MilliporeSigma is a business of Merck KGaA, Darmstadt, Germany

CONTAMINANT TESTING IN MARIJUANA: PESTICIDES, MYCOTOXINS AND RESIDUAL SOLVENTS

Katherine K. Stenerson, Olga I. Shimelis,

Michael Ye, Michael Halpenny

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

Cannabis in the United States The Current State of Things...

2 Residual Solvents 1 Pesticide Residues

3 Mycotoxins

QuEChERS extraction and cleanup

GC/MS/MS analysis

Evaluated two different cleanups

Solid Phase Microextraction (SPME)

Quick and simple method

Quantitative analysis using hemp extract

Agenda

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

5

• QuEChERS extraction & cleanup

• Analysis by GC/MS/MS

• Analyzed samples unspiked and spiked (50 ng/g)

1 g ground cannabis + 10

mL water

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

QuEChERS procedure used

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

What is Supel QuE Verde ???

Supelclean™ ENVI-Carb™ Y Specialized GCB that balances chlorophyll removal and analyte recovery

Supel QuE Z-Sep+ Removes unwanted pigment (color) and lipid (fat) interferences

Supelclean PSA Removes unwanted fatty acids, organic acids, polar pigments, sugars

6

•Z-Sep+

Zr & C18 functionalized silica

ZrO2

C18

ZrO2

C18

ZrO2

C18

ZrO2

C18

ENVI-Carb Y

Specially engineered graphitized carbon black

GC/MS Scan analysis of extract background in marijuana extracts

10 20 30

Time (min)

0.0

0E

+0

02.0

0E

+0

74.0

0E

+0

7

10 20 30

Time (min)

0.0

0E

+0

02

.00

E+

07

4.0

0E

+0

7

10 20 30

Time (min)

0.0

0E

+0

02

.00

E+

07

4.0

0E

+0

7

Supel QuE Verde PSA/C18/ENVI-Carb no cleanup

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

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

no cleanup Sum of all peak areas = 540,827,547

cannabinoids

0

10

20

30

40

50

60

70

80

90

100

110

120

Avg

. %

Reco

very

Pesticides

Supel QuE PSA/C18/ENVI-Carb

Supel QuE Verde

8

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%

Residual Solvents

Marijuana oil produced by extraction of cannabis flower buds

Extraction often uses organic 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

10

What is “SPME”?

Solid phase microextraction

• 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

11

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

Equilibrium of

analyte conc. in

fiber and sample

Details of Analysis

Samples:

Pure hemp oil, spiked at 10 µg/g (triplicate analyses)

Soybean oil blanks

Quantitation:

external standard

6-point calibration curve (6-100 µg/g) in soybean oil

Analysis:

GC/MS, full scan

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

Class per ICH guidelines

Residual Solvents Tested

Peak # Solvent Class

4 Acetone III

3 Acetonitrile II

8 Benzene I

9 Cyclohexane II

2 Ethanol III

10 Heptane III

7 Hexane II

5 Isopropanol III

1 Methanol II

6 Tetrahydrofuran II

11 Toluene II

12&13 Xylene (o,m,p) II 4 6 8 10 12 14 16 18 20 22

Time (min)

1 2

3

4

5 6

7 8,9

10

11

12

13

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)

Headspace SPME Method for Residual Solvents

Sample/matrix:

SPME Fiber:

5 g hemp oil in 10 mL vial

Carboxen®/PDMS, 75µm (CAR/PDMS)

Strong adsorbent fiber; provides retention of light compounds- down to C3.

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

Extraction: 5 min, headspace, 40°C

At 40°C, only a short extraction time is needed.

Desorption:

Fiber Postbake: 2 min, 320°C

Cleans fiber & prevents carryover

14

Method Calibration For Residual Solvents; HS SPME using CAR/PDMS Fiber

R² = 0.9858

R² = 0.9864 R² = 0.9806 R² = 0.9806

R² = 0.9806

R² = 0.9869

R² = 0.9936

0

500000

1000000

1500000

2000000

2500000

3000000

0 20 40 60 80 100 120

Resp

on

se (

ab

solu

te)

Conc. (ug/g)

methanol

THF

heptane

o xylene

isopropanol

Standards made using soybean oil

Overload starting at 70 ug/g for some compounds

0%

20%

40%

60%

80%

100%

120%

140%

% A

ccu

racy

7%

n=3

HS SPME Method; Measurement Accuracy & Reproducibility 10 ug/g spiking level in hemp oil

3% 9%

6% 5%

8%

9% 6%

7%

7% 6%

8%

Detected in unspiked hemp oil at 58.5 ug/g

% RSD

Method accuracy 80% for all compounds

Good reproducibility: RSDs < 10%

High level of hexane detected in unspiked hemp oil

Mycotoxins

Produced by fungi present on cannabis plants

They present acute and chronic toxicity risks to humans - including suspected carcinogenicity

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

17

Method # of Steps (minimum)

Storage Type of cartridges

Immunoaffinity (IAC)

Bind & elute 3 Refrigeration Mycotoxin specific

SPE Interference removal or bind & elute

1 or 3 Room temp. Mycotoxin specific

Methods for Sample Cleanup for Mycotoxin Analysis

Issues with IAC • Thermally unstable • Introduce many sources of

variability due to the complexity of the required procedure.

• Require many steps: utilize “Bind and Elute” principle

Benefits of SPE • Do not require refrigerated storage

• Simple procedure; can utilize chemical filtration approach

• Does not require dilution of sample with water prior to cleanup

4

3

2

1 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

Analysis of Aflatoxins in Cannabis

Sample preparation & cleanup of aflatoxins in cannabis using SPE

19

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

20

RESULTS

AFLATOXINS IN Cannabis

0

20

40

60

80

100

120

140

160

180

200

2.5 3 3.5 4 4.5

co

un

ts

Time (minutes)

aflatoxin G2

aflatoxin G1

afaltoxin B2

afaltoxin B1

Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

Spiking level 24.4 ppb 6.1 ppb 24.4 ppb 6.1 ppb

Recovery (%) 102 109 108 127

RSD% (n=3) 8 12 3 9

Matrix matched calibration standards were used

• Ascentis Express Phenyl-Hexyl

10 cm x 2.1 mm, 2.9 um particle size

• NH4formate/formic acid water-methanol gradient

• 10µL injection

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

Cannabis Matrix Effects

21

0

500

1000

1500

2000

2500

3000

3500

4000

0.0000 0.5000 1.0000

co

un

ts

concentration (ng/mL)

Aflatoxin B1

in cannabis

extract

in solvent

0

100

200

300

400

500

600

700

800

900

0.0000 0.1000 0.2000 0.3000

co

un

ts

concentration (ng/mL)

Aflatoxin B2

in cannabis

extract

in solvent

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

Summary & Conclusions

Residual solvents in hemp (and cannabis) extract can be quickly and quantitatively analyzed by HS-SPME

• 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

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

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

3

2

1

23

Acknowledgments

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

Many Thanks to….

Thank you for your attention