Complete Workflow for Comprehensive Cannabis Terpenes Analysis

Workflow for the Analysis of Terpenes in Hemp and Cannabis

Introduction: Analysis of Terpenes in Cannabis and Hemp

Terpenes are a class of compounds responsible for the particular aroma or fragrance of the cannabis flower. The search of cannabis customers for a desired aroma or effect makes it important to determine the terpene profile of a cannabis strain. Terpenes have been proposed to enhance the therapeutic properties of cannabinoids, by what is referred to as the entourage effect. In the absence of current universal regulatory requirements for terpenes testing, this application note is to serve as a guide to the customers developing their own analysis for the desired terpene. Herein we propose a fast and efficient way to determine the terpene profile of hemp or cannabis flower using a simple sample preparation technique based on liquid extraction. This approach reduces the sample preparation time, solvent consumption, and the time to obtain analytical results. This workflow analyzes 31 cannabis-related terpenes by GC/MS in full scan mode and the use certified reference standards. Tridecane is used as the internal standard as it is not found in the samples and elutes in the middle of the chromatogram. The full scan spectra of the reference standard mix, calibration standards, and samples were searched and matched against the NIST GC/MS spectra library. The method can be easily modified and adapted to include additional terpenes of interest for their identification and quantitation with the same approach.

Chemical Structures of Terpene Analytes

Sample Preparation and Internal Standard Solution Preparation

Instructions for Hemp Flower Extract Preparation

Step	Instructions
1	Weigh 1.0 g ± 0.001 g of ground hemp flower sample into a 50 mL polypropylene centrifuge tube
2	Add 10 mL of GC grade ethyl acetate to each tube and cap
3	Sonicate for 15 minutes
4	Centrifuge the sample tube at 1252 x g for 5 minutes
5	Transfer 1 mL of supernatant to a 2 mL autosampler vial and cap

Instructions for the Preparation of Internal Standard Solutions

Step	Instructions
1	Add 3.31 µL of Tridecane into a 25 mL volumetric flask
2	Bring up to volume with GC grade ethyl acetate
3	Transfer 1 mL of Internal Standard (ISTD) solution into a 2 mL GC autosampler vial
4	Place in an appropriate autosampler location to perform sandwich injection addition of Internal Standard
	This makes a 100 ppm [µg/mL] ISTD solution

Instructions for the Preparation of Working Standard Solutions

Step	Instructions
1	Transfer 500 µL of Cannabis Terpenes Standard Mix A CRM [2000 µg/mL] to a 10 mL amber vial
2	Transfer 500 µL of Cannabis Terpenes Standard Mix B CRM [2000 µg/mL] to the same 10 mL amber vial above
3	Add 1.00 mL of GC grade ethyl acetate to the same 10 mL amber vial and cap
	This makes a 500 ppm [µg/mL] working standard solution to prepare dilutions for the instrument calibration curve using the calibration curve standard dilution scheme. Store the working standard solutions and any remaining CRM mixes according to the labeling guidance.

Calibration Curve Standard Dilution Scheme

Calibration curve standard dilution scheme. Working standard solution (WS): 500 µg/mL in GC-grade ethyl acetate Cal 1: To prepare, add 15 µL of Cal 7 (50 ppm) to 985 µL of GC-grade ethyl acetate

Level	Conc. [µg/mL, ppm]	µL of Solution	Solution	µL Ethyl Acetate to add
Cal 1	0.75	15	Cal 7	985
Cal 2	1	2	WS	998
Cal 3	2	4	WS	996
Cal 4	5	10	WS	990
Cal 5	10	20	WS	980
Cal 6	25	50	WS	950
Cal 7	50	100	WS	900
Cal 8	70	140	WS	860
Cal 9	100	200	WS	800

GC-MS Instrument Conditions

GC Conditions
Column	SLB®-5ms 20 m x 0.180 mm ID; 0.18 µM film thickness
Detector	GC-MS with 9 mm Drawout Lense
Inlet	300 °C
Column Temperature	45 °C for 2 minutes, then 10 °C/min to 140 °C, Hold at 140 °C for 0.5 minutes then 30°C/min to 300 °C Hold at 300 °C for 2 minutes
Flow	0.75 mL/min Constant Flow
Carrier gas	Helium
Liner	Single Taper FocusLiner™ with glass wool
Injection	2.0 µL – Pulsed Split 50:1
Sample Diluent	Ethyl Acetate
Sample Preparation	Solid-Liquid Extraction with Ethyl Acetate (1 g Cannabis + 10 mL Ethyl Acetate
Standard Solution	Dilutions of Cannabis Terpenes Mix A and Mix B in Ethyl Acetate (0.75- 100 ppm) with 100 ppm Tridecane as ISTD Sandwich Injection
Sample	1-gram hemp flower containing less than 0.3% THC

