LCMS-8040 Application Pesticides in Cannabis

Je Dahl,1 Julie Kowalski,2 Jason Zitzer,3 and Gordon Fagras3 1Shimadzu Scientic Instruments; Columbia, Maryland 2Restek Corporation; Bellefonte, Pennsylvania 3Trace Analytics; Spokane, Washington Pesticides in Cannabis

Background: Medicinal and recreational use of After hydration of the samples, 15 mL acetonitrile cannabis has increased rapidly in recent years. with 1% acetic acid was added to each followed Like other crops, cannabis is susceptible to by shaking for 30 minutes. To each sample was insects, mold, and chemical residue contamina- added the contents of an AOAC QuEChERS tion. Pesticides and antifungals have been applied Packet (Restek part 26237) and the samples were to cannabis to increase yields however these vigorously mixed for 2 minutes and centrifuged. substances may cause human harm if they are consumed by users. Sensitive and selective Dispersive SPE was used to clean up the sample detection of these residues is necessary for con- extracts for LCMS analysis. Several formulations sumer protection. QuEChERS extraction and of cleanup reagents were tested for optimum LC-MS analysis offers effective and efficient combination of matrix removal and recovery. The detection of such chemical residues. best formulation was a combination of PSA, C18,

Method: Cannabis samples were provided by licensed growers in Spokane, Washington, and samples were prepared and analyzed in a certified lab in that state. Pesticide-free organically-grown cannabis was used for spiking studies and calibration curves. A variety of cannabis samples offered for retail sale were analyzed for pesticides as well.

Cannabis dried flower samples were homogenized by an automated grinder and weighed portions of 1.5 grams were used for each analysis. Each ground sample was hydrated using 15 mL of 1% acetic acid in water and agitated for 30 minutes. Figure 1: Moldy cannabis flower. The white As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and powder is mold growth which can flourish in recovery less than 50% or greater than 120%. results are shown in the table. The most com- high humidity growing operations. One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples. Figure 2: Representative dried cannabis flower samples (anonymized) tested for pesticides.

Page 2

Electrospray ionization in continuous polarity with good peak shape and retention time repro- switching mode was used for detection. Optimized ducibility. Individual chromatograms from selected MRM settings were used for each compound and pesticides are shown in the figure. The calibration at least one quantifier and one qualifier transition curve was prepared in spiked matrix over the was selected. The retention times were found and range of 3.91 to 500 ppb, as dried flower weight. used to program the MRM segments for optimum Individual calibration curves for selected com- duty cycle. pounds are shown as well.

Cannabis dried flower samples were homogenized by an automated grinder and weighed portions of 1.5 grams were used for each analysis. Each ground sample was hydrated using 15 mL of 1% acetic acid in water and agitated for 30 minutes. As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and recovery less than 50% or greater than 120%. results are shown in the table. The most com- One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples.

Pesticides in Cannabis

Hydrate 15 mL 15 mL 1% Add AOAC 1% acetic acid acetic acid in QuEChERS vortex/shake ACN, vortex & salts, vortex & 30 min shake 30 min shake 2 min

Grind 1.5 g test portion

Spin 2 min 3000 RCF, clean up 1 mL supernatant with dSPE Figure 3: QuEChERS extraction of dried cannabis flower for pesticide analysis.

(x100,000) 1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6 Electrospray ionization in continuous polarity with good peak shape and retention time repro- switching mode was used for detection. Optimized ducibility. Individual chromatograms from selected 0.5 MRM settings were used for each compound and pesticides are shown in the figure. The calibration 0.4 at least one quantifier and one qualifier transition curve was prepared in spiked matrix over the

0.3 was selected. The retention times were found and range of 3.91 to 500 ppb, as dried flower weight. used to program the MRM segments for optimum Individual calibration curves for selected com- 0.2 duty cycle. pounds are shown as well. 0.1

0.0 Results and Discussion: A representative chro- The total recovery for each compound, as mea- 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 min matogram of a prepared, spiked cannabis sample sured by comparing the peak area for a 50 ppb 150715.P200x28_75_J17L5-50_075.lcd is shown in the figure. The total run time was 15 spiked sample with a spiked process blank, which Figure 4: Representative chromatogram of pesticide mix spiked in cannabis matrix at an min. and chromatographic peaks were observed represents 100% recovery and no matrix effects. intermediate level (31 ppb).

