GC-FID ANALYSIS OF FOOD SAMPLES MADE OF HEMP

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GC-FID ANALYSIS OF FOOD SAMPLES MADE OF HEMP AJINKYA BHARAT LALGE 1, PETER MENDEL 1, TOMAS VYHNANEK 1, VACLAV TROJAN 1, HELENA FISEROVA 1, LUDEK HRIVNA 2, EVA MRKVICOVA 3 LADISLAV HAVEL 1 1

1 GC-FID ANALYSIS OF FOOD SAMPLES MADE OF HEMP AJINKYA BHARAT LALGE 1, PETER MENDEL 1, TOMAS VYHNANEK 1, VACLAV TROJAN 1, HELENA FISEROVA 1, LUDEK HRIVNA 2, EVA MRKVICOVA 3 LADISLAV HAVEL 1 1 Department of Plant Biology 2 Department of Food Technology 3 Department of Animal Nutrition and Forage Production Mendel University in Brno Zemedelska 1, Brno CZECH REPUBLIC ajinkya.lalge@mendelu.cz Abstract: The aim of the work was to analyse different cannabinoids occurring in the food samples. The chromatographic data for the cannabinoids of interest namely Δ ⁹-Tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) were determined using gas chromatography. Hemp seed powder, hemp oil and hemp protein in various degrees of concentration were used for the preparation of the food sample to be analysed. Hemp has a botanical relationship with drug/medicinal cannabis. Cannabis has psychotropic substances, which are wrongly attributed to hemp. The psychoactive compound THC was found in very low concentrations in the food samples. Key Words: cannabis, hemp, cannabinoids, gas chromatography INTRODUCTION The hemp plant Cannabis sativa L. is an annual plant in the Cannabaceae family. It has been an important source of food, fiber, medicine and psychoactive/religious drug since prehistoric times. Cannabis is mentioned as a medication in ancient Egyptian medical texts: Ramesseum III Papyrus (1700 B.C.), Eber's Papyrus (1600 B. C.), the Berlin Papyrus (1300 B.C.), and the Chester Beatty VI Papyrus (1300 B.C.) (Russo 2007, Maniche 1989). Two main types of Cannabis sativa L. must be distinguished, the drug and non-drug types. The first is also known as marijuana, hashish or cannabis tincture and contains Δ9 Tetrahydrocannabinol (THC) (Figure 1) in concentrations between 1 20%, high enough to exhibit psychoactivity. The second type of Cannabis sativa L. is industrial hemp with THC concentrations < 0.3% so it has no psychoactive properties (Ross et al. 2000, Holler et al. 2008). Structure of main cannabinoids Figure 1 Shows the structure of main cannabinoids. Legend: CBD - Cannabidiol, CBN Cannabinol, THC- Δ9 Tetrahydrocannabinol Hempseed possesses excellent nutritional value. It is very rich in essential fatty acids and other polyunsaturated fatty acids. It has almost as much protein as soybean and is rich in vitamin E and minerals such as phosphorus, potassium, sodium, magnesium, sulphur, calcium, iron, and zinc 600 P age

2 (Callaway 2004). Cannabinoids are terphenolic secondary metabolites produced in sessile and stalked trichromes by cannabis plant. More than 100 cannabinoids have been discovered and studied until now (Turner et al. 1980). The most interesting and most studied compounds of this class are THC and CBD. The largest proportion of THC can be found on the surface of the seed coat. As a consequence, only very low THC concentrations are found inside of the seeds (less than 2 mg/kg with drug hemp and less than 0.5 mg/kg with fibre hemp) (Ross et al. 2000, Small and Marcus 2003). Cannabis is analysed for several different purposes. Identification and quantification of the main cannabinoids is the main objective for the analysis of hemp food products. There are various methods available for analysing cannabinoids such as High performance liquid chromatography (HPLC) and gas chromatography. HPLC can be used to determine the decarboxylated form of THC, CBD and CBN. A rapid and simple method was optimized for the determination of THC, CBD and CBN contents in the food sample by gas chromatography with a flame ionisation detector using squalene as an internal standard. MATERIALS AND METHODS PCH Fisons instrument equipped with a flame ionisation detector (FID) was used for analysis of cannabinoids. The instrument was equipped with DB5 non-polar capillary column with 5% phenyl, 95% dimethylarylensiloxan (30 m length, 0.25 mm internal diameter, film thickness 0.25 μm). The temperature gradient started at 150 C and increased at a rate of 3 C/min until 270 C. The thermostat injection temperature was 250 C and the FID detector temperature was set to 270 C. The instrument was calibrated with three points from 0.05 to 0.5% CBD (cannabidiol, 1mg / ml in methanol, Sigma), CBN (cannabinol, 1 mg/ml in methanol, Sigma), and THC (10 mg/ml ethanol - Ipomed). They were established retention times cannabinol (CBN), cannabidiol (CBD) tetrahydrocannabidol (THC) and squalene (Table 1). Table 1 Shows the retention time (tr) and the relative retention time (Rtr) of analytes. Analyte tr (min) Rtr Cannabidiol Tetrahydrocannbidol cannabinol Squalene Extractive solution used was 35 ml of squalene in 100 ml of hexane. 0.1 gm of each hemp food sample was homogenised with liquid nitrogen. After homogenisation, 5 ml of the extractive solution was added to the samples placed in a volumetric flask. The volumetric flask was then covered with aluminium foil and placed in an ultra sound for 20 mins. The extract was collected and centrifuged at 3000 rpm for 5 mins at 4 C. The liquid phase was separated and stored in dark storage bottles at -20 C in a freezer. The samples to be analysed were provided by the Department of Food Technology of Mendel University in Brno. Different hemp components like protein powder, hemp grits and hemp flour were provided. The oil sample was not analysed. The hemp component was used in different proportions in the food to be analysed (Table 2). Each sample was analysed three times and an average values of mg/g of the cannabinoids were calculated. 601 P age

