Abstract
Background:
Cannabis sativa is a psychoactive plant indigenous to Central and South Asia, traditionally used both for recreational and religious purposes, in addition to folk medicine. Cannabis is a rich source of natural compounds, the most important of which are commonly known as cannabinoids that cause a variety of effects through interaction with the endocannabinoid system.
Materials and Methods:
In this study, a high-performance liquid chromatography-ultraviolet/photodiode array (PDA) method was developed and validated for the analysis of 15 cannabinoids in cannabis plant materials and cannabis-based marketed products. These cannabinoids are cannabidivarinic acid, cannabidivarin, cannabidiolic acid, cannabigerolic acid, cannabigerol, cannabidiol, delta-9-tetrahydrocannabivarin, delta-9-tetrahydrocannabivarinic acid, cannabinol, delta-9-tetrahyrocannabinol, delta-8-tetrahyrocannabinol, cannabicyclol, cannabichromene, delta-9-tetrahyrocannabinolic acid A, and cannabichromenic acid. The separation was carried out using a reversed-phase Luna® C18(2) column and a mobile phase consisting of 75% acetonitrile and 0.1% formic acid in water. A PDA detector was used, and data were extracted at λ=220 nm. Principal component analysis of cannabis four varieties was performed.
Results:
The method was linear over the calibration range of 5–75 μg/mL with R 2>0.999 for all cannabinoids. This method was sensitive and gave good baseline separation of all examined cannabinoids with limits of detection ranging between 0.2 and 1.6 μg/mL and limits of quantification ranging between 0.6 and 4.8 μg/mL. The average recoveries for all cannabinoids were between 81% and 104%. The measured repeatability and intermediate precisions (% relative standard deviation) in all varieties ranged from 0.35% to 9.84% and 1.11% to 5.26%, respectively.
Conclusions:
The proposed method is sensitive, selective, reproducible, and accurate. It can be applied for the simultaneous determination of these cannabinoids in the C. sativa biomass and cannabis-derived marketed products.
Keywords: Cannabis sativa, cannabinoids, HPLC-UV/PDA, validation, marketed products
Introduction
Cannabis sativa is a chemically complex species based on its numerous reported natural secondary metabolite constituents. 1 Since ancient times, cannabis has been used for its medicinal properties. 2 The bioactive compounds from cannabis are mostly concentrated in female inflorescence. 3
There are more than 500 secondary metabolites, of which over 129 cannabinoids have been identified in cannabis, and the number is still growing. 4,5
Cannabinoids are terpenophenolic compounds specific to cannabis. 6 Delta-9-tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD) are the most well-known cannabinoids for their therapeutic properties. 6,7 Δ 9 -THC content in the dried inflorescence (leaves and buds) is used to determine the plant's psychoactivity. Quantitative and qualitative analysis of cannabis can be employed to characterize the plant phenotype and phytocannabinoid profile. 8
Based on the major cannabinoid content (mainly THC and CBD), cannabis can be divided into three major chemotypes, as most recently recommended by the United States Pharmacopeia (USP) expert panel, based on the total CBD and THC content as THC-dominant chemotype, THC/CBD intermediate chemotype, and CBD-dominant chemotype. 9 In the THC-dominant chemotype, the ratio of total THC content to total CBD content is not less than (NLT) 5:1. In the THC/CBD intermediate chemotype, the ratio of the total THC content to total CBD content is NLT 0.2:1 and not more than (NMT) 5:1, while the CBD-dominant chemotype, the ratio of the total THC content to total CBD content is NMT 1:5. 9 There is an additional chemotype based on cannabigerol (CBG) content named as CBG-dominant chemotype, characterized by a significant amount of total CBG.
A significant rise in the number of laboratories performing cannabinoid analyses of cannabis plant materials and marketed products has occurred in recent years as a result of the legalization of cultivation of cannabis for medicinal and recreational purposes by many states. For the identification and quantification of terpenes, cannabinoids, and other substances found in the cannabis plant, a range of analytical methods is reported in the literature.
The majority of cannabinoid analytical techniques require expensive, and highly specialized equipment. 10,11 The most commonly used methods include liquid chromatography with tandem-mass spectrometry (MS), 12 –15 gas chromatography with flame ionization detectors (GC/FID), 16,17 (although other methods are also available with other detectors, such as electrochemical sensors), high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection, 18,19 high-performance thin-layer chromatography-MS, 20,21 ultra-performance liquid chromatography, 21 and matrix-assisted laser desorption ionization spectrometry (MALDI-MS). 22
Gas chromatography with flame ionization (GC/FID) or MS detection (GC/MS) is the most commonly used chromatographic technique in the analysis of cannabinoids. 16,17,23,24 In GC/MS, the analytes are fragmented using a standardized electron ionization process, allowing the use of compound libraries for compound identification. Without using derivatization, GC methods were used to measure the total quantity of cannabinoids (the sum of the acidic and neutral cannabinoids). Due to the high temperature at the injection port, the acidic cannabinoids undergo decarboxylation immediately after injection. 24 –26 As a result, GC cannot identify acidic cannabinoids without first derivatizing them. 23 Unlike GC, HPLC allows the differentiation between acidic and neutral cannabinoids 27 without derivatization before analysis. 2,28 Thus, HPLC provides a more comprehensive chemical report of cannabis samples compared to GC.
The quantification of the cannabinoid profile and content of cannabis preparations becomes increasingly important as the use of cannabis is rising dramatically. Consequently, to ensure the safety of consumers, analytical methods for cannabis quality control have to be implemented to ensure the safety and efficacy of these cannabis products.
This study focuses on the development and validation of a simple HPLC method for the identification and quantification of the major and minor cannabinoids in four chemotypes of cannabis inflorescences (THC, CBD, THC/CBD, and CBG chemotypes) and marketed products that can be easily reproduced and applied.
Materials and Methods
Reagents and standards
HPLC grade acetonitrile, methanol (MeOH; purity >99.9%), HPLC water, and formic acid (>98%) were purchased from Fisher Scientific (Waltham, MA, USA). 4-Androstene-3,17-dione (purity >99%) was used as the internal standard (I.S.) and was purchased from Sigma-Aldrich.
Standard solutions of pure Δ 9 -THC, delta-9-tetrahyrocannabinolic acid A, CBD, cannabidiolic acid (CBDA), CBC, CBG, cannabinol (CBN), and delta-8-tetrahyrocannabinol (Δ 8 -THC) were isolated in our laboratory and prepared as 1.0 mg/mL in acetonitrile. The other seven cannabinoid standards were purchased from the Cayman Chemical Company (Ann Arbor, MI, USA): cannabidivarin (CBDV), cannabidivarinic acid (CBDVA), tetrahydrocannabivarin (THCV), tetrahydrocannabivarinic acid (THCVA), cannabichromenic acid (CBCA), cannabicyclol (CBL), and cannabigerolic acid (CBGA). These standards were obtained as 1.0 mg/mL solutions in acetonitrile. The purities of all the standards were above 99%, as determined by HPLC. All standards were stored at −20°C and allowed to come to room temperature in a desiccator before use. MeOH is required for the dilution of standards and the extraction of samples.
Plant materials and marketed products
Cannabis sativa plants of four chemotypes (high THC, THC/CBD, high CBD, and high CBG) were grown at the University of Mississippi. The harvested flower buds were dried and ground before analysis. The marketed products were supplied by the National Marijuana Initiative and submitted to our laboratory for analysis of cannabinoid content.
Standard solutions preparation
From newly opened vials containing individual cannabinoids with a concentration of 1.0 mg/mL of each cannabinoid, 500 μL of each cannabinoid was mixed in a 10 mL glass scintillation vial and evaporated to dryness under a gentle flow of N2 gas. Five milliliters of MeOH was then added to make a stock standard solution of 200 μg/mL of each cannabinoid (stock A). Serial dilutions were made from stock A to make 10 μg/mL of stock B and 1 μg/mL of stock C standard solutions. These solutions were used to prepare the individual points of calibration curves.
Calibration curves preparation
Five concentrations ranging from 5 to 75 μg/mL were prepared from the above stock standard solutions by taking different volumes of stock solution, and then 100 μL of I.S. (1.0 mg/mL) was added to each concentration of the cannabinoid mixture in GC vials and the volume was adjusted to 1.0 mL with MeOH. The calibration curves were prepared in triplicate on separate days and constructed by plotting the concentrations versus the ratios of peak areas (peak area of the analyte to that of the I.S.).