Page 3 Pesticides in Cannabis

Summary: An LC-MS method for detection of fortification standards as appropriate. A multi- (x1,000) Methamidophos (x1,000) Thiabendazole (x10,000) Diethofencarb 142.00>94.00(+) 202.00>175.00(+) 268.10>226.10(+) pesticides in cannabis with QuEChERS extraction residue pesticide mix was used (Restek part 142.00>125.10(+) 202.00>131.05(+) 268.10>124.00(+) 7.5 4.0 was developed. number 31971). 7.5 3.0 5.0 5.0 Background: Medicinal and recreational use of After hydration of the samples, 15 mL acetonitrile 2.0 2.5 2.5 cannabis has increased rapidly in recent years. with 1% acetic acid was added to each followed 1.0

Like other crops, cannabis is susceptible to by shaking for 30 minutes. To each sample was 0.0 0.0 0.0 insects, mold, and chemical residue contamina- added the contents of an AOAC QuEChERS 1.1 1.2 1.3 1.4 1.5 5.4 5.5 5.6 5.7 5.8 7.2 7.3 7.4 7.5 7.6 tion. Pesticides and antifungals have been applied Packet (Restek part 26237) and the samples were (x10,000) Cycluron (x10,000) Promecarb (x10,000) Flufenacet 1.25 199.10>89.10(+) 2.0 208.10>109.00(+) 364.10>152.00(+) to cannabis to increase yields however these vigorously mixed for 2 minutes and centrifuged. 199.10>46.00(+) 208.10>151.00(+) 3.0 364.10>194.05(+) 1.00 substances may cause human harm if they are 1.5 0.75 2.0 consumed by users. Sensitive and selective Dispersive SPE was used to clean up the sample 1.0 detection of these residues is necessary for con- extracts for LCMS analysis. Several formulations 0.50 1.0 0.5 sumer protection. QuEChERS extraction and of cleanup reagents were tested for optimum 0.25

LC-MS analysis offers effective and efficient combination of matrix removal and recovery. The 0.00 0.0 0.0 detection of such chemical residues. best formulation was a combination of PSA, C18, 7.3 7.4 7.5 7.6 7.7 7.5 7.6 7.7 7.8 7.9 7.9 8.0 8.1 8.2

(x10,000) (x1,000) (x1,000) 2.5 Fluoxastrobin Kresoxym-methyl Zoxamide 459.20>427.00(+) 7.5 314.00>267.10(+) 336.10>187.00(+) Method: Cannabis samples were provided by 459.20>188.10(+) 314.00>116.00(+) 7.5 336.10>159.00(+) licensed growers in Spokane, Washington, and 2.0 samples were prepared and analyzed in a certified 1.5 5.0 5.0 1.0 lab in that state. Pesticide-free organically-grown 2.5 2.5 cannabis was used for spiking studies and calibra- 0.5 tion curves. A variety of cannabis samples offered 0.0 0.0 0.0 for retail sale were analyzed for pesticides as well. 8.2 8.3 8.4 8.5 8.3 8.4 8.5 8.6 8.7 8.6 8.7 8.8 8.9 9.0

(x10,000) Pyridaben (x1,000) Myclobutanil (x10,000) Piperonyl Butoxide 2.5 365.15>147.00(+) 4.0 289.20>70.10(+) 356.20>177.00(+) Cannabis dried flower samples were homogenized 365.15>309.05(+) 289.20>125.05(+) 356.20>149.00(+) 2.0 7.5 356.20>119.10(+) by an automated grinder and weighed portions of 3.0 1.5 1.5 grams were used for each analysis. Each 5.0 2.0 ground sample was hydrated using 15 mL of 1% 1.0 1.0 2.5 acetic acid in water and agitated for 30 minutes. 0.5 As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and 0.0 0.0 0.0 recovery less than 50% or greater than 120%. results are shown in the table. The most com- 10.9 11.0 11.1 11.2 11.3 7.9 8.0 8.1 8.2 8.3 10.3 10.4 10.5 10.6 10.7 150715.P200x28_48_J17L5-U_048 One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- Figure 5: Individual representative chromatograms for selected pesticides. effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples.

