, held for 20 min. The injection volume was 2.0 µL, with split vent flow of
138
50 mL/min and splitless time of 1.0 min. Separation was performed on a TG-5SilMS column
139
(30 m × 0.25 mm × 0.25 µm, Thermo Scientific, San Jose CA) using helium as the carrier gas
140
at a constant flow rate of 1.2 mL/min. The column temperature was programmed to start at
141
40oC for 1.5 min, increased to 90°C at 25°C/min, held for 1.5 min, ramped to 180°C at
142
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
143
min, with a total run time of 35 min. The transfer line and ion source temperatures were 280
144
and 300°C, respectively. The mass spectrometer was operated in electron impact (EI)
145
ionization mode using timed selected reaction monitoring (t-SRM) using retention time
146
windows of 0.9 min. Three MS/MS transitions were selected for each pesticide and optimized
147
for sensitivity and selectivity were determined by collision energy experiments. The MS/MS
148
transitions and their respective collision energies, chromatographic retention times, as well as
6 ACS Paragon Plus Environment
Page 6 of 29
Page 7 of 29
Journal of Agricultural and Food Chemistry
149
the molecular and chemical information of each pesticide are provided in Supporting Table
150
S1.
151
The pesticides were identified based on retention times, the presence of the precursor-
152
product transition ions, and their respective ion ratios using the built-in TraceFinder pesticide
153
identification software of the TSQ 8000 system. The software consists of a master method
154
containing retention times and transition parameters for 600 pesticides. Quantitation was
155
performed using the peak area ratio responses of the analyte in the matrix-matched standards
156
to that of the internal standard using linear or quadratic curve produced using the built-in
157
TraceFinder software.
158
For the pesticides that have high abundance and meet sensitivity requirement, the
159
transitions were directly exported to a new method. For those pesticides that were not in the
160
master method or had low abundances, the retention times and transitions were created and
161
optimized using the build-in auto selected reaction monitoring (AutoSRM) program. The
162
AutoSRM created a full-scan method for precursor ion selection and selected ion monitoring
163
(SIM) methods with collision energy (CE) setting at 10, 20 and 30 eV for product ion
164
selections, followed by further CE optimization for the selected product ions. The retention
165
time window of 0.9 min was used for the t-SRM considering the diversity of matrices
166
evaluated in the study. The t-SRM data for a particular pesticide was acquired at a constant
167
cycle time at its expected retention time, therefore increasing dwell time to ensure optimum
168
number of data points for each peak and constant sensitivity.
169
Sample Preparation and Recovery Studies
170
The recovery studies were conducted by fortifying 100 µL of 100, 250, 1000 and 5000
171
ng/mL spiking standard solutions to 50 mL polypropylene centrifuge tubes containing 1.0 ± 0.05 g
172
blank botanical matrices to achieve the fortification levels of 10, 25, 100, and 500 µg/kg,
173
respectively. The spiked samples were thoroughly vortexed and allowed to stand for 10 min to
7 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Page 8 of 29
174
allow absorption of pesticides in the matrices. The fortification step was eliminated for the blank
175
matrix and incurred samples. The dried plant material was hydrated with HPLC-grade water (10
176
mL) for 15 min and acetonitrile (10 mL) containing internal standard (60 µg/L of tris-(1,3-
177
dichloroisopropyl)-phosphate) followed by the addition of 4 g anhydrous magnesium sulfate and 1 g
178
sodium chloride for salting-out acetonitrile extraction. Samples were shaken vigorously at 1000
179
strokes/min for 1 min using GenoGrinder (SPEX Sample Prep, Metuchen, NY) followed by
180
centrifugation at 4200 × g for 5 min. The resulting supernatants were further processed using solid
181
phase extraction (SPE)
182
The acetonitrile supernatant (1.25 mL) was loaded on the Carbon-X/PSA or C18/Carbon-
