Introduction
Hemp is a strain of Cannabis sativa that has multiple industrial uses including paper, plastics, woven goods, and even food. While certain strains of Cannabis sativa are well-known for their use as a recreational drug due the presence of the psychoactive compound tetrahydrocannabinol (THC), hemp strains are defined by the U.S. federal government as those that contain less than 0.3% THC.1 Additionally, hemp strains typically contain more cannabidiol (CBD),2 which was recently approved by the FDA to treat certain types of epilepsy, and is currently being investigated as a medical treatment for other afflictions. Due to the regulation of THC in Cannabis sativa as a Schedule 1 drug,3 hemp has often been difficult to deal with from a regulatory standpoint. However, with the advent of federal guidelines emerging on the definition of hemp, there is a need for reliable analytical tools to meet the regulatory requirements for pesticide testing in Cannabis. Action limits for each pesticide vary between jurisdictions, but can be as low as 10 ppb.4,5
The JEOL JMS-TQ4000GC triple-quadrupole GC-MS/MS system offers high speed and high sensitivity for quantitation of trace or residual pesticides. The TQ4000 combines a unique short collision cell with JEOL’s patented ion accumulation and timed detection technology to provide high sensitivity and selectivity, as well as the fastest selected reaction monitoring (SRM) switching speed available (up to 1000 transitions per second). JEOL msPrimo and Escrime software provide all of the tools needed to develop optimized methods for target compound quantitation and report generation. In this application note, we describe a sensitive method for analyzing pesticides in a hemp strain of Cannabis sativa matrix using the SRM capabilities of our triple quadrupole system.
Experimental
Approximately 1 gram of dried hemp flower buds (provided by Think 20 Labs, Inc.) was extracted into 10 mL of acetonitrile by sonication for 15 minutes. The extract was centrifuged at approximately 2500 rpm for 10 minutes, followed by 10X dilution. One mL of the diluted extract was put through a dSPE cleanup step using Restek Q-sep QuEChERS dSPE Tubes (AOAC 2007.01 method6, PN# 26125) and following the provided dSPE instructions. The supernatant was used as the matrix for each sample. Each spiked sample was created by adding 10 µL of prepared pesticide standard to 90 µL of the matrix. Samples were analyzed on the JMS-TQ4000GC using the parameters and SRM channels outlined in Tables 1 - 3 below. Optimal product- and precursor-ion pairs and optimized collision energies for each pesticide were determined using built-in SRM optimization tools. Each sample was run in triplicate with the exception of the 1 ppb samples for which 8 replicates were done to calculate the instrument detection limit (IDL) where possible.
Results
Figure 1 shows the total ion current chromatogram (TICC) with labeled peaks, and Figure 2 shows several selected SRM chromatograms. Table 4 lists the data acquired for 46 pesticides analyzed by GC-MS/MS analysis. There were 35 pesticides observed at 1 ppb or less, which translates to 10 ppb on the plant. The IDL and %CV were not calculated for samples that could not be observed at 1 ppb. For samples with isomers (e.g., chlordane), the best performing isomer was used for reporting. All samples below showed good linearity, even up to 100 ppb. Example calibration curves are shown in Figure 3. Although some matrix effects were observed, system performance was generally good with very few pesticides affected by matrix interference.
Conclusions
The JMS-TQ4000GC is an excellent platform for fast, sensitive analysis of a wide range of pesticides in hemp matrix. Using built-in SRM optimization tools, optimal ion transitions and collision energies for each pesticide were determined in the presence of the matrix. The SRM method provided high sensitivity and selectivity, and reduced matrix effects without a complicated extraction method. Thirty-five pesticides were observed at one ppb or lower with good linearity, which translates to ten ppb on the flower and is sufficient to meet the action limits of jurisdictions of interest.
References
(1) United States Deprartment of Agriculture. Establishment of a Domestic Hemp Production Program; United States of America, 2019.
(2) Swanson, T. E. Controlled Substances Chaos: The Department of Justice’s New Policy Position on Marijuana and What It Means for Industrial Hemp Farming in North Dakota. North Dekota Law Rev. 2014, 90 (3), 599–622.
