better living through (sensory) chemistry: gas ... · a new tool for analysis of terpenes and...
TRANSCRIPT
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
#emeraldconference© 2018 Emerald Conference
Acknowledgments
• Amanda Rigdon, Ken Snoke, Wes Burk, Cliff Beneventi, and others!• Emerald Scientific
• Julie Kowalski• Trace Analytics
#emeraldconference© 2018 Emerald Conference
Jack’s flight details forThe 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
Deuterium Lamp CCD Detector
Gas Chromatograph
~33 cm
~ 75 cm
~43 cm
Absorbance Spectrum
VUV Analytics VGA-100120 to 240nm
1 to 90 spectra/sec
Deuterium Lamp CCD Detector
Gas Chromatograph
~33 cm
~ 75 cm
~43 cm
Absorbance Spectrum
VUV Analytics VGA-100120 to 240nm
1 to 90 spectra/sec
VUV Absorbance Spectra (125 to 240 nm)
AcetoneBenzene
ButaneDichloromethane
Propane Butane
Pentane Hexane
VUV Absorbance Spectra125 to 240 nm
• 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
And more…
PDE is Permissible
Daily Exposure
Animal carcinogens and other
possible toxic effects
FDA 2012 companion document for the International Conference on Harmonisationof Technical Requirements for Registration of Pharmaceuticals for Human Use
Flame Ionization Detector
Headspace Transfer Line
Static Headspace Autosampler
The job of the static headspace instrument is to get solvent
(red circles) into gas phase for transfer to GC column.
GC Column
Gas Chromatograph
Slow equilibration
times.
GC run time of 60 min.
Static Headspace Conditions for GC-VUV
• Gerstel MPS2
• Syringe temperature: 90°C
• 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
Heating and shaking
Sampling and injection
Original sample
Equilibrated sample
Headspace phase
Sample phase
Water and Excipient
Solvents
+
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
• 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
Run time 8 min
TetralinMethanol
Cyclohexane
Methylcyclohexane
Toluene
m- and p-Xylenes
Dichloromethane
cis-1,2-Dichloroethene
o-XyleneCumene
Water
OxygenStatic Headspace GC-VUV
Class 2 Residual Solvents, USP Limit100 mg Citric Acid, 2 mL Water
125 – 160 nm
8 min
18
Dichloromethane
Static Headspace – GC-VUV
Class 2 Residual Solvents100 mg Citric Acid
2 mL Water
R2 = 0.9968
USP Limit
2x
0.5x02.5x
0.1x
Class 2 Residual Solvents100 mg Citric Acid
2 mL Water
Chlorobenzene
Ethylbenzene
m- and p-Xylenes
o-Xylene
2 x USP LimitUSP Limit
0.5 x USP Limit0.25 x USP Limit0.10 x USP Limit
Blank
1,1,1-Trichloroethane
Carbon tetrachloride
Benzene 1,2-Dichloroethane
3.19 min
Class 1 Residual SolventsStatic Headspace – GC-VUV
125 – 160 nm125 – 240 nm170 – 240 nm140 – 160 nm
Benzene
<<< 170 to 240 nm >>>
1,2-Dichloroethane
Vacuum Ultraviolet Absorbance Spectra
<<< 120 to 170 nm >>>
1,1,1-Trichloroethane
Carbon tetrachloride
Benzene
1,2-Dichloroethane3.19 min
Class 1 Residual SolventsStatic Headspace – GC-VUV
125 – 240 nm (full absorbance range)170 – 240 nm (aromatic absorbance range)Spectral Filter
1,1,1-Trichloroethane
Carbon tetrachloride
Benzene
1,2-Dichloroethane
130 – 145 nm125 – 160 nm145 – 155 nm
< - - - - - - - - - >
< - - - >< - >
No good spectral filter for 1,2-dichloroethane?
Benzene1,2-Dichloroethane
Benzene
1,2-Dichloroethane
VUV spectral deconvolution results in pure chromatographic peaks…
• Throat sprays• Phenol anesthetic
• Kids’ medicine• Acetominophen
• Herbal
• Allergy relief• Claritin®
• Immune support tablet• Similar to Airborne®
• Hangover relief• NSAID and caffeine
Samples Analyzed
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?
