Simple Protocol for Analysis of 17 Cannabinoids in Hemp and Cannabis

Workflow for cannabinoid analysis in hemp and cannabis

Workflow for cannabinoid analysis in hemp and cannabis

 

1.0 Introduction: Analysis of cannabinoids in cannabis and hemp for potency

Many countries throughout the world have started to legalize recreational and medicinal cannabis. In the United States, testing cannabis and cannabis products for potency is required. Emerging interest in the therapeutic benefits of minor cannabinoids has increased the demand for analytical methods capable of separating 17 cannabinoids. The cannabinoids targeted for testing consisted of those listed by the AOAC in standard method performance requirements (SMPRs) for dried plant material (low and high THC varieties), chocolates, and concentrates plus three additional cannabinoids of interest. 

A complete HPLC workflow has been developed to simplify cannabinoid profiling and potency determination. This workflow offers the following:

    • Step-by-step instructions for sample preparation and analysis of 17 cannabinoids

    • Rapid high-resolution gradient method: 6 minutes on C18 column (2 µm)

    • Rapid gradient method: 6 minutes on C18 (2.7 µm)

    • Low pressure isocratic method: 8 minutes on C8 column (2.7 µm)

    • Low flow rate for reduced solvent consumption  

    • Compatibility with mass spectrometry

 

An orthogonal analysis of a high CBD hemp strain was performed using two different stationary phases. The instructions provided below were used to prepare the hemp extract and standard solutions for cannabinoid quantitation. The Low Pressure Isocratic and Rapid High-Resolution methods were selected to determine potency (total THC and CBD) of the hemp flower.

 

1.1 Chemical structure of 17 cannabinoids

Chemical structure of 17 cannabinoids

 

2.0 Sample preparation of standard solutions, peak identification solution, and hemp extract

2.1 Preparation of hemp flower extract

        Step Instructions
1 Homogenize 5 g hemp flower (particle size ≤ 1mm).

Note: Frozen ball-milling is the preferred method of homogenization without sample degradation. Low temperature homogenization prevents degradation of analytes and produces uniform particle sizes. It is essential to ensure sampling protocol adheres to local guidelines and provides an accurate representation of the bulk material. (See reference: Guidance for State Medical Cannabis Testing Programs)
2 Weigh 0.5 ±0.01 g on a calibrated microbalance and transfer sample into a 50 mL polypropylene centrifuge tube
3 Dispense 20 mL ethanol into tube and vortex briefly, incubate sample on horizontal shaker for 30 mins at 250 rpm.
4 Centrifuge sample at 4000 rpm for 5 mins to pellet plant material.
5 Carefully pour supernatant into amber 50 mL volumetric flask and set aside for second extraction.
6 Perform second extraction of material with 20 mL ethanol and add extract to amber 50 mL volumetric flask containing contents of the first extraction.
7 Fill flask to mark with ethanol and mix well.
8 Perform 1:10 and 1:100 dilution of sample with methanol
9 Filter samples directly into HPLC vials with 0.2 µm PTFE membrane

Note: Sample filtering can be performed using a Millex syringe filter or Samplicity G2 Filtration System for high throughput applications.

 

2.2 Preparation of mobile phases

Mobile Phase Instructions
5 mM Ammonium Formate + 0.1% Formic Acid 1. Prepare 0.1% formic acid by adding 1 mL formic acid to 1 L water, mix well. Alternatively, use pre-formulated 0.1% formic acid water solution
2. Add 315.3 mg ammonium formate to 0.1% formic acid and mix well.
0.1% Formic Acid in Acetonitrile Add 1 mL formic acid to 1L acetonitrile and mix well. Alternatively, use pre-formulated 0.1% formic acid acetonitrile solution.