183
X/PSA SPE column (topped with approximately 100-200 mg anhydrous sodium sulfate and
184
preconditioned using two column volumes ~12 mL of acetone), and subsequently the SPE column
185
was eluted with approximately 12 mL acetone. The acetonitrile extracts from ginseng, tea, and
186
gingko biloba were cleaned up using 500 mg Carbon-X/500 mg PSA, whereas the spearmint and
187
saw palmetto were cleaned up using a second C18 SPE cartridge attached on top of the Carbon
188
X/PSA dual layer cartridge. The QuEChERS acetonitrile extract (approximately 10 mL) from the
189
black pepper was carefully transferred to a clean glass test tube with a cap and gently rinsed with 5
190
mL acetonitrile-saturated hexane. The hexane was removed and the process was repeated again and
191
the hexane-rinsed acetonitrile extract was loaded onto the two cartridge setup for C18/Carbon-
192
X/PSA cleanup. The eluting acetone extract including the loading acetonitrile supernatant were
193
collected in a 15 mL glass centrifuge tube and the cleaned extract was concentrated to
194
approximately 100 µL at 50oC under a gentle stream of nitrogen using a nitrogen evaporator (N-
195
Evap, Organomation, Associates, Berlin, MA). Special attention was made to avoid the evaporation
196
of the extracts to go to complete dryness to prevent losses of the pesticide analytes. To the reduced
197
extract, 500 µL toluene, 50 µL QC standards, and 25 mg anhydrous magnesium sulfate were added.
198
The mixture was vortexed for 30 sec, centrifuged at 1258 × g for 5 min. The resulting supernatant
8 ACS Paragon Plus Environment
Page 9 of 29
Journal of Agricultural and Food Chemistry
199
was transferred into an amber glass autosampler vial and stored at - 20 oC prior to GC-MS/MS
200
analysis.
201
The working standards were obtained by diluting the intermediate standard solution mixture
202
with matrix blanks (containing the internal standard, tris-(1,3-dichloroisopropyl) phosphate) to
203
achieve working concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 ng/mL. The matrix blank
204
samples used for preparation of the matrix-matched standard were pre-screened for presence of any
205
target pesticide residue prior to use for the matrix-matched standard preparation. The matrix blanks
206
were prepared by extracting blank matrices following the same sample preparation protocol as the
207
fortification study.
208
Determination of Matrix-Dependent Instrument Quantitation Limit (MDIQL)
209
Matrix-matched standards at concentrations of 2, 5, 10, 20 and 50 ng/mL, each with
210
eight replicates were analyzed and the results were used for the determination of the matrix
211
dependent-instrument quantitation limit (MDIQL). The peak area ratio response of a pesticide
212
to that of the internal standard was plotted against the corresponding matrix-matched standard
213
curve and the concentration was calculated. The lowest level of pesticide in the standard that
214
produces results within ± 25% of the expected accuracy for the qualify ion with minimum of
215
one confirmation ion was used for MDIQL determination of that pesticide. The MDIQL were
216
calculated according to the U.S. Environmental Protection Agency (EPA) procedure used to
217
determine the method limit of quantitation (13). The standard deviations (SD) of the
218
responses were calculated from the eight injections for each pesticide and the MDIQL was
219
calculated at 3 × SD× 3 × dilution factor. [i.e., 2.998 × SD (critical t 0.010 = 2.998 at degree of
220
freedom (df) = 7) × 3 × dilution factor] (13). Means and standard deviations of the recoveries
221
in the fortified samples, matrix dependent instrument quantitation limit (MDIQL) were
222
determined using Microsoft Excel 2010.
223
Analysis of Pesticide Residues in Commercially Available Botanical Samples
9 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
224
Commercially available tea samples (green tea, black tea and oolong tea) and ginseng
225
samples were analyzed for pesticide residues using the sample preparation method in
226
combination with GC-MS/MS analysis developed in the current study.
