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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
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
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
Analyte
Adsorbed
Silica Rod
Liquid Polymer
Aqueous
Solution
Vial
Time
Adsorption Mechanism for SPME
Rapid uptake
onto fiber
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
Resp
on
se
Cal stds.
quantitate
unknown
21
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
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
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
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