Mass Spectrometer Conditions

MS Conditions
Tuning	Auto-tune
Acquisition	Full Scan Mode (EI); 40-400 amu
Solvent Delay	4 min
MS Source Temperature	300 °C
Quadrupole Temperature	150 °C
Electron Energy	70 eV
Dwell Time	50 ms

TIC Chromatogram of Terpenes Standards Mix

Detection and Calibration

GC-MS Detection and Calibration of α-Pinene

Overlaid EIC of nine α-Pinene standards

Three injections of 50 mg/mL α-Pinene standard

Peak	Compound	Retention Time (min)	Quant (m/z)	Qual 1 (m/z)	Qual 2 (m/z)	Gain
1	α-Pinene	5.260	93	105	121	10

Level	α-Pinene (μg/mL)
1	0.75ppm
2	1ppm
3	2ppm
4	5ppm
5	10ppm
6	25ppm
7	50ppm
8	70ppm
9	100ppm

Standard Repeatability (0.75 μg/mL/)	Peak Area
STD1 Injection 1	3052
STD1 Injection 2	3262
STD1 Injection 3	3098
Mean	3137.60
Standard Deviation	110.37
RSD (%)	3.52

Calibration curve for α-Pinene standards from 0.75 µg/ml to 100 µg/ml

Linearity, LOD and LOQ
Concentration (μg/mL)	ISTD Resp. Ratio
0.75	0.016
1	0.032
2	0.079
5	0.183
10	0.374
25	0.916
50	1.814
70	2.499
100	3.589
LOD	0.25 μg/mL
LOQ	0.75 μg/mL

GC-MS Detection and Calibration of Camphene

Overlaid EIC of nine Camphene standards

Three injections of 50 mg/mL Camphene standard

Peak	Compound	Retention Time (min)	Quant (m/z)	Qual 1 (m/z)	Qual 2 (m/z)	Gain
2	Camphene	5.551	93	121	79	10

Level	α-Pinene (g/mL)
1	0.75ppm
2	1ppm
3	2ppm
4	5ppm
5	10ppm
6	25ppm
7	50ppm
8	70ppm
9	100ppm

Standard Repeatability (0.75 μg/mL/)	Peak Area
STD1 Injection 1	1702
STD1 Injection 2	1660
STD1 Injection 3	1632
Mean	1665
Standard Deviation	35.41
RSD (%)	2.13

Calibration curve for α-Terpinene standards from 0.75 µg/ml to 100 µg/ml

Linearity, LOD and LOQ
Concentration (μg/mL)	ISTD Resp. Ratio
0.75	0.007
1	0.017
2	0.043
5	0.107
10	0.234
25	0.616
50	1.256
70	1.7726
100	2.5716
LOD	0.25 μg/mL
LOQ	0.75 μg/mL

GC-MS Detection and Calibration of α-Terpinene

Overlaid EIC of nine α-Terpinene standards

Three injections of 50 mg/mL α-Terpinene standard

Peak	Compound	Retention Time (min)	Quant (m/z)	Qual 1 (m/z)	Qual 2 (m/z)	Gain
5	α-Terpinene	6.726	121	136	105	10

Level	α-Pinene (g/mL)
1	0.75ppm
2	1ppm
3	2ppm
4	5ppm
5	10ppm
6	25ppm
7	50ppm
8	70ppm
9	100ppm

Standard Repeatability (0.75 μg/mL/)	Peak Area
STD1 Injection 1	1702
STD1 Injection 2	1660
STD1 Injection 3	1632
Mean	1665
Standard Deviation	35.41
RSD (%)	2.13

Calibration curve for α-Terpinene standards from 0.75 µg/ml to 100 µg/ml

Linearity, LOD and LOQ
Concentration (μg/mL)	ISTD Resp. Ratio
0.75	0.007
1	0.017
2	0.043
5	0.107
10	0.234
25	0.616
50	1.256
70	1.7726
100	2.5716
LOD	0.25 μg/mL
LOQ	0.75 μg/mL