227
10 ACS Paragon Plus Environment
Page 10 of 29
Page 11 of 29
Journal of Agricultural and Food Chemistry
RESULTS AND DISCUSSION
228 229 230
Instrument Performance and Method Optimization A standard mixture containing the 227 pesticides in toluene (5,000 ng/mL) was
231
analysed using the GC-MS/MS and the chromatograms and precursor-to-product ion
232
transitions of each pesticide were examined using the TraceFinder software. The 227
233
pesticides evaluated in this study along with their retention times, transitions and collision
234
energy are provided in Supporting Table S1. The quantitative calibration and linearity
235
evaluation for each pesticide were performed using matrix-matched standards in the
236
concentration range of 1-1000 ng/mL (r2 > 0.99). The linearity ranges for the majority of
237
pesticides in all matrices except for the black pepper were in the range of 1 to 1000 ng/mL
238
with regression coefficients greater than 0.99, except for allethrin, captan, dicofol,
239
dioxathion, ethoxyquin, isoproturon, lufenuron, metalaxyl, and trichlorfon. Of the 227
240
pesticides fortified in the black pepper extract, the chromatographic peaks of 50 pesticides
241
were not present and could not be identified even at concentration up to 1000 µg/L. This is
242
probably contributed to the high concentrations of chemical components still present in the
243
black pepper extract that are overwhelming the presence and interfering the chromatographic
244
separation of the less concentrated pesticide analytes.
245
Recovery of Pesticides
246
Recovery studies were conducted by fortifying the 227 pesticides into blank matrices
247
of green tea, ginseng, gingko leaves, saw palmetto, spearmint and black pepper, to
248
concentrations of 10, 25, 100 and 500 µg/kg. The number of pesticides with recoveries of 1-
249
49%, 50-69%, 70-120%, and >121%, as well as the average recovery and relative standard
250
deviation at each fortification level for each matrix are presented in Table 1. Individual
251
pesticide recoveries in each matrix are provided in Supporting Table S2. For green tea,
252
ginseng, gingko leaves, saw palmetto and spearmint, a majority (> 90%) of the fortified
11 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
253
pesticides showed an average recovery of 76 - 99%, with standard deviations of 4 - 12%.
254
The number of pesticides with acceptable recoveries within the 70 - 120% range at the 10 and
255
500 µg/kg fortification levels were 170, 181, 202, 165, 154 and 170, 197, 209, 196, 199 for
256
green tea, ginseng, gingko biloba, saw palmetto, and spearmint, respectively. These average
257
recovery levels for the fortified pesticides are comparable with those reported previously by
258
Hayward et al. (11, 12). The number of non-detected pesticides decreased with increasing
259
fortification levels. At the 10 µg/kg fortification, 16, 12, 13, 29 and 29 pesticides were not
260
detected in green tea, ginseng, gingko, saw palmetto and spearmint, respectively, whereas 0,
261
1, 3, 11, 9 pesticides were not detected at the 500 µg/kg for the respective matrices. The non-
262
detects of allethrin, captan, dioxathion, ethoxyquin, isoproturon, lufenuron, metalaxyl and
263
trichorfon in some matrices at the low fortification levels were due to low instrument
264
responses of analytes in the matrices. The low recoveries were likely due to poor extraction
265
efficiency during acetonitrile extraction, adsorption to the sorbent during SPE clean-up,
266
chemical instability or decomposition, and matrix interference during chromatographic
267
analysis. These pesticides include acephate, anthraquinone, cyprodinil, chlorothalonil
268
ethoxyquin, fludioxinil, folpet, thiabendazole and phenmedipham, fluridone, prochloraz,
269
pyrimethanil, pentachloroaniline and pyraclostrobin. (10, 14-16). The high recoveries (145 -
270
2000%) of dicofol in green tea and non-detection or low recoveries in the other matrices may
271
be attributed to its response change during the batch analysis. This was observed by
272
monitoring the pesticide responses in the 20 ng/mL matrix-matched standard at the early,
273
middle and end stages of the sequence as peak area responses tended to increase for green tea
274
and ginseng and decrease for gingko biloba, saw palmetto, and spearmint matrices.
