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6851
AND
MARION MILLER
An analytical method was developed for the determination of eleven agrochemicals [abamectin (as
B1a), bifenazate, bifenthrin, carfentrazone-ethyl, cymoxanil, hexythiazox, imidacloprid, mefenoxam,
pymetrozine, quinoxyfen, and trifloxystrobin] in dried hops. The method utilized polymeric and NH2
solid phase extraction (SPE) column cleanups and liquid chromatography with mass spectrometry
(LC-MS/MS). Method validation and concurrent recoveries from untreated dried hops ranged from
71 to 126% for all compounds over three levels of fortification (0.10, 1.0, and 10.0 ppm). Commercially
grown hop samples collected from several field sites had detectable residues of bifenazate, bifenthrin,
hexythiazox, and quinoxyfen. The control sample used was free of contamination below the 0.050
ppm level for all agrochemicals of interest. The limit of quantitation and limit of detection for all
compounds were 0.10 and 0.050 ppm, respectively.
KEYWORDS: Pesticides; residue method; hops; liquid chromatography-mass spectrometry; LC-MS/MS
INTRODUCTION
Department of Environmental Toxicologys IR-4/Trace Analytical Laboratory with the desire to develop a multiresidue method
to screen for several of the most commonly applied pesticides
on dried hop cones (Table 1). The target limit of quantitation
(LOQ) was set at 0.1 ppm for all compounds. The rationale for
the LOQ was to have a method that could quantitate the majority
of compounds below the tolerances set by the U.S. EPA.
Analysis of the target compounds in raw agricultural commodities can be accomplished by various means including gas
chromatography (GC), high performance liquid chromatography
(HPLC), gas chromatography coupled to a mass spectrometer
(GC-MS) and liquid chromatography coupled to a tandem mass
spectrometer (LC-MS/MS) (616). In fact, many of the target
compounds have been successfully extracted and determined
together from high moisture crops in previously developed multi
residue methods (17, 18). Many of the methods in the literature
are quite sensitive and selective for the compounds of interest,
but were not intended for use with such a complicated matrix
Table 1. Compound-Specific U.S. EPA Tolerances on Dried Hops
compound
tolerance (ppm)
citation
abamectina
bifenazate
bifenthrin
carfentrazone-ethyl
cymoxanil
hexythiazox
imidacloprid
mefenoxamb
pymetrozine
quinoxyfen
trifloxystrobin
0.20
15.0
10.0
0.05
1.0
2.0
6.0
20.0
6.0
3.0
11.0
40CFR180.449
40CFR180.572
40CFR180.442
40CFR180.515
40CFR180.503
40CFR180.448
40CFR180.472
40CFR180.408
40CFR180.556
40CFR180.588
40CFR180.555
6852
DPb (V)
FPc (V)
EPd (V)
CEPe (V)
CEf (V)
CXPg (V)
RTh (min)
305.1
198.2
181.1
346.1
128.0
228.1
208.9
219.9
105.2
197.0
186.1
1
16
1
66
6
11
21
11
21
36
11
370
360
370
350
290
340
370
330
370
370
370
9
10
6.5
12
8.5
11.5
10.5
10.5
12
10.5
12
38
32
18
18
12
32
18
12
14
14
30
38
15
21
27
13
19
21
19
31
47
21
8
4
4
6
4
4
4
4
4
4
4
9.92
5.25
10.51
5.64
4.01
7.38
3.59
4.84
3.17
7.45
6.11
890.5
301.0
440.1i
412.0
198.9
353.1
256.0
280.1
217.9
307.9
409.1
a
Q1 and Q3 represent compound-specific transitions monitored. b Declustering potential. c Focusing potential. d Entrance potential into first quadrupole. e Collision cell
entrance potential. f Collision energy. g Collision cell exit potential. h Retention time i [NH4]+ adduct.
0.10 (n ) 9)
1.0a (n ) 6)
102 ( 9
113 ( 11b
87 ( 7
97 ( 10
85 ( 5
86 ( 9
104 ( 12
101 ( 8
98 ( 19
97 ( 8
100 ( 10
95 ( 3
95 ( 6
83 ( 5
94 ( 5
88 ( 4
91 ( 9
97 ( 7
103 ( 3
88 ( 7
91 ( 3
96 ( 2
10a (n ) 6)
89 ( 5
87 ( 2
74 ( 2
102 ( 2
83 ( 2
86 ( 2
97 ( 2
93 ( 3
89 ( 2
95 ( 1
95 ( 3
a
Values are mean percent recovered ( standard deviation; n is the number
of replicates. b For bifenazate at the 0.1 ppm level n ) 6.
6853
compound
cascadea
CTZa
Galenaa
Nuggeta
Willamettea
abamectin
bifenazate
bifenthrin
carfentrazone-ethyl
cymoxanil
hexythiazox
imidacloprid
mefenoxam
pymetrozine
quinoxyfen
trifloxystrobin
<0.10, <0.10
2.08, 0.25
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, 0.60
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, 0.45
<0.10, <0.10
<0.10, <0.10
1.03, 0.79
<0.10, 0.14
<0.10, <0.10
<0.10, <0.10
0.12, 0.31
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
0.15, 0.35
<0.10, <0.10
<0.10, <0.10
2.40, 0.36
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
0.16, 0.16
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
0.13, <0.10
<0.10, <0.10
<0.10, <0.10
2.15, 0.327
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
0.76, 0.32
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
5.49, 1.47
<0.10, 0.10
<0.10, <0.10
<0.10, <0.10
0.23, 0.32
<0.10, <0.10
<0.10, <0.10
<0.10, <0.10
<0.10, 0.12
<0.10, <0.10
Values represent an average of duplicate analyses from each sampling year. The first value is the 2006 sample, and the second value is the 2007 sample.
