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Analysis of Pesticides in a Hemp Matrix

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

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