Development of Tetrahydrocannabinol Content Analysis by HPLC and Physicochemical Properties of Cannabis Female Inflorescences and Leaves
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Abstract
Introduction and Objectives: Due to the absence of standard specification for the quality control of cannabis female inflorescences and leaves, this study aimed to evaluate their physicochemical properties, establish chemical profiles, develop quantitative analytical methods and validate these methods. The findings will support the formulation of monographs of cannabis female inflorescences and cannabis leaves for the Thai Herbal Pharmacopoeia.
Methods: A total of 23 cannabis female inflorescences samples and 16 cannabis leaf samples were analyzed. Chemical identity was assessed using tetrahydrocannabinol (THC) and cannabidiol (CBD) as reference standards. Physicochemical properties were evaluated, and a high performance liquid chromatography (HPLC) method was developed and validated for quantifying THC content.
Results: Colorimetric reactions revealed the presence of cannabinoids and terpenoids. Thin layer chromatography confirmed the presence of THC and CBD.For female inflorescences, the mean ± standard deviation values for moisture, total ash, acid-insoluble ash, water extractive, ethanol extractive and THC contents were 8.00 ± 0.68, 12.94 ± 2.03, 1.96 ± 0.96, 18.51 ± 3.33, 17.64 ± 2.59 and 1.95 ±1 .51 %w/w, respectively. For leaves, the corresponding values were 7.83 ± 0.74,17.38 ± 3.07, 2.48 ± 1.70, 26.04 ± 2.54,14.88 ± 3.39 and 0.39 ± 0.23 %w/w, respectively. The developed HPLC method demonstrated high specificity and linearity across the concentration range of 2 to 10 µg/mL (R2=0.9998). Recovery rates ranged form 97.71% to 103.15%, precision (%RSD) from 0.08% to 0.36%, with LOD and LOQ values of 0.08 and 0.25 µg/mL, respectively.
Discussion: Our study on the chemical profile of female cannabis inflorescences and leaves showed varying levels of THC and CBD, while CBD was detected in only a few samples. This variation may be due to differences in strain, cultivation location, and harvesting time. Within the same cultivation site, the female inflorescences contained more THC than the leaves, aligning with previous findings that female inflorescences are the primary site of cannabinoid accumulation in cannabis plants. Chemical profiling using HPLC enabled the separation of eight standard compounds. Physicochemical quality assessment can serve as a guideline for quality control of medicinal cannabis. Moreover, the development of an HPLC method to quantify THC in female inflorescences and leaves showed high specificity, with excellent linearity, recovery, and precision, meeting accepted pharmaceutical analysis standards. The use of a gradient HPLC system also significantly reduced analysis time.
Conclusion: The physicochemical specifications and the analytical method developed in this study can be used to help establish national standard specification for cannabis female inflorescences and leaves in the Thai Herbal Pharmacopoeia.
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