?
Water
Oxygen
Static Headspace – GC-VUV
Ethanol
Water
Oxygen
Static Headspace – GC-VUV
Sample spectrum (from Hangover)Library spectrum (ethanol)
Shatter• ~ 20-50 mg concentrate in a
headspace vial
• Full evaporation technique mitigates matrix effects
• Makes quantification easier (more accurate?)
Butane
iso-Pentane
Water
Ethanol
Acetone
2-Methylpentane
3-Methylpentane
HexaneBenzene
Heptane
Toluene
m-Xylenep-Xylene
Ethylbenzene
iso-Butane
o-Xylene
α-Pinene
β-Myrcene
β-Pinene
Limonene
Linalool
β-Caryophyllene
α-Humulene
Terpinolene
Ocimene Caryophyllene oxide
Butane α-Pinene
β-Myrcene
β-Pinene
Limonene
Linalool
β-Caryophyllene
Toluene
HeptaneHexane
m-Xylenep-Xylene
α-Humulene
• 5 µL steam-distilled cannabis flower extract and 2 µL gasoline
• FET static headspace – GC-VUV
• 20 min equilibration time
• 17 min GC time
Monoterpenes and Monoterpenoids
α-Pinene β-PineneEucalyptol
Linalool
β-Caryophyllene α-Humulene
(-)-Guaiol
Sesquiterpenes and Sesquiterpenoids
p-Cymene
Flavor, fragrance, medicine, “entourage effect” for cannabis
0
0.1
0.2
0.3
0.4
0.5
3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9
Det
ecto
r R
esp
on
se
Time (min)
1. α-Pinene 12. Terpinolene2. Camphene 13. Linalool3. β-Myrcene 14. Isopulegol4. β-Pinene 15. Geraniol5. 3-Carene 16. β-Caryophyllene6. α-Terpinene 17. α-Humulene7. cis-Ocimene 18. cis-Nerolidol8. Limonene 19. trans-Nerolidol9. p-Cymene 20. Guaiol10. trans-Ocimene 21. α-Bisabolol11. γ-Terpinene
12
3
4
5 6
7
8
9
10
11
12
13
14
15
1617
18
19 2021
125-160 nm
170-240 nm
Last analyte elutes before 9 min!
Static Headspace GC-VUV of Cannabis Terpenes Standard
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
9369
41
121 136
93
69
41
121 136
β-Myrcene
β-Pinene
Electron ionization mass spectra of coeluting terpene isomers
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
130 140 150 160 170 180 190 200 210 220 230 240
No
rmal
ized
Ab
sorb
ance
Wavelength (nm)
VUV Absorbance Spectra of Monoterpene Isomersα-Pinene β-Pinene β-Myrcene
Limonene cis-Ocimene trans-Ocimene
Spectral Deconvolution of Coeluting Peaks
Summary:• XXX
• XXX
• XXX
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
4.44 4.46 4.48 4.5 4.52 4.54 4.56 4.58 4.6 4.62 4.64 4.66
Det
ecto
r R
esp
on
se
Time (min)
125-240 nm Limonene
α-Terpinene p-Cymene
cis-Ocimene trans-Ocimene
Region 1 – Single Analyte Spectral Matching
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0.22
130 140 150 160 170 180 190 200 210 220 230 240
Ab
sorb
ance
Wavelength (nm)
Summed Retention Region Spectrum
Summed Target Spectrum Best Fit; R^2 = 0.99890
alpha-Terpinene
α-Terpinene
Region 2 – Summation of Spectra from Coelution #1
0
0.03
0.06
0.09
0.12
0.15
0.18
0.21
0.24
0.27
0.3
0.33
130 140 150 160 170 180 190 200 210 220 230 240
Ab
sorb
ance
Wavelength (nm)
Summed Retention Region Spectrum
Summed Target Spectra Best Fit; R^2 = 0.99923
alpha-Terpinene
cis-Ocimene
α-Terpinene
cis-Ocimene
Region 3 – Summation of Spectra from Coelution #2
