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. 2021 Nov 16;6(47):32186–32197. doi: 10.1021/acsomega.1c04992

Contents of Cannabinoids in Hemp Varieties Grown in Maryland

Xiaoyan Chen †, Hua Deng †, Janai A Heise ‡, David P Puthoff ‡, Nabeel Bou-Abboud †,§, Hongtao Yu †, Jiangnan Peng †,§,*
PMCID: PMC8637966  PMID: 34870039

Abstract

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Coincident with the cannabis legalization and the increased interest in the medicinal use of the plant, the cannabis marketplace and farming have seen tremendous growth. It is reported that there are more than 2000 cannabis varieties available to customers. However, the data that is available to the growers and breeders regarding the cannabinoid contents of various varieties remains low. Here, a high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous separation and determination of 11 cannabinoids. A total of 104 hemp bud materials belonging to 20 varieties were collected from farms in the state of Maryland and analyzed with the HPLC method. The contents of the 11 cannabinoids in various varieties were compared and discussed, highlighting the varieties that showed a high yield of cannabinoids and good consistency that are more appropriate for cannabinoid production.

Introduction

Cannabis sativa L., or Cannabis, is a member of the Cannabaceae family and is cultivated all over the world.1 It is one of the world’s oldest domesticated crops that had been used medically and nonmedically for thousands of years until, in the 1930s, it was classified as an illicit drug.2−4 Informally, drug types of cannabis are commonly referred to as marijuana that is primarily grown and used for its medicinal or recreational psychoactive properties, while hemp strains are grown as a rich source of fibers and other materials.5,6 There are more than 500 compounds identified from cannabis plants, among which over 100 are different phytocannabinoids.7,8 The two most known and researched cannabinoids are Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Δ9-THC is the primary psychoactive component of cannabis, the content level of which represents the key factor differentiating between hemp and medical cannabis. CBD is a major cannabinoid known as a pharmacologically active and nonintoxicating substance.6

The genus Cannabis has an inconclusive taxonomic organization, and there have been debates on the Cannabis species.9,10 It is accepted that Cannabis is monotypic, and all observable subpopulations, subspecies, and strains are varieties of one single species, C. sativa L.1,2 Through centuries of natural and artificial selection, cannabis has evolved to have huge genetic diversity and a large number of cultivated varieties.11 Cannabinoid composition shows much variation across different cannabis varieties.12,13 A range of studies can be found investigating the factors that affect cannabinoid production, such as the parts, sex, and maturity of the plant.14−17 However, there is a limited amount of data available regarding the cannabinoid profiles in various hemp varieties.

Though C. sativa L. had been listed as a “Schedule I” substance since the year 1970 in the United States, there were discrepancies between state laws and federal legislation, and many states have passed legislation designed to allow clinical research and medicinal use.4,18 California became the first state to legalize medical marijuana use in 1996. Colorado and Washington then became the first states to legalize recreational marijuana use in 2012.6 At the federal level, the Agriculture Improvement Act of 2018 (the 2018 Farm Bill) was signed into law in December 2018 and removed hemp or cannabis plants and derivatives that contain no more than 0.3 percent Δ9-THC on a dry weight basis from the Controlled Substances Act (CSA).19 Worldwide, an increasing number of countries have also begun to allow possession and use of cannabis in recent years.20

Driven by the cannabis/hemp legalization and increased interest in the medicinal use of the plant, the cannabis marketplace and industry have seen a boom.21 Coincident with this, hemp farming has exploded, and many farmers across the United States are looking to grow CBD hemp.22−24 According to USDA Farm Service Agency, U.S. industrial hemp planted acreage increased from zero in 2013 to 32 464 in 2018 to 146 065 by mid-2019.25