275
QuEChERS typically uses acetonitrile as the extraction solvent followed by dispersive
276
SPE with PSA and magnesium sulfate (9). The use of PSA helps remove organic acids, polar
277
pigments, some sugars, and fatty acids prior to GC-MS/MS analysis of pesticides. This
12 ACS Paragon Plus Environment
Page 12 of 29
Page 13 of 29
Journal of Agricultural and Food Chemistry
278
method works well for high moisture vegetal-based matrices but does not work well for food
279
matrices containing high levels of pigments and fats, which are prevalent in many botanical
280
products. Therefore cleanup is essential for satisfactory method performance as well as
281
general maintenance of the GC-MS/MS, particularly the liner, column, and ion source. The
282
effective use of graphitized carbon black (GCB) for SPE was reported by Fillion et al. (21) to
283
remove non-polar pigments such as carotenoids and chlorophyll (22). Pang et al. (23) applied
284
a similar approach using an acetonitrile salt-out extraction step, followed by SPE cleanup
285
with GCB/PSA whereas Lozano et al. (24) applied a modified QuEChERS (Quick, Easy,
286
Cheap, Rugged and Safe) procedure for the analysis of pesticides in tea samples. GCB has
287
non-specific retention not only to chlorophyll, but also to planar pesticides containing
288
aromatic moieties, resulting in lower recoveries. Typically, the eluting solvent contains
289
toluene as a competing planar agent to desorb the pesticides from the GCB sorbent although
290
some of the matrix components may also be eluted as well (25). Wong et al. (15) applied
291
dispersive SPE with C8 to dried ginseng sample extracts followed by dual layer SPE with
292
GCB and PSA to remove these matrix components that may interfere with the analysis of
293
pesticides. The use of GCB for the sample clean-up of plant-based materials for pesticide
294
analysis has been well established but newer carbon-based materials such as silica-dispersed
295
carbon (26) and alumina (27) and multiwalled carbon nanotubes (28), have been recently
296
introduced as chromatographic media for separation processes or as sorbents in removing
297
chemical co-extractives from the sample matrix.
298
cleanup efficiency of using carbon-coated alumina (Carbon X) followed by elution using
299
acetone or ethyl acetate without the use as toluene was comparable to those using the
300
GCB/PSA acetone/toluene SPE procedures in black and green teas. The SPE approach using
301
a combination of Carbon X/PSA or C18/Carbon X/PSA seems to work well with ginseng,
302
Gingko biloba, saw palmetto, spearmint, and green tea in this current study.
Hayward et al. (12) reported that the
13 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
303
However, when this promising approach was applied to black pepper, less than 50%
304
of the pesticides fortified in black pepper showed a 70 - 120% recovery range, specifically
305
with an average recovery range of 66 - 79% at the 10 and 25 µg/kg spike levels. Additional
306
clean-up involving the rinsing of the acetonitrile extracts with acetonitrile-saturated hexane to
307
further eliminate lipophilic co-extracts of the black pepper matrix was also implemented as
308
described by Przybylski and Segard (29). Significant signal loss was observed for a large
309
number of target pesticides and chromatographic signals could not be observed even at the
310
1000 ng/mL level for 50 pesticides. This may be due to the low ionization efficiency in the
311
mass analyzer caused by the overwhelming presence of interferring co-extractives from the
312
black pepper matrix. Black pepper, similar to other botanicals, contains high levels of
313
unsaturated amides, flavonoids, fatty esters, steroids, propenlyphenols and alkaloids (17).
