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INTRODUCTION

The increased use of both medical and recreational cannabis in combination with its expanding legal acceptance in many US states has led to increased demand for cannabis safety and quality control testing. Analytical testing typically includes cannabinoids profiling, potency, mycotoxins, terpenes, residual solvents, metals, and pesticide residue analysis. Pesticides are of particular interest as they are widely used in the cultivation of cannabis plants to safeguard against harmful insects and to promote crop yields. In addition to pesticides, cannabis must also be tested for mycotoxins. A robust and rapid test is critical and a single simultaneous test for pesticides and mycotoxins is ideal. Multi-residue compound detection is routinely performed using tandem quadrupole mass spectrometry (MS/MS) in combination with Liquid Chromatography (LC) and Gas Chromatography (GC). Tandem quadrupole MS is the detector of choice as it provides high sensitivity and selectivity for simultaneous analysis of hundreds of pesticides at low ng/g (ppb) levels in a single analysis. In this study, we present the use of a simple sample extraction and dSPE cleanup where the resulting extract is analyzed by UPLC-MS/MS and/or GC-MS/MS to rapidly monitor pesticides and mycotoxins in cannabis matrix to meet California regulations (Figure 1). With the variety of residues to be monitored as well as the continued possibility of new ones being added, method generation can be a tedious task. In this study, methods for LC-MS/MS were utilized from a software database and method manager, Quanpedia™, eliminating the need for method development for the California pesticide and mycotoxin lists (Figure 2).

ROUTINE ANALYSIS OF CANNABIS FOR PESTICIDES AND MYCOTOXINS USING UPLC-MS/MS

min3.80 4.00 4.20 4.40 4.60 4.80

%

0

100

F10:MRM of 2 channels,ES+225.1 > 127.1

10Nov2018_16

1.004e+006

Mevinphos4.37

4.69*

min6.500 6.600 6.700 6.800 6.900

%

0

100

F28:MRM of 4 channels,ES+304.1 > 97.1

10Nov2018_16

6.669e+005

Fenhexamid6.70

min9.25 9.50 9.75 10.00 10.25 10.50 10.75

%

0

100

F72:MRM of 2 channels,ES+748.5 > 142.1

10Nov2018_16

7.588e+005

Spinetoram10.02

9.76

min6.200 6.300 6.400 6.500

%

0

100

F59:MRM of 2 channels,ES+388.1 > 301.1

10Nov2018_16

2.200e+006

Dimethomorph6.36

min7.200 7.300 7.400 7.500 7.600 7.700 7.800

%

0

100

F52:MRM of 3 channels,ES+365 > 229

10Nov2018_16

3.630e+005

Coumaphos7.47

min11.00 11.50 12.00 12.50

%

0

100

F47:MRM of 2 channels,ES+351.9 > 124.9

10Nov2018_16

3.020e+005

Chlorpyrifos11.73

Mevinphos

Fenhexamid

Spinetoram

Dimethomorph

Coumaphos

Chlorpyriphos

Figure 3. Representative MRM chromatograms for (1) mevinphos iso-mers, (2) dimethomorph, (3) fenhexamid, (4) coumaphos, (5) spineto-ram, (6) chlorpyriphos spiked at a level of 0.10 μg/kg in cannabis flower .

Figure 4. Representative MRM chromatograms for aflatoxins B1, B2, G1, G2 and ochratoxin A spiked at a level of 0.02 µg/kg in cannabis matrix.

SAMPLE PREPARATION Initial Extraction • 0.5 g ground cannabis bud weighed into 50 mL centri-

fuge tube • 5 mL acetonitrile added • Process with Geno Grinder for 2min @ 1500 rpm • Remove 1 mL aliquot for dSPE

dSPE • 2 mL tube with 150 mg MgSO4, 50 mg PSA, 50 mg C18,

7.5 mg graphitized carbon • Shake dSPE tube for 1 min • Centrifuge • Transfer supernatant to autosampler vial for analysis by

LC-MS/MS and GC-MS/MS • Recoveries for most compounds were in the range of 80-

120%. Matrix effects were significantly reduced when dSPE was performed following the initial acetonitrile extraction.