Preparation of extracts of cannabis plant materials and marketed products
Samples of cannabis buds and leaves were dried separately for 24 h at 40°C in a ventilated oven and then powdered using coffee grinder (particle size of the powder was <200 μm). The powdered plant samples were weighed (50 mg each) into 20 mL glass scintillation vials and extracted with 10 mL of I.S. (100 μg/mL) solution by sonication for 20 min at 30°C. Each sample was filtrated through a 45 μm syringe filter. Appropriate dilutions were made with MeOH, when necessary. The homogeneity of the plant material was confirmed by extraction of two samples of the intermediate variety in six replicates.
The e-cigarette semiliquid extracts were weighed (25 mg each) into 20 mL glass scintillation vials and dissolved with 10 mL MeOH. The vials were sonicated for 20 min at 30°C. Each sample was filtrated through a 45 μm syringe filter, and then 50 μL of the methanolic solution was transferred to GC-vial and 1.0 mL of I.S. (100 μL/mL) was added. Quantitation was based on the ratio of peak areas of each analyte to the peak area of the I.S. All experiments were performed in triplicate.
Method validation
To determine the suitability of the developed analysis conditions, tests of the system suitability, specificity, linearity, range, accuracy, precision, detection limit, and quantitation limit were conducted with reference to the International Conference on Harmonization (ICH) Tripartite Guideline for Validation of Analytical Procedures. 29
Specificity
Specificity is the ability of the analytical method to distinguish between the cannabinoids and the other components in the sample matrix according to ICH guideline. 29 In the case of HPLC method, it is assured by distinct baseline separation of analytes' peaks from any other interfering peak. Chromatograms were checked for interfering peaks and sufficient separation of analytes. PDA was used not only to extract peak responses at λ=220 nm but also for full ultraviolet (UV) spectral acquisition to compare with reference standards for peak identification and peak purity.
Linearity
To evaluate the linearity of the method, mixed standard solutions of 15 standard cannabinoids were prepared by diluting the stock standard solution with the MeOH to obtain different concentrations of (5–75 μg/mL), and the I.S. at 100 μg/mL in each concentration. Three injections of each concentration were analyzed under the same conditions. Linear regression analysis was used to evaluate the linearity of the calibration curve by using the correlation coefficient (R 2 ).
Sensitivity
Limits of detection (LOD) and limits of quantification (LOQ) of each cannabinoid were determined based on S/N=3 LOD and S/N=10 for LOQ.
Accuracy
The accuracy of the method was determined by recovery studies for the 15 cannabinoids from the placebo matrix. Accuracy and precision were calculated as % recovery and % relative standard deviation (RSD), respectively. Exact amounts of reference substances of 15 cannabinoids were mixed with the placebo matrix prepared by exhaustive extraction of cannabis plant material using hexanes followed by ethanol in such a way that the spiked samples, after the preparation process, yielded solutions containing each analyte at three concentration levels, low, medium, and high (10, 25, and 50 μg/mL). At each concentration level, three samples were prepared and analyzed. The percentage recovery and the RSD were calculated for each replicate sample.
An additional spike recovery experiment was performed for plant material expected to contain higher percentages of six major cannabinoids (THC, tetrahydrocannabinolic acid [THCA], CBD, CBDA, CBG, and CBGA) at 20% w/w, followed by 10×dilution, and analysis.
Repeatability and intermediate precision
The method precision was evaluated by the analysis of two samples from each chemotype of plant material and two e-cigarettes (a total of 10 samples), and each sample was analyzed in triplicate, while the instrument performance was evaluated by spiking placebo plant material with mixtures of 15 cannabinoids at three different levels (10, 25, and 50 μg/mL). Six replicates from each concentration were analyzed on three separate days. 29 Repeatability and intermediate precisions (% RSD) were required to be <15%, as previously reported by others. 30 –32
Instrumentation and analytical method
A Waters Alliance HPLC system with a 2695 separation module equipped with 2996 PDA detector was used. Empower 3® software was used for instrument control, data acquisition, and processing. A Phenomenex Luna® C18(2) (250 mm×4.6 mm×3 μm) analytical column with a security guard C18 column (20 mm×4.6 mm×5 μm) was employed to achieve reversed phase separation.
The column was maintained at 40°C, with a mobile phase flow rate of 1 mL/min from 0 to 10 min and then the flow rate was increased to 1.2 mL/min from 10.1 to 30 min. The injection volume was 10 μL, and all the standards and samples were injected in triplicates. Acetonitrile with 0.1% formic acid (75%): water with 0.1% formic acid (25%) was used as the mobile phase. During each run, the PDA was set to acquire data from 190 to 400 nm and the chromatograms were visualized at 220 nm.
Statistics
All tests were performed in triplicates. Data are expressed as mean±standard deviation. Slopes and intercepts of calibration graphs were calculated by linear regression. Correlation coefficient (R 2 ) values were obtained by using the Microsoft Office Excel program. Data processing and chemometric analysis were performed using MetaboAnalyst 5.0 web server. The principal component analysis (PCA) was used to reduce dimensionality of the data set, as well as to identify natural groups or clusters of samples within the data set.
Results and Discussion
An HPLC method was developed and validated following the (ICH) Tripartite Guideline 29 for the identification and quantification of 15 cannabinoids in C. sativa, the structures of which are shown in Figure 1. The method was optimized to achieve the best separation with satisfactory retention times. 4-Androstene-3,17-dione was selected as the I.S. since it was found experimentally that its retention time falls at the beginning of the chromatogram (Rt =4.65 min), and it is not present in C. sativa plant extracts or the marketed products.
FIG. 1.
Chemical structure of 15 cannabis cannabinoids and internal standard.
Optimization of extraction and chromatographic parameters
Different extraction solvents were tested to produce the highest extraction efficiency, including ethyl acetate, ethanol, MeOH, and chloroform:MeOH (1:9) mixture. Single extraction with MeOH gave the best recovery compared to the other solvents, which is in agreement with previously reported study. 33 To prove that a single extraction is sufficient, the marc left after the first extraction was extracted for a second time using 10 mL (I.S.) in MeOH. The analysis of the marc showed that the cannabinoids' content is less than the limit of quantitation, as illustrated in Supplementary Figure S1. Homogeneity results were expressed as % RSD in Supplementary Table S1. The results confirmed the plant samples' homogeneity with % RSD value <15.
Four different HPLC columns with different dimensions and lengths were evaluated. Phenomenex Luna C18(2) (250 mm×4.6 mm×3 μm) was selected as it provided the best baseline separation for the tested 15 cannabinoids. The mobile phase was optimized to achieve the optimum separation between the target cannabinoids without any interference with any component of the plant matrix. Cannabinoids in samples were identified based on comparing their retention times with those of the reference standards, as well as comparing their full spectra (for the PDA) with those of the standards. Figure 2 shows a representative chromatogram of cannabinoid standard solution mixture.
FIG. 2.
HPLC-UV chromatogram of 15 cannabinoids and internal standard extracted at 220 nm. HPLC-UV, high-performance liquid chromatography-ultraviolet.
Method validation parameters
Resolution (Rs), retention factor (k′), selectivity (α), and column efficiency (N) of the 15 cannabinoids were determined and the results are summarized in Table 1. Based on the method, the peaks of analyzed cannabinoids showed good resolution (>1.5), except THCA (1.27).
Table 1.
System Suitability Parameters
| No. | Cannabinoids | Resolution (Rs) | Retention factor (k′) | Selectivity (α) | Column efficiency (N) |
|---|---|---|---|---|---|
| 1 | CBDVA | 10.05 | 1.67 | 2.05 | 1.46×104 |
| 2 | CBDV | 3.14 | 1.95 | 1.17 | 1.61×104 |
| 3 | CBDA | 8.57 | 2.81 | 1.44 | 1.84×104 |
| 4 | CBGA | 1.86 | 3.00 | 1.07 | 2.66×104 |
| 5 | CBG | 2.02 | 3.19 | 1.06 | 3.02×104 |
| 6 | CBD | 2.29 | 3.42 | 1.07 | 2.95×104 |
| 7 | THCV | 2.65 | 3.69 | 1.08 | 3.10×104 |
| 8 | THCVA | 10.01 | 4.86 | 1.32 | 3.46×104 |
| 9 | CBN | 2.70 | 5.22 | 1.07 | 3.05×104 |
| 10 | Δ 9 -THC | 9.88 | 6.72 | 1.29 | 3.15×104 |
| 11 | Δ 8 -THC | 1.64 | 7.00 | 1.04 | 3.09×104 |
| 12 | CBL | 5.93 | 8.11 | 1.16 | 3.41×104 |
| 13 | CBC | 3.08 | 8.74 | 1.08 | 3.09×104 |
| 14 | THCA | 1.27 | 9.01 | 1.03 | 2.91×104 |
| 15 | CBCA | 6.70 | 10.06 | 1.17 | 3.61×104 |
Δ 8 -THC, delta-8-tetrahydrocannabinol; Δ 9 -THC, delta-9-tetrahydrocannabinol; CBC, cannabichromene; CBCA, cannabichromenic acid; CBD, cannabidiol; CBDA, cannabidiolic acid; CBDV, cannabidivarin; CBDVA, cannabidivarinic acid; CBG, cannabigerol; CBGA, cannabigerolic acid; CBL, cannabicyclol; CBN, cannabinol; THCA, tetrahydrocannabinolic acid; THCV, tetrahydrocannabivarin; THCVA, tetrahydrocannabivarinic acid.
