Better Living Through (Sensory) Chemistry: Gas Chromatography - Vacuum Ultraviolet Spectroscopy as a New Tool for Analysis of Terpenes and Residual Solvents in Cannabis Products Jack Cochran, Lindsey Shear-Laude, Alex Hodgson VUV Analytics
© 2018 Emerald Conference #emeraldconference Acknowledgments
• Amanda Rigdon, Ken Snoke, Wes Burk, Cliff Beneventi, and others! • Emerald Scientific
• Julie Kowalski • Trace Analytics
© 2018 Emerald Conference #emeraldconference Jack’s flight details for The Emerald Conference… Presentation Outline
• Introduction to vacuum ultraviolet spectroscopy and VGA-100
• Analysis of residual solvents and terpenes with GC-VUV
• Possibility of combined solvents and terpenes analysis Vacuum Ultraviolet Detector for GC
• Absorbance spectrometer • 120 to 240 nm (“everything” absorbs in the 120 to 200 nm range) • Detects things that are problematic for other detectors • Formaldehyde, formic acid, water, carbon dioxide, carbon monoxide, etc. • Good detectability • Low tens to low hundreds pg depending on compound • Qualitative and quantitative analysis • Absorbance spectral library and Beer-Lambert Law (A ∝ c) • “Separation” of coeluting analytes, including isomers • Spectral filters and spectral deconvolution VUV Analytics VGA-100 Gas Chromatograph 120 to 240nm 1 to 90 spectra/sec
~ 75 cm
~43 cm Deuterium Lamp CCD Detector
Absorbance Spectrum
~33 cm VUV Analytics VGA-100 Gas Chromatograph 120 to 240nm 1 to 90 spectra/sec
~ 75 cm
~43 cm Deuterium Lamp CCD Detector
Absorbance Spectrum
~33 cm VUV Absorbance Spectra (125 to 240 nm) Benzene Acetone
Dichloromethane Butane VUV Absorbance Spectra 125 to 240 nm
Propane Butane
Pentane Hexane • Sample fails quality assurance testing if results exceed table limits • Residual solvent results of more than 5000 ppm for class three solvents, 50 ppm for class two solvents, and 2 ppm for class one solvents fail • Certified labs must test for the solvents in table at a minimum Animal carcinogens and other possible toxic effects
PDE is Permissible Daily Exposure
FDA 2012 companion document for the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use
And more… The job of the static headspace instrument is to get solvent (red circles) into gas phase for Headspace transfer to GC column. Transfer Line Static Headspace Flame Ionization Detector Autosampler
GC Column
Gas Chromatograph Slow equilibration times.
GC run time of 60 min. Static Headspace Conditions for GC-VUV
• Gerstel MPS2 Sampling • Syringe temperature: 90°C Heating and and injection shaking • Incubation temperature: 80°C • Incubation time: 10 min • Agitator: 250 rpm • 10 sec on, 1 sec off • Injection volume: 250 µL • Injection speed: 200 µL/sec Headspace phase
Sample phase
Solvents Original Equilibrated sample + sample Water and Excipient GC-VUV for Residual Solvents
• Agilent 6890 GC • 2mm ID Topaz straight inlet liner, 250°C, split ratio 2.5 • 30m x 0.25mm x 1.40µm Rxi-624Sil MS • He 4 mL/min constant flow Run time 8 min • 35°C (1 min), 30°C/min to 245°C • VUV Analytics VGA-100 • Transfer line and flow cell 275°C • Nitrogen makeup gas ~0.36 psi • Acquisition range 125 to 240 nm • Acquisition rate 4.5 spectra/sec Cyclohexane Static Headspace GC-VUV Oxygen Class 2 Residual Solvents, USP Limit 100 mg Citric Acid, 2 mL Water Water
m- and p-Xylenes
