CUSTOMER APPLICATION NOTE 000108

Quantitation of seventeen cannabinoids

in dried cannabis, hemp and vape oils
by LC-MS/MS
Complete CANNABIS TESTING SOLUTIONS for the
Canadian market from one trusted source
Authors: Garnet McRae and Jeremy E.
Melanson, National Research Council
of Canada

Keywords: Cannabinoids, Cannabis, Hemp,

Vape oils, LC-MS/MS, Quantitation, Validation

Goal
Demonstrate accurate and precise quantitation of
seventeen cannabinoids extracted from cannabis and
hemp dried plant materials, in addition to vape oils with
LC-MS/MS detection on a Thermo Scientific™ Vanquish™
Flex UHPLC System coupled to a Thermo Scientific™ quality demands, and the ever-changing regulatory
TSQ Altis™ Triple Quadrupole Mass Spectrometer. requirements.1-3 Liquid chromatography with UV detection
(LC-UV) has been used predominantly for this application,
Introduction however it generally lacks performance when analyzing a
Regulated cannabis markets have seen sharp increases large suite of cannabinoids or those at very low abundance
in production as jurisdictions around the world continue (e.g., minor neutral and acidic cannabinoids).
to legalize cannabis for medicinal and adult recreational
use. At the same time, the hemp industry produces large By contrast, liquid chromatography—tandem mass
amounts of hemp for CBD production, textiles and other spectrometry (LC-MS/MS) is capable of specific and
uses that must adhere to Δ9-THC regulatory limits. The sensitive quantitation of low level analytes in complex
need for reliable analytical methods able to accurately matrices, and is well-suited for the analysis of large sets of
quantitate cannabinoids in cannabis, hemp, and vape oils cannabinoids in cannabis, hemp, and vape oils. While more
has increased in step with the increases in production, complex and costly than LC-UV, LC-MS/MS is a viable
alternative when accurate quantification is required for Vape oil sample preparation: vape oils were removed from
regulatory purposes. The method outlined below is able the cartridges and sampled (25 mg). Sample extraction
to quantitate the major cannabinoids, Δ9-THC, Δ9-THCA, was performed twice using 5 mL of a mixture of methanol
CBD and CBDA, and also the lower concentration minor and chloroform (90:10, v/v) with centrifugation of the
cannabinoids, Δ8-THC CBN, CBNA, CBG, CBGA, CBC, samples after each extraction. The supernatants were
CBCA, THCV, THCVA, CBDV, CBDVA, CBL and CBLA.4 retained, combined, mixed and an aliquot was diluted
Detailed method parameters are provided and performance appropriately with methanol. For example, a 1/250 dilution
characteristics are described. of supernatant allows quantitation over a range of 1.0 to
1000 mg/g (0.1 to 100%) cannabinoid content.
Experimental
Calibration standards (STD) and quality control (QC) After extraction and dilution, aliquots (100 µL) of the
samples, containing all 17 cannabinoids (Cerilliant®), were diluted supernatants and each STD and QC sample were
prepared in methanol over a range of 10–10,000 ng/mL transferred to HPLC vials containing glass inserts followed
(Table 1). by the addition of internal standard (50 µL, 500 ng/mL
THC-d3, CBD-d3, CBN-d3 and THCA-d3 in methanol).
Cannabis and hemp sample preparation: dried cannabis
and hemp samples were cryo-ground to a homogeneous Analysis was originally performed on a high-end legacy
powder, using a SPEX Freezer/Mill™, and subsampled LC-MS/MS platform and later transferred to the TSQ Altis
(100 mg). Sample extraction was performed twice using system with a Vanquish UHPLC system. UPLC gradient
5 mL of a mixture of methanol and water (80:20, v/v). separation was performed on a Thermo Scientific™
The samples were centrifuged after each extraction, with Accucore™ C18 column, with a guard column of the same
both supernatants retained and combined. The combined phase and mobile phases consisting of 0.1% formic acid in
supernatants were mixed and an aliquot was diluted water and acetonitrile at a flow rate of 0.5 mL/min (Tables 2
appropriately with methanol. For example, a 1/300 dilution and 3). The mass spectrometer was operated in positive
of supernatant allows quantitation over a range of 0.3 to ion mode and selective reaction monitoring mode (SRM).
300 mg/g (0.03 to 30%) cannabinoid content. Two ion transitions were monitored for each cannabinoid
for quantitation and confirmation (Table 4 and 5).
Table 1. Calibration standards and QC samples
Standard/QC Conc. (ng/mL)
STD-7 10,000
STD-6 9,000
STD-5 6,000
STD-4 1,000
STD-3 100
STD-2 20
STD-1 10
STD-0 0
QC-3 8,000
QC-2 1,500
QC-1 30