314
The combined use of the hexane rinses and SPE cleanup with the C18/Carbon X/PSA
315
sorbents ought to be capable of removing high levels of co-matrix chemical components such
316
as fatty acid esters, sterols, polyphenolic, pigments, and non-polar compounds in the black
317
pepper extract. However, black pepper extracts consist of essential oils containing primarily
318
of terpenes and terpenoids compounds, such as β-caryophylline, limonene, β-pinene, and
319
piperine (18, 19). Since many synthetic pyrethroid and chlorinated pesticides have been
320
derived from terpene- and terpenoid-based (as well as other classes of) natural products (20),
321
the chemical analysis of pesticides in botanicals containing essential oils and oleoresins is
322
further complicated since the chemical and physical properties of both natural and synthetic
323
constituents are similar and difficult to separate from one another using conventional
324
chromatographic techniques. From a previous study (11), these botanicals, especially spices,
325
whose desirable features are due to the presence of these essential oils, are also posing
326
problems for pesticide analysis. Effective schemes need to be investigated to address and
327
resolve these problems for a majority of these botanicals that consist of these essential oils.
14 ACS Paragon Plus Environment
Page 14 of 29
Page 15 of 29
328 329
Journal of Agricultural and Food Chemistry
Matrix Dependent Instrument Quantitation Limit (MDIQL) The MDIQLs were established using the lowest concentration of matrix-matched
330
standards that produced within 25% of the expected levels with presence of at least one of the
331
confirmation ions. The results obtained are shown in Supporting Table S1 and summarized
332
in Table 1. More than 90% of analytes in all matrices (except black pepper) have MDIQL of
333
20 µg/kg) in all matrices possibly due to a lack of stable
335
product ions or thermal instability of the pesticide. The high MDIQLs of dicofol were due to
336
its response changes throughout the analysis. The MDIQL of thermally labile lufenuron
337
varied between matrices, from 10 µg/kg in ginseng and gingko to >80 µg/kg in saw palmetto
338
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).
15 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
16 ACS Paragon Plus Environment
Page 16 of 29
Page 17 of 29
Journal of Agricultural and Food Chemistry
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
17 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
18 ACS Paragon Plus Environment
Page 18 of 29
Page 19 of 29
Journal of Agricultural and Food Chemistry
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
19 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
424
REFERENCES
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
1.
Huggett, D.B; Block, D.S.; Khan, I.A; Allgood, J.C.; Benson, W.H. Environmental contaminants in the botanical dietary supplement ginseng and potential human risk. Hum. Ecol. Risk Assess. 2000, 6 (5), 767-776.
2.
Huggett, D.B; Khan, I.A.; Allgood, J.C.; Blosck, D.S.; Schlenk, D. Organochlorine pesticides and metals in select botanical dietary. Bull. Environ. Contam. Toxicol. 2001, 66, 150-155.
3.
Durgnat, J.M.; Heuser, J.; Andrey, D.; Perrin, C. Quality and safety assessment of ginseng extracts by determination of the contents of pesticides and metals. Food Addit. Contam. 2005, 22 (12), 1224-1230.
4.
Kong, M.F.; Chan, S.; Wong, Y.C.; Wong, S.K.; Sin, D.W.M. Interlaboratory comparison for the determination of five residual organochlorine pesticides in ginseng root samples by gas chromatography. J. AOAC Int. 2007, 90 (4), 1133-1141.
5.
Wong, J.W.; Hennessy, M.K.; Hayward, D.G.; Krynitsky, A.J.; Cassias, I.; Schenck, F.J. Analysis of organophosphorus pesticides in dried ground ginseng root by capillary gas chromatography-mass spectrometry and –flame photometric detection. J. Agric. Food Chem. 2007, 55 (4), 1117-1128.
6.
Hayward, D.G.; Wong, J.W. Organohalogen and organophosphorous pesticide method for ginseng root – a comparison of gas chromatography-single quadrupole mass spectrometry with high resolution time-of-flight mass spectrometry. Anal Chem, 2009, 81 (14), 5716-23.
7.
Wong, J.W.; Zhang, K.; Tech, K.; Hayward, D.G.; Krynitsky, A.J.; Cassias, I.; Schenck, F.J.; Banerjee, K.; Dasgupta, S.; Brown, D.J. 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, 5884-5896.
8.
U.S. Food and Drug Administration. Pesticide Analytical Manual, 3rd ed.; U.S. Department of Health and Human Services, Public Health Service: Rockville, MD, 1999; Vol. 1.
9.