1

Figure 2. Rapid implementation of LC, GC, MS and data processing methods using Quanpedia method database.

PESTICIDES AND MYCOTOXINS ANALYSIS BY UPLC-MS/MS

Canada and individual US states have defined different re-quirements for pesticide residue testing in cannabis. The list of pesticides varies from state to state as well as from country to country. The composition and complexity of the matrix varies widely across different cannabis strains. The combination of long lists of pesticides with variable and complex matrices pre-sents a significant challenge in method development. Linear calibration curves (R

2>0.990) for all pesticides were

obtained over the range tested 0.025 to 0.50 μg/kg. Repre-sentative MRM chromatograms for selected pesticides are displayed in Figure 3.

For detail on GC MS/MS analysis for pesticides in cannabis, please see Wednesday Poster 154

The LC-MS/MS analysis of mycotoxins can be combined with the analysis of pesticide residues in a single analytical injection, allowing trace level detection of aflatoxins B1, B2, G1, G2, and ochratoxin A. The calibration curves for all mycotoxins were linear (R

2>0.990) over the range tested 0.005 to 0.10 μg/kg

Figure 4 shows the chromatograms of cannabis matrix spiked at 0.02 μg/kg which is the action level set by the State of California for mycotoxins testing.

Figure 5. Recoveries for most of the pesticides were in the range of 80% to 120% (n=6)

Figure 6. Matrix suppression at the 200 µg/kg level; the red bars indicate suppression observed without dSPE and the blue bars indicate suppression after dSPE cleanup. The shaded area indicates the compounds that co-elute with cannabis resin constituents

1.Kim Tran, Kari Organtini et al. Analysis of Residual Pesticides and Mycotoxins in Can-nabis Using UPLC-MS/MS and GC-MS/MS to Meet California Regulatory Require-ments Waters application note 720006465EN http://www.waters.com/webassets/cms/library/docs/720006465en.pdf

Waters Quanpedia method database was used to auto-matically create the LC, GC, MS and data processing methods (See Figure 2 for the various target pesticides to be monitored using the transitions.) Users can quickly generate pre-defined LC-MS/MS and GC-MS/MS methods in just three steps, which greatly re-duces the level of potential error and the complexity in-volved in method development for large numbers of target analytes. As a result, it decreases the amount of work, time, and re-sources required for laboratories to set up methods. Addi-tionally, Quanpedia also contains functionality to quickly adjust retention times associated with a method eliminat-ing the lengthy process of manually adjusting MRM time windows due to retention time shifts. The LC-MS/MS method contained 67 compounds (62 pes-ticides and 5 mycotoxin) and the GC-MS/MS method con-tained 54 compounds, fully covering the California re-quirements for pesticide and mycotoxin residue analysis.

Linearity

Figure 7. . Representative example of a quantitation curve for fenhexamid demonstrating a linear range from 25 to 500 µg/kg (2.5 to 50 µg/kg in vial concentration) cannabis resin constitu-ents

RESULTS AND DISCUSSION

LC Conditions UPLC: ACQUITY™ UPLC™ H-Class Separation mode: Gradient Column: XBridge C18 2.1 x 150 mm, 2.5 µm Solvent A: 5 mM Ammonium formate with 0.020 % formic acid in water Solvent B: Methanol Flow rate: 0.400 mL/min Column temp.: 50 °C Injection volume: 5 µL

MS Conditions MS: Xevo™ TQ-Smicro Ionization mode: ESI + / ESI - Capillary voltage: 3.0 kV (+); 2.5 kV (-) Cone Voltage: Various V Collision Energy: Various eV Desolvation temp: 550 °C Source temp.: 150 °C Desolvation Gas Flow: 800 (L/Hr) Cone Gas: 50 (L/Hr)