Page 4

Cannabis dried flower samples were homogenized by an automated grinder and weighed portions of 1.5 grams were used for each analysis. Each ground sample was hydrated using 15 mL of 1% acetic acid in water and agitated for 30 minutes. As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and recovery less than 50% or greater than 120%. results are shown in the table. The most com- One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples.

Pesticides in Cannabis

Area Ratio(x10) Area Ratio(x10) Area Ratio(x10)

2.0 Methamidophos Thiabendazole Diethofencarb r2 > 0.997 1.0 r2 > 0.999 5.0 r2 > 0.998

1.0 0.5 2.5

0.0 0.0 0.0 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio

Area Ratio(x10) Area Ratio(x10) Area Ratio(x10) 2.0 Cycluron 3.0 Promecarb Flufenacet r2 > 0.997 r2 > 0.999 r2 > 0.996 2.0 5.0 1.0 1.0 2.5

0.0 0.0 0.0 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio

Area Ratio(x10) Area Ratio(x10) Area Ratio(x10) 1.5 Fluoxastrobin Kresoxym-methyl 1.5 Zoxamide 2 2 2 r > 0.995 1.0 r > 0.997 r > 0.998 2.5 1.0

0.5 0.5

0.0 0.0 0.0 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio

Area Ratio(x10) Area Ratio Area Ratio(x100) 1.5 2.0 Pyridaben 5.0 Myclobutanil Piperonyl Butoxide 2 2 2 r > 0.993 r > 0.997 1.0 r > 0.998

1.0 2.5 0.5

0.0 0.0 0.0 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio 0 10 20 30 Conc. Ratio 150715.P200x28.lcb Figure 6: Individual representative calibration curves for selected pesticides.