The LOD for individual cannabinoids ranged between 0.2 and 1.6 μg/mL, while the LOQ was between 0.6 and 4.8 μg/mL (Table 2). The calibration curves were linear between 5 and 75 μg/mL with R 2 values of ≥0.999 for all cannabinoids, as shown in Table 2 and Supplementary Figure S2.
Table 2.
Regression Equation, Linearity Range, Correlation Coefficient, Limits of Detection, and Limits of Quantification for the Fifteen Cannabinoids Analyzed
| No. | Cannabinoids | Rt (min) | Regression equation | R 2 | Linearity range (μg/mL) | LOD (μg/mL) | LOQ (μg/mL) |
|---|---|---|---|---|---|---|---|
| 1 | CBDVA | 6.85 | y=0.0322x−0.024 | 0.9999 | 5–75 | 1.3 | 3.9 |
| 2 | CBDV | 7.56 | y=0.0217x−0.0175 | 0.9997 | 5–75 | 1.3 | 3.9 |
| 3 | CBDA | 9.71 | y=0.0257x−0.0213 | 0.9997 | 5–75 | 0.3 | 0.9 |
| 4 | CBGA | 10.16 | y=0.0227x−0.0189 | 0.9996 | 5–75 | 1.6 | 4.8 |
| 5 | CBG | 10.66 | y=0.0159x−0.0157 | 0.9995 | 5–75 | 0.4 | 1.2 |
| 6 | CBD | 11.26 | y=0.0206x−0.0147 | 0.9998 | 5–75 | 0.3 | 0.9 |
| 7 | THCV | 11.92 | y=0.0216x−0.015 | 0.9998 | 5–75 | 0.2 | 0.6 |
| 8 | THCVA | 14.78 | y=0.0227x+0.026 | 0.9909 | 5–75 | 1.5 | 4.5 |
| 9 | CBN | 15.59 | y=0.0376x−0.0272 | 0.9998 | 5–75 | 0.5 | 1.5 |
| 10 | Δ 9 -THC | 19.26 | y=0.0207x−0.0246 | 0.9997 | 5–75 | 0.8 | 2.4 |
| 11 | Δ 8 -THC | 19.93 | y=0.0157x−0.0246 | 0.9994 | 5–75 | 1.1 | 3.3 |
| 12 | CBL | 22.63 | y=0.0256x−0.0271 | 0.9998 | 5–75 | 0.7 | 2.1 |
| 13 | CBC | 24.06 | y=0.0212x−0.0301 | 0.9997 | 5–75 | 1.1 | 3.3 |
| 14 | THCA | 24.75 | y=0.0241x−0.0468 | 0.9995 | 5–75 | 1.5 | 4.5 |
| 15 | CBCA | 28.99 | y=0.0071x−0.015 | 0.9996 | 5–75 | 1.6 | 4.8 |
LOD, limit of detection; LOQ, limit of quantitation; R 2, correlation coefficient; Rt , retention time.
Precision of the analytical procedure was expressed as % RSD. For high CBG variety, the % RSD values ranged from 1.48% to 10.16%, while the high THC variety showed % RSD ≤13.63%. The values of % RSD in high CBD and intermediate varieties were ≤10.55% and ≤14.58%, respectively. The e-cigarettes possessed % RSD value ≤14.46% (Table 3).
Table 3.
The Cannabinoid Concentration % (w/w) of Eight Plant Materials of Four Varieties and Two E-Cigarettes in Triplicates
| Cannabinoids | High CBG variety | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBG1 R1 | HCBG1 R2 | HCBG1 R3 | Mean | SD | RSD % | HCBG2 R1 | HCBG2 R2 | HCBG2 R3 | Mean | SD | RSD % | |
| CBDA | 0.129 | 0.122 | 0.121 | 0.124 | 0.004 | 3.43 | 0.118 | 0.103 | 0.113 | 0.111 | 0.007 | 6.70 |
| CBGA | 1.428 | 1.330 | 1.313 | 1.357 | 0.062 | 4.58 | 1.937 | 1.620 | 1.790 | 1.782 | 0.159 | 8.90 |
| CBG | 0.042 | 0.051 | 0.046 | 0.047 | 0.005 | 10.16 | 0.117 | 0.109 | 0.108 | 0.111 | 0.005 | 4.39 |
| CBD | 0.030 | 0.030 | 0.029 | 0.030 | 0.000 | 1.48 | 0.041 | 0.037 | 0.040 | 0.039 | 0.002 | 5.42 |
| Cannabinoids | High THC variety | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HTHC1 R1 | HTHC1 R2 | HTHC1 R3 | Mean | SD | RSD % | HTHC2 R1 | HTHC2 R2 | HTHC2 R3 | Mean | SD | RSD % | |
| CBDA | 0.209 | 0.211 | 0.206 | 0.209 | 0.002 | 1.13 | 0.245 | 0.238 | 0.242 | 0.242 | 0.003 | 1.42 |
| CBGA | 0.095 | 0.083 | 0.094 | 0.091 | 0.007 | 7.29 | 0.100 | 0.102 | 0.102 | 0.102 | 0.001 | 1.13 |
| CBG | 0.031 | 0.030 | 0.032 | 0.031 | 0.001 | 4.77 | 0.037 | 0.037 | 0.039 | 0.037 | 0.001 | 3.35 |
| CBD | bLOQ | bLOQ | bLOQ | – | – | – | bLOQ | bLOQ | bLOQ | – | – | – |
| CBN | 0.244 | 0.249 | 0.250 | 0.248 | 0.004 | 1.43 | 0.302 | 0.290 | 0.308 | 0.300 | 0.009 | 3.12 |
| Δ 9 -THC | 0.432 | 0.421 | 0.446 | 0.433 | 0.012 | 2.88 | 0.542 | 0.519 | 0.569 | 0.543 | 0.025 | 4.66 |
| Δ 8 -THC | 0.541 | 0.533 | 0.537 | 0.537 | 0.004 | 0.82 | 0.614 | 0.573 | 0.618 | 0.602 | 0.024 | 4.07 |
| CBC | 0.047 | 0.047 | 0.046 | 0.047 | 0.000 | 0.58 | 0.049 | 0.049 | 0.052 | 0.050 | 0.002 | 3.67 |
| THCA | 5.760 | 5.693 | 5.773 | 5.742 | 0.043 | 0.75 | 6.579 | 6.191 | 6.552 | 6.441 | 0.217 | 3.36 |
| CBCA | 0.198 | 0.169 | 0.223 | 0.197 | 0.027 | 13.63 | 0.193 | 0.187 | 0.229 | 0.203 | 0.022 | 11.05 |
| Total THC | 6.192 | 6.114 | 6.219 | 6.18 | 0.05 | 0.88 | 7.121 | 6.71 | 7.121 | 6.98 | 0.24 | 3.40 |
| Total CBD | 0.209 | 0.211 | 0.206 | 0.21 | 0.001 | 1.21 | 0.245 | 0.238 | 0.242 | 0.24 | 0.002 | 1.45 |
| Total THC/CBD ratio a | 29.63 | 28.98 | 30.19 | 29.60 | 0.61 | 2.05 | 29.07 | 28.19 | 29.43 | 28.89 | 0.63 | 2.19 |
| Cannabinoids | High CBD variety | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBD1 R1 | HCBD1 R2 | HCBD1 R3 | Mean | SD | RSD % | HCBD2 R1 | HCBD2 R2 | HCBD2 R3 | Mean | SD | RSD % | |