Various analytical methods have been developed for the determination of cannabinoids in plant materials and human matrices, including RP-HPLC-UV, UHPLC-MS/MS, and GC-FID.26−29 These analytical approaches were well summarized in a review by Citti et al.30 A concern with LC-UV is its inadequate resolution for the chromatographic separation of the cannabinoids such as CBG with CBD or CBDA with CBGA.27,28,31,32 We report an HPLC-UV method here that achieves a complete separation between the 11 cannabinoids. In 1973, Small and Beckstead identified three chemical types of cannabis based on ratios of THC and CBD in a comprehensive survey and reported a series of varieties with a high CBD/THC ratio (THC <0.3%).33 Numerous systematic biochemical studies of cannabis plants have been reported over the past dozen years.34−39 In the early surveys in cannabinoid content, interest was primarily on medical cannabis (marijuana), and a large number of papers had concentrated on the classification and the differentiation of various chemical types of cannabis, referred to as chemotype or chemical phenotype.34,36,38,40−51 Until recently, hemp began to attract much interest in the wake of legalization.13,52−62 The data regarding the cannabinoid profile is available only for a small part of hemp varieties that is on the market.

Since cannabinoids were found to concentrate in female flowers and to achieve the highest level at the budding stage, this study focused on the buds of hemp.4,7,13,63 This paper intends to provide an overview of commonly occurring cannabinoids, including cannabidivarin (CBDV), (-)-trans-Δ9-tetrahydrocannabivarin (THCV), CBD, cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabinol (CBN), Δ9-THC, Δ8-THC, CBC, and (-)-Δ9-(trans)-tetrahydrocannabinolic acid A (THCA) in hemp varieties grown in Maryland. Our findings will help the growers make wise choices when selecting the varieties to grow and be useful for developing new varieties as well.

Results and Discussion

Calibration Curves and Parameters of Validation

The resolutions Rs of all cannabinoid peaks of interest are >1.2, indicating that the method proposed here is selective. A typical chromatogram of hemp extract and the chromatogram of standards produced from the developed method are shown in Figure 1. The regression equation, calibration range, LOD, and LOQ for each cannabinoid are listed in Table 1. The calibration curves are presented in Supporting Information S2. The correlation coefficient (R2) was obtained between 0.9994 and 0.9997, which showed a good linear relationship of the developed method. For the 11 cannabinoids under investigation, the LOD and LOQ ranged from 0.01 to 0.11 and 0.04 to 0.36 μg/mL, respectively, with an injection volume of 5 μL.

Figure 1.

Figure 1

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(a) Typical HPLC/UV (220 nm) chromatogram of hemp extract produced from the developed method. (b) HPLC chromatographic separation of the mixture of standard cannabinoids.

Table 1. Regression Equations and Calibration Ranges, LOD, and LOQ for the 11 Cannabinoids.

cannabinoid regression equation coefficient of determination R2 calibration range (μg/ml)a LOD (μg/ml)a LOQ (μg/ml)a
CBDV Y = 2445.05X 0.9995 50–0.1 0.01 0.04
THCV Y = 2179.88X 0.9994 50–0.4 0.11 0.36
CBD Y = 2303.26X 0.9996 50–0.3 0.07 0.30
CBG Y = 2255.69X 0.9995 50–0.4 0.08 0.31
CBDA Y = 2669.64X 0.9997 50–0.3 0.07 0.22
CBGA Y = 2849.37X 0.9995 50–0.2 0.06 0.20
CBN Y = 4202.93X 0.9994 50–0.2 0.03 0.11
Δ9-THC Y = 2173.57X 0.9996 50–0.2 0.06 0.16
Δ8-THC Y = 1760.81X 0.9996 50–0.2 0.06 0.20
CBC Y = 2433.96X 0.9995 50–0.2 0.04 0.14
THCA Y = 2553.98X 0.9996 50–0.4 0.07 0.17
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a

These parameters correspond to an injection volume of 5.0 μl.

As demonstrated in Table 2, the results of precision and accuracy showed good reproducibility for the quantification of the 11 cannabinoids in hemp plants with intra- and interday variations of 0.4–5.4 and 0.4–5.7%, respectively. In addition, the compounds showed percentage recoveries ranging from 78.4–104.6%. These results demonstrated that this HPLC/UV method had satisfactory accuracy, precision, and reproducibility for the quantitative determination of 11 cannabinoids in cannabis plants.

