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Determination of 5 Hemp-Based Cannabinoids & 12 Standard Cannabinoids by Hand-Portable HPLC

This application note demonstrates the ability of the Axcend Focus LC® portable High-Performance Liquid Chromatography system (HPLC) to detect and separate 5 principal cannabinoids present in 50 mg of a hemp-variant of cannabis flowers (dry weight), namely Cannabidiolic Acid (CBDA), Cannabigerol Acid (CGBA), Tetrahydrocannabinolic Acid (THCA), Cannabidiol (CBD), and Cannabichromene (CBC). For comparison, 12 additional cannabinoids were also determined in a Restek* standard, with run-times for both analyses of ~18 minutes with a 50:50 mixture of ACN 0.3 M NH4Ac buffer pH 4.6 (Mobile Phase A) and MeOH 50% ACN (Mobile Phase B). 

 

Materials and Methods

Hemp Sample Preparation

  • 50 mg of cut Hemp flower inserted into a 5 mL glass tube
  • 1 mL of Methanol added
  • Shaken for 2 min
  • Filtered through a 0.2 µm filter

Run Conditions

Detection Wavelength UV at 235 nm
Column Length 10 cm
Internal Diameter 150 µm
Packing 1.7 µm C8
Injection Volume 40 nL
Flow Rate 0.97 µL/min

 

 

Results

Hemp Cannabinoid Detection

Cannabinoid Image 1Asset 4

Figure 1. UV detection of 5 cannabinoids in a hemp-variant of cannabis at 235 nm using the Axcend Focus LC. Elution Order — 6.5 min: CBDA; 7 min: CBGA; 8.5 min: THCA; 11 min: CBD; and 18 min: CBC. Flow rate: 0.97 µL/min; Gradient %B: 0 min—3%, 1 min—25%, 10 min—25%, 17 min—60%, 18 min—95%, 20 min—95%; Equilibration Time: 4 min; Mobile Phase A (50% H2O 50% ACN 0.3 M NH4Ac buffer pH 4.6); Mobile Phase B (50% MeOH 50% ACN); Column: 10 cm x 150 µm i.d.; Phenomenex Kinetex 1.7 µm C8.

 

Analysis of 12 Cannabinoids Standard

Due to its small size, low-weight, hand-portability, and lower solvent usage, the Axcend Focus LC is uniquely suited for field and laboratory analysis of cannabinoid compounds. As shown below, we also detected and identified 12 separate cannabinoids in a Restek standard.

Cannabinoid Image 2Asset 5

Figure 2. UV detection of 12 cannabinoids in a Restek standard at 235 nm using the portable Axcend Focus LC. Elution Order shown above and below. Flow rate: 0.97 µL/min; Gradient %B: 0 min—3%, 1 min—25%, 10 min—25%, 17 min—60%, 18 min—95%, 20 min—95%; Equilibration Time: 4 min; Mobile Phase A (50% H2O 50% ACN 0.3 M NH4Ac buffer pH 4.6); Mobile Phase B (50% MeOH 50% ACN); Column: 10 cm x 150 µm i.d.; Phenomenex Kinetex 1.7 µm C8.

 

Name Abbr Average RT (min) RT RSD Amount (mg/ml) Amt % RSD Dry Weight % R2 Linear Range (mg/ml)
Cannabidiolic Acid CBD-A 6.12 0.53 5.36 0.85 10.72 0.9999 0.07-1.0
Cannabigerol Acid CBG-A 6.90 0.51 0.08 4.14 0.17 0.9999 0.07-1.0
Cannabidivarin CBDV 7.19 ND ND NA ND NA NA
Tetrahydrocannabinolic Acid THC-A 8.89 0.48 0.15 1.61 0.30 0.9998 0.07-1.0
Tetrahydrocannabivarin THCV 9.98 ND ND NA ND NA NA
Cannabidiol CBD 11.50 0.39 0.22 2.67 <0.02 0.9997 0.07-1.0
Cannabigerol CBG 11.94 ND <0.02 NA <0.02 0.9993 0.07-1.0
Cannabinol CBN 14.28 ND ND NA ND NA NA
Tetrahydro-cannabinol (Δ9) Δ9 THC 15.78 ND <0.02 NA <0.02 0.9999 0.07-1.0
Δ8-Tetrahydro-cannabinol   Δ8 THC 16.09 ND <0.02 NA <0.02 0.9997 0.07-1.0
Cannabicyclol CBL 16.99 ND ND NA ND NA NA
Cannabichromene CBC 17.93 0.21 ND NA NA NA NA

* - Cannabinoid samples provided by Restek Corporation.

Table 1. UV detection of 12 cannabinoids in a Restek standard at 235 nm using the portable Axcend Focus LC.

 

Axcend Focus LC UV detection is also available from Axcend® at 255 and 275 nm in fixed wavelengths or in variable wavelengths between 200—400 nm.

 

 

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