The Quantitative Determination of Phytocannabinoids in Hemp Oils Using HPLC with UV Detection: Page 2 of 4

June 20, 2018
Volume: 
1
Issue: 
2
Figure 2
Figure 2: Hemp oil 1, black label, label claim: 23 mg per serving; 100 servings per 100 mL; calculation of label claim: 23,000 µg/mL or 2.3%. Hemp oil 2, blue label, 500 mg per 30 mL, calculation of label claim: 16,666 µg/mL or 1.7%. Hemp oil 3, green label, 15 mg per 1 serving per 0.5 mL = 15 mg/0.5 mL, calculation of label claim: 30,000 µg/mL or 3.0%.
Abstract / Synopsis: 

Medical marijuana generally possesses high levels of the psychotropic tetrahydrocannabinol (Δ9-THC) and lower levels of the nonpsychotropic can nabidiol (CBD). Pain mitigation and reduced severity of nausea and seizures are just a few of the therapeutic benefits reported by medical cannabis patients. The main source of CBD-rich oil is carbon dioxide or butane extraction of industrial hemp. Hemp is a robust crop containing high quantities of CBD and minor quantities of other cannabinoids. Like cannabis, hemp oil may be analyzed easily and effectively for its cannabinoid content. Presented herein is a procedure for the quantitative determination of 11 important cannabinoids, including CBD, in hemp oil using high performance liquid chromatography (HPLC) with ultraviolet (UV) detection.

Experimental

Instrument and Chromatographic Conditions

Instrument: Shimadzu LC-2030C UV (Cannabis Analyzer for Potency)

Column: Shimadzu NexLeaf CBX for Potency (150 mm x 4.6 mm, 2.7-μm dp)

Guard column: Shimadzu NexLeaf CBX Guard

Mobile-phase A: 0.09% phosphoric acid in water 

Mobile-phase B: 0.09% phosphoric acid in acetonitrile

Gradient: B concentration 70% (initial) →95% (8 min)

Flow rate: 1.5 mL/min

Column temperature: 35 °C

Injection volume: 5 μL

Detection: 220 nm

Hemp Oil Sample Preparation

Hemp oils are typically rich in CBD, with relatively minor concentrations of other cannabinoids. All cannabinoid targets have a linear dynamic range, above which the detector response ceases to be linear with concentration. Accurate quantitation relies on the detector response to the analyte lying within the calibration range. Therefore, two dilution factors were used, depending on the quantitative goal. One dilution factor yielded the appropriate detector sensitivity to the array of minor cannabinoids. A second, higher dilution factor was established for the most accurate quantitation of the major CBD component so that it’s response was within the established quantitative dynamic range established for that analyte. In practice, it was found that the two approaches yielded quantitative values for CBD that agreed within 0.2%.

A. Quantitative Total Cannabinoids

  1. Add 400 µL of isopropanol to a 2-mL glass vial
  2. Add 10 µL of hemp oil sample and completely dissolve
  3. Agitate for 30 s
  4. Add 400 µL of methanol to the mixture
  5. Agitate for 30 s
  6. Filter the mixture through a 0.2-µm PTFE syringe filter into a high performance liquid chromatography (HPLC) vial
  7. (Note: Total dilution factor 81X)

B. Quantitative CBD Only

  1. Add 800 µL of methanol to a 2-mL glass vial
  2. Add 200 µL of the part A mixture
  3. Agitate for 30 s
  4. (Note: Total dilution factor 405X)

Five hemp oils were tested in this study; they were purchased from various mail-order vendors. The appearance and label information for three of the five appear in Figure 2, referenced as black, blue, and green. (See upper right for Figure 2, click to enlarge; Figure 2: Hemp oil 1, black label, label claim: 23 mg per serving; 100 servings per 100 mL; calculation of label claim: 23,000 µg/mL or 2.3%. Hemp oil 2, blue label, 500 mg per 30 mL, calculation of label claim: 16,666 µg/mL or 1.7%. Hemp oil 3, green label, 15 mg per 1 serving per 0.5 mL = 15 mg/0.5 mL, calculation of label claim: 30,000 µg/mL or 3.0%.) The two other samples tested but not pictured are referred to as red and yellow.

References: 
  1. E.B, Russo, Br. J. Pharmacol. 163, 1344–1364 (2011).
  2. C.M. Andre, J.F. Hausman, and G. Guerriero, “Cannabis Sativa: the Plant of the Thousand and One Molecules,” Frontiers in plant science, frontiersin.org (2016).
  3. US Food and Drug Administration, Warning Letters: https://www.fda.gov/newsevents/publichealthfocus/ucm484109.htm (FDA, Rockville, Maryland).
  4. M.A. ElSohly and D. Slade, Life Sci. 78, 539–548 (2005).

Craig Young, MS, is the HPLC product manager for Shimadzu Scientific Instruments in Columbia, Maryland. Bob Clifford, PhD, is the general manager for Shimadzu Scientific Instruments. Direct correspondence to: [email protected]

How to Cite This Article

C. Young and B. Clifford, Cannabis Science and Technology 1(2), 38-43 (2018).