Table 2. Intraday Precision (RSD) (n = 4), Interday Precision (RSD) (n = 3), and Individual Percentage Recovery (n = 3) of 11 Cannabinoids Obtained by RP-HPLC/UV.

cannabinoid intraday (%) interday (%) spiking level (μg/mL) recovery (%) cannabinoid intraday (%) interday (%) spiking level (μg/mL) recovery (%)
CBDV 2.9 5.7 0.39 97.9 CBN 0.4 1.3 0.39 92.7
0.96 96.9 0.96 94.4
2.06 94.9 2.06 94.4
THCV 5.4 3.4 0.39 81.3 Δ9-THC 2.1 1.9 0.39 95.2
0.96 78.4 0.96 98.2
2.06 90.8 2.06 95.6
CBD 1.4 2.3 0.39 96.6 Δ8-THC 0.6 0.4 0.39 98.2
0.96 86.3 0.96 95.3
2.06 93.7 2.06 101.8
CBG 3.0 3.2 0.39 104.6 CBC 1.8 4.5 0.39 98.0
0.96 91.2 0.96 92.5
2.06 95.0 2.06 97.7
CBDA 1.9 5.5 11.90 95.8 THCA 1.7 2.8 0.39 93.6
18.50 101.8 0.96 99.0
22.73 98.6 2.06 95.9
CBGA 1.1 2.8 0.39 101.8          
0.96 99.3    
2.06 94.8    
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Results of the Quantitative Determination of CBDA, CBD, THCA, and THC in Hemp Bud Materials from 20 Varieties

The quantitative data of 11 cannabinoid contents in 104 hemp samples expressed in mg/g dry weight are shown in the Supporting Information S3 and Figure 2. Table 3 presents the mean values of the 11 cannabinoid contents in each variety. ANOVA analyses showed a significant difference (p < 0.05) in content for all cannabinoids except for Δ9-THC between varieties (Supporting Information S3). Large variations within a single variety were extensively observed that were revealed by the standard deviation (SD) in Table 3 and the spread of box and whisker plots in Figure 2, which was also indicated by the coefficient of variation (CV > 30%) in Supporting Information S4.64 This low uniformity in a variety may be explained by the well-known fact that most available cannabis varieties are highly unstable in terms of genetics, morphological trait, and chemical profile, which is especially true of hybrid strains.13,65,66 Additionally, environmental and other factors, including soil conditions, day length, temperature, and plant placement, also affect cannabinoid yield in hemp.23,36 It should be realized that the variance within and between the varieties observed here is a combined effect of genetic diversity, environmental conditions, and workers’ operation, considering that the samples were collected from the field instead of being cultivated in a facility under a controlled environment.

Figure 2.

Figure 2

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Box and whisker plot graph of (a) CBD, (b) CBDA, (c) total CBD, (d) Δ9-THC, and (e) THCA content in 104 samples from 20 varieties. In each box, the low whisker represents the minimum, and the upper whisker represents the maximum. The concentrations below the LOQ are considered as 0 in this graph. Please see the values for a particular sample in Supporting Information S3.

Table 3. Average Content (mg/g) of 11 Cannabinoids for Each Variety (Mean ± SD).

