HPLC-UV-HRMS analysis of cannabigerovarin and cannabigerobutol, the two impurities of cannabigerol extracted from hemp

Highlights

• Cannabigerovarin (CBGV) and cannabigerobutol (CBGB) are the two major impurities in cannabigerol (CBG) extracted from hemp.

• CBGV and CBGB were identified by high-resolution mass spectrometry.

• CBGV and CBGB were synthesized and fully characterized (NMR, FT-IR, UV, HRMS).

• A simple and selective HPLC-UV method was developed and validated.

• The method was applied to three batches of commercial CBG marketed by certified companies.

Abstract

A sensitive and straightforward HPLC-UV method was developed for the simultaneous quantification of the two main impurities in “pure” commercial cannabigerol (CBG) samples. The identification of such impurities, namely cannabigerovarin (CBGV) and cannabigerobutol (CBGB), the propyl and butyl homologs of CBG, respectively, was accomplished employing the high-resolution mass spectrometry (HRMS) technique, and subsequently confirmed by comparison with the same compounds obtained by chemical synthesis. Complete spectroscopic characterization (NMR, FT-IR, UV, and HRMS) of both impurities is reported in the present work. The method was validated in terms of linearity, which was assessed in the range 0.01–1.00 μg/mL, sensitivity, selectivity, intra- and inter-day accuracy and precision, and short-term stability, which all satisfied the acceptance criteria of the ICH guidelines. Application of the method to the analysis of four commercial CBG samples highlighted a certain variability in the impurity profile that might be ascribed to the hemp variety of the starting plant material. With these new analytical standards in hand, it would be interesting to investigate their concentrations in different hemp varieties and expand the scope of a phytocannabinomics approach for a comprehensive profiling of this remarkable class of natural compounds.

Introduction

Cannabis sativa L. is a plant producing over 150 different phytocannabinoids, of which only a few have been isolated and fully characterized to date. Considerable efforts have been devoted to the investigation of the chemistry of the main psychotropic compound of cannabis, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), and its pharmacology; in comparison, very little has been done on the front of the non-psychotropic phytocannabinoids, such as cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and so forth. CBD has certainly gained much more attention in the last five years due to the discovery of a plethora of pharmacological properties that have prompted industrial companies to start a business on the extraction of CBD from the plant to market it as a pure active ingredient of pharmaceutical grade. Since its approval as a drug for the treatment of severe forms of infant epilepsy under the commercial name of Epidiolex (GW Pharmaceuticals, UK), CBD has been extensively extracted from cannabis/hemp for pharmaceutical purposes and marketed as a pure substance. However, recent studies revealed two main impurities in such commercial products with chemical structures similar to that of CBD but with a different length of the side alkyl chain [1,2]. The most abundant impurity, cannabidivarin (CBDV), has a three-term linear side chain. In contrast, the second impurity, cannabidibutol (CBDB), has a four-term chain and was characterized for the first time by our research group [1,2].

Similarly, CBG has recently attracted the interest of both the industrial and scientific community, as shown by the increase in the number of publications per year on PubMed [3]. Although CBG is generally present as a minor component (<10 % of the total cannabinoid fraction) compared to either the dominant CBD in the most common fiber-type cannabis varieties or to THC in drug-type cannabis, CBG-predominant cultivars (with >85 % CBG) have also been reported in the literature [4]. Such a very attractive chemotype has started to be bred [4] due to the remarkable pharmacological properties of this phytocannabinoid especially as antimicrobial, anti-inflammatory, cytotoxic, and antidepressant agent, besides its non-psychotropic nature [5]. CBG-rich cannabis varieties are therefore genetically selected for the extraction of CBG to make a pure marketable substance.

Commercial CBG is generally labelled as ≥98 % pure, bearing two main impurities, one of which is cannabigerovarin (CBGV) and another compound with the hypothetical structure of a cannabigerol with a butyl side chain, commonly named CBG-C₄. Some companies do not even mention the chemical nature of the impurities in their certificate of analysis. However, to the best of the authors’ knowledge, the butyl homolog of CBG has been neither identified nor characterized up to now. The aim of the present work was to characterize the impurities of commercial CBG and provide a simple method for their qualitative and quantitative determination. Such impurities were first putatively identified by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS) as the propyl and butyl homologs of CBG, namely CBGV and cannabigerobutol (CBGB) in line with the common nomenclature given to this type of variants with respect to olivetol-derived phytocannabinoids [1,2,6]. The former was first reported in 1975 [7], and its chemical and pharmacological properties have been investigated [[8], [9], [10]], although not extensively, whereas the latter has never been characterized to date. Only two records from Berman et al. mention the presence of a minor peak putatively identified as CBG-C₄ in the same cannabis extracts, but it was not chemically characterized [11,12]. An ad hoc stereoselective synthesis of the pure E-isomers of both CBGV and CBGB and their complete chemical, physical characterization by spectroscopic techniques (HRMS, NMR, FT-IR, and UV spectroscopy) allowed for the identification of the two main impurities in “pure” CBG samples. In particular, the present work represents the first reported full chemical characterization of the butyl homolog of CBG, which will enable to determine such compound in different hemp varieties and perhaps study a potential correlation among the several cannabinoid homologs with different length of the alkyl chain [6,13,14]. Lastly, a straightforward and fast HPLC method coupled to UV detection was developed and validated according to ICH guidelines (Q3A) in terms of selectivity, linearity, sensitivity, intra- and inter-day accuracy and precision, carry-over, and stability [15].