(3) United States Drug Enforcement Administration. The Controlled Substances Act https://www.dea.gov/controlled-substances-act (accessed Mar 19, 2020).
(4) Health Canada. Mandatory cannabis testing for pesticide active ingredients - List and limits https://www.canada.ca/en/public-health/services/publications/drugs-health-products/cannabis-testing-pesticide-list-limits.html (accessed Mar 19, 2020).
(5) Dodson, L.; Laprade, N. M. The Natalie M. Laprade Maryland Medical Cannabis Commission’s (MMCC) Technical Authority for Medical Cannabis Testing; 2019.
(6) Official Methods of Analysis. Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. Association of Official Agricultural Chemists: 2007.01.
Table 1: Gas Chromatograph Parameters
|
GC
|
7890B (Agilent)
|
|
Column
|
ZB-5MSPlus, 30.0 m, 0.25 mm i.d., 0.25 µm
(Phenomenex, Cat#:7HG-G030-11)
|
|
Inlet liner
|
Zebron Plus 4 mm Single Taper w/Wool on bottom
(Phenomenex Cat#: AG2-0A11-05)
|
|
Inlet Temp.
|
260 °C
|
|
Carrier Gas Type, Flow
|
He, 1.000 mL/min constant flow
|
|
Mode
|
Pulsed Splitless
|
|
Pulsed Pressure, Time
|
206.84 kPa, 0.550 min
|
|
Purge Flow
|
30 mL/min, 1.0 min
|
|
Septum Purge Flow
|
3.0 mL/min
|
|
Saver flow, Time
|
15 mL/min, 5.0 min
|
|
Injection Volume
|
1.0 µL
|
|
Oven Program
|
80 °C (0.75 min) → 35 °C/min → 190 °C → 5 °C/min → 240 °C → 20 °C/min → 300 °C (6 min)
|
Table 2: Mass spectrometer parameters
|
JMS-TQ4000GC
|
|
Ion Source Temp.
|
250 °C
|
|
Interface Temp.
|
300 °C
|
|
Ionization Mode
|
EI+, 70 eV, 100 µA
|
|
Measurement Mode
|
SRM, High Sensitivity
|
|
Target Cycle Time
|
Approx. 330 ms
|
|
Acquisition Rate
|
2.778 Hz
|
|
Channel Time
|
20 – 100 ms
|
|
Relative EM Voltage
|
900 V
|
|
Collision Gas
|
N2, 10%
|
Table 3: SRM channel data
|
Compound
|
Quantitative ion
|
Referenced ion 1
|
Referenced ion 2
|
Collision Energy
|
|
Precursor
|
Product ion
|
Precursor
|
Product ion
|
Precursor
|
Product ion
|
Quantitative ion
|
Referenced ion 1
|
Referenced ion 2
|
|
ion m/z
|
m/z
|
ion m/z
|
m/z
|
ion m/z
|
m/z
|
|
Acephate
|
136
|
94
|
136
|
42
|
77
|
51
|
10
|
15
|
15
|
|
Azoxystrobin
|
344
|
156
|
388
|
345
|
388
|
360
|
30
|
20
|
10
|
|
Bifenazate
|
258
|
196
|
258
|
199
|
300
|
196
|
15
|
10
|
25
|
|
Bifenthrin
|
181
|
165
|
181
|
166
|
181
|
164
|
30
|
20
|
30
|
|
Boscalid
|
140
|
112
|
140
|
76
|
342
|
140