Tridecane ISTD Response Reproducibility

Level	Ret. Time [min]	Tridecane ISTD Response
1	11.050	196767
2	11.050	197806
3	11.051	197399
4	11.051	202600
5	11.051	207489
6	11.049	212668
7	11.049	217217
8	11.050	220822
9	11.050	220802
% RSD	0.006 %	4.82 %

Overlaid EIC chromatograms of 9 Tridecane ISTD Injections from calibration curve

TIC of Extracted Hemp Sample

Chromatogram of the Extracted Compound from Hemp Sample

Targeted MS Library Created from Cannabis Terpene CRM Mix A & B

MS Spectra of Peak at 13.082 min α-Humulene

Tabulated Results for 31 Cannabis Terpenes and IS

Peaks	Compound	CAS No.	Ret. Time [min]	Lib Match Factor	r2	Range [μg/mL]	Detected in hemp sample?
1	α-Pinene	80-56-8	5.257	98.99	0.99991	0.75-100	Y
2	Camphene	79-92-5	5.553	99.00	0.99990	0.75-100	Y
3	β-Pinene	127-91-3	6.049	99.17	0.99995	1.50-200	Y
4	3-Carene	13466-78-9	6.589	99.15	0.99992	1.50-200	Y
5	α-Terpinene	99-86-5	6.726	99.62	0.99987	0.75-100	Y
6	p-Cymene	99-87-6	6.856	99.58	0.99987	0.75-100	Y
7	Limonene	138-86-3	6.938	99.21	0.99997	1.50-200	Y
8	γ-Terpinene	99-85-4	7.422	99.35	0.99994	0.75-100	Y
9	Terpinolene	586-62-9	7.872	99.21	0.99990	0.75-100	N
10	L-Fenchone	7787-20-4	7.930	99.47	0.99989	0.75-100	Y
11	Linalool	78-70-6	8.101	99.37	0.99915	0.75-100	Y
12	Fenchol	2217-02-9	8.426	99.43	0.99996	0.75-100	Y
13	Camphor	76-22-2	8.866	99.57	0.99997	1.50-200	Y
14	Isoborneol	124-76-5	9.126	99.45	0.99983	0.75-100	Y
15	(+)-Borneol	464-43-7	9.259	99.36	0.99968	0.75-100	Y
16	DL-Menthol	89-78-1	9.331	99.52	0.99988	0.75-100	Y
17	α-Terpineol	10482-56-1	9.596	99.34	0.99942	0.75-100	Y
18	Citronellol	106-22-9	10.036	99.05	0.99961	0.75-100	Y
19	Pulegone	89-82-7	10.234	99.55	0.99956	0.75-100	Y
20	Geraniol	106-24-1	10.386	98.21	0.99946	0.75-100	Y
21	Geranyl Acetate	105-87-3	12.145	98.64	0.99980	0.75-100	Y
22	α-Cedrene	469-61-4	12.699	99.62	0.99997	0.75-100	N
23	β-Caryophellene	87-44-5	12.728	99.68	0.99953	0.75-100	Y
24	α-Humulene	6753-98-6	13.082	98.90	0.99975	0.75-100	Y
25	Nerolidol I	7212-44-4	13.636	98.73	0.99913	1.50-200	Y
26	Nerolidol II	7212-44-4	13.845	99.19	0.99905		Y
27	Cedrol	77-53-2	14.226	99.46	0.99978	0.75-100	Y
28	β-Eudesmol	473-15-4	14.471	99.60	0.99945	0.75-100	Y
29	α-Bisabolol	23089-26-1	14.594	99.62	0.99965	0.75-100	Y
30	Phytol I	7541-49-3	16.145	92.91	0.99975	0.75-100	Y
31	Phytol II	7541-49-3	16.225	92.91	0.99950		Y
ISTD	Tridecane	629-50-5	11.050	96.17	******	100 ppm

Conclusion

A fast, efficient, and highly reproducible analytical method was developed for the quantitative analysis of 31 cannabis-related terpenes using GC-MS. Supelco^® chromatography consumables, solvents, supplies, analytical reagents, and certified reference standards in combination with GC-MS provide an efficient way to analyze cannabis products for terpenes to meet the current cannabis product labeling requirements. Low quantitation levels of 0.75 µg/g (750ppb) are easily achieved. Also obtaining all the consumables and reagents from one supplier ensures that time is well spent in analyzing and running samples rather than finding multiple sources for the required analytical supplies. The use of a 20m x 0.18mm x 0.18 μM GC column provides the greatest chromatographic resolution of terpene analytes and facilitates the analysis of additional analytes in the future, if necessary.