Methylcyclohexane 125 – 160 nm Toluene
cis-1,2-Dichloroethene
Dichloromethane o-Xylene Tetralin Methanol Cumene 8 min Dichloromethane
R2 = 0.9968 2x
USP Limit Class 2 Residual Solvents 100 mg Citric Acid 2 mL Water 02.5x 0.5x Static Headspace – GC-VUV 0.1x
18 Class 2 Residual Solvents 2 x USP Limit 100 mg Citric Acid USP Limit 2 mL Water m- and p-Xylenes 0.5 x USP Limit 0.25 x USP Limit Ethylbenzene 0.10 x USP Limit Blank Chlorobenzene
o-Xylene Class 1 Residual Solvents Static Headspace – GC-VUV 1,1,1-Trichloroethane
125 – 160 nm 125 – 240 nm 170 – 240 nm 140 – 160 nm Carbon tetrachloride Benzene 1,2-Dichloroethane
3.19 min Vacuum Ultraviolet Absorbance Spectra
1,2-Dichloroethane Benzene
<<< 120 to 170 nm >>> <<< 170 to 240 nm >>> Class 1 Residual Solvents Static Headspace – GC-VUV
125 – 240 nm (full absorbance range) Spectral Filter 170 – 240 nm (aromatic absorbance range)
1,1,1-Trichloroethane Benzene
3.19 min Carbon tetrachloride 1,2-Dichloroethane 1,1,1-Trichloroethane 1,2-Dichloroethane Benzene < ------> < - - - > 130 – 145 nm < - > 125 – 160 nm 145 – 155 nm
Carbon tetrachloride No good spectral filter for 1,2-dichloroethane? 1,2-Dichloroethane
Benzene VUV spectral deconvolution results Benzene in pure chromatographic peaks…
1,2-Dichloroethane Samples Analyzed • Throat sprays • Phenol anesthetic • Kids’ medicine • Acetominophen • Herbal • Allergy relief • Claritin® • Immune support tablet • Similar to Airborne® • Hangover relief • NSAID and caffeine https://firstaidshottherapy.com/
Hangover Drug Facts - Uses
• For temporary relief of minor aches and pains associated with a hangover
• Helps restore mental alertness or wakefulness when experiencing fatigue or drowsiness associated with a hangover
• Also for temporary relief of headaches or body aches and pains alone Hangover Drug Facts - Ingredients
• Choline salicylate (NSAID) • Pain reliever • Caffeine • Pain reliever aid • Citric acid, sodium citrate, sodium benzoate • Natural flavors, purified water • Sucralose (sweetener)
• Anything else? Oxygen
Water Static Headspace – GC-VUV ? Oxygen
Water Static Headspace – GC-VUV
Ethanol Sample spectrum (from Hangover) Library spectrum (ethanol) • ~ 20-50 mg concentrate in a headspace vial Shatter • Full evaporation technique mitigates matrix effects • Makes quantification easier (more accurate?) Water Acetone
Ethanol iso-Pentane
Butane
m-Xylene p-Xylene 2-Methylpentane Toluene iso-Butane 3-Methylpentane Benzene Hexane Heptane Ethylbenzene o-Xylene β-Myrcene β-Pinene Limonene
β-Caryophyllene
α-Pinene
Terpinolene
α-Humulene Linalool
Ocimene Caryophyllene oxide β-Myrcene
• 5 µL steam-distilled cannabis flower extract and 2 µL gasoline
• FET static headspace – GC-VUV β-Pinene • 20 min equilibration time • 17 min GC time Limonene β-Caryophyllene
Butane α-Pinene α-Humulene
Linalool Heptane m-Xylene Hexane Toluene p-Xylene
Sesquiterpenes and Monoterpenes and Monoterpenoids Sesquiterpenoids
α-Pinene β-Pinene Eucalyptol β-Caryophyllene α-Humulene
p-Cymene Linalool
Flavor, fragrance, medicine, “entourage effect” for cannabis (-)-Guaiol 0.5 7 125-160 nm 11 Static Headspace GC-VUV of 8 170-240 nm 3 Cannabis Terpenes Standard 6 9 0.4 5
