Journal:Simultaneous quantification of 17 cannabinoids in cannabis inflorescence by liquid chromatography–mass spectrometry
| Full article title | Simultaneous quantification of 17 cannabinoids in cannabis inflorescence by liquid chromatography–mass spectrometry |
|---|---|
| Journal | Separations |
| Author(s) | Hewavitharana, Amitha K.; Gloerfelt-Tarp, Francine; Nolan, Matthew; Barkla, Bronwyn J.; Purdy, Sarah; Kretzschmar, Tobias |
| Author affiliation(s) | Southern Cross University, New South Wales Department of Primary Industries |
| Primary contact | Email: a dot hewavitharana at pharmacy dot uq dot edu dot au |
| Year published | 2022 |
| Volume and issue | 9(4) |
| Article # | 85 |
| DOI | 10.3390/separations9040085 |
| ISSN | 2297-8739 |
| Distribution license | Creative Commons Attribution 4.0 International |
| Website | https://www.mdpi.com/2297-8739/9/4/85/htm |
| Download | https://www.mdpi.com/2297-8739/9/4/85/pdf (PDF) |
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Abstract
With an increasing appreciation for the unique pharmacological properties associated with distinct, individual cannabinoids of Cannabis sativa, there is demand for accurate and reliable quantification for a growing number of them. Although recent methods are based on highly selective chromatography–mass spectrometry technology, most are limited to a few cannabinoids, while relying on unnecessarily sophisticated and expensive ultra-high-performance liquid chromatography and tandem mass spectrometry. Here we report an optimized, simple extraction method followed by a reliable and simple high-performance liquid chromatography (HPLC) method for separation. The detection is performed using a time-of-flight mass spectrometer that is available in most natural products research laboratories. Due to the simplicity of instrumentation, and the robustness resulting from a high resolution in the chromatography of isobaric cannabinoids, the method is well-suited for routine phytocannabinoid analysis for a range of applications. The method was validated in terms of detection and quantification limits, repeatability, and recoveries for a total of 17 cannabinoids. Detection limits were in the range 11–520 pg when using a 1 µL sample injection volume, and the recovery percentages ranged from 85% to 108%. The validated method was subsequently applied to determine cannabinoid composition in the inflorescences of several medicinal Cannabis sativa varieties.
Keywords: cannabinoids, phytocannabinoids, LC-MS, Cannabis sativa, tetrahydrocannabinol, THC, cannabidiol, CBD
Introduction
Cannabis sativa L. (Cannabis) is one of the oldest domesticated plants, cultivated for the purpose of food, fiber, psychoactives, and medicine. In recent years, its medicinal properties have gained a renewed interest given its therapeutic potential in the treatment of a variety of disease conditions. To date, over 100 different cannabinoids have been identified, with Δ9-tetrahydrocannabinol (THC), well known for its psychoactive properties, and others, such as cannabidiol (CBD), recognized for their therapeutic and medicinal value. [1,2]
Historically, cannabis samples were analyzed mostly in a legal or regulatory context for the purpose of determining Δ9-THC content. However, as more cannabinoids were identified and their pharmacological potential was examined, the quantification of cannabinoids other than Δ9-THC gained importance. Additional cannabinoids of interest now include Δ9-tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabivarin (THCV), Δ9-tetrahydrocannabivarinic acid (THCVA), cannabidivarin (CBDV), and cannabidivarinic acid (CBDVA). [2,3] A recent study on virus-neutralizing capabilities of naturally occurring cannabinoids (in acid form) even found that CBDA and CBGA are capable of binding to the spike protein of SARS-CoV-2, therefore making for possible candidates for the treatment and prevention of COVID-19. [4] Clearly, accurate methods of quantification are needed to determine the concentrations of both major and minor cannabinoids of pharmacological potential in cannabis inflorescence, the source of cannabinoids in the plant.
Despite the rising demand and importance of accurate quantification of cannabinoids, the number of studies with a clear focus on analytical method development remains relatively small. This might be in part due to regulatory constraints, which necessitate specific licenses to handle this scheduled drug and limit the availability of standard compounds and test samples. Due to this, the cannabis testing industry remains poorly established, with only a relatively limited number of laboratories offering analytical services and a lack of standardized protocols for extraction and analysis. [5]
Two review articles published recently [6,7] contain critical evaluations of methods used for cannabinoid analysis and the recent trends; therefore, a detailed comparison of methods is not included here. Chromatographic techniques are generally used for the separation of cannabinoids. Liquid chromatography (LC) has gained preference over gas chromatography (GC) as LC avoids conversion of the acid forms of cannabinoids to their neutral forms, which occurs at the high temperatures used in GC. [8] Until recently, the detection of column-separated cannabinoids was predominantly carried out using UV spectrophotometry, which provides low specificity and makes baseline separation of all cannabinoids imperative. However, complete separation of structurally similar cannabinoids, especially isomeric compounds such as Δ8-THC and Δ9-THC, is challenging. [3] Due to the lack of specificity of UV detection, any unknown compound/s co-eluting at the same retention time as the target cannabinoid can cause overestimation of its concentration.
