D-776
Cannabinoid Determination and Identification by HPLC
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1.0 Purpose
This procedure describes the reversed phase HPLC method for the determination of
Cannabidivarin (CBDV), Cannabidiolic Acid (CBDA), Cannabigerol (CBG), Cannabidiol
(CBD), Cannabinol (CBN), A-9-Tetrahydrocannabinol (A-9-THC), Cannabichromene (CBC),
and Tetrahydrocannabinolic Acid (THCA) in raw materials and finished products.
Additionally, it describes the ID of 8 other cannabinoids
2.0 Scope
This procedure applies to the assay and identification of CBDV, CBDA, CBG, CBD, CBN, A-
9-THC, CBC and THCA in raw materials and finished products, validated under Protocols
MV-LAB-19-044, MV-LAB-19-087 and MV-LAB-19-112. In addition, 8 other cannabinoids
have been identified by retention time and referenced to CBD (RRT).
3.0 Responsibility
3.1 It is the responsibility of QC and Analytical Chemists who have verified their ability to
execute this procedure to follow this procedure.
3.2 ‘It is the responsibility of QC Laboratory Management to implement this procedure,
ensure that the procedure is being followed.
3.3. ‘It is the responsibility of QC Laboratory Management and/or Analytical Development
to keep this procedure aligned with current practices.
4.0 Definitions
4.1 HPLC/UV — High Pressure Liquid Chromatography w/ Ultraviolet Detection
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Cannabinoid Determination and Identification by HPLC
4.2 QC — Quality Control
4.3 CofA — Certificate of Analysis
tt RRT — Relative response time
4.5 IPA — Isopropyl alcohol
4.6 HCO2H — Formic acid
5.0 References
5.1 MV-LAB-19-044, Protocol, Cannabinoid Determination & ID By HPLC Using UV/Vis
Spectroscopy
5.2 MV-LAB-19-087, Protocol, Cannabigerol and Cannabidiolic Acid Determination and
Identification using HPLC/UV
5.3 MV-LAB-19-112, Protocol, Cannabidivarin (CBDV), Cannabinol (CBN) and
Cannabichromene (CBC) Determination and Identification using HPLC/UV
5.4 USP General Chapter <621> Chromatography
6.0 Reagents, Supplies, Glassware and Equipment
6.1 Reagents
6.1.1 Water, MilliQ
6.1.2 ACN, HPLC Grade
6.1.3 IPA, ACS Grade or Better
6.1.4 Ammonium Formate, ACS Grade or Better
6.1.5 Formic Acid, ACS Grade or Better
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Cannabinoid Determination and Identification by HPLC
6.1.6 CBDV, CBDA, CBG, CBD, CBN, A-9-THC, CBC, THCA Reference Standard
Solution(s), 1 mg/ml (Restek, Cerilliant or Equivalent)
6.1.7 CBD Bulk Isolate (Extract Labs or Equivalent, Qualified In-House)
6.2 Supplies and Glassware
6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa
6.2.2 Vacuum filtration apparatus with 0.454 membrane filters
62.3 Class “A” volumetric pipets, flasks and graduated cylinders
6.2.4 Glass media bottles
6.2.5 Plastic Luer lock syringes
6.2.6 Nylon syringe filters, 0.45
6.2.7 Plastic transfer pipets
6.2.8 Teflon stir bars
6.3 Equipment
6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
and UV detector with a chromatographic data handling system
6.3;2 Analytical Balance
6:3:3 Stir Plate
7.0 Procedure
7.1 Mobile Phase Preparation
TAs Mobile Phase A (5 mM Ammonium Formate, 0.1% Formic Acid)
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Cannabinoid Determination and Identification by HPLC
7.1.1.1 Combine 1000 mL of water with 1.0 mL of formic acid. Add about
0.3153g of ammonium formate. Stir until dissolved then vacuum
filter.
7.1.2 Mobile Phase B (ACN, 0.1% Formic Acid)
7.1.3 Combine 1000 mL of Acetonitrile with 1.0 mL of formic acid. Mix well.
7.1.4 Extraction Solvent = Diluent = IPA
7.2 Standard Prep
7.2.1 CBD — Using a volumetric pipet, transfer 1.0 ml of the (ambient temperature)
CBD Reference Standard Solution into a 25 ml volumetric flask. QS to volume
with Diluent, mix well, this is the CBD Working Standard (Std A). The
nominal concentration of the CBD Working Standard is 40 ug/ml. Prep an
additional standard (Std B) as a standard check.
