CONTAMINANT TESTING IN MARIJUANA: PESTICIDES, MYCOTOXINS AND RESIDUAL SOLVENTS

CONTAMINANT TESTING IN MARIJUANA: PESTICIDES, MYCOTOXINS AND RESIDUAL SOLVENTS Katherine K. Stenerson, Olga I. Shimelis, Michael Ye, Michael Halpenny ...
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CONTAMINANT TESTING IN MARIJUANA: PESTICIDES, MYCOTOXINS AND RESIDUAL SOLVENTS Katherine K. Stenerson, Olga I. Shimelis, Michael Ye, Michael Halpenny

MilliporeSigma is a business of Merck KGaA, Darmstadt, Germany

Cannabis in the United States The Current State of Things... Recreational Use  Legal in 4 US states (Colorado, Washington, Oregon, Alaska) Medical Use  Legal in 25 states Testing  Contaminants  Pesticides  Mycotoxins  Residual solvents  Heavy metals  Profiling and content in plant material  Cannabinoids

 Terpenes  No standardized methods currently exist

Agenda

1

Pesticide Residues

 QuEChERS extraction and cleanup

2

Residual Solvents

 Solid Phase Microextraction (SPME)

 GC/MS/MS analysis

 Quick and simple method

 Evaluated two different cleanups

 Quantitative analysis using hemp extract

3

Mycotoxins

 Extract cleanup using SupelTox SPE  Alternative to immunoaffinity  Simpler and easier method

Pesticide Residues

 Analyte list chosen based on current testing; includes different classes of pesticides: • • • •

Triazole fungicides Organophosphorus Organochlorine Pyrethroid

 Marijuana sample provided courtesy of Dr. Hari H Singh*.

*Program Director at the Chemistry & Physiological Systems Research Branch of the National Institute on Drug Abuse at the National Institute of Health.

Analysis of Pesticide Residues in Marijuana

• QuEChERS extraction & cleanup • Analysis by GC/MS/MS • Analyzed samples unspiked and spiked (50 ng/g) QuEChERS procedure used

1 g ground cannabis + 10 mL water

5

Add 10 mL of acetonitrile, shake for 10 min at 2500 rpm

Add citrate salts and shake for 1 min

Centrifuge, 5000 rpm/5 min

Transfer 1 mL of supernatant to 2 mL tube containing cleanup sorbent. Shake for 2 min

Centrifuge, 5000 rpm/5 min

Draw off supernatant for GC/MS/MS analysis

Evaluated two sorbents for cleanup 1. PSA/C18/GCB 2. Supel QuE Verde

What is Supel QuE Verde ??? Supelclean™ ENVI-Carb™ Y

Supel QuE Z-Sep+

Supelclean PSA

•Z-Sep+ Zr & C18 functionalized silica ZrO2 C18 ZrO2 C18 ZrO2 C18 ZrO2 C18 6

Specialized GCB that balances chlorophyll removal and analyte recovery Removes unwanted pigment (color) and lipid (fat) interferences Removes unwanted fatty acids, organic acids, polar pigments, sugars ENVI-Carb Y Specially engineered graphitized carbon black

0.00E+002.00E+074.00E+07

GC/MS Scan analysis of extract background in marijuana extracts no cleanup Sum of all peak areas = 540,827,547

10

20

30

PSA/C18/ENVI-Carb Sum of all peak areas = 449,294,987

10

20

Supel QuE Verde no cleanup

30 T ime (min)

0.00E+00 2.00E+07 4.00E+07

0.00E+002.00E+074.00E+07

Time (min)

Supel QuE Verde Sum of all peak areas = 281,530,256

10

20

30 T ime (min)

cannabinoids

PSA/C18/ENVI-Carb

Analyte Recovery Avg. overall % recovery  PSA/C18/ENVI-Carb: 71%  Supel QuE Verde: 75%