 

2.3 Preparation of standard solutions

        Step Instructions
1 To a 10 mL amber volumetric flask, add 1 mL of the following CRMs:
# Cannabinoid Product Number mg/mL
1 CBDA C144 1.0
2 THCA T-093 1.0
3 CBG
C-141 1.0
4 CBD
C-045 1.0
5 CBN
C-046
1.0
6 9-THC T-005 1.0
 
Fill to mark with methanol and shake well for a final solution of 100 µg/mL each analyte.
   
Perform the following dilution scheme to obtain a 6-point calibration curve:
  mg/mL Dilution Factor
  100 1
  25 4
  5 5
  1 5
  0.5 2
  0.25 2
Note: Standard solutions can also be prepared gravimetrically to improve accuracy and reduce lot-to-lot variation.

 

2.4 Preparation of peak identification solution

        Step Instructions      
1 To a 20 mL amber volumetric flask, add 1 mL of the following CRMs:      
# Cannabinoid Product Number mg/mL      
1 CBDVA C-152 1.0      
2 CBDA C144 1.0      
3 CBGA C-142 1.0      
4 THCVA
T-111 1.0      
5 THCA T-093 1.0      
6 CBNA C-153 1.0      
7 CBCA C-150 1.0      
8 CBLA C-171 0.5      
9 CBDV C-140 1.0      
10 CBG C-141 1.0      
11 CBD C-045 1.0      
12 THCV T-094
1.0      
13 CBN C-046 1.0      
14 9-THC
T-005 1.0      
15 8-THC T-032 1.0      
16 CBC C-143 1.0      
17 CBL C-154 1.0      
       
2 Fill to mark with methanol and shake well for a final solution of 50 µg/mL each analyte (25 µg/mL CBLA).      

 

3.0 Chromatography

3.1 System and method parameters (Rapid High-Resolution, Rapid Gradient, and Low Pressure Isocratic methods)

Method         Low Pressure Isocratic
        Rapid High-Resolution             Rapid Gradient
Column Ascentis® Express C8 (2.7 µm) 150x3.0 mm Ascentis® Express C18 (2 µm) 150x2.1 mm Ascentis® Express C18 (2.7 µm) 150x3.0 mm
Mobile Phase A 5 mM Ammonium Formate + 0.1% Formic Acid in Water
Mobile Phase B 0.1% Formic Acid in Acetonitrile
Mobile Phase Conditions Isocratic: 27:73, A:B 75% to 90% B in 2 min; held at 90% B for 5 min 75% to 85% B in 2 min; held at 85% B for 5 min
Flow Rate (mL/min) 0.7 0.4 0.8
Column Temp. (°C) 30 25 25
UV Detection (nm)   228  
Injection (µL) 5 3 5
Max Pressure (Bar) 220 540 250
Instrument Agilent 1290 Infinity II LC System; Quaternary pump; 0.12 mm ID tubing; 10 mm Max-Light Cartridge Cell 1.0 µL

 

3.2 Chromatograms: standard solutions

Low Pressure Isocratic Method: Standard Solution and Blank

Rapid High-Resolution Gradient Method: Standard Solution and Blank

Rapid Gradient Method: Standard Solution and Blank

 

