A reliable and validated LC-MS/MS method for the simultaneous quantification of 4 cannabinoids in 40 consumer products

Qingfang Meng, Beth Buchanan, Jonathan Zuccolo, Mathieu-Marc Poulin, Joseph Gabriele, David Charles Baranowski

PLoS ONE, 2018, 13, 5, e0196396.

https://doi.org/10.1371/journal.pone.0196396

 

Abstract

In the past 50 years, Cannabis sativa (C. sativa) has gone from a substance essentially prohibited worldwide to one that is gaining acceptance both culturally and legally in many countries for medicinal and recreational use. As additional jurisdictions legalize Cannabis products and the variety and complexity of these products surpass the classical dried plant material, appropriate methods for measuring the biologically active constituents is paramount to ensure safety and regulatory compliance. While there are numerous active compounds in C. sativa the primary cannabinoids of regulatory and safety concern are (-)-Δ9- tetrahydrocannabinol (THC), cannabidiol (CBD), and their respective acidic forms THCA-A and CBDA. Using the US Food and Drug Administration (FDA) bioanalytical method validation guidelines we developed a sensitive, selective, and accurate method for the simultaneous
analysis CBD, CBDA, THC, and THCA-A in oils and THC & CBD in more complex matrices. This HPLC-MS/MS method was simple and reliable using standard sample dilution and homogenization, an isocratic chromatographic separation, and a triple quadrupole mass spectrometer. The lower limit of quantification (LLOQ) for analytes was 0.195 ng/mL over a 0.195±50.0 ng/mL range of quantification with a coefficient of correlation of >0.99. Average intra-day and inter-day accuracies were 94.2±112.7% and 97.2±110.9%, respectively. This method was used to quantify CBD, CBDA, THC, and THCA-A in 40 commercial hemp products representing a variety of matrices including oils, plant materials, and
creams/cosmetics. All products tested met the federal regulatory restrictions on THC content in Canada (<10 μg/g) except two, with concentrations of 337 and 10.01 μg/g. With respect to CBD, the majority of analyzed products contained low CBD levels and a CBD: CBDA ratio of <1.0. In contrast, one product contained 8,410 μg/g CBD and a CBD: CBDA ratio of >1,000 (an oil-based product). Overall, the method proved amenable to the analysis of various commercial products including oils, creams, and plant material and may be diagnostically indicative of adulteration with non-hemp C. sativa, specialized hemp cultivars, or unique manufacturing methods.

Introduction

Cannabis sativa is one of three generally recognized plant species of Cannabis [1]. C. sativa has been used for industrial textiles, food production (hemp), medicinal, and illicit psychoactive properties (marihuana) for several thousand years [2]. The biological potential of the plant has been investigated for the treatment of pain, glaucoma, nausea, asthma, depression, insomnia and neuralgia [3,4], multiple sclerosis [5], and inflammatory diseases [6,7], epilepsy [8], and movement disorders [9]. Not until the mid-20th century were the cannabinoids responsible for the biological effects of C. sativa first identified [10,11].

C. sativa contains a family of approximately 60 structurally similar cannabinoids [12], however the majority of research to date has focused upon the psychoactive Δ9-tetrahydrocannabinol (THC) and the structurally similar non-psychoactive cannabidiol (CBD). THC is a ligand for cannabinoid receptor-1 and -2 (CB1 and CB2), which regulate a variety of basic physiological processes such as appetite, mood, memory, and inflammation [13]. As such, activation of CB1 and CB2 can yield broad neurological manifestations that are further complicated by the dissimilar molecular effects of THC or CBD [14]. Specifically, THC is a partial agonist of CB1 and CB2 whereas CBD is a negative allosteric modulator and so the overall physiological effect of C. sativa is often related to both THC and CBD content [15,16]. While THC and CBD are the most relevant cannabinoids to mammalian biology, C. sativa produces both in their inactive acidic forms [17,18].

The acidic forms of CBD and THC are cannabidiolic acid (CBDA) and Δ9-tetrahydrocannabinolic acid A (THCA-A), respectively. CBDA and THCA are psychologically inactive precursors that may be converted to CBD and THC via decarboxylation [19]. This conversion is promoted by heat, however the extent of conversion is dependant on the heating method [20,21]. Therefore, analytical methods that include thermal sample manipulations (e.g. gas chromatography) require chemical derivatization to evaluate CBDA and THCA-A independently of CBD and THC [22]. This has given rise to numerous liquid chromatography-based methods to evaluate both acidic and non-acidic forms.

LC-MS/MS test development has been prolific in THC forensic analysis of hair, blood, urine, and sweat [23±26]. In contrast, the application of LC-MS/MS method to the complex and diverse matrices often encountered in the food and supplements marketplace is not strongly established. Citti and colleagues developed and tested a liquid chromatography and ultraviolet spectroscopy (LC-UV) method to evaluate hemp seed oils for multiple cannabinoids [27]. However, the low sensitivity of UV spectroscopy and lack of specificity detracts from its broad applicability in complex samples. Similarly, Carcieri and colleagues developed an LC mass spectrometry (LC-MS/MS) method to test inter-lot variability in medicinal preparations of olive oil-based formulations containing Cannabis [28]. This investigation identified high variability in THC and CBD between lots, however, the described sample LLOQ of 100 μg/mL is insufficient to verify if products conform to hemp regulations (10 μg/g). Yang and colleagues investigated three brands of consumer-grade hemp seeds using four different procedures to extract phytocannabinoids, and quantified total THC and CBD [29]. In almost all cases, THC concentrations were reported as higher than the legal limit [30]. Given the hypothesized absence of a cannabinoid biosynthetic pathway within the seeds [31,32], the elevated THC levels observed likely arise from remaining husks or contamination by other organs. This method was specific to seeds and did not account for the complex matrices encountered in consumer goods.

Here, a sensitive, simple, and reliable method for the simultaneous quantification of THC, CBD, THCA-A and CBDA in a variety of finished commercial products is presented. The LC-MS/MS method was validated and applied to 40 commercially available hemp products including solids, oils, creams, and capsules. To our knowledge, this is the first report of a A reliable and validated LC-MS/MS method for the simultaneous quantification of 4 cannabinoids in 40 consumer products method that quantifies these primary cannabinoids in a variety of materials and is amenable to safety, quality, stability, and consistency testing.

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pone.0196396