Identification of an Unknown Constituent in Hemp-Derived Extract Using Reversed-Phase Orthogonal Methodology

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: [email protected] 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-tetrahydrocannabinol (Δ9 THC), although 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 varieties classified as hemp [7,8]. for a significant number of infused cannabis products [14]. Factors which influence Cannabis is a complex plant with over Cannabinoids accumulate in the secretory inaccurate results may be attributed 400 chemical entities of which more than cavity of the glandular trichomes, which are to inadequate sampling procedures, 60 are cannabinoid compounds [1]. Even mainly found in female flowers and in most inconsistent manufacturing techniques, or though cannabis has been used and aerial portions of the plant. They have also inaccurate testing methods. cultivated by mankind for at least 6000 been detected in low quantity in other parts years [2], our current knowledge regarding of the plants including the seeds [9] roots [10] In an attempt to capitalise on the need for its pharmacological properties is based on and pollen [11]. Adding to the complexity, the improved testing methods, some analytical studies which have taken place only since concentration of phytocannabinoids depends instrumentation manufacturers are actively the end of the nineteenth century. Cannabis on the age, variety, growth conditions, promoting ‘turn-key’ chromatographic testing has significant potential for enlarging the nutrition, humidity, light intensity, harvest solutions. This one-size-fits-all standardised library of naturally occurring bioactive time and storage conditions of the plant chromatographic separation may work well for metabolites. To date, several phytochemicals [12]. Many cannabinoids are present as non- the separation of pure cannabinoid reference have been described in cannabis derived enzymatically decarboxylated acids, which are standards however may not be adequate for extract [3] including essential mono, poly converted into their corresponding neutral product variability inherent during routine and saturated fatty acids, cannabinoids, form after harvest and upon heating [13]. sample testing. Laboratories that purchase these solutions are left with the potential terpenes, plant sterols, vitamin E and Cannabis testing laboratories provide challenge of re-developing these ‘turn- chlorophyll. Many of these compounds are important information regarding chemical key’ chromatographic methods in order to capable of optimising health and wellness composition of the cannabis plant for separate, quantify and identify compounds of alone, however the interplay of these active the same purpose that quality assurance interest within a particular batch or formulation and inactive compounds as synergists laboratories provide purity results to ensure [15]. Adding to this challenge, several of the can produce an enhanced physiological the safety of pharmaceutical products. In the important cannabinoids are isobars (identical effect including inhibition of side effects, absence of federal guidance, states have in chemical composition and nominal mass) improved absorption, bacterial defence, led cannabis testing laboratories to develop and are therefore distinguishable only by high- and the ability to impact multiple molecular proprietary chromatographic methods in resolution MS/MS analysis (Table 1). targets [4,5].The ‘entourage effect’ is a term order to meet state testing specifications introduced in cannabinoid science in 1998 to for raw materials, extracts, and/or infused A multi-levelled approach to understanding, represent the novel endogenous synergistic products such as beverages, edibles and identifying, quantifying, and controlling cannabinoid molecular regulation route topicals. Within the past few years, studies bioactive compounds should include [6]. More recently, a similar phenomenon including those performed by the US Food orthogonal or fundamentally complementary referred to by some as the ‘hemptourage and Drug Administration, have reported the chromatographic methodology, as is often effect’, has been applied to low THC occurrence of inaccurate potency reporting employed in pharmaceutical testing [17]. 