no. variety CBDA CBD total CBD THCA Δ9-THC total THC CBGA CBG total CBGa CBC CBDV THCV Δ8-THC CBN
1 T1 trump 136.04 ± 11.44 5.42 ± 3.27 124.72 ± 11.17 5.15 ± 0.54 0.70 ± 0.45 5.21 ± 0.38 2.88 ± 0.77 N/Ab 2.62 ± 0.62 0.58 ± 0.23 N/A 1.10 ± 0.99a N/A N/A
2 T2 trump 133.83 ± 11.56 9.42 ± 1.33 126.79 ± 9.85 3.55 ± 0.04 1.10 ± 0.07 4.21 ± 0.08 1.34 ± 0.26 N/A 1.17 ± 0.23 1.04 ± 0.11 1.18 ±0.01 1.55 ± 1.09a N/A N/A
3 cherry 133.07 ± 32.56 7.33 ± 2.72 128.41 ± 29.22 5.07 ± 1.46 0.89 ± 0.31 5.34 ±1.22 2.58 ±1.20 N/A 2.49 ± 1.19 0.66 ± 0.15 N/A 0.91 ± 0.96a N/A N/A
4 magic bullet 102.95 ± 34.50 4.12 ± 1.26 94.41 ± 29.69 4.06 ± 1.58 0.47 ± 0.10 4.03 ±1.34 3.21 ± 1.63 N/A 3.08 ± 1.45 0.43 ± 0.03 N/A 0.46 ± 0.48a N/A N/A
5 cherry kandy 27.17 ± 10.86 3.61 ± 1.86 27.44 ± 10.50 0.65 ± 0.26 0.28 ± 0.19 0.85 ± 0.38 0.86 ± 0.47 N/A 0.75 ± 0.41 0.21 ± 0.71a N/A N/A N/A N/A
6 box 48.22 ± 21.44 5.02 ± 2.51 47.31 ± 20.47 1.37 ± 0.65 0.50 ± 0.26 1.70 ± 0.74 1.68 ± 0.69 N/A 1.63 ± 0.69 0.62 ± 0.33 N/A N/A N/A N/A
7 bubblegum 30.74 ± 8.36 4.07 ± 1.78 31.03 ± 8.94 0.72 ± 0.19 0.35 ± 0.14 0.98 ± 0.28 0.91 ± 0.59 N/A 0.89 ± 0.66 0.49 ± 0.53 N/A N/A N/A N/A
8 red kross 34.44 ± 10.48 2.80 ± 1.24 33.00 ± 9.46 4.00 ± 4.59 1.31± 1.84a 4.82 ± 5.26 0.81 ± 0.47 N/A 0.71 ± 0.42 N/A N/A N/A N/A N/A
9 baox 94.87 ± 30.27 2.83 ± 0.73 86.00 ± 26.98 3.56 ± 1.17 0.37 ± 0.13 3.50 ± 0.99 5.73 ± 3.54 N/A 5.23 ± 3.33 0.30 ± 0.15a 0.37 ± 0.34 N/A N/A N/A
10 cherry wine 40.62 ± 24.92 1.73 ± 1.51 37.35 ± 22.10 2.22 ± 2.29 0.55 ± 1.25 2.49 ± 3.17 1.89 ± 1.64 N/A 1.66 ± 1.44 N/A N/A N/A N/A N/A
11 wife 82.99 ± 28.75 2.07 ± 0.76 74.85 ± 25.48 3.33 ± 1.09 0.31 ± 0.08 3.23 ± 1.04 4.32 ± 0.61 N/A 4.09 ± 0.71 N/A N/A N/A N/A N/A
12 stray kat 54.83 ± 5.29 2.89 ± 0.37 50.97 ± 4.78 1.69 ± 0.19 0.24 ± 0.04 1.72 ± 0.19 2.97 ± 0.67 N/A 2.61 ± 0.58 0.58 ± 0.06 0.27 ± 0.19 N/A N/A N/A
13 mystery 79.75 ± 15.02 6.15 ± 0.43 76.09 ± 13.57 2.23 ± 0.49 0.59 ± 0.02 2.55 ± 0.45 0.60 ± 0.07 N/A 0.53 ± 0.06 0.98 ± 0.34 0.67 ± 0.17 1.94 ± 1.11 N/A N/A
14 materhorn 4.69 ± 1.65 0.65 ± 0.25 4.76 ± 1.69 0.25 ± 0.22a N/A 0.34 ± 0.36 74.32 ± 8.27 1.98 ± 0.69 67.23 ± 7.70 0.43 ± 0.24 N/A N/A N/A N/A
15 goliath 113.46 ± 14.63 6.91 ± 1.27 106.41 ± 13.78 3.66 ± 0.45 0.85 ± 0.12a 4.05 ± 0.49 2.16 ± 1.09 0.75 ± 0.21 2.65 ± 1.07 0.79 ± 0.06 1.09 ± 0.12 1.27 ± 0.44 N/A N/A
16 lindorea 134.68 ± 4.24 7.80 ± 0.91 125.91 ± 4.58 4.32 ± 0.29 0.91 ± 0.11 4.70 ± 0.17 3.00 ± 0.69 0.59 ± 0.06 3.22 ± 0.66 1.16 ± 0.36 1.16 ± 0.20 6.03 ± 0.98 N/A N/A
17 eclipse 130.15 ± 6.72 5.66 ± 1.06 119.80 ± 6.96 4.14 ± 0.09 0.59 ± 0.11 4.22 ± 0.15 2.03 ± 0.45 0.54 ± 0.09 2.32 ± 0.30 1.83 ± 0.52 1.05 ± 0.03 2.57 ± 1.56 N/A N/A
18 jupiter 107.44 ± 14.86 3.71 ± 0.91 97.93 ± 13.61 3.99 ± 0.47 0.35 ± 0.09 3.85 ± 0.46 2.93 ± 0.66 N/A 2.57 ± 0.58 0.35 ± 0.08 N/A 0.57 ± 0.62a N/A N/A
19 cherry #5 162.33 ± 21.19 6.42 ± 0.69 148.78 ± 18.23 6.36 ± 0.98 0.75 ± 0.09 6.33 ± 0.84 5.35 ± 3.15 N/A 5.06 ± 3.01 0.82 ± 0.14 N/A 0.69 ± 0.38a N/A N/A
20 otto 2 sweeten 80.56 ± 27.82 1.62 ± 0.56 72.27 ± 24.78 3.12 ± 1.18 N/A 2.84 ± 1.14 2.80 ± 1.03 N/A 2.46 ± 0.90 0.25 ± 0.20a N/A N/A N/A N/A
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a