|
10
|
25
|
20
|
|
Carbaril (decomp)
|
144
|
115
|
144
|
116
|
89
|
63
|
25
|
15
|
15
|
|
Carbaril (intact)
|
144
|
115
|
144
|
116
|
89
|
63
|
25
|
15
|
15
|
|
Carbofuran
|
164
|
149
|
164
|
103
|
149
|
103
|
15
|
25
|
20
|
|
Chlordane (cis)
|
375
|
266
|
373
|
266
|
373
|
264
|
20
|
25
|
25
|
|
Chlordane (trans)
|
373
|
266
|
373
|
264
|
375
|
266
|
25
|
20
|
20
|
|
Chlorfenapyr
|
59
|
31
|
247
|
227
|
59
|
41
|
5
|
15
|
5
|
|
Chlorpyrifos
|
197
|
169
|
199
|
171
|
197
|
134
|
15
|
15
|
25
|
|
Chlorpyrifos-d10
|
200
|
172
|
260
|
167
|
260
|
139
|
20
|
25
|
30
|
|
Cinerin I
|
150
|
108
|
123
|
79
|
123
|
81
|
10
|
20
|
10
|
|
Cinerin II
|
107
|
91
|
121
|
93
|
121
|
77
|
10
|
5
|
25
|
|
Clofentezine
|
137
|
102
|
137
|
75
|
139
|
102
|
10
|
25
|
15
|
|
Cyfluthrin I
|
226
|
206
|
206
|
151
|
206
|
150
|
15
|
25
|
25
|
|
Cyfluthrin II
|
226
|
206
|
163
|
127
|
163
|
91
|
20
|
10
|
15
|
|
Cyfluthrin III
|
226
|
206
|
163
|
127
|
163
|
91
|
20
|
10
|
15
|
|
Cyfluthrin IV
|
226
|
206
|
163
|
127
|
163
|
91
|
15
|
10
|
15
|
|
Cypermethrin I
|
163
|
127
|
181
|
152
|
163
|
91
|
10
|
25
|
20
|
|
Cypermethrin II
|
163
|
127
|
181
|
152
|
163
|
91
|
10
|
25
|
15
|
|
Cypermethrin III
|
163
|
127
|
181
|
152
|
163
|
91
|
10
|
25
|
15
|
|
Cypermethrin IV
|
163
|
127
|
181
|
152
|
163
|
91
|
10
|
25
|
15
|
|
Diazinone
|
137
|
84
|
199
|
135
|
199
|
93
|
15
|
10
|
15
|
|
Dichlorvos
|
109
|
79
|
185
|
93
|
79
|
47
|
10
|
15
|
10
|
|
Dimethoate
|
93
|
63
|
87
|
42
|
87
|
46
|
10
|
10
|
20
|
|
Ethoprophos
|
158
|
97
|
158
|
114
|
97
|
79
|
15
|
10
|
20
|
|
Etofenprox
|
163
|
107
|
163
|
135
|
135
|
107
|
20
|
10
|
10
|
|
Etoxazole
|
141
|
113
|
300
|
270
|
204
|
176
|
15
|
30
|
10
|
|
Fenoxycarb
|
116
|
88
|
186
|
157
|
186
|
158
|
10
|
15
|
10
|
|
Fipronil
|
213
|
143
|
367
|
213
|
213
|
178
|
25
|
30
|
20
|
|
Compound
|
Quantitative ion
|
Referenced ion 1
|
Referenced ion 2
|
Collision Energy
|
|
Precursor
|
Product ion
|
Precursor
|
Product ion
|
Precursor
|
Product ion
|
Quantitative ion
|
Referenced ion 1
|
Referenced ion 2
|
|
ion m/z
|
m/z
|
ion m/z
|
m/z
|
ion m/z
|
m/z
|
|
Fludioxonil
|
248
|
127
|
248
|
154
|
248
|
182
|
30
|
25
|
20
|
|
Imazalil
|
173
|
145
|
215
|
173
|
173
|
109
|
20
|
10
|
25
|
|
Jasmolin I
|
164
|
109
|
123
|
79
|
123
|
81
|
10
|
20
|
10
|
|
Jasmolin II
|
121
|
93
|
121