7.2.2 (Minor Component) CBDV, CBDA, CBG, CBN, A-9-THC, CBC and THCA —
Using a volumetric pipet, transfer 1.0 ml of the (ambient temperature) required
Reference Standard Solution into a 50-mL volumetric flask. QS to volume with
Diluent, mix well. This is the Minor Component Stock Standard (Stock Std A).
Finally, transfer 6.0 mL of the Minor Component Stock Standard into a 50ml
volumetric flask. QS to volume with Diluent, mix well. This is the Minor
Component Working Standard (Std A). The nominal concentration of the Minor
Component Working Standard is 2.4 ug/ml in each cannabinoid. (Alternatively,
inject only the THC standard and quantify using the relative response factors
provided in Section 7.8 below.) Prep an additional standard (Std B) as a
standard check, starting with the stock preparation.
7.2.3 Alternative dilution schemes are permitted.
7.3 Sample Preparation- Assay (Prepare a Single Preparation)
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Cannabinoid Determination and Identification by HPLC
7.3.1 Specific sample testing details are provided in each products profile. If a
specific testing details section is not available, then follow preparation
procedure as described below.
7.3.2 CBD Stock Sample Solution
7.3.2.1. Determine the size of the volumetric flask to be used for the CBD
Stock Sample Solution (V,,,.):
Sample Strength (% CBD) V.-. (mL)
<5 25
5-10 50
11-30 100
7.3.2.2 Calculate the sample weight needed for the Stock Sample Solution:
40 X Veey
%CBD
7.3.2.3 Place the empty, volumetric flask on the balance and press Tare.
Transfer the amount of sample calculated above into the volumetric
flask taking care not to get sample on the sides of the flask. Record the
weight of the sample. QS to volume with Extraction Solvent, and mix
well. This is the CBD Sample Stock Solution.
7.3.3. CBD Sample Solution
7.3.3.1 Using a glass pipet, transfer 5.0 mL of the Sample Stock Solution into
a 50-mL volumetric flask. QS to volume with Diluent, and mix well.
7.3.4 Minor Component Sample Solution
7.3.4.1 The intent of the Minor Component Sample preparation is to ensure
that the ratio of the standard concentration to the sample concentration
is ~0.1%, which corresponds with the LOQ of the method for both
THC and THCA.
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Cannabinoid Determination and Identification by HPLC
7.3.4.2 Weigh ~ 125mg sample into a 50-ml volumetric flask. Dilute to
volume with Diluent and mix well.
7.4 HPLC Parameters
7.4.1 Column: Agilent Poroshell 120 SB-C18, 4.6 x 150mm, 2.7 (Or Equivalent)
74.2 Column Temperature: 30°C
7.4.3 Flow rate: 1.5 ml/min
7.4.4 Wavelength: 228 nm (CBDV, CBD, CBN, THC, CBC, THCA, and CBG), 307
nm (CBDA), 285 nm (CBN and CBC) — Optional
7.4.5 Injection Volume: SyuL
7.4.6 Run Time: 11 min
7.4.7 Recommended 3-D Spectral Range (For Identification): 210nm to 350nm
7.4.8 Mobile Phase: Isocratic 25% A / 75% B
7.5 Recommended Sequence
7.5.1 Make at least 2 injections of the Diluent.
Y ioe FP Make five (5) injections of Working Standard A.
(mew Make two (2) injections of Working Standard B.
7.5.4 Make a single injection of each Sample Preparation.
Fe Make a single injection of the Standard Solution after every ten (10) sample
injections or at the end of a run.
7.6 System Suitability Requirements
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Cannabinoid Determination and Identification by HPLC
7.6.1 The %RSD of the first five (5) CBD standard A injections is NMT 2.0%.
7.6.2 The calculated recovery of Std A using Std B is 98-102% for CBD (Major
component) standards.
7.6.3 The %RSD of all CBD standard injections is NMT 3.0%.
7.6.4 The %RSD of the first five (5) Minor Component standard A injections is NMT
7.0%.
7.6.5 The calculated recovery of Std A using Std B is 93-107% for the Minor
Component Standards.