The concentrations below LOQ were considered as 0 during the caculation of the mean.

b

N/A indicates the content below the LOD.

CBD is the principal component of many hemp products, the most commonly known is CBD oil and is now ubiquitous in the U.S. marketplace for over-the-counter purchases.67,68 Besides being approved as a prescription drug to treat seizures associated with Lennox–Gastaut syndrome (LGS), Dravet syndrome (DS), or tuberous sclerosis complex (TSC) by the U.S. FDA, the rapidly-rising popularity of CBD is due to its perceived analgesic, anxiety relief, neuroprotective, and anticancer effects.68,69 A variety of positive roles of CBD have been demonstrated in preclinical research.25,70,71 Due to economic interests and legislation, high levels of CBD and low levels of THC are desirable characters for many growers and breeders.66 In vivo studies have demonstrated that CBDA is also pharmacologically useful as an anti-inflammatory, anti-emetic, anti-convulsant, and anti-cancerogenic agent.72

All 20 varieties investigated in the present study are varieties commonly grown for CBD except for Matterhorn. Matterhorn is bred specifically, delivering high yields of CBG. The CBD contents of the 20 varieties are presented in Supporting Information S3 and Figure 2a, where we can see that the highest amounts of CBD were found with the varieties of T2 trump (8.44–11.30 mg/g), Mystery (5.56–6.57 mg/g), Goliath (5.35–8.48 mg/g), Lindorea (6.53–8.63 mg/g), and Cherry #5 (5.81–7.56 mg/g), followed by Eclipse, Jupiter, Bubblegum, and Magic bullet (>2.0 mg/g). Though the high CBD content was also detected with T1 Trump (10.65 and 9.30 mg/g), Boax (8.12 and 6.85 mg/g), and especially Cherry (12.04 mg/g), high nonuniformities within a variety were found (Supporting Information S3).

The distribution of CBDA contents in the 20 varieties showed a similar pattern to that of CBD, as shown in Figure 2a,b. This phenomenon indicated that the variety that yielded high CBDA generally yielded high CBD as well, consistent with the fact mentioned above that CBD is formed from its precursor CBDA.

CBDA presented to be the most abundant cannabinoid in all varieties tested except for the CBG variety Matterhorn. Cherry #5 had the highest CBDA content ranging from 196.16 to 133.12 mg/g. The second-highest CBDA contents were observed in T1 Trump, T2 Trump, Lindorea, and Eclipse (>116.92 mg/g), which were slightly higher than that in Jupiter and Goliath (>89.65 mg/g). Similarly, in the case of CBD, high CBDA contents were detected in some samples of Cherry, Magic Bullet, Boax, Wife, and Otto 2 Sweeten, but low uniformities within a variety were observed.