Avg. overall % RSD  PSA/C18/ENVI-Carb: 8%  Supel QuE Verde: 5%

120 110 100

Supel QuE PSA/C18/ENVI-Carb Supel QuE Verde

Avg. % Recovery

90 80 70 60 50 40 30

20 10 0

8

Pesticides

 Marijuana oil produced by extraction of cannabis flower buds

 Extraction often uses organic solvents

Residual Solvents

 Some solvent can remain behind in the final extract  Testing can be done by headspace GC  Traditional headspace requires a separate analyzer connected to the GC  SPME can be used as an alternative; and can be automated with an X-Y-Z autosampler

What is “SPME”? Solid phase microextraction

10



Solvent-free extraction technique for nearly any sample or matrix



Alternative to headspace GC and solid phase extraction (SPE) techniques



Directly interfaced with GC analysis



Quantitative



Non-destructive to sample



Reusable (50-100+ times)



Inexpensive



Fast



Easy to automate SPME process

SPME Fiber Coating: The Business End • Not an exhaustive extraction technique • An equilibrium is set up between analytes dissolved in the sample (solution or gas phase) and in the liquid coating on the fiber. • The fiber coatings consist of: • Polymer films (e.g. PDMS)

• Particles + binder (e.g. carbons or DVB in PDMS)

Enlargement of the SPME fiber coating 11

Equilibrium of analyte conc. in fiber and sample

Details of Analysis

Residual Solvents Tested

 Samples:  Pure hemp oil, spiked at 10 µg/g (triplicate analyses)

Peak #

Solvent

Class

4

Acetone

III

3

Acetonitrile

II

8

Benzene

I

9

Cyclohexane

II

2

Ethanol

III

 GC/MS, full scan

10

Heptane

III

7

Hexane

II

 Supel-Q™ PLOT, 30 m x 0.32 mm I.D. capillary column

5

Isopropanol

III

1

Methanol

II

6 11 12&13

Tetrahydrofuran

II

Toluene

II

Xylene (o,m,p)

II

 Soybean oil blanks  Quantitation:  external standard  6-point calibration curve (6-100 µg/g) in soybean oil  Analysis:

12

3

1

2 4

Class per ICH guidelines

7

4

6

8

10 5

10

11

8,9

13

6

12 14 Time (min)

16

18

Oven: 50°C (5 min), 10°C/min to 230°C (5 min) Carrier: He, 2 mL/min constant flow Splitter open during injection/desorption (10:1)

20

22

Headspace SPME Method for Residual Solvents Sample/matrix:

SPME Fiber:

Extraction:

Desorption:

Fiber Postbake:

 5 g hemp oil in 10 mL vial

 Carboxen®/PDMS, 75µm (CAR/PDMS) Strong adsorbent fiber; provides retention of light compounds- down to C3.  5 min, headspace, 40°C At 40°C, only a short extraction time is needed.

 3 min, 320°C; split 10:1 High temp. used to efficiently and completely desorb analytes. High sensitivity of SPME requires split of 10:1 to prevent overload  2 min, 320°C Cleans fiber & prevents carryover

Method Calibration For Residual Solvents; HS SPME using CAR/PDMS Fiber 3000000 R² = 0.9858

Response (absolute)

2500000

R² = 0.9869

2000000 methanol THF

1500000

heptane R² = 0.9936

1000000

o xylene R² = 0.9806

R² = 0.9806

isopropanol

R² = 0.9864

500000

0 0

20

40

60

80

100

120

Conc. (ug/g)

 Standards made using soybean oil  Overload starting at 70 ug/g for some compounds 14

HS SPME Method; Measurement Accuracy & Reproducibility 10 ug/g spiking level in hemp oil n=3 % RSD 140%

% Accuracy

100%

80%

8%

7%

120% 6% 5% 3%

8%

7%

9%

6%

9%

60% 40% 20%

0%

Detected in unspiked hemp oil at 58.5 ug/g

7%

6%

 Method accuracy 80% for all compounds  Good reproducibility: RSDs < 10%  High level of hexane detected in unspiked hemp oil

 Produced by fungi present on cannabis plants  They present acute and chronic toxicity risks to humans - including suspected carcinogenicity

Mycotoxins

 Quantitative analysis involves extraction and cleanup followed by chromatographic separation (GC and HPLC)  Complex matrix of cannabis necessitates the need for extract cleanup  Extract Cleanup usually done using immunoaffinity; however SPE offers an easier alternative

Methods for Sample Cleanup for Mycotoxin Analysis Method Immunoaffinity (IAC)

Bind & elute

SPE

Interference removal or bind & elute

Issues with IAC

# of Steps (minimum)

Storage

Type of cartridges

3

Refrigeration

Mycotoxin specific

1 or 3

Room temp.