|
77
|
121
|
91
|
10
|
20
|
20
|
|
Kresoxim-methyl
|
116
|
89
|
206
|
116
|
206
|
131
|
20
|
10
|
10
|
|
Malathion
|
127
|
99
|
93
|
63
|
125
|
79
|
10
|
10
|
15
|
|
Metalaxyl
|
206
|
132
|
132
|
117
|
206
|
105
|
20
|
15
|
20
|
|
Methiocarb
|
168
|
153
|
168
|
109
|
153
|
109
|
10
|
15
|
10
|
|
Methomyl
|
105
|
88
|
58
|
31
|
105
|
58
|
5
|
5
|
10
|
|
Methyl parathion
|
263
|
109
|
125
|
79
|
125
|
47
|
15
|
10
|
15
|
|
MGK 264 I
|
164
|
93
|
164
|
121
|
164
|
77
|
15
|
10
|
30
|
|
MGK 264 II
|
164
|
67
|
164
|
80
|
164
|
98
|
10
|
25
|
15
|
|
Myclobutanil
|
179
|
125
|
150
|
123
|
179
|
90
|
20
|
20
|
30
|
|
Naled
|
145
|
109
|
185
|
93
|
145
|
113
|
10
|
15
|
20
|
|
Oxamyl
|
98
|
58
|
98
|
69
|
72
|
56
|
10
|
5
|
10
|
|
Paclobutrazol
|
236
|
125
|
125
|
89
|
236
|
132
|
20
|
25
|
20
|
|
Permethrin (cis)
|
183
|
153
|
183
|
168
|
183
|
165
|
20
|
20
|
20
|
|
Permethrin (trans)
|
183
|
153
|
183
|
168
|
163
|
91
|
20
|
20
|
15
|
|
Phosmet
|
160
|
133
|
160
|
105
|
160
|
77
|
15
|
20
|
20
|
|
Piperonyl butoxide
|
176
|
117
|
176
|
103
|
176
|
131
|
20
|
25
|
15
|
|
Prallethrin
|
123
|
81
|
123
|
79
|
105
|
77
|
10
|
20
|
15
|
|
Propiconazole I
|
173
|
109
|
173
|
145
|
259
|
191
|
25
|
15
|
10
|
|
Propiconazole II
|
173
|
109
|
173
|
145
|
259
|
191
|
25
|
15
|
10
|
|
Propoxur
|
110
|
63
|
152
|
110
|
110
|
64
|
25
|
10
|
20
|
|
Pyrethrin II
|
133
|
105
|
91
|
65
|
107
|
91
|
10
|
15
|
10
|
|
Pyridaben
|
147
|
117
|
147
|
105
|
147
|
132
|
20
|
10
|
15
|
|
Spiromesifen
|
272
|
254
|
272
|
209
|
272
|
226
|
5
|
15
|
10
|
|
Spiroxamine I
|
100
|
72
|
100
|
58
|
100
|
41
|
10
|
10
|
20
|
|
Spiroxamine II
|
100
|
72
|
100
|
58
|
100
|
41
|
10
|
10
|
20
|
|
Tebuconazole
|
250
|
125
|
125
|
89
|
125
|
99
|
25
|
20
|
20
|
|
Thiamethoxam
|
132
|
71
|
212
|
139
|
212
|
182
|
10
|
15
|
5
|
|
Trifloxystrobin
|
116
|
89
|
172
|
145
|
131
|
89
|
20
|
20
|
25
|
Figure 1: TIC chromatogram
Figure 2: Selected SRM chromatograms
Figure 3: Selected calibrations curves
Table 4: Performance data for tested pesticides
|
Compound
|
Range
(ppb)
|
Linearity
(R2)
|
% CV
|
LOQ
(ppb)
|
IDL (ppb)
|
|
Azoxystrobin
|
2.5 - 100
|
0.9886
|
N/A
|
2.5
|
N/A
|
|
Bifenazate
|
1- 100
|
0.9943
|
17.23
|
2.5
|
0.52
|
|
Bifenthrin
|
0.25 - 100
|
0.9928
|
3.36
|
0.5
|
0.10
|
|
Boscalid
|
0.25 - 100
|
0.9926
|
8.65
|
0.5
|
0.26
