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Using Solid Phase Microextraction for Cannabis Testing
This document discusses the application of solid phase microextraction (SPME) in cannabis testing, highlighting its advantages in analyzing terpenes and residual solvents. It outlines the methodology, including the preparation of samples and the analysis process, while emphasizing the lack of standardized testing methods in the cannabis industry. The document includes details on specific terpenes identified in cannabis samples and the quantitative accuracy of the SPME approach compared to traditional solvent extraction methods.
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Using Solid Phase Microextraction for Cannabis Testing
- 1. Using Solid Phase Microextractionfor Cannabis Testing By: Katherine Stenerson Principal Scientist, Workflow R&D MilliporeSigma
- 2. MilliporeSigma is a businessof 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. Whatis 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 (accessed2/24/2017) 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 SPMEFiber 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:
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- 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 SPMEProcess • 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 AdsorptionMechanism for SPME Rapid uptake onto fiber
- 20. 20 Number of Molesof 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 Nameis 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 forTerpenes 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 usedfor 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 ofDried 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 profilingof 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 indried 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 usedfor 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 differentSPME 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 theSpiking 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 CompoundCalibration 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-SPMEcompare 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 oilproduced 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 Methodfor 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 ForResidual 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 – Toolsfor 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 Informationon 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. HariH. 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…
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