Mycotoxin specific

• • •

Benefits of SPE

• •

• 17

Thermally unstable Introduce many sources of variability due to the complexity of the required procedure. Require many steps: utilize “Bind and Elute” principle Do not require refrigerated storage Simple procedure; can utilize chemical filtration approach Does not require dilution of sample with water prior to cleanup

Analysis of Aflatoxins in Cannabis

1 2

3

4

Used dried cannabis for extraction obtained courtesy of Dr. H. Hari Singh at NIH

Spiked at ppb levels with Aflatoxins B1, B2, G1, G2 Spiking done after extraction and prior to cleanup to evaluate recovery performance of SPE cleanup

Cleanup done using Supel™ Tox AflaZea SPE Simple interference removal approach

Final analysis by LC/MS/MS Using matrix-matched calibration curve

Sample preparation & cleanup of aflatoxins in cannabis using SPE Extraction of homogeneous sample (0.5 g in 10 mL) Shaken 90 minutes in 86:14 ACN:H20

Filter and spike

SPE – Supel Tox AflaZea

Dilution with water – no precipitate (200 uL to FV 1 mL)

LC-MS/MS analysis 19

RESULTS AFLATOXINS IN Cannabis 200



180

Ascentis Express Phenyl-Hexyl 10 cm x 2.1 mm, 2.9 um particle size

160 counts

140 120

aflatoxin G2

100

aflatoxin G1

80

afaltoxin B2

60

afaltoxin B1

40

20 0 2.5

3

3.5

4



NH4formate/formic acid watermethanol gradient



10µL injection



MS, ESI(+), MRM 331.3/189.0, 329.1/243.0, 315.9/259.0, 313.1/241.0

4.5

Time (minutes)

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

24.4 ppb

6.1 ppb

24.4 ppb

6.1 ppb

Recovery (%)

102

109

108

127

RSD% (n=3)

8

12

3

9

Spiking level

Matrix matched calibration standards were used

20

Cannabis Matrix Effects

4000

Aflatoxin B1

900

3500

800

3000

700

counts

in cannabis extract

2000

in solvent

1500

counts

600

2500

500 400 300

1000

200

500

100

0 0.0000

Aflatoxin B2

0.5000

1.0000

concentration (ng/mL)

0 0.0000

in cannabis extract in solvent 0.1000

0.2000

0.3000

concentration (ng/mL)

 Significant matrix effect observed  Use of matrix-matched standards required for accurate quantitation  Supel Tox AflaZea cleanup produced a clear extract but did not remove all interfering compounds 21

Summary & Conclusions

1

QuEChERS extraction and cleanup is a viable approach for analysis of pesticides in marijuana

2



Use of a cleanup sorbent containing GCB is recommended for chlorophyll removal



Compared to PSA/C18/GCB, Supel QuE Verde cleanup showed lower overall GC/MS background and better recovery of quinoxyfen, a planar pesticide

Residual solvents in hemp (and cannabis) extract can be quickly and quantitatively analyzed by HSSPME •

The method using the CAR/PDMS fiber could be extended to include analysis of lighter compounds such as butane



The SPME method offers an alternative to conventional headspace; and does not require the use of a separate headspace analyzer

3

SPE cleanup can be used in mycotoxin analysis of cannabis •

Supel Tox AflaZea offers a simpler alternative to IAC cleanup



Cleanup using AflaZea SPE allows for accurate detection of mycotoxins at ppb levels with the use of matrix-matched standards

Acknowledgments Many Thanks to….  Dr. Hari H. Singh, Program Director at the Chemistry & Physiological Systems Research Branch of the National Institute on Drug Abuse at the National Institute of Health for supplying the dried cannabis sample used for testing  Yong Chen and Bob Shirey of MilliporeSigma for many helpful discussions on SPME

23

Thank you for your attention

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