Table 2a

Vanquish Flex UHPLC Components Part number Specifics

Vanquish Autosampler VF-A10-A Split Sampler FT
Vanquish Pump VF-P10-A Binary Pump F (150 µL mixer)
Vanquish Column Compartment VH-C10-A Forced or still air heating/cooling option

2
Table 2b. Chromatography parameters Table 5. MS/MS parameters
Parameter Setting IS used Q1 Q3 RF CE
Column Accucore C18, 150 × 2.1 mm, 2.6 µm Name for quant (m/z) (m/z) (V) (eV)
Guard column Accucore C18, 10 × 2.1 mm, 2.6 µm CBDVA CBN-d3 313.2 191.1 95 26
Mobile phase A 0.1% Formic acid in water 313.2 233.1 95 19
Mobile phase B 0.1% Formic acid in ACN CBDV CBN-d3 287.2 165.1 58 22
Flow rate 0.5 mL/min 287.2 123.1 58 31
Run time 18.0 min CBDA THCA-d3 341.2 219.1 105 26
Column temperature 40°C 341.2 261.1 105 19
Injection volume 1 µL CBGA CBN-d3 343.2 219.1 72 22
Needle wash ACN:MeOH:Water:FA; 40:40:20:1 343.2 149.1 72 38
Autosampler temperature 4°C CBG CBN-d3 317.2 193.1 57 16
317.2 123.1 57 32
THCV CBN-d3 287.2 165.1 58 22
287.2 123.1 58 31
Table 3. UHPLC gradient CBD CBD-d3 315.2 193.1 62 22
Time (min) %A %B 315.2 123.1 62 31
0.0 40 60 THCVA CBN-d3 313.2 191.1 95 26
8.0 32 68 313.2 233.1 95 21
13.5 32 68 CBN CBN-d3 311.2 223.1 71 20
13.6 5 95 311.2 241.1 71 18
14.5 5 95 CBNA THCA-d3 337.2 235.1 156 28
14.6 40 60 337.2 253.1 156 23
18.0 40 60 Δ9-THC THC-d3 315.2 193.1 62 22
315.2 123.1 62 31
Δ8-THC THC-d3 315.2 193.1 62 22
Table 4. Mass spectrometer parameters 315.2 123.1 62 31
CBL CBN-d3 315.2 235.1 55 17
Parameter Setting
315.2 81.1 55 30
Scan type SRM
THCA THCA-d3 341.2 219.1 125 26
Ion source HESI
341.2 261.1 125 21
Polarity Positive
CBC CBN-d3 315.2 193.1 62 18
Spray voltage 4000 V
315.2 259.1 62 14
Sheath gas 50
CBLA THCA-d3 359.2 261.1 67 25
Aux gas 25
359.2 219.1 67 32
Sweep gas 2
CBCA THCA-d3 341.2 219.1 111 25
CID 1.5 mTorr
359.21 219.1 60 25
Ion transfer tube
325°C CBD-d3 — 318.2 196.1 62 22
temperature
Vaporizer temperature 280°C CBN-d3 — 314.2 223.1 71 20
THC-d3 — 318.2 196.1 62 22
THCA-d3 — 344.2 222.1 120 26
quantitation ion
confirmation ion

