Better Living Through (Sensory) Chemistry: Gas ... · a New Tool for Analysis of Terpenes and...

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