| CBDA | 4.386 | 4.723 | 4.332 | 4.480 | 0.212 | 4.73 | 6.993 | 7.056 | 7.671 | 7.240 | 0.375 | 5.18 |
| CBGA | 0.243 | 0.250 | 0.226 | 0.240 | 0.012 | 5.02 | 0.275 | 0.317 | 0.292 | 0.295 | 0.021 | 7.19 |
| CBN | ND | ND | ND | – | – | – | 0.023 | 0.021 | 0.024 | 0.022 | 0.001 | 5.60 |
| Δ 9 -THC | 0.047 | 0.045 | 0.044 | 0.045 | 0.002 | 4.18 | 0.052 | 0.057 | 0.055 | 0.055 | 0.003 | 4.65 |
| Δ 8 -THC | 0.035 | 0.041 | 0.035 | 0.037 | 0.003 | 9.01 | 0.033 | 0.034 | 0.040 | 0.036 | 0.004 | 10.55 |
| CBC | 0.038 | 0.044 | 0.041 | 0.041 | 0.003 | 7.41 | 0.049 | 0.055 | 0.046 | 0.050 | 0.005 | 9.26 |
| THCA | 0.138 | 0.139 | 0.130 | 0.136 | 0.005 | 3.52 | 0.145 | 0.140 | 0.151 | 0.145 | 0.005 | 3.67 |
| CBCA | 0.141 | 0.143 | 0.154 | 0.146 | 0.007 | 5.00 | 0.182 | 0.194 | 0.209 | 0.195 | 0.014 | 7.02 |
| Total THC | 0.185 | 0.184 | 0.174 | 0.18 | 0.006 | 3.36 | 0.197 | 0.197 | 0.206 | 0.20 | 0.005 | 2.60 |
| Total CBD | 4.386 | 4.723 | 4.332 | 4.48 | 0.212 | 4.73 | 6.993 | 7.056 | 7.671 | 7.24 | 0.375 | 5.17 |
| Total THC/CBD ratio a | 0.04 | 0.04 | 0.04 | 0.04 | 0.002 | 4.02 | 0.03 | 0.03 | 0.03 | 0.03 | 0.001 | 2.53 |
| Cannabinoids | Intermediate variety | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| IM1 R1 | IM1 R2 | IM1 R3 | Mean | SD | RSD % | IM2 R1 | IM2 R2 | IM2 R3 | Mean | SD | RSD % | |
| CBDVA | 0.054 | 0.057 | 0.052 | 0.054 | 0.002 | 4.13 | 0.030 | 0.025 | 0.028 | 0.028 | 0.002 | 8.45 |
| CBDV | 0.023 | 0.026 | 0.025 | 0.025 | 0.001 | 5.46 | 0.027 | 0.025 | 0.026 | 0.026 | 0.001 | 4.61 |
| CBDA | 3.679 | 3.828 | 3.651 | 3.719 | 0.095 | 2.56 | 3.671 | 3.468 | 3.378 | 3.505 | 0.150 | 4.28 |
| CBGA | 0.240 | 0.236 | 0.217 | 0.231 | 0.012 | 5.27 | 0.288 | 0.302 | 0.289 | 0.293 | 0.008 | 2.67 |
| CBG | 0.053 | 0.040 | 0.045 | 0.046 | 0.007 | 14.58 | 0.065 | 0.072 | 0.069 | 0.068 | 0.003 | 4.96 |
| CBD | 0.541 | 0.575 | 0.540 | 0.552 | 0.020 | 3.62 | 0.900 | 0.818 | 0.763 | 0.827 | 0.069 | 8.34 |
| THCV | bLOQ | bLOQ | bLOQ | – | – | – | 0.019 | 0.017 | 0.016 | 0.017 | 0.002 | 8.97 |
| CBN | 0.121 | 0.134 | 0.123 | 0.126 | 0.007 | 5.30 | 0.202 | 0.190 | 0.180 | 0.190 | 0.011 | 5.84 |
| Δ 9 -THC | 0.173 | 0.179 | 0.175 | 0.176 | 0.003 | 1.78 | 0.308 | 0.295 | 0.260 | 0.287 | 0.025 | 8.61 |
| Δ 8 -THC | 0.232 | 0.247 | 0.239 | 0.239 | 0.008 | 3.25 | 0.220 | 0.273 | 0.214 | 0.236 | 0.033 | 13.78 |
| CBC | bLOQ | bLOQ | bLOQ | – | – | – | 0.065 | 0.057 | 0.061 | 0.061 | 0.004 | 6.13 |
| THCA | 1.131 | 1.212 | 1.009 | 1.117 | 0.102 | 9.17 | 0.996 | 1.010 | 0.946 | 0.984 | 0.034 | 3.41 |
| Total THC | 1.304 | 1.391 | 1.184 | 1.29 | 0.104 | 8.04 | 1.304 | 1.305 | 1.206 | 1.27 | 0.057 | 4.47 |
| Total CBD | 4.22 | 4.403 | 4.191 | 4.27 | 0.115 | 2.69 | 4.571 | 4.286 | 4.141 | 4.33 | 0.219 | 5.05 |
| Total THC/CBD ratio a | 0.31 | 0.32 | 0.28 | 0.30 | 0.018 | 5.83 | 0.29 | 0.30 | 0.29 | 0.29 | 0.010 | 3.35 |
| Cannabinoids | E-Cigarette | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| EC1 R1 | EC1 R2 | EC1 R3 | Mean | SD | RSD % | EC2 R1 | EC2 R2 | EC2 R3 | Mean | SD | RSD % | |
| CBDA | 0.774 | 0.730 | 0.715 | 0.740 | 0.031 | 4.17 | ND | ND | ND | – | – | – |
| CBGA | 1.549 | 1.221 | 1.370 | 1.380 | 0.165 | 11.92 | 0.43 | 0.405 | 0.45 | 0.428 | 0.023 | 5.26 |
| CBG | 3.264 | 3.458 | 3.236 | 3.320 | 0.121 | 3.65 | 4.805 | 4.833 | 4.605 | 4.748 | 0.125 | 2.63 |
| CBD | bLOQ | bLOQ | bLOQ | – | – | – | 0.400 | 0.430 | 0.365 | 0.398 | 0.033 | 8.17 |
| THCV | 0.900 | 0.920 | 0.845 | 0.888 | 0.039 | 4.34 | 0.590 | 0.550 | 0.535 | 0.558 | 0.010 | 1.86 |
| CBN | 1.020 | 1.001 | 1.064 | 1.028 | 0.032 | 3.12 | 1.285 | 0.962 | 1.110 | 1.119 | 0.162 | 14.46 |
| Δ 9 -THC | 78.931 | 77.548 | 79.610 | 78.696 | 1.051 | 1.34 | 85.553 | 82.018 | 80.625 | 82.732 | 2.540 | 3.07 |
| CBC | 2.076 | 2.141 | 2.005 | 2.074 | 0.068 | 3.29 | 1.660 | 1.738 | 1.493 | 1.630 | 0.173 | 10.63 |
Total THC/CBD ratio=(Δ 9 -THC + THCA)/(CBD + CBDA).
bLOQ, below the limit of quantitation; ND, not detected; RSD, relative standard deviation; SD, standard deviation.
On the other hand, instrument performance was evaluated, and the repeatability values calculated as % RSD ranged from 0.46% to 5.61%, 0.35% to 9.84%, and 0.51% to 9.32%, on the first, second, and third validation days, respectively (Table 4). The intermediate precision values ranged from 1.11% to 5.26%. The fact that all precision values were <10% indicates a good precision of the method (Table 5).
Table 4.
