The Quantitative Determination of Phytocannabinoids in Hemp Oils Using HPLC with UV Detection: Page 3 of 4

June 20, 2018
Volume: 
1
Issue: 
2
Figure 3
Figure 3: (a) Hemp oil 1 (black), total cannabinoids (81x dilution). (b) Hemp oil 1 (black), CBD only (405x dilution). (c) Hemp oil 2 (blue), total cannabinoids (81x dilution). (d) Hemp oil 2 (blue), CBD only (405x dilution). (e) Hemp oil 3 (green), total cannabinoids (81x dilution). (f) Hemp oil 3 (green), CBD only (405x dilution).
Table I: Summary of CBD quantitative determination for five hemp oils
Table I: Summary of CBD quantitative determination for five hemp oils
Table II: Summary of total cannabinoids quantitative determination
Table II: Summary of total cannabinoids quantitative determination
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.

Qualitative Analysis of Hemp Oils

Chromatograms for hemp oils 1 (black), 2 (blue), and 3 (green) appear in Figure 3. (See upper right for Figure 3, click to enlarge; Figure 3: (a) Hemp oil 1 (black), total cannabinoids (81x dilution). (b) Hemp oil 1 (black), CBD only (405x dilution). (c) Hemp oil 2 (blue), total cannabinoids (81x dilution). (d) Hemp oil 2 (blue), CBD only (405x dilution). (e) Hemp oil 3 (green), total cannabinoids (81x dilution). (f) Hemp oil 3 (green), CBD only (405x dilution).) Peak labels appear for only those cannabinoids identified in the sample.

Quantitative Results Summary for Hemp Oils

The astute reader may correctly ask about the noncannabinoid constituents in the sample and how they may influence the chromatography. As the sample preparation presented here is essentially a simple dilution of the oil sample into injectable form, nothing is done to separate cannabinoids from terpenes, phenolics, or pigment compounds. Under the reversed-phase chromatography conditions used here, any pigment compounds emerge early in the chromatography.

The sum total terpene content of hemp and cannabis is 1–3% (2), comprising possibly hundreds of terpenoids. As for chemistry, many triterpenes and sesquiterpenes have similar polarities to the cannabinoids and are expected to retain on the analytical column to similar degrees as the cannabinoids. Within the detection limits of this work, any terpenoids are undetectable, essentially lost in the baseline.

Discussion

Tables I and II summarize the quantitative findings for the samples studied. Table I reflects the accurate quantitation of CBD using the higher dilution factor (405X). (See upper right for Table I, click to enlarge; Table I: Summary of CBD quantitative determination for five hemp oils.) Table II reflects the quantitation of the comprehensive target list. (See upper right for Table II, click to enlarge; Table II: Summary of total cannabinoids quantitative determination.)

As a general sample observation, hemp oils 1 (black) and 2 (blue) exhibited a transparent, weak-yellow or green coloration. Our assumption was that each of these is a product of multistep purification after extraction; for example, carbon dioxide or butane extraction followed by steam distillation. Notably, hemp oil 3 (green) was opaque brown or green and gritty in appearance. It also had the most intense smell—a distinctly “earthy” odor. Accordingly, our assumption was that the sample was the result of crude extraction only, with no further refinement.

It is important to note that it has been reported in the literature that the whole plant can be more beneficial to the consumer because it contains not only the cannabinoids, but also an array of terpenes providing a synergistic whole plant benefit. The whole plant can also provide essential fatty acids, plant sterols for lowering cholesterol, and antioxidants chlorophyll and vitamin E.

Hemp oils 1 (black) and 2 (blue) showed high ratios of CBD to total cannabinoids, both at 92%, and the lowest quantity of other cannabinoids. This finding supported the assumption, along with transparency and color, that these oils were the more highly purified samples. Both samples also tested close to label claim at 95% and 92%, respectively.

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).