Using Solid Phase Microextraction for

Cannabis Testing

By:

Katherine Stenerson

Principal Scientist, Workflow R&D

MilliporeSigma

MilliporeSigma is a business of Merck KGaA, Darmstadt, Germany

SOLID PHASE MICROEXTRACTION AND CANNABIS TESTING Presented by: Kathy Stenerson

MilliporeSigma

Bellefonte, PA

3

Who we are…

MilliporeSigma • The life science business of Merck KGaA, Darmstadt,

Germany

• EMD Millipore + Sigma Aldrich = MilliporeSigma

What we offer…

• 300,000 products

• Sigma, Aldrich, Supelco, EMDMillipore

Agenda

1. Background

2. What is Solid Phase Microextraction (SPME)?

3. SPME analysis of terpenes in cannabis

4. SPME analysis of residual solvents in hemp extract

5 Source: thecannabist/com (accessed 2/24/2017)

Recreational use legal in 8 states plus Washington, D.C.

Medical use legal in 28 states

Testing

Contaminants

Microbiological

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...

7

What is “SPME” ?

Manual SPME holder and inlet guide.

Assembled SPME fiber and holder with fiber immersed in a liquid sample.

• Solid Phase MicroExtraction

• Solvent-free extraction technique for nearly any sample or matrix

• Alternative to head-space GC and solid phase extraction (SPE) techniques

• Directly interfaced with GC analysis

• Non-destructive to sample

• Reusable (100+ times)

• Inexpensive

• Fast

• Easily automated

8

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

9

Types of SPME Fiber Coatings

Coating Type Polarity

7 µm Polydimethylsiloxane (PDMS) Absorbent Nonpolar 30 µm PDMS Absorbent Nonpolar 100 µm PDMS Absorbent Nonpolar 85 µm Polyacrylate (PA) Absorbent Polar 60 µm PEG (Carbowax) Absorbent Polar

Coating Type Polarity

85 µm Carboxen-PDMS Adsorbent Bipolar 65 µm PDMS-DVB Adsorbent Bipolar 55 µm/30 µm DVB/Carboxen-PDMS Adsorbent Bipolar

Particles – Adsorption:

Films – Absorption:

10

The SPME Concept

11

The SPME Concept

12

The SPME Concept

13

The SPME Concept

14

The SPME Concept

15

The SPME Concept

16

The SPME Concept

The SPME process

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Sample incubation

Sample usually heated

Agitation sometimes used

Sample extraction

Fiber placed into sample directly or into headspace

Sample is agitated

Temperature control essential for quantitation

Sample preparation

Sample placed into vial with septa cap

Additives may be used (water, salt, pH)

Fiber desorption

Fiber place in hot GC inlet

Thermal energy desorbs analytes

Analysis GC analysis similar to a liquid injection

Extraction

18

Automating the SPME Process

• Autosampler head equipped with SPME holder

• Magnet used to hold samples

• Transport to a heated agitator for extraction

• Insertion of SPME fiber into sample vial

• Thermal desorption of SPME fiber in GC inlet

Direct GC analysis

Moving sample

Selecting sample

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Analyte

Adsorbed

Silica Rod

Liquid Polymer

Aqueous

Solution

Vial

Time

Adsorption Mechanism for SPME

Rapid uptake

onto fiber

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Number of Moles of Analyte Extracted by Fiber (n)

n = KfsVf C0 •Kfs = Distribution constant between fiber and sample

•Vf = volume of fiber coating

•Co = initial concentration in sample

K affinity of analyte for stationary phase on fiber

Kfs= C∞fVf /C∞

sVs

Is SPME quantitative?...... YES!!!

Concentration

Resp

on

se

Cal stds.

quantitate

unknown

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Solid Phase Microextraction (SPME) for Terpenes and Residual Solvents

Terpenes

Isoprene unit

Name is derived from “turpentine”

Classified by the number of isoprene units in the structure

Cannabis contains >100 different terpenes and terpenoids

Distinct aromas and flavors resulting from different terpene profiles

Traditional test method uses solvent extraction and GC analysis

SPME an alternative approach for terpene analysis

Linalool α-Pinene

Why Terpenes?

