| Abstract [eng] |
Hemp (Cannabis sativa L.) accumulates plenty of chemical compounds which are beneficial for human health. For centuries it has been used for textile fiber production and in medicinal chemistry. Recently, more scientists got interested in hemp because of terpenophenolic compounds, called phytocannabinoids which are produced in trichomes of leaves. Pharmacological activity of phytocannabinoids has been proven by many studies and it is related to endocannabinoid system in humans. Due to growing demand of products with hemp it is important to create new analytical methods to determine the exact composition of phytocannabinoids in Cannabis sativa L. extracts and other products. The aim of this study is to optimize gas chromatography and liquid chromatography conditions for phytocannabinoid analysis and validate them, as well as perform Cannabis sativa L. extract analysis with validated methods and determine composition of phytocannabinoids. In this study conditions of phytocannabinoid analysis was optimized with three chromatographic systems: gas chromatography with time of flight mass spectrometry, gas chromatography with flame ionization detector and high performance liquid chromatography with diode array detector. Methods were validated according to ICH guidlines by evaluating specifity, precision, linearity, Limit of Detection and Limit of Quantification for each phytocannabinoid in each method. Gas chromatography with time of flight mass spectrometry method was validated for cannabidiol, cannabigerol, cannabinol, cannabichromene and cannabidivarin, gas chromatography with flame ionization detector method was validated for cannabidiol, cannabigerol, cannabinol and cannabidivarin, high performance liquid chromatography with diode array detector method was validated for cannabidiol, cannabigerol, cannabinol, canabichromene, cannabigerolic acid and cannabidiolic acid. For quantative analysis calibration curves were made for each validated cannabinoid in each method. From all three methods liquid chromatography method had the best values of validation parameters. It was found that limit of detection and limit of quantification values for high performance liquid chromatography method respectively were distributed in 1,36–2,96 µg/ml and 4,13–8,86 µg/ml intervals. For gas chromatography with flame ionization detector method limit of detection values were within the range of 1,71–2,71 µg/ml and limit of quantification values within the range of 5,19–8,20 µg/ml. Limit of detection and limit of quantification values for gas chromatography with time of flight mass spectrometry method were highest from all three methods, respectively they are within the ranges of 5,00–8,15 µg/ml and 15,15–24,69 µg/ml. Each method was applied for the phytocannabinoid analysis of Cannabis sativa L. extracts. It was found that the most abundant phytocannabinoid in all extracts is cannabidiol, followed by cannabidivarin. However, the amount of cannabidivarin was significantly lower. Cannabinol, canabigerol, cannabichromene and cannabigerolic acid were also identified and quantified in these extracts, yet in very small quantities. |