Intraday Accuracy and Precision for Cannabinoids of the Assay Method
| Cannabinoids | Day 1 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| High concentration (50 μg/mL) | Medium concentration (25 μg/mL) | Low concentration (10 μg/mL) | ||||||||||
| Average | SD | RSD % | Recovery % | Average | SD | RSD % | Recovery% | Average | SD | RSD % | Recovery % | |
| CBDVA | 42.95 | 0.26 | 0.61% | 86% | 23.14 | 0.1 | 0.44% | 93% | 9.7 | 0.13 | 1.36% | 97% |
| CBDV | 47.58 | 0.65 | 1.37% | 95% | 25.24 | 0.09 | 0.35% | 101% | 10 | 0.14 | 1.41% | 100% |
| CBDA | 43.51 | 0.54 | 1.25% | 87% | 23.7 | 0.17 | 0.73% | 95% | 10 | 0.21 | 2.06% | 100% |
| CBGA | 44.46 | 0.86 | 1.94% | 89% | 24.21 | 0.42 | 1.72% | 97% | 10.06 | 0.24 | 2.39% | 101% |
| CBG | 47.43 | 0.7 | 1.48% | 95% | 25.03 | 0.34 | 1.37% | 100% | 9.71 | 0.22 | 2.21% | 97% |
| CBD | 47.73 | 0.74 | 1.54% | 95% | 25.48 | 0.27 | 1.07% | 102% | 10.2 | 0.17 | 1.70% | 102% |
| THCV | 47.51 | 0.76 | 1.60% | 95% | 25.12 | 0.24 | 0.97% | 100% | 9.92 | 0.16 | 1.58% | 99% |
| THCVA | 41.98 | 0.35 | 0.83% | 84% | 22.61 | 0.14 | 0.60% | 90% | 9.41 | 0.13 | 1.35% | 94% |
| CBN | 46.4 | 0.38 | 0.82% | 93% | 24.29 | 0.11 | 0.43% | 97% | 9.7 | 0.11 | 1.15% | 97% |
| Δ 9 -THC | 47.04 | 0.66 | 1.39% | 94% | 24.58 | 0.47 | 1.90% | 98% | 9.64 | 0.19 | 2.02% | 96% |
| Δ 8 -THC | 46.55 | 1 | 2.14% | 93% | 23.96 | 0.63 | 2.64% | 96% | 9.31 | 0.38 | 4.07% | 93% |
| CBL | 46.4 | 0.76 | 1.63% | 93% | 24.43 | 0.35 | 1.43% | 98% | 9.57 | 0.15 | 1.57% | 96% |
| CBC | 44.96 | 0.9 | 2.00% | 90% | 23.53 | 0.36 | 1.52% | 94% | 9.2 | 0.52 | 5.65% | 92% |
| THCA | 42.26 | 0.44 | 1.04% | 85% | 22.72 | 0.64 | 2.80% | 91% | 8.9 | 0.6 | 6.69% | 89% |
| CBCA | 40.59 | 0.9 | 2.21% | 81% | 20.49 | 1.3 | 6.33% | 82% | 9.3 | 0.56 | 5.99% | 93% |
| Cannabinoids | Day 2 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| High concentration (50 μg/mL) | Medium concentration (25 μg/mL) | Low concentration (10 μg/mL) | ||||||||||
| Average | SD | RSD % | Recovery % | Average | SD | RSD% | Recovery % | Average | SD | RSD % | Recovery % | |
| CBDVA | 46.1 | 0.32 | 0.70% | 92% | 23.92 | 0.16 | 0.65% | 96% | 9.8 | 0.05 | 0.51% | 98% |
| CBDV | 47.91 | 0.38 | 0.79% | 96% | 24.57 | 0.17 | 0.71% | 98% | 9.79 | 0.11 | 1.08% | 98% |
| CBDA | 46.81 | 0.49 | 1.05% | 94% | 24.38 | 0.25 | 1.02% | 98% | 10.1 | 0.16 | 1.57% | 101% |
| CBGA | 45.9 | 0.84 | 1.83% | 92% | 23.93 | 0.45 | 1.89% | 96% | 9.86 | 0.15 | 1.53% | 99% |
| CBG | 47.93 | 0.97 | 2.02% | 96% | 24.24 | 0.5 | 2.07% | 97% | 9.6 | 0.14 | 1.47% | 96% |
| CBD | 48.09 | 0.31 | 0.64% | 96% | 24.76 | 0.14 | 0.58% | 99% | 10.06 | 0.1 | 1.01% | 101% |
| THCV | 48 | 0.37 | 0.77% | 96% | 24.4 | 0.16 | 0.65% | 98% | 9.78 | 0.13 | 1.33% | 98% |
| THCVA | 45.51 | 0.25 | 0.55% | 91% | 23.38 | 0.2 | 0.85% | 94% | 9.46 | 0.13 | 1.41% | 95% |
| CBN | 46.73 | 0.28 | 0.60% | 93% | 24.11 | 0.15 | 0.64% | 96% | 9.76 | 0.1 | 1.04% | 98% |
| Δ 9 -THC | 47.86 | 0.44 | 0.92% | 96% | 24.04 | 0.47 | 1.95% | 96% | 9.4 | 0.18 | 1.92% | 94% |
| Δ 8 -THC | 47.61 | 0.72 | 1.52% | 95% | 23.66 | 0.46 | 1.93% | 95% | 9.01 | 0.31 | 3.48% | 90% |
| CBL | 47.51 | 0.34 | 0.71% | 95% | 23.89 | 0.18 | 0.74% | 96% | 9.42 | 0.16 | 1.66% | 94% |
| CBC | 45.44 | 0.38 | 0.85% | 91% | 23.57 | 0.32 | 1.34% | 94% | 9.5 | 0.31 | 3.24% | 95% |
| THCA | 45.49 | 0.38 | 0.84% | 91% | 23.12 | 0.43 | 1.87% | 92% | 9.23 | 0.4 | 4.30% | 92% |
| CBCA | 42.07 | 2.36 | 5.61% | 84% | 22.47 | 2.21 | 9.84% | 90% | 9.2 | 0.5 | 5.43% | 92% |
| Cannabinoids | Day 3 | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| High concentration (50 μg/mL) | Medium concentration (25 μg/mL) | Low concentration (10 μg/mL) | ||||||||||
| Average | SD | RSD % | Recovery % | Average | SD | RSD % | Recovery % | Average | SD | RSD % | Recovery % | |
| CBDVA | 44.7 | 0.21 | 0.46% | 89% | 23.6 | 0.19 | 0.79% | 94% | 9.72 | 0.1 | 1.00% | 97% |
| CBDV | 48.52 | 0.48 | 0.98% | 97% | 25.31 | 0.33 | 1.30% | 101% | 10.06 | 0.09 | 0.88% | 101% |
| CBDA | 45.46 | 0.41 | 0.89% | 91% | 24.09 | 0.3 | 1.24% | 96% | 10.16 | 0.17 | 1.71% | 102% |
| CBGA | 45.58 | 0.9 | 1.97% | 91% | 24.26 | 0.43 | 1.79% | 97% | 10.08 | 0.29 | 2.84% | 101% |
| CBG | 48.33 | 0.92 | 1.89% | 97% | 24.83 | 0.44 | 1.78% | 99% | 9.74 | 0.19 | 1.93% | 97% |
| CBD | 48.7 | 0.41 | 0.85% | 97% | 25.51 | 0.27 | 1.06% | 102% | 10.37 | 0.18 | 1.74% | 104% |
| THCV | 48.49 | 0.72 | 1.48% | 97% | 25.13 | 0.31 | 1.24% | 101% | 9.97 | 0.09 | 0.87% | 100% |
| THCVA | 44.14 | 0.28 | 0.63% | 88% | 23.27 | 0.22 | 0.94% | 93% | 9.44 | 0.14 | 1.51% | 94% |
| CBN | 46.92 | 0.22 | 0.46% | 94% | 24.31 | 0.11 | 0.43% | 97% | 9.8 | 0.12 | 1.25% | 98% |
| Δ 9 -THC | 48.41 | 0.66 | 1.37% | 97% | 24.81 | 0.51 | 2.05% | 99% | 9.58 | 0.22 | 2.32% | 96% |
| Δ 8 -THC | 47.92 | 0.93 | 1.95% | 96% | 24.16 | 0.71 | 2.94% | 97% | 8.89 | 0.37 | 4.15% | 89% |
| CBL | 47.94 | 0.65 | 1.36% | 96% | 24.81 | 0.4 | 1.63% | 99% | 9.69 | 0.25 | 2.55% | 97% |
| CBC | 45.88 | 0.24 | 0.53% | 92% | 23.58 | 0.23 | 0.97% | 94% | 9.07 | 0.71 | 7.86% | 91% |
| THCA | 44.37 | 0.37 | 0.84% | 89% | 23.22 | 0.44 | 1.91% | 93% | 8.87 | 0.68 | 7.71% | 89% |
| CBCA | 42.63 | 1.44 | 3.37% | 85% | 22.76 | 1.42 | 6.25% | 91% | 8.94 | 0.83 | 9.32% | 89% |
Table 5.