3.3 System suitability: peak identification solutions

Analyte Low Pressure
Isocratic Method

Analyte Rapid High-Resolution
Gradient Method

Rapid Gradient
Method

Resolution % RSD (n=7) Resolution % RSD (n=7) Resolution % RSD (n=7)
CBDVA -
0.20 CBDVA - 0.26 - 0.39
CBDV 5.2 0.14 CBDV 4.0 0.28 4.6 0.38
CBDA 7.4 0.14 CBDA 9.7 0.27 9.0 0.35
CBGA 2.4 0.15 CBGA 2.6 0.27 1.8 0.36
CBG 4.5 0.21 CBG 2.5 0.29 3.0 0.38
CBD 1.6 0.14 CBD 2.7 0.37 3.1 0.45
THCV 2.1 0.47 THCV 5.3 0.30 5.0 0.39
THCVA 5.7 0.27 THCVA 10.8 0.29 9.0 0.25
CBN 6.4 0.26 CBN 4.0 0.30 3.4 0.36
CBNA 2.8 0.54 CBNA 8.0 0.48 6.6 0.59
∆-9-THC 6.8 0.36 ∆-9-THC 3.0 0.27 3.5 0.53
∆-8-THC
1.0 0.72 ∆-8-THC 1.9 0.30 1.6 0.55
THCA 5.5 0.39 CBL 6.2 0.28 5.8 0.53
CBL 1.7 0.30 CBC 2.0 0.30 1.6 0.44
CBC 1.8 0.17 THCA 2.1 0.26 1.4 0.39
CBCA 1.5 0.35 CBCA 4.4 0.32 4.0 0.71
CBLA
3.0 0.45 CBLA 4.1 0.37 3.6 0.69

 

Low Pressure Isocratic Method: 17 Cannabinoids in Peak Identification Solution

 

Rapid High-Resolution Method: 17 Cannabinoids in Peak Identification Solution

 

Rapid Gradient Method: 17 Cannabinoids in Peak Identification Solution

 

 

4.0 Results: Cannabinoid profile and potency of hemp flower

Low Pressure Isocratic Method

 

Analyte % Weight Overlay of Peak Identification Solution and Hemp Extract
CBDA 18.20
CBG 0.13
CBD
1.40
CBN ND
Delta 9 0.22
THCA 0.46
Total THC 0.62
Total CBD 17.36

 

Rapid High-Resolution Gradient Method

 

Analyte % Weight Overlay of Peak Identification Solution and Hemp Extract
CBDA 18.08
CBG 0.10
CBD
1.40
CBN ND
Delta 9 0.24
THCA 0.50
Total THC 0.68
Total CBD 17.26

 

4.1 Low pressure isocratic method: Linearity and range (0.25-100 µg/mL)

 

                                                                              Peak Area
       µg/mL        CBDA        CBG        CBD
       CBN        ∆9-THC        THCA
0.25 6.5 3.8 3.8 9.2 3.1 7.8
0.5 13.2 7.0 6.7 16.6 6.1 13.8
1.0 26.4 13.9 14.0 33.0 12.4 25.8
5.0 137.7 71.5 72.6 170.8 64.7 128.7
25.0 706.1 367.5 373.1 881.9 332.4 652.5
100.0 2813.0 1476.5 1512.1 3523.4 1339.7 2581.7

 

4.2 Rapid high-resolution gradient method: Linearity and range (0.25-100 µg/mL)

 

                                                                              Peak Area
       µg/mL        CBDA        CBG        CBD
       CBN        ∆9-THC       THCA
0.25 7.2 4.3 3.7 8.3 3.3 6.7
0.5 14.1 7.9 7.4 17.1 6.3 12.7
1.0 28.5 15.8 16.9 34.2 13.2 25.6
0.5 143.2 73.9 76.5 176.1 67.0 132.3
25.0 739.3 382.1 389.4 895.4 337.5 676.0
100.0 2935.2 1534.8 1564.1 3547.2 1350.6 2673.6

 

 

5.0 Conclusion: Accurate analysis of cannabinoids using HPLC

A complete HPLC workflow has been developed to simplify cannabinoid profiling and potency determination. This workflow offers simple sample preparation and a choice of three methods:

  • Step-by-step instructions for sample preparation and analysis of 17 cannabinoids
  • Rapid High-Resolution Gradient Method: 6 minutes on C18 column (2 µm)
  • Rapid Gradient Method: 6 minutes on C18 (2.7 µm)
  • Low Pressure Isocratic Method: 8 minutes on C8 column (2.7 µm)
  • Low flow rate for reduced solvent consumption
  • Compatibility with mass spectrometry

Using the Rapid High-Resolution Gradient and the Low Pressure Isocratic Methods in an orthogonal approach, the total THC was found to significantly exceed the 0.3% total THC limit imposed by the United States Department of Agriculture Interim Final Rule. Results with the Rapid Gradient Method were comparable (data not shown). Although ∆9-THC levels were low, a significant amount of the acidic form (THCA) was present. The hemp sample tested here illustrates the challenges in hemp production to obtain results in a timely manner for harvesting before THC content exceeds regulatory requirements. Rapid and accurate methods support timely analysis of hemp and cannabis.

