34 February / March 2018
Identification of an Unknown Constituent in Hemp-Derived Extract Using Reversed-Phase Orthogonal Methodology
by Catharine E. Layton*, Shawn C. Helmueller and Andrew J. Aubin Waters Corporation, 34 Maple St. Milford, MA, 01757, USA *Corresponding author:
Catharine_Layton@Waters.com
The analysis of Cannabis sativa L. extracts can pose significant challenges due to complexities derived from extraction efficiency, cultivar genetic influences, and environmental factors such as weather and growing conditions. With over 400 constituents in the cannabis plant as a whole, potentially synergistic bioactive relationships are actively being investigated. The ‘entourage effect’ is an enhanced effect derived from a combination of two or more bioactive compounds. When referencing this phenomenon in the discussion of cannabis, this term usually is applied to cannabis strains selectively bred for target ratios of the most popular synergistic cannabinoids; such as cannabidiol (CBD) and (−)-trans-Δ9
more recently, it has been applied to low Δ9 THC cannabis varieties classified as hemp. The goal of this manuscript is to demonstrate an approach for identification of an unknown constituent, present in a significant amount, in hemp-derived extract by employing orthogonal, reversed phase separation techniques on a fast, highly efficient Ultra-High Performance Liquid Chromatography (UHPLC) platform.
Introduction
Cannabis is a complex plant with over 400 chemical entities of which more than 60 are cannabinoid compounds [1]. Even though cannabis has been used and cultivated by mankind for at least 6000 years [2], our current knowledge regarding its pharmacological properties is based on studies which have taken place only since the end of the nineteenth century. Cannabis has significant potential for enlarging the library of naturally occurring bioactive metabolites. To date, several phytochemicals have been described in cannabis derived extract [3] including essential mono, poly and saturated fatty acids, cannabinoids, terpenes, plant sterols, vitamin E and chlorophyll. Many of these compounds are capable of optimising health and wellness alone, however the interplay of these active and inactive compounds as synergists can produce an enhanced physiological effect including inhibition of side effects, improved absorption, bacterial defence, and the ability to impact multiple molecular targets [4,5].The ‘entourage effect’ is a term introduced in cannabinoid science in 1998 to represent the novel endogenous synergistic cannabinoid molecular regulation route [6]. More recently, a similar phenomenon referred to by some as the ‘hemptourage effect’, has been applied to low THC
cannabis varieties classified as hemp [7,8].
Cannabinoids accumulate in the secretory cavity of the glandular trichomes, which are mainly found in female flowers and in most aerial portions of the plant. They have also been detected in low quantity in other parts of the plants including the seeds [9] roots [10] and pollen [11]. Adding to the complexity, the concentration of phytocannabinoids depends on the age, variety, growth conditions, nutrition, humidity, light intensity, harvest time and storage conditions of the plant [12]. Many cannabinoids are present as non- enzymatically decarboxylated acids, which are converted into their corresponding neutral form after harvest and upon heating [13].
Cannabis testing laboratories provide important information regarding chemical composition of the cannabis plant for the same purpose that quality assurance laboratories provide purity results to ensure the safety of pharmaceutical products. In the absence of federal guidance, states have led cannabis testing laboratories to develop proprietary chromatographic methods in order to meet state testing specifications for raw materials, extracts, and/or infused products such as beverages, edibles and topicals. Within the past few years, studies including those performed by the US Food and Drug Administration, have reported the occurrence of inaccurate potency reporting
for a significant number of infused cannabis products [14]. Factors which influence inaccurate results may be attributed to inadequate sampling procedures, inconsistent manufacturing techniques, or inaccurate testing methods.
In an attempt to capitalise on the need for improved testing methods, some analytical instrumentation manufacturers are actively promoting ‘turn-key’ chromatographic testing solutions. This one-size-fits-all standardised chromatographic separation may work well for the separation of pure cannabinoid reference standards however may not be adequate for product variability inherent during routine sample testing. Laboratories that purchase these solutions are left with the potential challenge of re-developing these ‘turn- key’ chromatographic methods in order to separate, quantify and identify compounds of interest within a particular batch or formulation [15]. Adding to this challenge, several of the important cannabinoids are isobars (identical in chemical composition and nominal mass) and are therefore distinguishable only by high- resolution MS/MS analysis (Table 1).
A multi-levelled approach to understanding, identifying, quantifying, and controlling bioactive compounds should include orthogonal or fundamentally complementary chromatographic methodology,
as is often employed in pharmaceutical testing [17]. -tetrahydrocannabinol (Δ9 THC), although
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