3
Results and discussion and 15 minor cannabinoids, while a vape oil sample
This method has been described previously, and is chromatogram (Figure 5) shows the presence of neutral
presented here in a modified form. Full validation results are cannabinoids only. The acidic cannabinoids form both
available in the corresponding peer reviewed publication.4 the molecular ion [M+H]+ and a water loss species [M+H-
Calibration standard ion chromatograms (Figure 1) show H2O]+ in positive ionization mode. Monitoring either or both
separation of all cannabinoid critical pairs (within ±2 m/z), species can be used to exploit differences in sensitivity
while cannabis sample chromatograms dominated by to enhance the accuracy of quantitation of closely eluting
THCA (Figure 2) and CBDA (Figure 3) show the ability of cannabinoids.
the method to efficiently quantitate low levels of minor
cannabinoids in the presence of high levels of major Representative calibration curves (Figures 6 and 7) show
cannabinoids. A hemp sample chromatogram (Figure 4) linearity, while precision (RSD) and accuracy (Acc.) using
shows chromatographic separation of a sample containing QC samples, a cannabis certified reference material (CRM)
16 cannabinoids with CBD as a major component and hemp CRM, are shown in Table 6.

Table 6. Statistical results for calibration curves, QC samples and matrix CRM samples*
QC-1 QC-2 QC-3 Cannabis HEMP
30 ng/mL 1500 ng/mL 8000 ng/mL CRM CRM
Analyte r 2 RSD (%) Acc. (%) RSD (%) Acc. (%) RSD (%) Acc. (%) RSD (%) Acc. (%) RSD (%) Acc. (%)
CBDVA 0.9997 1.2 108.4 1.7 105.5 1.0 99.8 4.1 101.0 1.6 100.1
CBDV 0.9997 1.5 107.3 1.4 103.1 0.5 94.8 3.9 100.4 1.4 94.4
CBDA 0.9996 1.1 102.1 1.6 100.8 1.6 93.4 5.1 101.1 1.5 97.1
CBGA 0.9998 0.8 101.3 2.1 99.7 1.8 91.7 5.1 94.9 1.7 95.7
CBG 0.9954 1.2 112.8 0.9 109.4 0.9 92.0 4.8 100.3 2.0 96.1
THCV 0.9973 2.7 110.6 0.9 105.4 1.2 91.1 4.9 92.6 1.9 104.8
CBD 0.9992 1.5 108.2 1.0 101.2 0.4 94.7 4.7 101.3 1.2 96.2
THCVA 0.9992 1.9 110.5 2.2 109.3 0.8 104.5 3.8 106.5 1.1 97.3
CBN 0.9996 1.9 103.2 0.7 100.7 1.0 96.7 4.3 112.2 1.9 90.4
CBNA 0.9982 2.5 111.6 0.9 113.2 1.1 95.2 4.0 108.1 1.8 107.0
Δ9-THC 0.9990 1.4 105.2 0.9 103.6 1.0 93.9 4.9 96.2 1.6 102.1
Δ8-THC 0.9993 0.7 106.6 0.3 103.7 1.1 94.3 N/AP N/AP N/AP N/AP
CBL 0.9995 0.7 105.4 0.6 101.7 0.9 95.1 N/AP N/AP 1.4 90.5
Δ9-THCA 0.9995 1.3 106.4 0.7 102.5 1.0 94.2 4.4 95.9 1.1 92.0
CBC 0.9984 2.3 104.0 0.9 101.8 0.8 101.0 3.1 100.0 2.1 90.4
CBLA 0.9992 2.9 103.3 1.8 104.6 1.5 103.9 N/AP N/AP 1.5 103.7
CBCA 0.9990 2.4 106.2 4.2 104.5 1.9 107.8 5.1 97.3 3.1 98.4
*This data was originally acquired on a high-end legacy LC-MS/MS platform, and was later transferred to the TSQ Altis which provided equal or better method
performance.

4
9.0e5
counts
4
8.0e5 1. CBDVA 7. CBD/CBD-d3 13. CBL
2. CBDV 8. THCVA 14. Δ9-THCA
3. CBDA 9. CBN/CBN-d3 15. CBC
7.0e5 4. CBGA 10. CBNA 16. CBLA
5. CBG 11. Δ9-THC/Δ9-THC-d3 17. CBCA
6. THCV 12. Δ8-THC
6.0e5

1
5.0e5
5 13
3
4.0e5

3.0e5 8 9
2 6
2.0e5 11 14
7 12

1.0e5 10 15 16 17

0.0e0
-5.0e4
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

Time (minutes)

Figure 1. LC-MS/MS ion chromatogram of a calibration standard containing 17 cannabinoids