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
130 140 150 160 170 180 190 200 210 220 230 240
Ab
sorb
ance
Wavelength (nm)
Summed Retention Region Spectrum
Summed Target Spectra Best Fit; R^2 = 0.99962
Limonene
p-Cymene
Limonene
p-Cymene
Region 4 – Single Analyte Spectral Matching
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
130 140 150 160 170 180 190 200 210 220 230 240
Ab
sorb
ance
Wavelength (nm)
Summed Retention Region Spectrum
Summed Target Spectrum Best Fit; R^2 = 0.99771
trans-Ocimene
trans-Ocimene
Static Headspace GC-VUV Linearity of Terpenes
y = 0.0343xR² = 0.9995
0
4
8
12
16
20
0 100 200 300 400 500
Pea
k A
rea
Concentration (ppm)
α-Pineney = 0.046x
R² = 0.9978
0
5
10
15
20
25
0 100 200 300 400 500
Peak
Are
a
Concentration (ppm)
Limonene
y = 0.0073xR² = 0.997
0
1
2
3
4
0 100 200 300 400 500
Peak
Are
a
Concentration (ppm)
Linalooly = 0.0308xR² = 0.9974
0
3
6
9
12
15
18
0 100 200 300 400 500Pe
ak A
rea
Concentration (ppm)
α-Humulene2.5 to 500 ppm
Essential Oils
Summary:• XXX
• XXX
• XXX
0
0.2
0.4
0.6
0.8
1
1.2
3 4 5 6 7 8
Eucalyptus
0
0.2
0.4
0.6
0.8
1
1.2
1.4
3 4 5 6 7 8
Peppermint
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
3 4 5 6 7 8
Lavender
0
0.2
0.4
0.6
0.8
1
1.2
1.4
3 4 5 6 7 8
Neroli
0
0.5
1
1.5
2
2.5
3 4 5 6 7 8
Sweet Orange
0
0.2
0.4
0.6
0.8
1
1.2
1.4
3 4 5 6 7 8
Tea Tree
1
21
2
3 12
3
1
3
2
3
41
1
23
1. α-Pinene2. Limonene3. Eucalyptol*
1. cis-Ocimene2. trans-Ocimene3. Linalool
1. β-Pinene2. Limonene3. Linalool
1. Limonene2. Eucalyptol* 3. Menthol 1*4. Menthol 2*
*non-target
1. Limonene 1. α-Pinene2. α-Terpinene 3. γ-Terpinene
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
• 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
Run time 12.6 min
α-Pinene
β-Myrcene
β-Pinene
Limonene
Linaloolβ-Caryophyllene
α-Humulene
Terpinolene
Caryophyllene oxide
Sample 7
Sample 17
Sample 15
Fenchol
Steam-Distilled Cannabis ExtractsGC-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
Terpenes (mg/mL) in Steam-Distilled Extracts of Cannabis (GC-VUV)
Sample 5
α-Pinene
β-Myrcene
β-Pinene
Limonene
Linalool β-Caryophyllene
α-HumuleneCaryophyllene oxide
Fenchol
Sample 16
Steam-Distilled Cannabis ExtractsGC-VUV
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5
Det
ecto
r R
esp
on
se
Time (min)
1
2
3
4
8
5,6 712
9-1114
16
27
28
1. α-Pinene
2. Camphene
3. β-Pinene
4. β-Myrcene
5. α-Phellandrene
6. 3-Carene
7. α-Terpinene
8. Limonene
9. cis-Ocimene
10. p-Cymene
11. Eucalyptol
13 1517
18
19
20
21,22
23,24
25 26 29
30,31
32
12. trans-Ocimene
13. γ-Terpinene
14. Terpinolene
15. Fenchone
16. Linalool
17. Fenchol
18. Isopulegol
19. Camphor
20. Isoborneol
21. Menthol
22. Borneol
23. Citronellol
24. Nerol
25. cis-Citral
26. Thymol
27. β-Caryophyllene
28. α-Humulene
29. trans-Nerolidol
30. Caryophyllene oxide
31. Guaiol
32. α-Bisabolol
Overlay of 17 Steam-Distilled Cannabis Extracts
3.5
min
11.5
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
#emeraldconference© 2018 Emerald Conference