Figure 2. Total ion chromatogram of 20 pg/L (equivalent to 0.1 ppm) calibration standard: pymetrozine (3.17 min), imidacloprid (3.59 min), cymoxanil
(4.01 min), mefenoxam (4.84 min), bifenazate (5.25 min), carfentrazone-ethyl (5.64 min), trifloxystrobin (6.11 min), hexythiazox (7.38 min), quinoxyfen
(7.45 min), abamectin (9.92 min), and bifenthrin (10.51 min).
unfiltered sample extract was loaded to the SPE. Mild vacuum was
applied and the eluant was collected in a 45 mL conical tube at a flow
rate of 1-2 drops per second. Once the extract was loaded to the
SPE, the 50 mL disposable tube was rinsed with 5 mL of MeCN and
was added to the SPE. Following the rinse, the filter cake above the
SPE packing was rinsed with an additional 5 mL of MeCN. The eluted
sample was transferred to a TurboVap tube and the 45 mL conical
tube was rinsed with 5 mL of acetone and pooled with the MeCN
fraction. The extract was then concentrated to 0.5 mL with dry
nitrogen and water bath at 35 C using a TurboVap II workstation
(Caliper Life Science, Hopkinton, MA). The concentrated sample was
dissolved into 5 mL of ethyl acetate/hexane (50:50, v/v) and subjected
to a second cleanup by SPE. Mega Bond Elut-NH2 SPE columns (5
g/20 mL, Varian Inc., Harbor City, CA) were conditioned with 15 mL
of ethyl acetate/hexane (50:50, v/v). When the solvent reached the top
of the packing, the sample was loaded to the SPE. Mild vacuum was
applied and eluant was collected in a 45 mL conical tube at a flow rate
of 1-2 drops per second. The TurboVap tube from the previous
concentration step was rinsed twice with 5 mL of ethyl acetate/hexane
(50:50, v/v) and the rinse solvent was added to the SPE. An SPE
reservoir was attached to the SPE and sample elution was continued
with an additional 25 mL of ethyl acetate/hexane (50:50, v/v). Once
the ethyl acetate/hexane aliquot was completely loaded to the SPE,
the TurboVap tube from the previous concentration step was rinsed
with 5 mL of acetone/hexane (50:50, v/v). The resulting sample was
transferred to a clean TurboVap tube and the 45 mL conical tube was
placed back into the vacuum manifold. The final elution step was completed with 45 mL of acetone/hexane (50:50, v/v) and the resulting
sample was pooled with the previous fraction in the TurboVap tube.
Sample extracts were then concentrated to near dryness using the
TurboVap II workstation mentioned above. The final sample was
dissolved into an appropriate volume with 10 mM ammonium acetate/
methanol (10:90, v/v) and filtered through a 0.2 m Acrodisc syringe
filter (Pall Corporation, Ann Arbor, MI) prior to analysis by LC-MS/
MS. For determinations at 0.1 ppm, final sample volume was 5 mL
(0.2 g/mL).
Sample Analysis. Sample analysis was conducted with a PerkinElmer Series 200 autosampler and binary micropumps (Perkin-Elmer,
6854
Figure 3. Total ion chromatogram of Idaho control. No residues were detected above 0.05 ppm for expected retention times of target compounds. Refer
Figure 4. Total ion chromatogram of 0.1 ppm recovery from Idaho control sample: pymetrozine (3.14 min), imidacloprid (3.56 min), cymoxanil (3.97 min),
mefenoxam (4.82 min), bifenazate (5.24 min), carfentrazone-ethyl (5.62 min), trifloxystrobin (6.07 min), hexythiazox (7.38 min), quinoxyfen (7.45 min),
abamectin (9.82 min), and bifenthrin (10.45 min).
Shelton, CT) coupled to an Applied Biosystem API-2000 tandem mass
spectrometer via a atmospheric pressure chemical ionization source
(APCI) (Applied Biosystem, Palo Alto, CA). The APCI source was
6855
Figure 5. Total ion chromatogram of 2007 CTZ sample: 0.79, 0.31, 0.35, and 0.14 ppm of bifenazate (5.25 min), hexythiazox (7.38 min), quinoxyfen
(7.46 min), and bifenthrin (10.54 min), respectively.
spectrometer was operated in multiple reactant monitoring mode
(MRM). See Table 2 for compound conditions. Chromatographic
separation was accomplished with a Agilent Zorbax Eclipse Plus C18
column (100 3.0 mm i.d., 3.5 m particle size, Agilent Technologies,
Palo Alto, CA). Initial mobile phase composition was 80:20 (v/v) 10
mM ammonium acetate/methanol with a flow rate of 600 L/min. The
mobile phase program consisted of 0-0.5 min 80:20, 0.5-3 min ramp
gradient to 10:90, 3.0-8.0 min hold 10:90, 8.0-8.1 min ramp gradient
to 5:95, 8.1-14.0 min hold 5:95, 14.0-14.1 min ramp gradient back
to 80:20, 14.1-17.0 min hold 80:20. Injection volume was 10 L.
Sample residues were quantified using a linear standard curve method
(R (2) ) 0.98 or better for all compounds). See Figure 1 for the basic
sample flowchart used for analysis.
RESULTS AND DISCUSSION
6856
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
ACKNOWLEDGMENT
(20)
LITERATURE CITED
(21)
(22)
(23)
(24)
Received for review March 27, 2008. Revised manuscript received May
29, 2008. Accepted May 30, 2008.
JF8009624