Anastassiades, M.; Lehotay, S.J.; Stajnbaher, D.; Schenck, F.J. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and "dispersive solid-phase extraction" for the determination of pesticide residues in produce. J. Agric. Food Chem. 2003, 86 (2), 412-431.
10.
Chen, Y.; Al-Taher, F.; Juskelis, R.; Wong, J.W.; Zhang, K.; Hayward, D. G.; Zweigenbaum, J.; Stevens, J.; Cappozzo, J. Multiresidue pesticide analysis of dried botanical dietary supplements using an automated dispersive SPE cleanup for
20 ACS Paragon Plus Environment
Page 20 of 29
Page 21 of 29
471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519
Journal of Agricultural and Food Chemistry
QuEChERS and high-performance liquid chromatography-tandem mass spectrometry. J. Agric. Food Chem. 2012, 60(40), 9991-9999. 11.
Hayward, D.G; Wong, J.W.; Shi, F.; Zhang, K.; Lee, N.S.; DiBenedetto, A.L. Multiresidue pesticide analysis of botanical dietary supplements using salt-out acetonitrile extraction, solid-phase extraction cleanup column, and gas chromatography-triple quadrupole mass spectrometry. Anal. Chem. 2013, 85, 4686−4693.
12.
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.
13.
Title 40- Protection of Environment. Part 136 - Guidelines establishing test procedures for the analysis of pollutants, Appendix B: Definition and procedure for the determination of the method detection limit – revision 1.11. Code of Federal Regulations; U.S. Government Printing Office: Washington, DC, 2014; http://www.gpo.gov/fdsys/pkg/CFR-2014-title40-vol23/xml/CFR-2014-title40-vol23part136.xml
14.
Chen, G.; Cao, P.; Liu, R. A multi-reside method for fast determination of pesticides in tea by ultra performance liquid chromatography-electrospray tandem mass spectrometry combined with modified QuEChERS sample preparation procedures. Food Chemistry, 2011, 125, 1406-1411.
15.
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.
16.
Koesukwiwat, U.; Lehotay, S.J.; Miao, S.; Leepipatpiboon, N. High throughput analysis of 150 pesticides in fruits and vegetables using QuEChERS and low-pressure gas chromatography-time-of flight mass spectrometry. J of Chromatogr. A 2010, 1217, 6692-6703.
17.
Lija-Escaline, J.; Senthil-Nathan, S.; Thanigaivel, A.; Pradeepa, V.; VasanthaSrinivasan, P.; Ponsankar, A.; Edwin, E.S.; Selin-Rani, S.; Abdel-Megeed, A. Physiological and biochemical effects of botanical extract from Piper nigrum Linn (Piperaceae) against dengue vector Aedes aegypti Liston (Diptera: Culicidae). Parasitol. Res. 2015, 114(11), 4239-4249.
18.
Orav, A.; Stulova, I.; Kailas, T.; Müürisepp, M. Effect of storage on the essential oil composition of Piper nigrum L. fruits of different ripening states. J. Agric. Food Chem. 2004, 52, 2582-2586.
19.
Kapoor, I. P. S.; Singh, B.; Singh, G.; DeHeluani, C. S.; DeLampasona, M. P.; Catalan, C. A. N. Chemistry and in vitro antioxidant activity of volatile oil and
21 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568
oleoresins of black pepper (Piper nigrum). J. Agric. Food Chem. 2009, 57, 53585364. 20.
Cantrell, C. L.; Dayan, F. E.; Duke, S. O. Natural products as sources for new pesticides. J. Nat. Prod. 2012, 75, 1231-1242.
21.
Fillion, J.; Hindle, R.; Lacroix, M.; Selwyn, J. Multiresidue determination of pesticides in fruit and vegetables by gas chromatography-mass selective detection and liquid chromatography with fluorescence detection. J. AOAC Int. 1995, 78(5), 12521266.
22.
Hennion, M.C. Graphitized carbons for solid-phase extraction. J. Chromatogr. A 2000, 885(1-2), 73-95.