Page 5 Pesticides in Cannabis

Summary: An LC-MS method for detection of fortification standards as appropriate. A multi- 3-hydroxycarbofuran 19.5 Fenhexamid 126 Nitenpyram 19.6 pesticides in cannabis with QuEChERS extraction residue pesticide mix was used (Restek part Acephate 4.6 Fenobucarb 8.3 Novaluron 4.9 was developed. number 31971). Acetamiprid 4.1 Fenoxycarb 10.6 Omethoate 4.5 Aldicarb 4.1 Fenpyroximate 1.3 Oxadixyl 5.4 Aldicarb Sulfoxide 17.8 Fenuron 4.3 Oxamyl 2.1 Background: Medicinal and recreational use of After hydration of the samples, 15 mL acetonitrile Aldoxycarb 4.3 Fipronil 9.7 Paclobutrazol 11.8 cannabis has increased rapidly in recent years. with 1% acetic acid was added to each followed Aminocarb 2.1 Flonicamid 25.8 Penconazole 27.4 Like other crops, cannabis is susceptible to by shaking for 30 minutes. To each sample was Azoxystrobin 4.5 Fluazinam 7.4 Picoxystrobin 1.1 insects, mold, and chemical residue contamina- added the contents of an AOAC QuEChERS Benalaxyl 2.6 Fludioxonil 38.5 Piperonyl Butoxide 1.2 tion. Pesticides and antifungals have been applied Packet (Restek part 26237) and the samples were Bendiocarb 4.3 Flufenacet 5.0 Pirimicarb 2.1 Bifenazate 2.2 Fluometuron 8.6 Prochloraz 12.9 to cannabis to increase yields however these vigorously mixed for 2 minutes and centrifuged. Bitertanol 13.4 Fluoxastrobin 5.1 Promecarb 9.9 substances may cause human harm if they are Boscalid 52.8 Fluquinconazole 119 Prometon 19.1 consumed by users. Sensitive and selective Dispersive SPE was used to clean up the sample Bromuconazole Isomer 1 67.4 Flusilazole 62.0 Propamocarb 9.5 detection of these residues is necessary for con- extracts for LCMS analysis. Several formulations Bromuconazole Isomer 2 67.4 Flutolanil 5.0 Propargite 2.3 sumer protection. QuEChERS extraction and of cleanup reagents were tested for optimum Bupirimate 199 Flutraifol 20.5 Propiconazole 70.6 Butafenacil 2.5 Formetanate 2.4 Propoxur 2.1 LC-MS analysis offers effective and efficient combination of matrix removal and recovery. The Carbaryl 19.5 Fuberdiazole 5.0 Pymetrozine 6.7 detection of such chemical residues. best formulation was a combination of PSA, C18, Carbendazim 5.7 Furalaxyl 2.3 Pyracarbolid 2.3 Carbetamide 2.2 Furathiocarb 1.2 Pyraclostrobin 10.2 Method: Cannabis samples were provided by Carbofuran 2.3 Hexaconazole 21.5 Pyridaben 11.5 Carboxin 4.7 Hexaflumuron 3.0 Pyriproxyfen 1.3 licensed growers in Spokane, Washington, and Carfentrazone-ethyl 45.3 Hexythiazox 11.2 Quinoxyfen 3.1 samples were prepared and analyzed in a certified Chloantraniliprole 16.8 Imazalil 180 Rotenone 13.3 lab in that state. Pesticide-free organically-grown Chlorotoluron 4.8 Imidacloprid 33.7 Siduron 20.5 cannabis was used for spiking studies and calibra- Chloroxuron 2.4 Indoxacarb 36.9 Spinetoram 6.1 tion curves. A variety of cannabis samples offered Clethodim Isomer 1 50.2 Ipconazole 23.2 Spinosad A 7.4 Clofentazine 51.3 Iprovalicarb 18.8 Spinosad D 12.3 for retail sale were analyzed for pesticides as well. Clothianidin 9.2 Isoprocarb 4.6 Spirotetramat 10.9 Cyazofamid 4.5 Isoproturon 2.6 Sulfentrazone 8.2 Cannabis dried flower samples were homogenized Cycluron 11.4 Kresoxym-methyl 10.9 Tebuconazole 5.5 by an automated grinder and weighed portions of Cyproconazole 62.5 Linuron 33.9 Tebufenozide 5.2 Cyromazine 90 Mandipropamid 7.7 Tebufenpyrad 62.5 1.5 grams were used for each analysis. Each Dicrotophos 9.1 Mefenacet 2.7 Tebuthiuron 1.1 ground sample was hydrated using 15 mL of 1% Diethofencarb 2.4 Mepanipyrim 92.6 Temephos 24.5 acetic acid in water and agitated for 30 minutes. Difenoconazole 62.5 Mepronil 31.6 Tetraconazole 22.5 As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and Diflubenzuron 38.8 Metaflumizone 2.2 Thiacloprid 2.1 recovery less than 50% or greater than 120%. results are shown in the table. The most com- Dimethoate 4.4 Metalaxyl 4.1 Thiamethoxam 4.3 Dimethomorph 50.1 Metconazole 43.1 Thidiazuron 25.1 One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, Dimoxystrobin 2.3 Methabenzthiazuron 2.5 Thiobencarb 5.1 showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- Diniconazole 41.3 Methamidophos 4.9 Thiophanate-methyl 4.9 effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- Dinotefuran 4.4 Methiocarb 9.6 Triadimefon 33.6 variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a Diuron 4.5 Methomyl 4.4 Triadimenol 8.9 considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to Emamectin B1a 1.6 Methoprotryne 21.2 Trichlorfon 7.2 MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) Epoxiconazole 40.1 Methoxyfenozide 5.0 Tricyclazole 9.1 detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- Eprinomectin 5.6 Metobromuron 15.5 Trifloxystrobin 2.5 number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Ethiofencarb 9.1 Metribuzin 15.4 Triflumizole 2.8 Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, Ethiprole 33.9 Mevinphos 18.2 Triflumuron 52.7 ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total Etoxazole 1.3 Mexacarbate 4.2 Triticonazole 89.0 Fenamidone 10.1 Monocrotophos 181 Vamidothion 1.1 to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to Fenarimol 125 Monolinuron 4.2 Zoxamide 10.3 better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of Fenazaquin 1.4 Myclobutanil 9.0 The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling Fenbuconazole 12.4 Neburon 150 quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- Table 1: LCMS-8040 Compound list and estimated quantitation limits (ppb) on the basis of dried sale and presented to us for analysis were anony- cially available cannabis samples. flower weight