Two samples from the varieties of Cherry and Cherry Wine showed very high amounts of CBD (30.19 and 15.72 mg/g). These high CBD contents probably were caused by the conversion of CBDA to CBD during post-harvest processing, given the fact that the two samples were air-dried instead of being lyophilized like the other samples. Acidic cannabinoids such as CBGA, THCA, CBDA, CBCA, and CBGA were considered to be the final products of enzymic biosynthesis in cannabis.73 CBGA is the central precursor of various cannabinoids, which produces THCA, CBDA, and CBCA by oxido-cyclization by each related synthase. The phytocannabinoid acids then undergo spontaneous decarboxylation to be further converted into their corresponding neutral derivatives, which occur both in the living plant and upon heating after harvesting.74 A series of studies regarding the kinetics of decarboxylation have been reported. Citti et al. found that at 80 and 90 °C, the CBDA in hemp seed oil was transformed to CBD at the rate constant of 3.37 × 10–5 and 1.01 × 10–4 S–1.75 At 100 °C or above, other processes could be involved, leading to the loss of CBD.75 Wang et al. observed that the rate constants for THCA decarboxylation were always approximately twice those of CBDA or CBGA, which were nearly identical.76 Moreover, Taschwer et al. proved that temperature higher than 50 °C led to an accelerated and complete decarboxylation of THCA in cannabis plant materials.77 These findings suggest that the CBDA amounts should be taken into account when growers and breeders choose the varieties appropriate for CBD production, which applies to the selection of CBG hemp as well. In general, a decarboxylation step is carried out in CBD oil manufacturing.78 Therefore, the total CBD, THC, and CBG contents were calculated for each sample following a reported method13

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Cherry #5 showed the highest total CBD content in the range of 124.31–177.90 mg/g, followed by T1 Trump (105.75–137.81 mg/g), T2 Trump (115.68–139.63 mg/g), Lindorea (119.47–129.75 mg/g), and Eclipse (111.24–128.29 mg/g) (Figure 2c and Supporting Information S3). These varieties are appropriate for CBD production.

According to the 2018 Farm Bill, hemp-derived products with less than 0.3% Δ9-THC are lawful.19 Results displayed that mean levels of Δ9-THC for all listed varieties fell below the value of cutoff (Table 3). It is worth noting that the USDA released the final rule regulating the production of hemp in January 2021, mandating that hemp must be tested for total Δ9-THC content.79 In terms of total THC, the mean contents of the 12 varieties, including T1 trump (0.52%), T2 trump (0.42%), Cherry (0.53%), Magic Bullet (0.40%), Red Kross (0.48%), Baox (0.35%), Wife (0.32%), Goliath (0.41%), Lindorea (0.47%), Eclipse (0.42%), Jupiter (0.39%), and Cherry #5 (0.63%) exceeded the 0.3% limit, as shown in Table 3.

Results of the Quantitative Determination of Minor Cannabinoids

In addition to THC and CBD-dominant products, there is a growing market of products featuring other cannabinoids.21 For example, cannabigerol (CBG)- and cannabinol (CBN)-rich oils have been seen.21,80 CBG and CBC were displayed to be partial agonists of both CB1R- and CB2R-dependent signaling in a recent study, which had previously been reported to show anti-nociceptive, anxiolytic-like, and disease-ameliorating anti-inflammatory effects in vivo.81 CBDV displayed selective demonstrable affinity and activity at CB2R in cell culture assays and anti-convulsant effects in rodent models.81,82

Matterhorn had the highest levels of CBGA and CBG, with concentrations of 62.65–80.82 and 1.34–2.93 mg/g, respectively (Figure 3a,b). The Matterhorn variety had a more than 6-fold higher CBGA level than the other varieties. Besides Matterhorn, Goliath, Lindorea, and Eclipse showed high contents of CBG compared with the other varieties. The contents are in the range of 0.47–0.98 mg/g (Goliath), 0.51–0.59 mg/g (Lindorea), and 0.41–0.63 mg/g (Eclipse). Results presented here with regard to CBG are different from a previous study that showed CBG was not detected in all 13 varieties under investigation.13 Baox and Cherry #5 showed a broad range of total CBG, 0.60–10.48 and 0.67–10.78 mg/g, respectively. Other varieties that showed a significant level of total CBG were Wife (3.20–4.94 mg/g) and Lindorea (2.44–4.07 mg/g) (Figure 3c), as was the case with CBGA.