The results confirmed the high total CBD content, with the majority found to be in the acidic form (CBDA). The results from all methods were in agreement and demonstrated a high degree of selectivity despite the abundance of matrix components.

These methods can easily be adopted to quantitate cannabinoids in plant material spanning concentrations of 0.05–100% by weight. The short run times and low solvent use makes these methods cost-effective for high-throughput potency testing.

 

Materials

  Product List    PN
Columns Ascentis® Express C18 150x2.1 mm, 2 µm 50814-U
Ascentis® Express C8 150x3.0 mm, 2.7 µm 53853-U
Ascentis® Express C18 150x3.0 mm, 2.7 µm 53816-U
Ascentis® Express Guard Cartridge Holder
53500-U
Ascentis® Express C18, 2 µm Guard Cartridge 50822-U
Ascentis® Express C8, 2.7 µm Guard Cartridge 53511-U
Ascentis® Express C18, 2.7 µm Guard Cartridge 53504-U
     
Reference Materials Cannabidivarinic acid (CBDVA) C-152
Cannabidiolic acid (CBDA) C-144
Cannabigerolic acid (CBGA) C-142
Tetrahydrocannabivarinic acid (THCVA)
T-111
∆9-tetrahydrocannabinolic acid (THCA) T-093
Cannabinolic acid (CBNA) C-153
Cannabichromenic acid (CBCA) C-150
Cannabicyclolic acid (CBLA) C-171
Cannabidivarin (CBDV) C-140
Cannabigerol (CBG) C-141
Tetrahydrocannabivarin (THCV) T-094
Cannabinol (CBN)
C-046
∆9-tetrahydrocannabinol (∆9-THC) T-005
∆8-tetrahydrocannabinol (∆8-THC) T-032
Cannabichromene (CBC) C-143
Cannabicyclol (CBL) C-154
     
Water, solvent, chemicals Ultrapure water from Milli-Q® system or bottled water Milli-Q® IQ 7005 or 101262
Acetonitrile Solution (0.1% formic acid) 900686
Water Solution (0.1% formic acid) 900687
Ammonium Formate, LiChropur 70221
Formic Acid, LC-MS LiChropur 533002
Ethyl Alcohol 459828
Methanol 900688
  Product List   PN
Filters Millex Hydrophilic Syringe Filters  0.20 µm SLLG033
     
Additional Lab Ware and Accessoires Samplicity G2 Filtration System SAMP2SYSB
50 mL Polypropylene Centrifuge Tubes CLS430828
BRAND BLAUBRAND Volumetric Flask, Glass Stopper, Amber Glass Z327050
BRAND BLAUBRAND Volumetric Flask, Glass Stopper, Amber Glass Z327026
BenchMixer Shaker/Vortexer Z742705
Corning LSE Compact Centrifuge CLS6755
Corning LSE Rotor 6x50 mL Tubes CLS480136
BRAND Seripettor Bottle-top Dispenser Z627577
Certified Vial Kit - Amber 29653-U
Whatman Mini-UniPrep G2 Filter Vial WHAGN203APEORGSP
Chemical Duty Pump, 115 V/60 Hz WP6111560
Pipette 10-100 µL EP4924000258
Pipette 100-1000 µL EP4924000282
Pipette 500-5000 µL EP4924000304
Pipette tips 2-200 µL box Z640220
Pipette tips 50-1000 µL box Z640247
Pipette tips 100-5000 µL box Z640271
Corning Glass Media Storage Bottles (1L) CLS13991L