7.6.6 The %RSD of all Minor Component standard injections is NMT 8.0%.
7.6.7 Adjustments allowed in USP <621> are permitted.
7.7 Calculations
cBD(Oo% ~“) ee mE| X Coq X Vs“Sttaockc k x S m o g S x S Ven B i.) x . 100
7.7.1 Ru Sample Peak Area
Rs Mean Standard Peak Area
Cs CBD Standard Concentration (ug/mL)
Vstock Stock Sample Solution Volume (mL)
SW Sample Weight (mg)
Vspt Sample Solution Volume (mL)
Agspi Aliquot of Stock Sample used to prepare Sample (mL)
THC(%~) = & X Cop X vee x BE x St x 100) + (0.877 x THCA)
Le <n Pas
Re sw 1000pg = Aggy
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Ru Sample Peak Area
Rs Mean Standard Peak Area
Csa THC Standard Concentration (ug/mL)
Vstock Stock Sample Solution Volume (mL)
SW Sample Weight (mg)
Vsp| Sample Solution Volume (mL)
Agsp1 Aliquot of Stock Sample used to prepare Sample (mL)
THCA Assay of THCA calculated as in Section 7.7.1 (% =)
Ww
7.7.3. Mi; nor Component | % ~wy) =—R x Cog X VeSttaocc k 4, mgTAO _,, V“i x RRF x 100
w Rs SW 1000pg Aden
Ru Sample Peak Area
Rs Mean Standard Peak Area
Csta Standard Concentration (ug/mL)
Vstock Stock Sample Solution Volume (mL)
SW Sample Weight (mg)
Vsp| | Sample Solution Volume (mL)
Agdspi Aliquot of Stock Sample used to prepare Sample (mL)
RRF Relative Response Factor (Use THC Peak Area @ 228 nm)
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Cannabinoid Determination and Identification by HPLC
Minor Component (% “) = Ru Vstack —TMy 9Yel_ y pppx
SW Ad spi
100
7.8 Relative Response Factors for Minor Component Assays
Phytocannabinoid Wavelength Established RRF
THC 228 1.0000
CBDV 228 0.8388
CBDA 307 2.5189
CBG 228 0.8162
CBN 228 0.3948
CBN 285 0.5741
CBC 228 0.4141
CBC 285 1.1690
THCA 228 0.5273
7.9 Relative Retention Times
Phytocannabinoid Established RRT
CBDVA 0.617
CBDV 0.671
CBDA 0.864
CBGA 0.924
CBG 0.948
CBD 1.000
THCV hIES
CBN 1.533
d-9-THC 1.929
CBNA 1.981
d-8-THC 1.988
CBL 2.281
CBC 2.440
THCA 2.667
CBCA 3.086
CBLA 3.164
Note: CBCA and CBLA are typically not observed in samples, and elute after the
prescribed 11 minute method run time. If desired, the method run time may
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Cannabinoid Determination and Identification by HPLC
be extended to 13 minutes in order to confirm the presence/absence of these
species.
7.10 Reporting Results
7.10.1 The expanded uncertainty for CBD is 5.8%.
7.10.2 The expanded uncertainty for THC and THCa is 9.5%.
7.10.3 The expanded uncertainty for all other minor components is 14.8%.
7.10.4 The Coverage factor for all analytes is 2.
7.10.5 Report results along with the expanded uncertainty and coverage factor in the
following format (example for CBD):
7.10.5.1 108% of Label Claim, U =+5.8% k=2
7.11 Column Wash & Storage
7.11.1 Flush / store the column with / on 75% ACN / 25% Water.
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Cannabinoid Determination and Identification by HPLC :
8.0 Chromatograms
8.1 Typical CBD Working Standard Chromatogram
23AS010A
80+ 2o
wt ©
705 3O
60-
°
50- -
Z
30-
20;
oS
10+ © {| ,
o| —_S BIR I
: Li ' if bi ' t ' L i EI Li 1 t
05 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105 11
Time [min]
8.2 Typical CBD Sample Chromatogram
220490
23
saad 3O
70+
60- @
50- 7
4
30+
20"
2
10- ©oe =
0- A Be at — ‘8
05 1 15 2 25 3 35 4 45 § 55 6 65 7 75 8 85 9 95 10 105 11
Time [min]
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8.3 Typical Minor Component Working Standard Chromatogram
a 23AS011 A
o
10 be
9-
84
77+
> &
=.
- LO
3 2© oF
2- o
0- th nna
al T T T t T T T T F T T 7 T T T T T T T 7 T t
05 1 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105 11
Time [min]
8.4 Typical Minor Component Sample Solution Chromatogram
R47897
1n4l RBT SOs csai) ©=
— os a)
9-
8-
a t
zo 5 ats &
Ee 4- AH a a“ rpoen
D> © %
~ = co
iO ">
0.5 15 2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105 11
Time [min]
9.0 Revision History
| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 06/26/19 | New N/A C. Perry | - | - |
| 1 | 10/26/21 | Revised to include ISO 17025 requirements. CC- 02/23/23 Add instruction to check the product profile for test details, make CC-23-0092 S Sanawian it easier to see text in example chromatograms. | 21-0396 | J. Sassman |