Interday Accuracy and Precision for Cannabinoids of the Assay Method
| Cannabinoids | High concentration (50 μg/mL) | Medium concentration (25 μg/mL) | Low concentration (10 μg/mL) | Average | SD | RSD % |
|---|---|---|---|---|---|---|
| CBDVA | 90.10% | 94.65% | 97.67% | 94.14% | 0.04 | 4.05% |
| CBDV | 95.60% | 99.17% | 98.59% | 97.79% | 0.02 | 1.96% |
| CBDA | 91.42% | 96.61% | 100.67% | 96.23% | 0.05 | 4.81% |
| CBGA | 90.84% | 96.15% | 99.22% | 95.40% | 0.04 | 4.44% |
| CBG | 95.53% | 98.03% | 96.37% | 96.64% | 0.01 | 1.32% |
| CBD | 95.94% | 99.99% | 101.04% | 98.99% | 0.03 | 2.72% |
| THCV | 95.68% | 98.56% | 98.26% | 97.50% | 0.02 | 1.63% |
| THCVA | 88.66% | 92.49% | 94.41% | 91.85% | 0.03 | 3.18% |
| CBN | 93.24% | 96.68% | 97.37% | 95.77% | 0.02 | 2.31% |
| Δ 9 -THC | 95.17% | 96.89% | 94.81% | 95.62% | 0.01 | 1.16% |
| Δ 8 -THC | 94.51% | 95.02% | 91.11% | 93.55% | 0.02 | 2.27% |
| CBL | 94.28% | 96.28% | 94.70% | 95.09% | 0.01 | 1.11% |
| CBC | 90.57% | 94.22% | 93.67% | 92.82% | 0.02 | 2.12% |
| THCA | 88.83% | 91.96% | 91.22% | 90.67% | 0.02 | 1.80% |
| CBCA | 83.15% | 87.23% | 92.33% | 87.57% | 0.05 | 5.26% |
The proposed HPLC-UV/PDA method showed good recovery as the intraday average recoveries for individual cannabinoids spiked in the placebo marijuana samples ranged from 81% to 97%, 82% to 102%, and 89% to 104%, on the three consecutive days, while the interday recovery values ranged from 88% to 99% (Tables 4 and 5).
The results of the high spike recovery experiment at 20% (w/w) content of the six major cannabinoids (THC, THCA, CBD, CBDA, CBG, and CBGA) displayed % recovery ranging from 91.6% to 111.7%, as shown in Supplementary Table S2.
The chromatograms of the tested 15 cannabinoids show distinct baseline separation between the target cannabinoids and the plant matrix components, indicating the specificity of the method, as shown in Figures 2 and 3. The UV spectrum of each analyzed cannabinoid and its retention time in the real samples are in good agreement with those of standard cannabinoids, confirming the specificity of our method (Figs. 3 and 4).
FIG. 3.
HPLC-UV chromatogram of the four varieties (A) IM, intermediate chemovar; (B) HCBD, high CBD chemovar; (C) HCBG, high CBG chemovar; and (D) HTHC, high THC chemovar (extracted at λ=220 nm). CBD, cannabidiol; CBG, cannabigerol; THC, tetrahydrocannabinol.
FIG. 4.
UV spectra of 15 cannabinoids and the internal standard in the range of 190–400 nm obtained in the HPLC system.
To determine the degree of carryover, one MeOH blank was injected after each run (calibration standards or sample solutions). The blank did not show any peak from any of the analytes or the I.S. at a signal-to-noise ratio ≥3.
Analysis of cannabis plant material and marketed products
Figure 4 shows the spectral absorbance profiles of the 15 cannabinoids and the I.S. Neutral cannabinoids (CBDV, CBG, CBD, THCV, CBL, Δ 9 -THC, and Δ 8 -THC) exhibit a similar spectral profile that differs from the acidic ones (CBDVA, CBDA, CBDA, THCVA, and THCA). The UV spectrum could distinguish between the neutral and acidic cannabinoids, but may not be sufficiently reliable to confirm cannabinoids within the same class. Also, other cannabinoids (CBN, CBC, and CBCA) have substantially different spectral profiles based on their structures.
The chromatograms of the four cannabis plant chemotypes are shown in Figure 3. Each tested analyte in the sample was identified/confirmed by comparing its retention time and UV spectra with the standard compounds. The developed HPLC method was applied for 78 samples, including 73 cannabis plant material and five e-cigarette samples (Table 6).
Table 6.
High-Performance Liquid Chromatography Results in % w/w for Plant Samples (1–73) and Five E-Cigarettes (74–78)
| Sample number a | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | |
| HCBD1 | HCBD2 | HCBD3 | HCBD4 | HCBD5 | HCBD6 | HCBD7 | HCBD8 | HCBD9 | HCBD10 | HCBD11 | HCBD12 | HCBD13 | |
| CBDVA | 0.01 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.01 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 |
| CBDV | bLOQ | bLOQ | bLOQ | bLOQ | bLOQ | bLOQ | ND | bLOQ | bLOQ | bLOQ | bLOQ | ND | bLOQ |
| CBDA | 3.73 | 4.91 | 4.70 | 4.79 | 5.21 | 5.10 | 4.37 | 2.68 | 3.07 | 3.57 | 3.76 | 5.45 | 5.08 |
| CBGA | 0.08 | 0.18 | 0.11 | 0.09 | 0.11 | 0.22 | 0.08 | 0.02 | 0.03 | 0.05 | 0.08 | 0.11 | 0.13 |
| CBG | 0.01 | 0.02 | 0.02 | 0.02 | 0.01 | 0.03 | 0.02 | 0.01 | 0.01 | 0.01 | 0.01 | 0.02 | 0.09 |
| CBD | 0.27 | 0.35 | 0.31 | 0.36 | 0.28 | 0.38 | 0.43 | 0.23 | 0.28 | 0.37 | 0.20 | 0.50 | 0.44 |
| THCV | 0.01 | 0.01 | 0.01 | 0.01 | bLOQ | 0.01 | 0.01 | 0.01 | 0.01 | bLOQ | bLOQ | 0.00 | bLOQ |
| THCVA | 0.05 | 0.03 | 0.04 | 0.04 | ND | 0.02 | ND | ND | 0.06 | 0.04 | ND | 0.04 | 0.04 |
| CBN | ND | ND | ND | ND | 0.04 | bLOQ | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | bLOQ | 0.00 |
| Δ 9 -THC | 0.02 | 0.02 | 0.02 | 0.02 | 0.01 | 0.03 | 0.02 | 0.01 | bLOQ | 0.01 | 0.00 | 0.02 | 0.01 |
| Δ 8 -THC | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | 0.01 |
| CBL | ND | 0.01 | ND | ND | ND | 0.01 | ND | ND | ND | ND | ND | ND | bLOQ |
| CBC | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | 0.01 | 0.02 | bLOQ | 0.01 | 0.01 | ND | 0.03 | 0.02 |
| THCA | 0.15 | 0.17 | 0.18 | 0.11 | 0.14 | 0.23 | 0.10 | 0.06 | 0.06 | 0.07 | 0.10 | 0.14 | 0.14 |
| CBCA | 0.14 | 0.22 | 0.21 | 0.21 | 0.25 | 0.25 | 0.18 | 0.11 | 0.13 | 0.15 | 0.17 | 0.26 | 0.26 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBD14 | HCBD15 | HCBD16 | HCBD17 | HCBD18 | HCBD19 | HCBD20 | HCBD21 | HCBD22 | HCBD23 | HCBD24 | HCBD25 | HCBD26 | |
| CBDVA | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | 0.03 | 0.01 | bLOQ | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
| CBDV | bLOQ | ND | bLOQ | bLOQ | bLOQ | bLOQ | bLOQ | ND | ND | bLOQ | ND | ND | 0.01 |
| CBDA | 3.67 | 3.49 | 4.14 | 4.02 | 4.22 | 4.73 | 2.20 | 2.02 | 1.57 | 1.97 | 1.54 | 2.30 | 2.63 |
| CBGA | 0.04 | 0.04 | 0.07 | 0.07 | 0.08 | 0.06 | 0.04 | 0.04 | 0.03 | 0.03 | 0.02 | 0.07 | 0.07 |
| CBG | 0.05 | 0.01 | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | bLOQ | 0.01 | 0.01 | ND | 0.01 | 0.01 |
| CBD | 0.25 | 0.33 | 0.46 | 0.39 | 0.39 | 0.49 | 0.20 | 0.22 | 0.13 | 0.51 | 0.16 | 0.26 | 0.27 |
| THCV | 0.01 | 0.01 | bLOQ | 0.01 | 0.01 | ND | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | ND | ND |
| THCVA | 0.07 | ND | 0.03 | 0.03 | ND | 0.08 | ND | ND | ND | ND | ND | ND | ND |
