In the quickly changing landscape of cannabis testing, laboratories are faced with the challenge of providing accurate results for a variety of different tests on challenging matrices. The wide variety of matrices encountered, including both plants, foods, and oily extracts, have complex chemical compositions which include pigments, fats, proteins and sugars. The sample preparation methods used must provide analyses free of these interferences for accurate and robust testing. In this seminar, we will present simple and effective sample preparation and analysis methods using solid phase microextraction (SPME) for determination of residual solvents and terpenes. SPME is a solvent-less extraction technique that is quantitative, sensitive, cost effective, and easy to automate. We will describe how to use SPME to cleanly, accurately and quantitatively determine residual solvents from hemp extract and terpenes from cannabis plant material. For both applications, descriptions of the method procedure along with data showing accuracy and reproducibility will be presented.

1. Using Solid Phase
Microextraction for
Cannabis Testing
By:
Katherine Stenerson
Principal Scientist, Workflow
R&D
MilliporeSigma

2. MilliporeSigma is a
business of Merck
KGaA,
Darmstadt, Germany
SOLID PHASE MICROEXTRACTION
AND CANNABIS TESTING
Presented by: Kathy Stenerson
MilliporeSigma
Bellefonte, PA

3. 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

4. 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. 5
Source: thecannabist/com (accessed 2/24/2017)
Recreational use legal in 8
states plus Washington, D.C.
Medical use legal in 28 states

6. 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. 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. 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. 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. 10
The SPME Concept

11. 11
The SPME Concept

12. 12
The SPME Concept

13. 13
The SPME Concept

14. 14
The SPME Concept

15. 15
The SPME Concept

16. 16
The SPME Concept

17. The SPME process
17
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

18. 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

19. 19
Analyte
Adsorbed
Silica Rod
Liquid Polymer
Aqueous
Solution
Vial
Time
Adsorption Mechanism for SPME
Rapid uptake
onto fiber

20. 20
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
Response
Cal stds.
quantitate
unknown

21. 21
Solid Phase Microextraction (SPME)
for Terpenes and Residual Solvents

22. 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

23. Linaloolα-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

24. 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

25. 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
carryover6
Sample: 0.5 g dried cannabis in
10 mL vial
1
SPME fiber: 50/30 µm
DVB/CAR/PDMS
2
Incubation: 30 min, 40 °C3
Extraction: 20 min,
headspace, 40 °C
4
Desorption: 3 min, 270 °C5
Postbake: 3 min, 270 °C6

26. 26
HS-SPME Analysis of Dried Cannabis
0 10 20 30 40
Time (min)
0.00E+001.00E+082.00E+08
Abundance
0 10 20 30 40
Time (min)
0.00E+002.00E+08
Abundance
100 µm PDMS
DVB/CAR/ PDMS
Difference in SPME fibers
Difference in fiber
selectivity

27. 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. 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

29. 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
carryover6
Sample: 0.1 g dried, ground
cannabis* + 8 mL water in 20 mL
vial
1
SPME fiber: 100 µm PDMS2
Incubation: 5 min, 40 °C,
w/agitation
3
Extraction: 10 min, headspace,
40 °C, w/agitation
4
Desorption: 3 min, 270 °C5
Postbake: 5 min, 270 °C6
*Spiked with terpenes at 0.16 – 10.3 mg/g

30. 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

31. 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. 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
Response(areacts)
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
Response(areacts)
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
Response(areacts)
Conc. (mg/g)
Linalool
 Standards made by spiking low-terpene
cannabis
 Calibration range reflects terpene levels
in various cannabis varieties

33. 33
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

34. 34
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

35. 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

36. 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)

37. 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. 38
Method Calibration For Residual Solvents; HS SPME using
CAR/PDMS Fiber
R² = 0.9858
R² = 0.9864
R² = 0.9806R² = 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
Response(absolute)
Conc. (ug/g)
methanol
THF
heptane
o xylene
isopropanol
 Standards made using
soybean oil
 Overload starting at 70 ug/g
for some compounds

39. 0%
20%
40%
60%
80%
100%
120%
140%
%Accuracy
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

40. 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

41. 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. 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…

43. Many Thanks to
You!

44. Questions?

45. Thank You