Page 6

Cannabis dried flower samples were homogenized by an automated grinder and weighed portions of 1.5 grams were used for each analysis. Each ground sample was hydrated using 15 mL of 1% acetic acid in water and agitated for 30 minutes. As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and recovery less than 50% or greater than 120%. results are shown in the table. The most com- One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples.

Pesticides in Cannabis

3-hydroxycarbofuran 0.74 Fenobucarb 0.85 Neburon 0.41 Acephate 0.94 Fenoxycarb 0.75 Nitenpyram 0.88 Acetamiprid 0.87 Fenpyroximate 1.16 Novaluron 1.43 Aldicarb 0.83 Fenuron 0.74 Omethoate 0.96 Aldicarb Sulfoxide 1.02 Fipronil 0.93 Oxadixyl 0.67 Aldoxycarb 0.98 Flonicamid 0.91 Oxamyl 0.95 Aminocarb 0.86 Fluazinam 0.98 Paclobutrazol 0.67 Azoxystrobin 0.71 Fludioxonil 0.73 Penconazole 0.50 Benalaxyl 0.66 Flufenacet 0.70 Picoxystrobin 0.80 Bendiocarb 0.71 Fluometuron 0.65 Piperonyl Butoxide 0.82 Bifenazate 7.08 Fluoxastrobin 0.66 Pirimicarb 0.63 Bitertanol 1.00 Fluquinconazole 0.46 Prochloraz 0.57 Boscalid 0.52 Flusilazole 0.63 Promecarb 0.73 Bromuconazole Isomer 1 0.43 Flutolanil 0.64 Prometon 0.34 Bromuconazole Isomer 2 0.54 Flutraifol 0.80 Propamocarb 0.75 Bupirimate 0.27 Formetanate 0.78 Propargite 0.57 Butafenacil 0.80 Fuberdiazole 0.44 Propiconazole 1.00 Carbaryl 0.55 Furalaxyl 0.67 Propoxur 0.75 Carbendazim 0.27 Furathiocarb 0.84 Pymetrozine 0.75 Carbetamide 0.84 Hexaconazole 0.84 Pyracarbolid 0.68 Carbofuran 0.59 Hexaflumuron 0.81 Pyraclostrobin 0.66 Carboxin 0.68 Hexythiazox 0.43 Pyridaben 0.27 Carfentrazone-ethyl 0.82 Imazalil 0.27 Pyriproxyfen 0.74 Chloantraniliprole 1.01 Imidacloprid 1.19 Quinoxyfen 0.49 Chlorotoluron 0.67 Indoxacarb 0.79 Rotenone 0.71 Chloroxuron 0.73 Ipconazole 0.76 Siduron 0.65 Clethodim Isomer 1 0.42 Iprovalicarb 0.74 Spinetoram 0.59 Clofentazine 0.37 Isoprocarb 0.66 Spinosad A 0.47 Clothianidin 1.02 Isoproturon 0.59 Spinosad D 0.47 Cyazofamid 0.85 Kresoxym-methyl 0.64 Spirotetramat 0.81 Cycluron 0.52 Linuron 0.65 Sulfentrazone 4.74 Cyromazine 0.56 Mandipropamid 0.89 Tebuconazole 0.71 Dicrotophos 0.93 Mefenacet 0.59 Tebufenozide 0.72 Diethofencarb 0.94 Mepanipyrim 0.34 Tebuthiuron 0.71 Diflubenzuron 0.82 Mepronil 0.72 Temephos 0.84 Dimethoate 0.77 Metaflumizone 0.67 Tetraconazole 0.61 Dimethomorph 0.84 Metalaxyl 0.72 Thiacloprid 0.64 Dimoxystrobin 0.72 Metconazole 0.70 Thiamethoxam 1.08 Electrospray ionization in continuous polarity with good peak shape and retention time repro- Diniconazole 0.89 Methabenzthiazuron 0.65 Thidiazuron 0.43 switching mode was used for detection. Optimized ducibility. Individual chromatograms from selected Dinotefuran 1.03 Methamidophos 0.74 Thiobencarb 0.66 MRM settings were used for each compound and pesticides are shown in the figure. The calibration Diuron 0.64 Methiocarb 0.81 Thiophanate-methyl 0.68 at least one quantifier and one qualifier transition curve was prepared in spiked matrix over the Emamectin B1a 0.62 Methomyl 0.79 Triadimefon 0.79 was selected. The retention times were found and range of 3.91 to 500 ppb, as dried flower weight. Epoxiconazole 0.75 Methoprotryne 0.27 Triadimenol 0.89 used to program the MRM segments for optimum Individual calibration curves for selected com- Eprinomectin 1.30 Methoxyfenozide 0.81 Trichlorfon 1.64 duty cycle. pounds are shown as well. Ethiofencarb 0.70 Metobromuron 0.65 Tricyclazole 0.54 Ethiprole 1.20 Metribuzin 0.63 Trifloxystrobin 0.75 Etoxazole 0.73 Mevinphos 0.81 Triflumizole 0.61 Results and Discussion: A representative chro- The total recovery for each compound, as mea- Fenamidone 0.86 Mexacarbate 0.60 Triflumuron 0.62 matogram of a prepared, spiked cannabis sample sured by comparing the peak area for a 50 ppb Fenazaquin 0.54 Monocrotophos 0.67 Triticonazole 0.52 is shown in the figure. The total run time was 15 spiked sample with a spiked process blank, which Fenbuconazole 0.73 Monolinuron 0.62 Vamidothion 0.77 min. and chromatographic peaks were observed represents 100% recovery and no matrix effects. Fenhexamid 0.79 Myclobutanil 0.83 Zoxamide 0.71