| CBN | ND | 0.04 | 0.04 | 0.01 | 0.02 | ND | 0.01 | bLOQ | 0.01 | 0.02 | 0.01 | 0.02 | 0.02 |
| Δ 9 -THC | 0.01 | 0.01 | 0.02 | 0.02 | 0.01 | 0.02 | ND | 0.01 | bLOQ | 0.02 | bLOQ | 0.01 | 0.01 |
| Δ 8 -THC | ND | ND | ND | bLOQ | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| CBL | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| CBC | 0.01 | 0.01 | 0.02 | 0.01 | 0.01 | 0.02 | bLOQ | bLOQ | ND | 0.02 | ND | bLOQ | 0.01 |
| THCA | 0.10 | 0.07 | 0.09 | 0.11 | 0.10 | 0.10 | 0.04 | 0.03 | 0.04 | 0.03 | 0.02 | 0.04 | 0.05 |
| CBCA | 0.17 | 0.16 | 0.20 | 0.19 | 0.20 | 0.23 | 0.08 | 0.07 | 0.06 | 0.07 | 0.04 | 0.09 | 0.10 |
| 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 | 36 | 37 | 38 | 39 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBD27 | HCBD28 | HCBD29 | HCBD30 | HCBD31 | IM1 | IM2 | IM3 | IM4 | IM5 | IM6 | IM7 | HTHC1 | |
| CBDVA | 0.02 | 0.03 | 0.01 | 0.01 | 0.02 | 0.02 | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | 0.01 | ND |
| CBDV | 0.02 | 0.03 | 0.01 | bLOQ | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | bLOQ | bLOQ | bLOQ |
| CBDA | 3.97 | 4.61 | 3.11 | 2.48 | 5.00 | 5.04 | 3.19 | 3.80 | 3.31 | 3.67 | 2.83 | 2.39 | 0.05 |
| CBGA | 0.10 | 0.12 | 0.11 | 0.10 | 0.19 | 0.31 | 0.18 | 0.24 | 0.22 | 0.23 | 0.11 | 0.11 | 0.08 |
| CBG | 0.01 | 0.02 | 0.02 | 0.01 | 0.03 | 0.04 | 0.02 | 0.04 | 0.04 | 0.05 | 0.03 | 0.02 | 0.01 |
| CBD | 0.18 | 0.26 | 0.45 | 0.39 | 0.72 | 0.50 | 0.41 | 0.61 | 0.50 | 0.86 | 0.49 | 0.31 | bLOQ |
| THCV | ND | bLOQ | 0.00 | 0.00 | 0.01 | 0.04 | 0.02 | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 |
| THCVA | ND | 0.07 | ND | ND | ND | 0.03 | 0.01 | 0.01 | 0.01 | bLOQ | ND | ND | 0.34 |
| CBN | 0.04 | 0.04 | 0.02 | 0.01 | 0.01 | 0.07 | 0.07 | 0.12 | 0.10 | 0.17 | 0.08 | 0.04 | 0.10 |
| Δ 9 -THC | 0.01 | 0.02 | 0.03 | 0.02 | 0.03 | 0.41 | 0.38 | 0.39 | 0.31 | 0.38 | 0.22 | 0.11 | 0.77 |
| Δ 8 -THC | ND | ND | 0.01 | ND | 0.01 | 0.25 | 0.16 | 0.23 | 0.22 | 0.23 | 0.20 | 0.14 | 0.17 |
| CBL | ND | ND | ND | ND | ND | ND | ND | ND | ND | 0.01 | ND | ND | ND |
| CBC | bLOQ | 0.01 | 0.01 | 0.01 | 0.03 | 0.02 | 0.02 | 0.03 | 0.02 | 0.04 | 0.02 | 0.01 | 0.01 |
| THCA | 0.10 | 0.12 | 0.10 | 0.08 | 0.11 | 3.05 | 1.59 | 1.93 | 1.68 | 1.49 | 0.99 | 0.64 | 5.20 |
| CBCA | 0.18 | 0.21 | 0.14 | 0.15 | 0.27 | 0.35 | 0.16 | 0.27 | 0.23 | 0.25 | 0.17 | 0.13 | 0.08 |
| 40 | 41 | 42 | 43 | 44 | 45 | 46 | 47 | 48 | 49 | 50 | 51 | 52 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HTHC2 | HTHC3 | HTHC4 | HTHC5 | HTHC6 | HTHC7 | HTHC8 | HTHC9 | HTHC10 | HTHC11 | HCBG1 | HCBG2 | HCBG3 | |
| CBDVA | ND | bLOQ | bLOQ | bLOQ | bLOQ | ND | bLOQ | bLOQ | ND | ND | ND | 0.02 | 0.02 |
| CBDV | bLOQ | 0.01 | 0.01 | bLOQ | bLOQ | bLOQ | bLOQ | bLOQ | ND | ND | 0.01 | 0.02 | 0.02 |
| CBDA | 0.05 | 0.05 | 0.04 | 0.03 | 0.02 | 0.03 | 0.02 | 0.02 | 0.01 | 0.01 | 0.07 | 0.19 | 0.14 |
| CBGA | 0.21 | 0.34 | 0.31 | 0.18 | 0.20 | 0.20 | 0.17 | 0.13 | 0.06 | 0.05 | 0.92 | 3.31 | 3.62 |
| CBG | 0.02 | 0.03 | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | bLOQ | bLOQ | 0.15 | 0.25 | 0.41 |
| CBD | 0.01 | 0.01 | bLOQ | bLOQ | bLOQ | bLOQ | 0.01 | 0.01 | bLOQ | bLOQ | 0.03 | 0.04 | 0.04 |
| THCV | 0.04 | 0.05 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.04 | 0.02 | 0.03 | ND | ND | ND |
| THCVA | 0.03 | 0.06 | 0.05 | 0.20 | 0.16 | 0.01 | 0.21 | 0.17 | 0.03 | ND | ND | 0.01 | 0.01 |
| CBN | 0.20 | 0.21 | 0.19 | 0.19 | 0.19 | 0.24 | 0.17 | 0.20 | 0.10 | 0.10 | ND | 0.01 | 0.01 |
| Δ 9 -THC | 0.80 | 0.72 | 0.67 | 0.70 | 0.68 | 0.74 | 0.66 | 0.74 | 0.30 | 0.27 | ND | ND | ND |
| Δ 8 -THC | 0.35 | 0.43 | 0.40 | 0.45 | 0.41 | 0.38 | 0.35 | 0.38 | 0.17 | 0.17 | ND | ND | ND |
| CBL | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | 0.03 |
| CBC | 0.06 | 0.02 | 0.02 | 0.02 | 0.03 | 0.03 | 0.03 | 0.04 | 0.02 | 0.02 | ND | ND | ND |
| THCA | 5.21 | 6.55 | 6.04 | 5.89 | 4.67 | 4.90 | 4.06 | 4.00 | 1.89 | 1.75 | ND | 0.02 | 0.03 |
| CBCA | 0.22 | 0.18 | 0.16 | 0.20 | 0.21 | 0.19 | 0.18 | 0.19 | 0.16 | 0.17 | ND | 0.06 | 0.08 |
| 53 | 54 | 55 | 56 | 57 | 58 | 59 | 60 | 61 | 62 | 63 | 64 | 65 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBG4 | HCBG5 | HCBG6 | HCBG7 | HCBG8 | HCBG9 | HCBG10 | HCBG11 | HCBG12 | HCBG13 | HCBG14 | HCBG15 | HCBG16 | |
| CBDVA | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | 0.03 | 0.01 | 0.01 | 0.02 | 0.01 | 0.01 | 0.01 | 0.01 |
| CBDV | 0.03 | 0.01 | 0.02 | 0.01 | 0.02 | 0.02 | 0.01 | 0.01 | 0.02 | 0.01 | ND | 0.01 | 0.01 |
| CBDA | 0.08 | 0.04 | 0.10 | 0.19 | 0.10 | 0.52 | 0.12 | 0.14 | 0.40 | 0.09 | 0.06 | 0.11 | 0.08 |
| CBGA | 2.84 | 0.57 | 1.30 | 3.55 | 1.83 | 4.96 | 1.46 | 1.93 | 4.58 | 2.44 | 1.26 | 2.95 | 1.22 |
| CBG | 0.29 | 0.22 | 0.20 | 0.29 | 0.16 | 0.34 | 0.17 | 0.08 | 0.29 | 0.17 | 0.20 | 0.19 | 0.18 |
| CBD | 0.03 | 0.02 | 0.35 | 0.41 | 0.03 | 0.07 | 0.04 | 0.03 | 0.05 | 0.03 | 0.02 | 0.03 | 0.02 |
| THCV | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| THCVA | ND | ND | ND | 0.02 | ND | 0.03 | ND | ND | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 |
| CBN | 0.01 | 0.02 | 0.01 | ND | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | |
| Δ 9 -THC | 0.02 | ND | ND | ND | 0.02 | 0.02 | ND | ND | 0.02 | ND | ND | 0.02 | 0.02 |
| Δ 8 -THC | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND | ND |
| CBL | ND | ND | ND | 0.02 | ND | ND | ND | ND | 0.02 | 0.02 | ND | 0.02 | ND |
| CBC | 0.05 | ND | ND | 0.03 | ND | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 | ND |
| THCA | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.03 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.03 |
| CBCA | 0.08 | ND | 0.05 | ND | 0.05 | 0.07 | ND | 0.06 | 0.07 | ND | ND | 0.07 | 0.06 |
| 66 | 67 | 68 | 69 | 70 | 71 | 72 | 73 | 74 | 75 | 76 | 77 | 78 | |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| HCBG17 | HCBG18 | HCBG19 | HCBG20 | HCBG21 | HCBG22 | HCBG23 | HCBG24 | E-CIG1 | E-CIG2 | E-CIG3 | E-CIG4 | E-CIG5 | |
| CBDVA | 0.02 | 0.02 | 0.02 | 0.02 | 0.01 | 0.03 | 0.01 | 0.03 | ND | ND | ND | 0.05 | 0.02 |
| CBDV | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.02 | 0.01 | 0.02 | ND | 0.32 | 0.34 | 0.24 | 0.07 |
| CBDA | 0.29 | 0.34 | 0.34 | 0.36 | 0.18 | 0.48 | 0.23 | 0.80 | ND | 0.13 | 0.15 | 0.78 | 0.03 |
| CBGA | 3.65 | 3.52 | 4.08 | 2.95 | 1.42 | 4.99 | 2.06 | 5.63 | ND | 0.12 | 0.11 | 0.46 | 0.86 |
| CBG | 0.25 | 0.28 | 0.02 | 0.17 | 0.08 | 0.30 | 0.09 | 0.35 | 1.97 | 3.52 | 2.26 | 0.01 | 1.42 |