|
|
Carbaril
|
2.5 - 100
|
0.9893
|
N/A
|
25
|
N/A
|
|
Carbofuran
|
1- 100
|
0.9902
|
12.52
|
2.5
|
0.38
|
|
Chlordane
|
1- 100
|
0.9975
|
11.73
|
1
|
0.35
|
|
Chlorfenapyr
|
1- 100
|
0.9959
|
11.85
|
1
|
0.36
|
|
Chlorpyrifos
|
0.25 - 100
|
0.9944
|
4.12
|
0.5
|
0.12
|
|
Chlorpyrifos-d10
|
2.5 - 100
|
0.9944
|
N/A
|
2.5
|
N/A
|
|
Cinerin I
|
25 - 100
|
0.9749
|
N/A
|
25
|
N/A
|
|
Clofentezine
|
0.5 - 100
|
0.9974
|
5.20
|
1
|
0.16
|
|
Cyfluthrin
|
0.5 - 100
|
0.9808
|
5.87
|
5
|
0.18
|
|
Cypermethrin
|
1- 100
|
0.9933
|
9.93
|
10
|
0.30
|
|
Diazinone
|
0.25 - 100
|
0.9954
|
6.98
|
0.5
|
0.21
|
|
Dichlorvos
|
0.25 - 100
|
0.9964
|
7.40
|
0.5
|
0.22
|
|
Dimethoate
|
1- 100
|
0.9920
|
13.25
|
2.5
|
0.40
|
|
Compound
|
Range
(ppb)
|
Linearity
(R2)
|
% CV
|
LOQ
(ppb)
|
IDL (ppb)
|
|
Ethoprophos
|
0.25 - 100
|
0.9947
|
6.38
|
2.5
|
0.19
|
|
Etofenprox
|
0.25 - 100
|
0.9961
|
6.60
|
2.5
|
0.20
|
|
Etoxazole
|
2.5 - 100
|
0.9947
|
N/A
|
2.5
|
N/A
|
|
Fenoxycarb
|
0.5 - 100
|
0.9947
|
5.77
|
10
|
0.17
|
|
Fipronil
|
0.5 - 100
|
0.9966
|
10.11
|
1
|
0.30
|
|
Fludioxonil
|
0.5 - 100
|
0.9935
|
9.75
|
0.5
|
0.29
|
|
Jasmolin I
|
5 -100
|
0.9904
|
N/A
|
25
|
N/A
|
|
Kresoxim-methyl
|
0.5 - 100
|
0.9958
|
9.13
|
0.5
|
0.27
|
|
Malathion
|
0.5 - 100
|
0.9909
|
11.89
|
5
|
0.36
|
|
Metalaxyl
|
0.5 - 100
|
0.9978
|
6.39
|
2.5
|
0.19
|
|
Methiocarb
|
1- 100
|
0.9925
|
15.18
|
2.5
|
0.46
|
|
Methomyl
|
2.5 - 100
|
0.9895
|
N/A
|
50
|
N/A
|
|
Methyl parathion
|
1- 100
|
0.9960
|
12.10
|
5
|
0.36
|
|
MGK 264
|
0.5 - 100
|
0.9983
|
7.06
|
0.5
|
0.21
|
|
Myclobutanil
|
0.25 - 100
|
0.9954
|
11.00
|
0.5
|
0.33
|
|
Naled
|
10 - 100
|
0.9899
|
N/A
|
25
|
N/A
|
|
Paclobutrazol
|
0.25 - 100
|
0.9942
|
9.09
|
0.5
|
0.27
|
|
Permethrin
|
1- 100
|
0.9957
|
16.45
|
2.5
|
0.49
|
|
Phosmet
|
0.5 - 100
|
0.9914
|
13.01
|
1
|
0.39
|
|
Prallethrin
|
5 -100
|
0.9929
|
N/A
|
25
|
N/A
|
|
Propiconazole
|
0.25 - 100
|
0.9948
|
12.41
|
1
|
0.39
|
|
Propoxur
|
0.25 - 100
|
0.9893
|
7.76
|
0.5
|
0.23
|
|
Pyridaben
|
0.5 - 100
|
0.9922
|
5.22
|
0.5
|
0.16
|
|
Spiromesifen
|
0.5 - 100
|
0.9857
|
8.88
|
1
|
0.27
|
|
Spiroxamine
|
0.25 - 100
|
0.9955
|
5.03
|
1
|
0.15
|
|
Tebuconazole
|
0.25 - 100
|
0.9951
|
9.67
|
1
|
0.29
|
|
Thiamethoxam
|
5 -100
|
0.9927
|
N/A
|
5
|
N/A
|
|
Trifloxystrobin
|
0.5 - 100
|
0.9952
|
9.32
|
1
|
0.28
|