Table 2: LCMS-8040 total recovery for each pesticide in dried flower matrix.

Page 7 Pesticides in Cannabis

Summary: An LC-MS method for detection of fortification standards as appropriate. A multi- pesticides in cannabis with QuEChERS extraction residue pesticide mix was used (Restek part Total Recovery was developed. number 31971). Total 25-50% 50-70% 70-120% >120% Background: Medicinal and recreational use of After hydration of the samples, 15 mL acetonitrile 151 18 49 81 3 cannabis has increased rapidly in recent years. with 1% acetic acid was added to each followed Like other crops, cannabis is susceptible to by shaking for 30 minutes. To each sample was insects, mold, and chemical residue contamina- added the contents of an AOAC QuEChERS Table 3: Number of compounds by total recovery. tion. Pesticides and antifungals have been applied Packet (Restek part 26237) and the samples were to cannabis to increase yields however these vigorously mixed for 2 minutes and centrifuged. substances may cause human harm if they are Sample Code Pesticides detected consumed by users. Sensitive and selective Dispersive SPE was used to clean up the sample A No pesticides detected detection of these residues is necessary for con- extracts for LCMS analysis. Several formulations B No pesticides detected sumer protection. QuEChERS extraction and of cleanup reagents were tested for optimum C No pesticides detected LC-MS analysis offers effective and efficient combination of matrix removal and recovery. The D No pesticides detected detection of such chemical residues. best formulation was a combination of PSA, C18, E No pesticides detected F No pesticides detected Method: Cannabis samples were provided by G 534 ppb Piperonyl butoxide licensed growers in Spokane, Washington, and samples were prepared and analyzed in a certified H 45 ppb Piperonyl butoxide lab in that state. Pesticide-free organically-grown I No pesticides detected cannabis was used for spiking studies and calibra- J 19 ppb Spinosad A, 21 ppb Spinosad D, 1.1 ppb Piperonyl butoxide tion curves. A variety of cannabis samples offered K 8.4 ppb Myclobutanil, 2.7 ppb Piperonyl butoxide for retail sale were analyzed for pesticides as well. L 21 ppb Myclobutanil