Figure 1. A workflow for multi-residue pesticide analysis by LC-MS/MS

REFERENCES

Method recovery was assessed by spiking pesticides at the 0.1μg/kg and 0.5μg/kg levels in a cannabis flower matrix and comparing the response to that observed from spiked matrix blanks (matrix matched standards). As shown in Figure 5, the recoveries observed for most pesticides were in the range of 80-120%. Matrix suppression was determined at the 200 μg/kg level by comparison of the response observed in matrix matched standards to response observed in solvent standards. Matrix suppression data are presented in Figure 6. The dSPE cleanup provided significant reduction of suppression for most compounds. Those compounds that co-elute with cannabis resin constituents (retention times from 9 to 12 minutes) showed the greatest suppression after dSPE cleanup. The recovery of daminozide, ochratoxin A from the PSA sorbent are decreased due to the interaction of this compound with the PSA sorbent. Analysis of these compounds should be performed before dSPE. For linearity and sensitivity evaluation, matrix matched calibration curves were generated and an example of the quantitation curve and respective MRM chromatograms for fenhexamid is shown in Figure 7.

CONCLUSION This simple sample extraction and dSPE cleanup method followed by UPLC-MS/MS and GC-MS/MS analysis provides a rapid, sensitive, and robust workflow for determination of the pesticides and mycotoxins in challenging cannabis matrix. Matrix suppression was significantly reduced using dSPE cleanup for many pesticides; thereby improving the data quality. This method is capable of meeting the action levels for the California pesticide list and mycotoxins in cannabis matrix.

100ppb spiked 3

Time4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80

%

0

100

10Nov2018_18 43: MRM of 5 Channels ES+ 331.1 > 245.1 (Aflatoxin G2)

4.43e44.63

10Nov2018_18 40: MRM of 4 Channels ES+ 329.1 > 243.1 (Aflatoxin G1)

1.14e54.78

10Nov2018_18 35: MRM of 4 Channels ES+ 315.1 > 287.1 (Aflatoxin B2)

3.02e44.94

10Nov2018_18 33: MRM of 4 Channels ES+ 313.1 > 285.1 (Aflatoxin B1)

6.30e45.07

10Nov2018_23 61: MRM of 3 Channels ES+ 404.1 > 239 (Orchatoxin A)

5.26e46.04Ochratoxin A

Aflatoxin G2

Aflatoxin G1

Aflatoxin B2

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Kim Tran,1 Kari Organtini,

1 Marian Twohig,

1 Peter Alden,

1 Michael S. Young,

1 Narendra Meruva,

1 Kenneth Rosnack,

1 Gordon Fujimoto,

2 Rebecca Stevens,

3 James Roush,

3 and Christopher J. Hudalla

3

1Waters Corporation, Milford, MA USA;

2Waters Corporation, Beverly, MA, USA;

3ProVerde Laboratories, Milford, MA, USA

PESTICIDES AND MYCOTOXINS ANALYSIS BY UPLC-MS/MS

Residuals < 2%

R2 = 0.999

25-500 µg/kg in sample

2.5-50 µg/kg in vial

25 µg/kg in sample

2.5 µg/kg in vial

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% Matrix Effect no dSPE

% Matrix Effect dSPE

Red bars = without dSPE Blue bars = with dSPE% Suppression

Time (min) %A %B Curve

0.00 98% 2% -

0.20 98% 2% 6

4.00 30% 70% 6

10.00 30% 70% 6

12.00 1% 99% 6

15.00 1% 99% 6

15.01 98% 2% 1

17.00 98% 2% 1

TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2019 Waters Corporation

Kim Tran,1 Kari Organtini,1 Marian Twohig,1 Peter Alden,1 Michael S. Young,1 Narendra Meruva,1 Kenneth Rosnack,1 Gordon Fujimoto,2 Rebecca Stevens,3 James Roush,3 and Christopher J. Hudalla3 1Waters Corporation, Milford, MA USA; 2Waters Corporation, Beverly, MA, USA; 3ProVerde Laboratories, Milford, MA, USA J. , Milford, MA, USA