Bicyclic monoterpene

Pine aroma

Associated therapeutic benefits include bronchodilator, anti-inflammatory, stimulant

Cyclic monoterpene

Citrus aroma

Associated therapeutic benefits include anti-depressant, antimutagenic

Acyclic monoterpene

Floral, citrus, candy aroma

Associated therapeutic values include sedative, anti-anxiety, anti-depressant

Acyclic monoterpene

Earthy, herbal-type aroma

Associated therapeutic values include analgesic, antiinflammatory, antibiotic

d-Limonene β-Myrcene

SPME Approaches for Terpenes in Cannabis

Qualitative analysis

• Useful for terpene profiling

• GC/MS spectra and retention indices used for peak identification

Quantitative analysis

• For quantitation of specifically identified terpenes

• Demonstrated here for pinene, limonene, and linalool; could be extended to include other terpenes

1

2

SPME parameters used for terpene profiling of unknown cannabis sample

Sample and vial size chosen to allow sufficient headspace for fiber and efficient extraction

1

Adsorbent SPME fiber, dual layer, with very strong Carboxen adsorbent

2 Incubation to bring sample to extraction temperature and allow terpenes into headsapce

3

Highest possible temperature used for efficient analyte desorption

5

Extraction time sufficient for uptake of entire terpene profile

4

Postbake ensures no carryover 6

Sample: 0.5 g dried cannabis in 10 mL vial

1

SPME fiber: 50/30 µm DVB/CAR/PDMS

2

Incubation: 30 min, 40 °C 3

Extraction: 20 min, headspace, 40 °C

4

Desorption: 3 min, 270 °C 5

Postbake: 3 min, 270 °C 6

26

HS-SPME Analysis of Dried Cannabis

0 10 20 30 40

Time (min)

0.0

0E

+00

1.0

0E

+08

2.0

0E

+08

Abundance

0 10 20 30 40

Time (min)

0.0

0E

+0

02

.00

E+

08

Ab

un

da

nce

100 µm PDMS

DVB/CAR/ PDMS

Difference in SPME fibers

Difference in fiber selectivity

Results: Terpene profiling of dried cannabis using HS-SPME

10 20 30

Time (min)

1

2 3 4

5

6

7

9

10

11

12

13

14 16

17

18

19

21,22

23

24

25 26

28

29

30

31

32

34

35

36

37

39,40

41

42

43

44 45

8

15 20 27

33

38

Identification of terpenes

MS spectral library match (NIST and Wiley)

Retention indices & comparison to published values

Comparison to published data for cannabis

Determination of retention index

• Using Kovat’s retention index system (KRI)

• Calculated against RT’s of n-alkanes run under same GC conditions

GC-MS Conditions

Non-polar Equity-1 column used

Oven profile: 60°C (2 min), 5 °C/min to 275°C (5 min)

Carrier gas: helium, 1 mL/min constant flow

MS: full scan, m/z 50-500

28

Terpenes identified in dried cannabis sample Peak #

R.T. (min) Name

RI calculated

1 8.57 hexanal

2 10.05 hexene-1-ol

3 10.89 2-heptanone

4 12.56 α-thujene 928

5 12.86 α-pinene 939

6 13.27 camphene 953

7 13.69 6-methyl-5-hepten-2-

one 966

8 14.09 β-pinene 979

9 14.27 β-myrcene 984

10 15.09 Δ-3-carene 1010

11 15.2 α-terpinene 1014

12 15.29 cymene 1018

13 15.6 d-limonene 1028

14 16.42 γ-terpinene 1056

15 16.6 trans-sabinene

hydrate 1062

16 16.72 cis-linalool oxide 1066

17 17.43 linalool 1087

18 18.04 d-fenchyl alcohol 1107

19 18.82 trans-pinocarveol 1135

20 19.59 borneol L 1161

21 19.81 1,8-methandien-4-ol 1168

22 19.81 p-cymen-8-ol 1168

23 19.92 terpinene-4-ol 1172

Peak # R.T. (min) Name

RI calculated

24 20.22 α-terpineol 1181

25 24.2 piperitenone 1322

26 24.76 piperitenone oxide 1344

27 25.85 α-ylangene 1384

28 25.97 α-copaene 1388

29 26.76 γ-caryophyllene 1419

30 27.01 α-santalene 1429

31 27.16 caryophyllene 1435

32 27.36 trans-α-bergamotene + unknown 1443

33 27.49 α-guaiene 1448

34 27.56 trans-β-farnesene 1451

35 27.98 humulene 1467

36 28.17 alloaromadendrene 1475

37 28.25 α-curcumene 1478

38 28.75 β-selinene 1497

39 28.97 α-selinene 1507

40 28.97 β-bisobolene 1507

41 29.13 α-bulnesene 1514

42 30.12 selina-3,7(11)-diene 1556

43 30.94 caryophyllene oxide 1590

44 31.5 humulene oxide 1614

45 32.48 caryophylla-3,8(13)-dien-5-ol A 1658

monoterpenes & monoterpenoids Sesquiterpenes & sesquiterpenoids

Most abundant terpene in

sample

May be due to specific variety

and/or nature of sample

SPME parameters used for quantitation of select terpenes from spiked cannabis matrix

Grinding sample and addition of water increases reproducibility.