Figure 3.

Figure 3

Figure 3

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Box and whisker plot graph of (a) CBGA, (b) CBG, (c) total CBG, (d) CBC, (e) CBDV, and (f) THCV content in 104 samples from 20 varieties. In each box, the low whisker represents the minimum and the upper whisker represents the maximum. The concentrations below the LOQ are considered as 0 in this graph. Please see the values for a particular sample in Supporting Information S3.

The highest CBC level was found in Eclipse, with the range of 1.16–2.42 mg/g followed by Lindorea (0.81–1.66 mg/g), Mystery (0.61–1.44 mg/g), and Trump T2 (0.95–1.19 mg/g) (Figure 3d). Based on a previous study that concluded CBC content was higher in less mature flowers, higher CBC levels are possible if buds are harvested earlier.13

The varieties with the highest CBDV contents were the T2 trump (1.18–1.20 mg/g) and Eclipse (1.02–1.09 mg/g), both of which showed good uniformities within variety (Figure 3e). The high CBDV contents were also detected in Lindorea and Goliath with the content ranges of 0.93–1.41 and 0.98–1.26 mg/g, however, with relatively high differences within variety in comparison to T2 Trump and Eclipse.

The highest THCV content was detected in Lindorea, which had a content range of 5.34–7.42 mg/g. Lower concentrations of 1.13–4.74 and 1.13–3.53 mg/g THCV were detected in Eclipse and Mystery (Figure 3f).

CBN and Δ8-THC were not found in most of the samples. Trace amounts of CBN were found in Cherry #5, Goliath, and Matterhorn (<0.20 mg/g).

In summary, low uniformity between varieties was extensively seen for the tested cannabinoids, which probably was a combined effect of genetic diversity, environmental conditions, and growers’ operation. Low uniformity within variety regarding cannabinoid content was observed as well, illustrating the need for improving genotype consistency of cannabis and optimizing the cultivation conditions.83,84 The results of this research provide reference information for the selection of hemp varieties for cannabinoid production purposes as to high yield and uniformity.

Materials and Methods

Plant Materials, Standards, Reagents, and Software

A total of 104 hemp bud samples belonging to 20 varieties were collected from nine farms in the state of Maryland during the 2019–2020 season. Three to 15 samples were obtained for each of the 20 varieties. Detailed sample information is listed in Supporting Information S3. The plants were collected at the phase of maturity when the farmers harvested. Fresh plant materials were transferred to the laboratory and kept frozen until lyophilization. Samples were dried by lyophilization for 20 h and kept in a freezer until analyzed except the samples 16 and 56 (Supporting Information S3) that were air-dried at room temperature.

Methanol (HPLC grade) was purchased from Honeywell (North Carolina). HPLC grade phosphoric acid (85% W/W) was from Sigma-Aldrich (Missouri). Reference standards of CBDV, THCV, CBD, CBG, CBDA, CBGA, CBN, Δ9-THC, Δ8-THC, CBC, and THCA were supplied as a mixture stock solution containing each cannabinoid at the concentration of 250 μg/mL in acetonitrile from Shimadzu Scientific Instruments (Maryland). Working standard solutions were prepared in the range from 0.1 to 50 μg/mL by proper dilution of the stock solution with methanol. All calibration standards were stored at −80 °C. The statistical analyses were performed with Excel for Microsoft 365 and IBM SPSS statistics 27.