4 1. α-Pinene 12. Terpinolene 17 12 2. Camphene 13. Linalool 16 1 3. β-Myrcene 14. Isopulegol 2 0.3 4. β-Pinene 15. Geraniol 10 5. 3-Carene 16. β-Caryophyllene 6. α-Terpinene 17. α-Humulene 7. cis-Ocimene 18. cis-Nerolidol 8. Limonene 19. trans-Nerolidol Last analyte elutes Detector Response Detector 0.2 9. p-Cymene 20. Guaiol before 9 min! 10. trans-Ocimene 21. α-Bisabolol 11. γ-Terpinene
13 0.1 19 20 14 18 21 15
0 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 Time (min) Monoterpene tR min Formula MW Sources
α-Pinene 3.87 C10H16 136.23 Coniferous trees, rosemary, eucalyptus
Camphene 4.03 C10H16 136.23 Camphor, neroli, valerian
β-Myrcene 4.21 C10H16 136.23 Thyme, cardamom, hops, cannabis
β-Pinene 4.25 C10H16 136.23 Nutmeg, rosemary, sage
Δ-3-Carene 4.43 C10H16 136.23 Turpentine, rosemary, cedar
α-Terpinene 4.50 C10H16 136.23 Allspice, juniper, marjoram
cis-Ocimene 4.53 C10H16 136.23 Basil, lavender, clary sage
Limonene 4.58 C10H16 136.23 Citrus fruits, mint trans-Ocimene 4.64 C10H16 136.23 Basil, lavender, clary sage
γ-Terpinene 4.77 C10H16 136.23 Citrus fruits, cumin, Syrian oregano
Terpinolene 4.98 C10H16 136.23 Allspice, citrus fruits, juniper 41 93 Electron ionization 69 β-Myrcene mass spectra of coeluting terpene isomers
121 136
93 β-Pinene 41
69
121 136 VUV Absorbance Spectra of Monoterpene Isomers 1 α-Pinene β-Pinene β-Myrcene 0.9 Limonene cis-Ocimene trans-Ocimene 0.8
0.7
0.6
0.5
0.4 Normalized Absorbance Normalized 0.3
0.2
0.1
0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm) Spectral Deconvolution of Coeluting Peaks
0.4 125-240 nm Limonene 0.35 α-Terpinene p-Cymene
0.3 cis-Ocimene trans-Ocimene
0.25
0.2
Detector Response Detector 0.15
0.1
0.05 Summary: • XXX 0 • XXX 4.44 • XXX4.46 4.48 4.5 4.52 4.54 4.56 4.58 4.6 4.62 4.64 4.66 Time (min) Region 1 – Single Analyte Spectral Matching
0.22
0.2
0.18
0.16
0.14
0.12
0.1 Absorbance 0.08 α-Terpinene
0.06
0.04 Summed Retention Region Spectrum Summed Target Spectrum Best Fit; R^2 = 0.99890 0.02 alpha-Terpinene 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm) Region 2 – Summation of Spectra from Coelution #1
Summed Retention Region Spectrum 0.33 Summed Target Spectra Best Fit; R^2 = 0.99923 0.3 alpha-Terpinene
0.27 cis-Ocimene
0.24
0.21
0.18
0.15 α-Terpinene Absorbance
0.12
0.09
0.06
0.03 cis-Ocimene
0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm) Region 3 – Summation of Spectra from Coelution #2
1.2 Summed Retention Region Spectrum
1.1 Summed Target Spectra Best Fit; R^2 = 0.99962
1 Limonene
0.9 p-Cymene
0.8
0.7
0.6
Absorbance 0.5
0.4 Limonene
0.3
0.2 0.1 p-Cymene 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm) Region 4 – Single Analyte Spectral Matching
0.5
0.45
0.4
0.35
0.3
0.25 Absorbance 0.2 0.15 trans-Ocimene 0.1 Summed Retention Region Spectrum Summed Target Spectrum Best Fit; R^2 = 0.99771 0.05 trans-Ocimene 0 130 140 150 160 170 180 190 200 210 220 230 240 Wavelength (nm) Static Headspace GC-VUV Linearity of Terpenes
α-Pinene Limonene 20 y = 0.0343x 25 y = 0.046x R² = 0.9995 R² = 0.9978 16 20
12 15
8 10
Peak Area Peak Peak Area Peak 4 5
0 0 0 100 200 300 400 500 0 100 200 300 400 500 Concentration (ppm) Concentration (ppm) 2.5 to 500 ppm Linalool α-Humulene 4 y = 0.0073x 18 y = 0.0308x R² = 0.997 15 R² = 0.9974 3 12