Cannabis dried flower samples were homogenized by an automated grinder and weighed portions of Table 4: Pesticides detected in commercially available cannabis samples presented for analysis. 1.5 grams were used for each analysis. Each ground sample was hydrated using 15 mL of 1% acetic acid in water and agitated for 30 minutes. As can be seen, only a few compounds have mized and measured for pesticide residues. The Samples were spiked with internal standards and recovery less than 50% or greater than 120%. results are shown in the table. The most com- One notable exception is bifenazate, which monly detected pesticide was piperonyl butoxide, showed a strong matrix enhancement effect. This and was detected over a wide range of concentra- effect was confirmed by spiking experiments in a tions. Piperonyl butoxide finds wide use in pesti- variety of cannabis specimens and should be cide formulations to enhance activity of the main and carbon in the ratio 50:50:7.5 in addition to pole mass spectrometer. The LC column was a considired whenever a signal for bifenazate is ingredient. Myclobutanil, an antifungal known to MgSO4 (Restek part 26243). The supernatants Restek Ultra-AQ C18 column (3 µm, 2×100 mm) detected. be used in cannabis cultivation, was detected in a were added to the dispersive SPE reagents, and a binary gradient was used of 5 mM ammo- number of samples as well. mixed vigorously, and centrifuged. The superna- nium formate with 0.1% formic acid (pump A) and Limits of quantitation were determined by measur- tants were removed and concentrated approxi- methanol (pump B). The flow rate was 0.5 mL/min, ing samples in triplicate at various levels. Signal- Conclusion: An effective method for detection of mately 5-fold by evaporation prior to analysis. the column temperature was 40 °C, and the total to-noise of at least ten to one and RSD of 20% or pesticides in cannabis is demonstrated using Each sample was centrifuged to remove particu- run time was 15 min. A divert valve was used to better were required at the limit of quantitation. QuEChERS sample extraction with dispersive lates before being placed in an autosampler vial. send the unretained and re-equilibration portion of The limits of quantitation are reported in the table. SPE cleanup and LC-MS analysis with a triple the gradient to waste to reduce intstrument fouling quadrupole mass spectrometer. The method was LCMS analysis was carried out on a Shimadzu and the injection volume was limited to 1 µL. A variety of cannabis samples offered for retail applied to detect a number of pesticides in comer- Prominence HPLC with LCMS-8040 triple quadru- sale and presented to us for analysis were anony- cially available cannabis samples.

Page 8

Results and Discussion: A representative chro- The total recovery for each compound, as mea- matogram of a prepared, spiked cannabis sample sured by comparing the peak area for a 50 ppb is shown in the figure. The total run time was 15 spiked sample with a spiked process blank, which min. and chromatographic peaks were observed represents 100% recovery and no matrix effects. Founded in 1875, Shimadzu Corporation, a leader in the development of advanced technologies, has a distinguished history of innovation built on the foundation of contributing to society through science and technology. We maintain a global network of sales, service, technical support and applications centers on six continents, and have established long-term relationships with a host of highly trained distributors located in over 100 countries. For information about Shimadzu, and to contact your local o ce, please visit our Web site at www.shimadzu.com

SHIMADZU SCIENTIFIC INSTRUMENTS, INC. Applications Laboratory 7102 Riverwood Drive, Columbia, MD 21045 Phone: 800-477-1227 Fax: 410-381-1222 URL http://www.ssi.shimadzu.com

For research use only. Not for use in diagnostic procedures.