35 Figure 1: Overlay of cannabinoid reference standard mixture and hemp extract separated using mobile phase at pH 10. The unknown constituent is marked by an asterisk. Chromatographic orthogonality is the use and leaves were ground, Table 2: SFE – BBES extraction and collection conditions of multiple separation mechanisms in order homogenised, and to gain additional analyte information. It is divided into five 4 lb Extraction Condition Flow Rate 170 g/min recognised that many cannabis laboratories bags. A sample was may not have access to high-end scientific analysed from each bag Pressure 344 bar o instrumentation therefore a simple approach with the average total Temperature 50 C to chromatographic orthogonality is most cannabinoid content Time 210 nm useful. Orthogonality was accomplished in this determined as 5.04 Collection Condition study using basic and acidic pH mobile phases wt%. Six extractions CS1 Pressure 158 bar o on the same chromatographic platform were performed at the CS1 Temperature 45 C CS2 Pressure 75 bar (i.e. column and instrument) to identify an 5 L scale via a solvent- CS2 Temperature 40oC unknown constituent in cannabis extract. free Bio-Botanical CS3 Pressure 53 bar Extraction System (SFE- CS3 Temperature 35oC BBES) (Waters, Milford, Experimental MA. USA). of 0.80 kV. An accessory fraction collector Hemp Extract Preparation The cyclone separator 1 (CS 1) extracts (Waters Fraction Manager – Analytical were combined and homogenised. Ethanol (WFM-A)) (Waters, Milford, MA. USA) was At a partnering cannabis testing laboratory, was used to clean CS 1 post extraction. added for automated collection of the peak of hemp extract was generated from a 20 lb feed Approximately 85 g raw extract were interest. Separations were achieved using an stock of Vermont grown hemp (Cannabis sativa added to 1.2 L of the ethanol wash, and ACQUITY CSH C , 130Å, 1.7µm, 2.1 mm x 50 L.) with seeds and stems removed. The buds 18 the solution stored at -20oC until analysis mm column (Waters, Milford, MA. USA) at a temperature of 30oC and a flow rate of 1.0 mL/ Table 1: Empirical formula of major cannabinoids and purification. Plant waxes were removed and monoisotopic mass [16]. from the homogenised solution by vacuum min. All data was collected and processed by filtration, and pigments were removed Empower® 3 Chromatography Data Software. Empirical Monoisotopic Cannabinoid through a patent pending clarification Formula Mass A ‘turn-key’ chromatographic separation strategy (Table 2). of the major cannabinoid reference Δ9 THC standards (Cerilliant, Round Rock, TX. CBD C H O 314 USA) cannabidivarian (CBD-V, C-140), CBC 21 30 2 Orthogonal Reversed-Phase tetrahydrocannabivarian (THC-V), Δ8 THC Chromatography cannabigerol (CBG, C-141), Δ9 –trans- THC-A Reversed-phase separations were performed tetrahydrocannabinol (Δ9-THC), Δ8 C22H30O4 358 CBD-A using an ACQUITY H-Class UPLC System –trans-tetrahydrocannabinol (Δ8-THC, THC-V (Waters, Milford, MA. USA) equipped with a T-032), cannabinol (CBN), Δ9 –trans- C H O 286 CBD-V 19 26 2 PDA (UV) detector at 228 nm, with a 4.8 nm tetrahydrocannabinolic acid (THC-A), resolution, and a 3D data λ range at 200-400 cannabidiol (CBD), cannabidiolic acid CBG-A C H O 360 22 32 4 nm. The UHPLC was also equipped with a (CBD-A), cannabigerolic acid (CBG-A) and CBG C H O 21 32 2 316 single quadrupole QDa (MS) detector with cannabichromene (CBC) prepared at 1.0 a programmed ESI (-) mass scan range of mg/mL in methanol was performed at pH 10 CBN C H O 310 21 32 2 100-600 Da, cone voltage at 15 V, capillary using 10mM ammonium bicarbonate (mobile o temperature of 500 C and capillary voltage phase A) and acetonitrile (mobile phase B). 36 February / March 2018 Acidic cannabinoids; CBD-A, THC-A, and CBG-A showed greater retention in acidic mobile phase (pH 3) than when separated under basic conditions (pH 10), while the retention of the neutral cannabinoids remained the same (Figure 2). The elution order of the unknown constituent was altered by the change in pH, therefore it was determined that the unknown constituent was susceptible

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