23.
Pang, G.F.; Fan, C. L.; Zhang, F.; Li, Y.; Chang, Q.Y.; Chang, Q. Y.; Cao, Y. Z.; Wang, Q. J.; Hu, X. Y.; Liang, P. High-throughput GC/MS and HPLC-MS/MS techniques for the multiclass, multiresidue determination of 653 pesticides and chemical pollutants in tea. J. AOAC Int. 2011, 94, 1253-1296.
24.
Lozano, A.; Rajski, Ł.; Belmonte-Valles, N.; Uclés, A.; Mezcua, M.; Fenandez-Alba, A. R. Pesticide analysis in teas and chamomile by liquid chromatography and gas chromatography tandem mass spectrometry using a modified QuEChERS method: validation and pilot survey in real samples. J. Chromatogr. A. 2012, 1268, 109-122.
25.
Zhimelis, O.; Yang, Y.; Stenerson, K.; Kaneko, T.; Ye, M. Evaluation of a solidphase extraction dual-layer carbon/primary secondary amine for clean-up of fatty acid matrix components from food extracts in multiresidue pesticide analysis. J. Chromatogr. A., 2007, 1165, 18-25.
26.
Haimovici, L.; Reiner, E. J.; Besevic, S.; Jobst, K. J.; Robson, M.; Kolic, T.; MacPherson, K. A modified QuEChERS approach for the screening of dioxins and furans in sediments. Anal. Bioanal. Chem. 2016, DOI 10.1007/s00216-016-9493-0.
27.
Zhao, P.; Wang, L.; Jiang, Y.; Zhang, F.; Pan C. Dispersive cleanup of acetonitrile extracts of tea samples by mixed multiwalled carbon nanotubes, primary secondary amine, and graphitized carbon black sorbents. J. Agric. Food Chem. 2012, 60, 40264033.
28.
Paek, C.; McCormick, A. V.; Carr, P. W. Preparation and evaluation of carbon coated alumnia as a high surface area packing material for high performance liquid chromatography. J. Chromatogr. A 2010, 1217, 6475-6483.
29.
Przybylski, C.; Segard, C. Method for routine screening of pesticides and metabolites in meat based baby-food using extraction and gas chromatography-mass spectrometry. J. Sep. Sci. 2009, 32, 1858-1867.
30.
Lacina, O.; Zachariasova, M.; Urbanova, J.; Vaciavikova, M.; Cajka, T.; and Hajslova, J. Critical assessment of extraction methods for the simultaneous determination of pesticide residues and mycotoxins in fruits, cereals, spices and oil
22 ACS Paragon Plus Environment
Page 22 of 29
Page 23 of 29
569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586
Journal of Agricultural and Food Chemistry
seeds employing ultra-high performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2012, 1262, 8-18. 31.
Yogendrarajah, P.; Poucke, C.V.; Meulenaer, B.D. and Saeger, S.D, Development and validation of a QuEChERS based liquid chromatography tandem mass spectrometry method for the determination of multiple mycotoxins in spices. J. Chromatogr. A 2013, 1297, 1-11.
32.
Ferrer, C.; Unterluggauer, H.; Fischer, R. J.; Fernández-Alba, A. R.; Masselter, S. Development and validation of a LC–MS/MS method for the simultaneous determination of aflatoxins, dyes and pesticides in spices. Anal Bioanal Chem. 2010, 397, 93-107.
33.
Huang, Z.Q.; Li, Y.J.; Chen, B. and Yao, S.Z. Simultaneous determination of 102 pesticide residues in Chinese teas by gas chromatography-mass spectrometry. J. Chromatogr. B 2007, 853, 154-162.
23 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
24 ACS Paragon Plus Environment
Page 24 of 29
Page 25 of 29
600 601 602 603 604 605 606 607 608 609 610 611 612 613 614
Journal of Agricultural and Food Chemistry
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.
25 ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
26 ACS Paragon Plus Environment
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
Journal of Agricultural and Food Chemistry
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
