Journal:Development and validation of a fast gas chromatography–mass spectrometry method for the determination of cannabinoids in Cannabis sativa L

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Full article title Development and validation of a fast gas chromatography–mass spectrometry method for the determination
of cannabinoids in Cannabis sativa L
Journal Journal of Food and Drug Analysis
Author(s) Cardenia, Vladimiro; Toschi, Tullia G.; Scappini, Simona; Rubino, Rosamaria C.; Rodriguez-Estrada, Maria T.
Author affiliation(s) University of Bologna, Enecta B.V.
Primary contact Email: tullia dot gallinatoschi at unibo dot it
Year published 2018
Volume and issue 26(4)
Page(s) 1283–92
DOI 10.1016/j.jfda.2018.06.001
ISSN 1021-9498
Distribution license Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
Website https://www.sciencedirect.com/science/article/pii/S1021949818301066
Download https://www.sciencedirect.com/science/article/pii/S1021949818301066/pdfft (PDF)

Abstract

A routine method for determining cannabinoids in Cannabis sativa L. inflorescence, based on fast gas chromatography coupled to mass spectrometry (Fast GC-MS), was developed and validated. To avoid the decarboxylation of the carboxyl group of cannabinoids, different derivatization approaches—i.e., silylation and esterification (diazomethane-mediated) reagents and solvents (pyridine or ethyl acetate)—were tested. The methylation significantly increased the signal-to-noise ratio of all carboxylic cannabinoids, except for cannabigerolic acid (CBGA). Since diazomethane is not commercially available, is considered a hazardous reactive, and requires one-day synthesis by specialized chemical staff, the process of silylation was used along the entire validation of a routine method. The method gave a fast (total analysis time < 7.0 min) and satisfactory resolution (R > 1.1), with a good repeatability (intraday < 8.38%; interday < 11.10%) and sensitivity (LOD < 11.20 ng/mL). The suitability of the fast GC-MS method for detection of cannabinoids in hemp inflorescences was tested; a good repeatability (intraday < 9.80%; interday < 8.63%), sensitivity (LOD < 58.89 ng/mg), and robustness (< 9.52%) was also obtained. In the analyzed samples, the main cannabinoid was cannabidiolic acid (CBDA; 5.19 ± 0.58 g/100 g), followed by cannabidiol (CBD; 1.56 ± 0.03 g/100 g) and CBGA (0.83 g/100 g). Δ9-tetrahydrocannabivarin (THCV) was present at trace levels. Therefore, the developed fast GC-MS method could be a valid, routine alternative for a fast, robust, and highly sensitive determination of the main cannabinoids present in hemp inflorescences.

Keywords: cannabinoids, cannabis, decarboxylation, fast gas chromatography, methylation

Graphical abstract

Fig0 Cardenia JofFoodDrugAnal2018 26-4.jpg

Introduction

Recently, the interest on Cannabis sativa L. has drastically increased. However, attention has primarily been given to addressing its psychoactive[1] and non-psychoactive compounds, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). In the past, the genus Cannabis was allocated into three main species: a drug-type (C. indica) with high levels of Δ9-THC, a fiber-type (C. sativa L.) with low levels of Δ9-THC, and an intermediate type C. ruderalis Janish.[2] More recently, the different Cannabis species have been divided into two broad types: C. sativa or “hemp” when referring to industrial use (fiber-type), and therapeutic “marijuana” (drug-type) when referring to varieties with a high level of Δ9-THC (>0.6%; w/w). To date, the main use for hemp is largely related to food; in fact, hemp seeds are generally used for producing oil and flour, and, depending on the country's local regulations, they may also be employed on the basis of their pharmacological properties.[3] However, hemp contains more than 500 different cannabinoids, of which about 10 have been classified according to their chemical structure, such as Δ9-tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabigerol (CBG), Δ8-tetrahydrocannabinol (Δ8-THC), Δ9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), cannabinol (CBN), cannabidiolic acid (CBDA), Δ9-tetrahydrocannabinolic acid (THCA), and cannabigerolic acid (CBGA).[4]

Hemp cannabinoids exhibit diverse biological effects. THCV displays various pharmacological profiles according to the type of molecular target (in vitro antagonistic/inverse agonistic effects and an in vivo agonism effect in an antinociception model).[5] The application of CBD for intractable pediatric epilepsy has also been recently studied.[6] On the other hand, CBC, which is particularly present in freshly harvested C. sativa, normalizes in vivo intestinal motility when intestinal inflammation occurs.[7] It should be pointed out that C. sativa does not produce Δ9-THC, CBD, CBG, and CBC, but their respective carboxylic acid forms (precursors) Δ9-THCA, CBDA, CBGA, and CBCA can undergo decarboxylation by heating or drying and thus exhibit their corresponding biological effects.[3] The galenic preparations of cannabis (such as medicinal oils), which are important for their possibility of being employed as a whole set of cannabinoids, are characterized by a high variability[8] and require a robust, simple quality control method for their titration.

Considering these and other biological effects of cannabinoids, their analysis in cannabis is of great interest and importance. There are several analytical methods for determining cannabinoids, most of which use gas chromatography coupled to mass spectrometry (GC-MS) or a flame ionization detector (GC-FID), or high-performance liquid chromatography coupled to mass spectrometry (LC-MS) or an ultraviolet detector (LC-UV).[2][3][4][9][10][11]

When GC-MS is used, the electron impact ionization (EI) generates mass spectra, which can be compared with those present in compound libraries for their identification. However, with LC-MS and electrospray (ESI) and atmospheric pressure chemical ionization (APCI), only molecular ions are generated, without other useful fragments for compound characterization; as such, expensive equipment able to perform tandem mass spectrometry (MS-MS) experiments is required.[12][13] As reported in literature, LC-MS sensitivity is lower than that of GC-MS.[4] However, there is a lot of criticism around the use of GC for cannabinoid analysis, since the high temperature of both injector and detector lead to decarboxylation of cannabinoid acids if not previously derivatized (e.g., using a process such as silylation).[14][15] Different silylation procedures have been reported for this scope; Purschke et al.[16] utilized N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), while other researchers have used the combination of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or MSTFA, with either trimethylchlorosilane (TMCS) or ethyl acetate.[17] However, no data are reported about esterification of cannabinoid carboxylic acids by diazomethane and the use of fast GC-MS for cannabinoid determination. Fast GC-MS has been demonstrated to provide the advantages of mass spectrometry boosted by the utilization of fast chromatography. In fact, the use of fast GC-MS drastically reduces the time of analysis without impairing sensitivity, resolution, and other analytical parameters (such as repeatability and reproducibility). Fast GC-MS has been successfully utilized for the determination of cholesterol oxidation products in 3.5 min[18], phytosterols and phytostanols in milk dairy products in less than 10 min[19], and heroin and cocaine in 3 min.[20]

To the best of our knowledge, no previous works have been published on the determination of cannabinoids in hemp inflorescences by fast GC-MS. The aim of this work was to develop and validate a fast GC-MS method for determining the main cannabinoids (THCV, CBD, CBC, CBDA, THCA, Δ9-THC, Δ8-THC, CBG, CBN, and CBGA) in hemp inflorescences, as related to different derivatization reagents (through silylation and esterification).

Materials and methods

Reagents and solvents

References

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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. Some grammar and punctuation was cleaned up to improve readability. In some cases important information was missing from the references, and that information was added. Everything else remains true to the original article, per the "NoDerivatives" portion of the distribution license.