2 9 Peak Area Peak Peak Area Peak 6 1 3 0 0 0 100 200 300 400 500 0 100 200 300 400 500 Concentration (ppm) Concentration (ppm) *non-target Eucalyptus Lavender Neroli 1.2 3 2 3 1.4 1 1. α-Pinene 1. cis-Ocimene 2 1. β-Pinene 1.8 3 2. Limonene 2. trans-Ocimene 1.2 2. Limonene 1 Essential Oils 2 3. Eucalyptol* 1.6 3. Linalool 3. Linalool 1 1.4 1 0.8 2 1.2 0.8 1 0.6 1 0.6 0.8 0.4 0.6 0.4 0.4 0.2 0.2 0.2
0 0 0 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8 Peppermint Sweet Orange Tea Tree 1.4 3 1 1.4 3 2.5 4 1. Limonene 1. Limonene 2 1. α-Pinene 2 1.2 2. Eucalyptol* 1.2 2. α-Terpinene 3. Menthol 1* 2 3. γ-Terpinene 1 1 4. Menthol 2* 1
0.8 0.8 1.5 1
0.6 0.6 1 0.4 0.4 Summary:
• XXX 0.5 0.2 0.2 • XXX 0 0 • XXX 0 3 4 5 6 7 8 3 4 5 6 7 8 3 4 5 6 7 8
Analysis of Steam-Distilled Cannabis Extracts
• Agilent 6890 GC • 4mm Precision split liner with wool, 250°C, 1 µL, split ratio 10 or 100 • 30m x 0.25mm x 0.25µm Rxi-1301Sil MS • He 2 mL/min constant flow Run time 12.6 min • 40°C (0.1 min), 16°C/min to 240°C • VUV Analytics VGA-100 • Transfer line and flow cell 275°C • Nitrogen makeup gas 0.25 psi • Acquisition range 125 to 240 nm • Acquisition rate 5 spectra/sec β-Myrcene Sample 7 Steam-Distilled Cannabis Extracts GC-VUV Limonene β-Pinene β-Caryophyllene α-Pinene Linalool Caryophyllene oxide α-Humulene
Sample 17
Terpinolene Fenchol
Sample 15 Terpenes (mg/mL) in Steam-Distilled Extracts of Cannabis (GC-VUV)
Terpene 5 11 3 7 17 15 9 6 12 α-Pinene 31.0 28.9 28.6 21.5 19.4 19.2 16.5 15.0 14.5 β-Pinene 39.1 31.6 26.0 21.8 25.3 18.9 14.6 18.6 15.6 β-Myrcene 79.2 95.1 77.0 56.9 57.2 67.2 40.9 34.1 48.3 Limonene 101 39.6 31.3 31.7 67.7 21.9 12.5 67.9 18.1 cis-Ocimene 2.30 4.22 3.04 2.09 4.41 2.24 2.53 1.59 5.74 trans-Ocimene 0.891 1.27 1.37 0.929 5.06 0.903 0.901 0.584 2.31 Terpinolene 0.531 1.41 1.09 0.683 7.37 1.390 1.30 0.252 8.51 Linalool 26.8 5.32 10.1 7.40 4.73 2.78 3.68 23.2 6.26 Fenchol 15.7 9.67 4.92 4.91 6.91 1.43 2.01 14.1 2.21 β-Caryophyllene 42.6 29.6 15.0 20.3 52.9 28.5 11.4 30.0 11.8 α-Humulene 9.03 6.07 3.44 44.2 9.31 7.17 2.76 6.48 2.62 Caryophyllene oxide 34.8 13.1 7.10 12.8 0.970 1.39 5.84 16.6 3.12 Steam-Distilled Cannabis Extracts Sample 5 GC-VUV
Limonene β-Myrcene
β-Caryophyllene β-Pinene Linalool α-Pinene Caryophyllene oxide Fenchol α-Humulene
Sample 16 Overlay of 17 Steam-Distilled Cannabis Extracts 1.6 4 1. α-Pinene 12. trans-Ocimene 23. Citronellol 2. Camphene 13. γ-Terpinene 24. Nerol 1.4 3. β-Pinene 14. Terpinolene 25. cis-Citral 4. β-Myrcene 15. Fenchone 26. Thymol 1.2 5. α-Phellandrene 16. Linalool 27. β-Caryophyllene 6. 3-Carene 17. Fenchol 28. α-Humulene 1 7. α-Terpinene 18. Isopulegol 29. trans-Nerolidol 8 8. Limonene 19. Camphor 30. Caryophyllene oxide 1 3 20. Isoborneol 0.8 9. cis-Ocimene 31. Guaiol 10. p-Cymene 21. Menthol 32. α-Bisabolol 11. Eucalyptol 22. Borneol Detector Response Detector 0.6 27
0.4 9-11 16 14 21,22 30,31 0.2 19 23,24 28 12 17 2 18 25 5,6 7 13 15 20 26 29 32 0 3.53.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.511.5 min Time (min) min Summary • GC-VUV is a new tool for analysis of residual solvents and terpenes in cannabis samples • Absorbance spectrum provides authoritative identification • VUV absorbance spectra deconvolution promotes accurate qualitative and quantitative work • Also opens the door for faster chromatography • Possibility of combo solvents/terpenes method with GC-VUV • Needs testing on “real world” products
© 2018 Emerald Conference #emeraldconference