2.0e5
counts
4 14
1.8e5

1.5e5

1.3e5
9-IS 11

1.0e5

7.5e4 7-IS
11-IS

5.0e4
5

2.5e4 8
3
9 10 17

0.0e0

-2.0e4
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

Time (minutes)

Figure 2. LC-MS/MS chromatogram of a cannabis sample with high THCA content

5
2.0e5
counts
3 1. CBDVA 7. CBD/CBD-d3 13. CBL
1.8e5 2. CBDV 8. THCVA 14. Δ9-THCA
3. CBDA 9. CBN/CBN-d3 15. CBC
4. CBGA 10. CBNA 16. CBLA
1.5e5 7 5. CBG 11. Δ9-THC/Δ9-THC-d3 17. CBCA
6. THCV 12. Δ8-THC
1.3e5
9 (IS)
4
1.0e5
14
11 (IS)
7.5e4 1 7-IS

5.0e4

5 17
2.5e4 11
2 15
0.0e0

-2.0e4
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

Time (minutes)

Figure 3. LC-MS/MS chromatogram of a cannabis sample with high CBDA content

1 3 7
9.0E+5

7.0E+5

5.0E+5
14

3.0E+5 9
4
11
2 13
1.0E+5 5 8 16 17
15
10

-1.0E+5
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

Time (minutes)

Figure 4. LC-MS/MS chromatogram of a hemp sample with high CBDA content

6
 5

11
2.8E+5

1. CBDVA 7. CBD/CBD-d3 13. CBL

2. CBDV 8. THCVA 14. Δ9-THCA
2.3E+5 3. CBDA 9. CBN/CBN-d3 15. CBC
4. CBGA 10. CBNA 16. CBLA
5. CBG 11. Δ9-THC/Δ9-THC-d3 17. CBCA
1.8E+5
6. THCV 12. Δ8-THC

1.3E+5
7

8.0E+4 9

6 15
3.0E+4 12

-2.0E+4
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0

Time (minutes)

Figure 5. LC-MS/MS chromatogram of a vape oil sample showing neutral cannabinoids without their corresponding acidic components and
high THC content

THC 315-193 0.9995 THCA 341-219 0.9990

5000 8000
% ISTD % ISTD

+ Calibration Standard + Calibration Standard

QC Sample 7000 QC Sample

4000
6000

5000
3000

4000

2000
3000

2000
1000

1000

ng/mL ng/mL
0 0
0 2000 4000 6000 8000 10000 12000 0 2000 4000 6000 8000 10000 12000

Figure 6. Calibration curve regression for THC and THCA

7
 CBD 315-193 0.9991 CBDA 341-219 0.9994
6000 14000
% ISTD % ISTD

+ Calibration Standard + Calibration Standard

QC Sample 12000 QC Sample
5000

10000
4000

8000

3000

6000

2000
4000

1000
2000

ng/mL ng/mL
0 0
0 2000 4000 6000 8000 10000 12000 0 2000 4000 6000 8000 10000 12000

Figure 7. Calibration curve regression for CBD and CBDA

Conclusion References
1. Sarma ND, Waye A, ElSohly MA, Brown PN, Elzinga S, Johnson HE, et al. Cannabis
The method described has been shown to provide Inflorescence for Medical Purposes: USP Considerations for Quality Attributes. J Nat
reproducible and accurate results for cannabinoids in Prod. 2020;83:1334-51.
dried cannabis, hemp, and vape oil samples. Separation 2. Cannabis Regulations, SOR/2018-144, (2018), Government of Canada, https://laws.
justice.gc.ca/eng/regulations/SOR-2018-144
of the key cannabinoids has been demonstrated in both
3. Industrial Hemp Regulations, SOR/2018-145, (2018), Government of Canada, https://
calibration solutions and extracted matrix samples. While laws.justice.gc.ca/eng/regulations/SOR-2018-144
the method has yet to be fully validated for more complex 4. McRae, G, Melanson, JE, Quantitative determination and validation of 17 cannabinoids
matrices such as edibles and matrices, the high specificity in cannabis and hemp using liquid chromatography-tandem mass spectrometry. Anal
Bioanal Chem. 2020;412(27):7381-93
of LC-MS/MS offers significant advantages for these
challenging samples types. Therefore, this method can
serve as viable starting point for such analyses.

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