1 Use of absorbent fiber 2 Incubation to bring sample to extraction temperature and allow terpenes into headsapce

3

Desorption temp. could be increased to 300C if necessary.

5

Extraction time sufficient for uptake of entire terpene profile

4

Postbake ensures no carryover 6

Sample: 0.1 g dried, ground cannabis* + 8 mL water in 20 mL vial

1

SPME fiber: 100 µm PDMS 2

Incubation: 5 min, 40 °C, w/agitation

3

Extraction: 10 min, headspace, 40 °C, w/agitation

4

Desorption: 3 min, 270 °C 5

Postbake: 5 min, 270 °C 6

*Spiked with terpenes at 0.16 – 10.3 mg/g

Why were different SPME parameters used?

SPME fiber: 100 µm PDMS

• Absorbent fiber

• More capacity than adsorbent DVB/CAR/PDMS fiber; less prone to overload

1

Sample Configuration

• Reduction in sample weight

• Addition of water

• Larger volume sample vial = more headspace

2

Reduced incubation & extraction times

• Reduced times give sufficient sensitivity without overload

3

100:1 split during desorption

• Prevent overload at higher concentrations

4

Goal: Reduce

overload of SPME

method from

higher levels of terpenes

Specifics of the Spiking Study

Dried cannabis* (unknown variety)

Studied three terpenes

α-pinene

d-limonene

Linalool

Spike concentrations determined by weight

Mimic levels reported in specific cannabis varieties

Analysis by GC-MS/Scan

Quantitation against 5-point curve prepared from dried cannabis

Terpene Spiking Study

*supplied courtesy of Dr. Hari H. Singh (National Institute on Drug Abuse at NIH)

32

HS-SPME Method Calibration

R² = 0.9804

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Resp

on

se (

area c

ts)

Conc. (mg/g)

d-Limonene

R² = 0.9886

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

0.00 0.50 1.00 1.50 2.00

Resp

on

se (

area c

ts)

Conc. (mg/g)

α-pinene

R² = 0.9993

0

50000

100000

150000

200000

250000

300000

350000

0.00 2.00 4.00 6.00 8.00

Resp

on

se (

area c

ts)

Conc. (mg/g)

Linalool

Standards made by spiking low-terpene cannabis

Calibration range reflects terpene levels in various cannabis varieties

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Spiking Study Results

Compound Calibration

Range

mg/g

Spike

Conc.

mg/g

Ave. amt.

measured

mg/g

Ave %

Accuracy

% RSD

(n=3)

α-pinene 0.16-1.67 1.08 1.11 103 0.9

d-Limonene 0.96-10.30 6.69 6.11 91 2.7

Linalool 0.54-5.73 3.72 3.62 97 3.0

Analysis of 3 spiked replicates

Accuracies of >90%

RSD values <5%

Determination of much lower terpene levels also possible

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How does HS-SPME compare to solvent extraction and GC/FID analysis?

Solvent extraction procedure

n=3

α-Pinene d-Limonene

Linalool

extraction

HS-SPME extraction

HS-SPME extraction

HS-SPME

Spike level (mg/g)

1.09 1.08 6.60 6.69 3.38 3.72

Avg. amt. measured (mg/g)

1.15 (1.8) 1.11 (0.9) 6.75 (1.9) 6.11 (2.7) 3.45 (3.4) 3.62 (3.0)

Avg. percent measured vs. spiked

106% 103% 102% 91% 102% 97%

Both methods accurate and reproducible

Sample prep easier and faster with SPME

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 can require a separate analyzer connected to the GC

SPME can be used as an alternative

Details of Analysis

Samples:

Hemp extract in 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 extract/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

38

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 extract/oil

3% 9%

6% 5%

8%

9% 6%

7%

7% 6%

8%

Detected in unspiked hemp extract at 58.5 ug/g

% RSD

Method accuracy 80% for all compounds

Good reproducibility: RSDs < 10%

High level of hexane detected in unspiked hemp extract

Summary – Tools for Testing

For the testing of terpenes and residual solvents in cannabis and cannabis oils, SPME offers:

Accurate and precise analysis for terpenes and residual solvents

Cleaner samples; less stress on instrumentation

Easy automation through the use of an X-Y-Z autosampler (such as the MPS 2)

Time savings: less “hands on” sample preparation time

Cost savings: less consumables used

A more “green” technique than conventional methods

Want More Information on SPME???

41

Visit our website: sigma-aldrich.com/SPME

If you have additional questions related to this presentation,

Contact katherine.stenerson@sial.com

42

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

Michael Halpenny of MilliporeSigma for his contributions to this work

Yong Chen and Bob Shirey of MilliporeSigma for many helpful discussions on SPME

Gerstel Corporation for their assistance in making this webinar possible

Many Thanks to….

And most importantly…

Many Thanks to You!

Questions?

Thank You