| CBD | 0.07 | 0.06 | 0.37 | 0.04 | 0.03 | 0.09 | 0.03 | 0.07 | 0.18 | 0.52 | 0.23 | 0.15 | 0.12 |
| THCV | 0.03 | ND | ND | ND | ND | 0.03 | ND | 0.01 | 0.35 | 0.63 | 0.50 | 0.15 | 0.37 |
| THCVA | ND | ND | 0.03 | 0.04 | 0.02 | 0.02 | 0.02 | 0.04 | 0.43 | 0.14 | 0.24 | 0.55 | 0.11 |
| CBN | ND | ND | ND | ND | 0.01 | 0.01 | ND | ND | 0.94 | 2.12 | 2.03 | 0.38 | 2.55 |
| Δ 9 -THC | ND | 0.02 | ND | ND | ND | ND | 0.02 | 0.02 | 73.67 | 65.22 | 73.29 | 55.86 | 60.55 |
| Δ 8 -THC | 0.02 | ND | ND | ND | ND | ND | ND | ND | 0.02 | 0.02 | 0.02 | 1.90 | 0.01 |
| CBL | 0.02 | 0.02 | ND | 0.03 | 0.02 | 0.02 | 0.01 | 0.02 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
| CBC | ND | 0.02 | ND | 0.02 | 0.02 | 0.02 | 0.02 | 0.04 | 0.88 | 1.22 | 1.05 | 0.02 | 0.76 |
| THCA | ND | 0.02 | ND | ND | ND | 0.02 | ND | ND | 0.02 | 1.24 | 0.02 | 17.00 | 2.10 |
| CBCA | 0.06 | 0.07 | 0.08 | 0.10 | 0.08 | 0.10 | 0.09 | 0.08 | 0.07 | 0.07 | 0.07 | 0.07 | 0.06 |
Each sample was injected as a triplicate.
E-CIG, electronic cigarette; HCBG, CBG-dominant variety; HTHC, THC-dominant variety; IM, THC/CBD intermediate variety.
Among the analyzed plant material samples, 42.5% belonged to CBD dominant, 9.6% to THC/CBD intermediate, and 15.1% to THC dominant. About 32.9% of plant material samples belong to CBG dominant (a cannabis chemotype contains high concentration of CBG/CBGA). As a promising field combining computer science and analytical chemistry, chemometrics has increasingly found application in natural product chemistry and has been used extensively for analytical data mining, graphical visualization, and class discrimination and prediction. 34 An important step in this research is data analysis where mathematical algorithms were used to extract useful information from huge data sets obtained from the HPLC method.
To clearly differentiate among the four groups of 73 cannabis plant samples, data were subjected to PCA. PCA is an unsupervised clustering technique commonly employed to reduce the complexity of multivariate data sets without losing important information, observe the variance in data sets, and visualize data clustering. The PCA score plots separated the cannabis samples into four different blocks, CBD-dominant (HCBD), THC/CBD-intermediate (IM), THC-dominant (HTHC), and CBG-dominant (HCBG) samples.
Figure 5 shows how a simple PCA effectively separates the 73 cannabis plant material data into four separate clusters. Both THC-dominant and THC/CBD-intermediate samples have negative PC1 scores, but they can be differentiated by PC2 in which THC-dominant samples have negative PC2 scores, whereas THC/CBD-intermediate samples have positive PC2 scores. The same trend is seen with CBG dominant and CBD dominant, which have positive PC1 scores, but CBG dominant has negative PC2 scores and CBD dominant has positive scores.
FIG. 5.
Principal component analysis score plots of 73 cannabis samples for HPLC data. Symbols: ● CBD dominant (HCBD); ■ CBG dominant (HCBG); ▲ THC dominant (HTHC); ⁕ THC/CBD intermediate (IM).
In cannabis plant samples, the sums of the concentration of Δ 9 -THC and its acidic precursor, THCA-A, were in the range of 0.02–0.26%, while the total CBD (CBD + CBDA) ranged between 1.7% and 5.95% in CBD dominant. In THC dominant, the total Δ 9 -THC ranged between 2.02% and 7.27% and total CBD ranged between 0.01% and 0.06%, while in THC/CBD intermediate, Δ 9 -THC was ranged between 0.75% and 3.46% and total CBD ranged between 2.70% and 5.54%.
The last chemotype, CBG dominant, has a high concentration of CBG as a total (CBG + CBGA) between 0.79% and 5.98% with low concentration of both total Δ 9 -THC and total CBD between 0.02% and 0.05% and 0.06% and 0.87%. In the five e-cigarette samples, the total Δ 9 -THC, total CBD, and total CBG ranged between 62.65% and 73.69%, 0.15% and 0.93%, and 0.47% and 3.64%, respectively. The content (% w/w) of individual cannabinoids in different chemotypes and e-cigarettes samples is displayed in Table 6. The analysis of e-cigarette samples has yielded some interesting results. In particular, the concentration of Δ 9 -THC detected was found to be quite high, ranging from 55.86% to 73.67%, while the concentration of CBG ranged from 0.1% to 3.52%. Notably, the “E-CIG4” sample had a significant concentration of THCA, with levels as high as 17%.
Conclusions
This work represents the development and validation of a simple HPLC-UV/PDA analytical method for reliable detection and quantification of 15 cannabinoids. The analytical method was validated following the criteria recommended by the ICH guidelines. The specificity, linearity, linear concentration range, LOD, LOQ, accuracy, and precision were determined for the method. In addition, the analytical method has shown to be specific, with acceptable precision and accuracy. The HPLC method was successfully applied to determine the cannabinoid profile of some cannabis plant material and cannabis marketed products such as electronic cigarettes.
Abbreviations Used
- Δ 8 -THC
delta-8-tetrahydrocannabinol
- Δ 9 -THC
delta-9-tetrahydrocannabinol
- CBC
cannabichromene
- CBCA
cannabichromenic acid
- CBD
cannabidiol
- CBDA
cannabidiolic acid
- CBDV
cannabidivarin
- CBDVA
cannabidivarinic acid
- CBG
cannabigerol
- CBGA
cannabigerolic acid
- CBL
cannabicyclol
- CBN
cannabinol
- GC/FID
gas chromatography with flame ionization detector
- GC/MS
gas chromatography with mass spectrometry
- HPLC
high-performance liquid chromatography
- ICH
International Conference on Harmonization
- I.S.
internal standard
- LOD
limits of detection
- LOQ
limits of quantification
- MS
mass spectrometry
- NLT
not less than
- NMT
not more than
- PCA
principal component analysis
- PDA
photodiode array
- RSD
relative standard deviation
- THCA
tetrahydrocannabinolic acid A
- THCV
tetrahydrocannabivarin
- THCVA
tetrahydrocannabivarinic acid
- UV
ultraviolet
Author Disclosure Statement
The authors declare no conflict of interest.
Funding Information
The project described was partially supported by the National Institute on Drug Abuse (contract No. N01DA-15-7793).
Supplementary Material
Cite this article as: Elhendawy MA, Radwan MM, Ibrahim EA, Wanas AS, Chandra S, Godfrey M, ElSohly MA (2023) Validation and quantitation of fifteen cannabinoids in cannabis and marketed products using high-performance liquid chromatography-ultraviolet/photodiode array method, Cannabis and Cannabinoid Research 9:4, e1091–e1107, DOI: 10.1089/can.2022.0335.
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