Extraction Procedure

The buds were ground with a mortar to a homogeneous fine powder, and 50 mg of it was accurately weighed in a vial. Methanol (5 mL) was added to the vial and kept at room temperature for 20 min, followed by sonication for 20 min. The solution was transferred into a 15 mL centrifuge tube and centrifuged at 3184 rcf for 5 min. The supernatant was filtered with a 0.45 μm Nylon syringe filter (Shimadzu Scientific Instruments, Maryland). A 20 μL aliquot of the filtrate was diluted to 1.0 mL by adding 980 μL of methanol and used for CBDA quantitative analysis. Another 100 μL aliquot was diluted to 1 mL by adding 900 μL of methanol and used for the quantitative analysis of CBDV, THCV, CBD, CBG, CBGA, CBN, Δ9-THC, Δ8-THC, CBC, and THCA.

HPLC Equipment and Chromatographic Conditions

A Shimadzu Prominence-i LC-2030C Plus system (Shimadzu, Maryland) equipped with a Deuterium (D2) UV diode array detector (DAD), an autosampler, a quaternary pump, an online degasser, and a column temperature controller was used in this study. Instrument control and data analysis were carried out using Shimadzu LabSolutions software. The chromatographic separation was performed on a NexLeaf CBX for Potency C18 column 2.7 μm, 150 mm × 4.6 mm column (Shimadzu Scientific Instruments, Maryland) coupled with a Zorbax Eclipse Plus-C18 2.1 × 12.5 mm2, 5 μm Narrow Bore Guard Column (Agilent Technologies, Folsom). The analytical column temperature was kept at 50 °C. The separation was achieved using 0.085% Phosphoric acid in water (A) and 0.085% Phosphoric acid in methanol (B) under gradient conditions (0–9 min, isocratic 68% B; 9.0–16.0 min, 68–92% B; 16.0–17.0 min 92–100% B) as the mobile phase at a flow rate of 1.6 mL/min within 18 min. The wavelength of detection was 220 nm.

Calibration

Calibration curves were constructed by plotting peak area against the corresponding amount (ng) of each standard after analyzing at five analyte levels. The limits of detection (LOD) and quantification (LOQ) for individual standards were set as the concentrations, where the ratio of the peak area of signals to noise of blank (S/N) were 3 and 10 times, respectively. The noise in LabSolutions is obtained with the ASTM method. The noise calculation range was set as an interval of 0.3 min from the vicinity of each peak. LOD and LOQ were experimentally obtained by diluting the concentration of standard solutions down to LOD and LOQ.

Method Validation

The linearity was assessed by linear regression. The selectivity of the method was checked by the resolution (Rs) of all cannabinoid peaks of 3 hemp samples.

The precision of the method was tested by the percentage relative standard deviation (% RSD) of intra- and interday variabilities. The intraday precisions of THCV, CBD, CBG, CBDA, CBGA, Δ9-THC, CBC, and THCA were determined by analysis of four samples of a plant material that were prepared and analyzed independently on the same day. The interday precisions of THCV, CBD, CBG, CBDA, CBGA, Δ9-THC, CBC, and THCA were determined by the analysis of three samples of this plant material that were prepared and analyzed on three different days. Because they were not detected in this plant material, the intraday and interday precisions of CBDV, CBN, and Δ8-THC were determined by analyzing spiked samples. All samples were run in triplicate. The accuracy of the method was determined through recovery experiments with 11 cannabinoids. An extract with a known content of cannabinoids was spiked with three different amounts of standards, and the recovery of each cannabinoid was calculated respectively by the use of the equation

graphic file with name ao1c04992_m004.jpg 4

All samples were analyzed by three replicate injections.

Acknowledgments

The authors are grateful to Dr. Willie May and Dr. Farin Kamangar for their support to this hemp program. The authors would like to thank Shimadzu Scientific Instruments, Inc. (Columbia, MD) for providing them with the HPLC equipment and technical support.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c04992.

  • Calibration curves, detailed sample information, and additional quantitative data (XLSX)

The authors declare no competing financial interest.

This paper was published ASAP on November 17, 2021. Due to a production error, parts e and f of Figure 3 were missing. The corrected version was reposted on November 18, 2021.

Supplementary Material

ao1c04992_si_001.xlsx (712.6KB, xlsx)

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