To overcome this specificity problem, the detection method of choice has been changing to mass spectrometry (MS). [6,7] As MS detection can identify molecules according to differences in mass, the chromatographic separation becomes less important. However, since some of the major cannabinoids—e.g., Δ8-THC, Δ9-THC, CBD, cannabichromene (CBC), and cannabicyclol (CBL)—have the same molecular mass, chromatographic separation is still required. Most of the methods using MS detection of cannabinoids are based on tandem mass spectrometry (MS-MS). [6,7] However, there is no real advantage of using this approach over basic MS, as cannabinoids of the same molar masses produce similar fragmentation patterns in the second MS event, thus not providing additional selectivity. Therefore, baseline separation by chromatography followed by simple MS detection is well suited for the routine analysis of cannabinoids.
According to a recent review of cannabinoid analysis, variability in extraction (solvent/s, method, time, and temperature) significantly contributes to differential analytical results. [6] Most of the recent analytical methods have used ethanol for extraction of both acidic and neutral cannabinoids [3,9], although the method used for extraction varied in terms of sample to solvent ratio, the extraction technique, and the duration of extraction. Therefore, a study of the effects of these variables on the amounts of cannabinoids extracted is warranted.
The aims of this study were to develop a simple yet effective method for the extraction of cannabinoids from cannabis inflorescences, and to develop a reliable, robust and simple liquid chromatography–mass spectrometry (LC–MS) method that can be used for routine analysis of 17 phytocannabinoids for which standards are available. In this study, a time-of-flight mass spectrometer (TOF-MS) was used as the detector. The acquisition of spectral data was by simple MS mode rather than MS-MS because there was no advantage in using tandem MS. As many natural products laboratories are equipped with a TOF instrument (primarily used for qualitative analysis), a quantification method based on a TOF-MS would facilitate adoption of this method. The extraction conditions were optimized to develop a simple and robust method to extract cannabinoids from inflorescence, without altering the composition of cannabinoids. A separation method that uses a low flow rate enabled the use of (high-performance liquid chromatography (HPLC) rather than requiring an expensive ultra-high-performance liquid chromatography (UHPLC) system. The chromatographic separation of the cannabinoids with the same molar mass was made sufficiently robust so that small changes in column and/or mobile phase do not cause the isobaric cannabinoids to co-elute.
Materials and methods
Materials
Proprietary medicinal cannabis samples of Cann Group Limited were supplied by New South Wales Department of Primary Industries (NSW DPI, Orange, Australia) under an Office of Drug Control (ODC) permit and received by Southern Cross University (SCU) under NSW Health authority. Samples of contrasting lines for THC/A and CBD/A content were used for method establishment and validation.
All cannabinoid reference standards were purchased from Novachem Pty. Ltd. (Heidelberg, Australia) as ampoules of 1,000 µg/mL solutions. The standards for (±)-Δ9-tetrahydrocannabinolic acid A (Δ9-THCA-A), tetrahydrocannabivarinic acid (THCVA), cannabidiolic acid (CBDA), cannabidivarinic acid (CBDVA), cannabigerolic acid (CBGA), cannabinolic Acid (CBNA), cannabichromenic acid (CBCA), (±)-cannabicyclol (CBL), and cannabicyclolic acid (CBLA) were supplied in acetonitrile. The standards for (−)-Δ9-tetrahydrocannabinol (Δ9-THC), (−)-Δ8-tetrahydrocannabinol (Δ8-THC), tetrahydrocannabivarin (THCV), cannabidiol (CBD), cannabidivarin (CBDV), cannabigerol (CBG), cannabinol (CBN), and cannabichromene (CBC) were supplied in methanol. Ibuprofen was purchased from Sigma-Aldrich Pty Ltd. (North Ryde, NSW, Australia). HPLC-grade ethanol (Scharlau, Scharlab S.L., Barcelona, Spain) was used for sample extractions, and LC-MS grade solvents (Honeywell Research Chemicals, Seelze, Germany) and MilliQ water (Merck Millipore, Billerica, MA, USA) were used to prepare mobile phase.
References
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.