3 Table 2 Shows the different concentrations of hemp powder used for each sample. Bread Sample wheat flour (g) hemp flour (g) hemp oil (ml) hemp grits (g) hemp protein (g) B B B B B B B B B B RESULTS AND DISCUSSIONS All the hemp samples were derived from the same source. The corresponding percentage values of cannabinoids for each sample were calculated (Table 3). The THC content of all the food samples analysed varied from 0 to 0.23% and were found to be below 0.3% whereas no CBN was found at all. CBN is the main chemical degradation product of Δ ⁹-Tetrahydrocannabinol (Harvey, 1990) which suggests that there was very little or no degradation at all. The food samples we analysed met the guidelines. Samples in the EU showed values ranged from 0.05% to 0.2% (El-Ghany 2002, Mechtler et al. 2004). The prescribed use of certified hemp planting seed by the EU and the increase of controls on manufacturers have obviously preserved low level of THC in EU food and feed products (Lachenmeier and Walch 2005). Expectedly no cannabinoids were detected in B 1 as no hemp component was used. Surprisingly, B 4 did not show presence of any cannabinoids. As seen from the figure 2. Sample B 6 in which grits were utilized showed the highest concentration of cannabinoids mg/g and mg/g for CBD and THC respectively whereas sample B 5 in which similar quantity of grits were present showed much lower value for cannabinoids. Interestingly the grits when measured did not show any measureable amounts of cannabinoids. Hemp seed has very low concentrations of THC and the largest proportions of THC is found on the seed coat (Leson et al. 2001). The sample B 9 and B 10 in which hemp protein component was used showed similar amounts of THC and CBD (Figure 2). The hemp protein when analysed showed lower values of cannabinoids than its final products. Similarly, with the analysed hemp flour the percentages of cannabinoids detected were lesser than the final product B 8. This could be either explained that the hemp component goes further decarboxylation during the baking process of the food samples (Heroudkova 2016). Hemp food products contain analysable amounts of THC. The presence of THC in the hemp containing food can raise concerns about psychoactive effects. According to the guidelines of European Union the hemp parts used for food production should not exceed 0.2% THC (Lachenmeier and Walch 2005, EU Commission 2008). According to a similar research conducted where 10% of hemp component was used the highest amount of THC and CBD found were mg/g and mg/g respectively (Heroudkova 2016). From a forensic point of view, the presence of THC in hemp containing food stuff raised not only the problem of psychoactive effects, but it also leads to concerns about the validity of positive results of drug tests (Elsohly 2003). 602 P age

4 Table 3 Cannabinoids content per sample. Sample CBD THC % mg/g % mg/g B B B B B B B , B B B Protein Grits Flour Legend: CBD - Cannabidiol, CBN Cannabinol, THC- Δ9 Tetrahydrocannabinol, B1-B10 see Table 2. Figure 2 Shows the percentage concentrations for each sample. 0,4 0,35 0,3 % Concentartion 0,25 0,2 0,15 0,1 0,05 0 Protein Grits Flour B 1 B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 Sample CBD THC CBN Legend: CBD - Cannabidiol, CBN Cannabinol, THC- Δ9 Tetrahydrocannabinol, B1-B10 see Table 2. CONCLUSION Hemp can be an excellent source of food and nutrition. Hemp containing food products are seeing a revival. The gas chromatography method used in the experiment was good for the detection of the main cannabinoids. The value of psychoactive compound was below or at par with the EU guidelines. In the future, we would analyse the food products made from different parts of hemp. 603 P age

5 ACKNOWLEDGEMENTS The research was financially supported by the IGA FA MENDELU No. TP 4/2015. REFERENCES Callaway, J.C Hempseed as a nutritional resource: An overview. Euphytica, 140(1): El-Ghany, M.E.A Molekulargenetische Diversität einer monözischen und einer diözischen Hanfsorte und Analyse des Fasergehaltes von verschiedenen Hanfformen. Dissertation Martin-Luther- Universität, Halle-Wittenberg, Germany. ElSohly, M.A Practical Challenges to Positive Drug Tests for Marijuana. Clinical Chemistry, 49(7): EU Comission, Comission Regulations. Available at: E0002 [ ]. Harvey, D.J Stability of cannabinoids in dried samples of cannabis dating from around Journal of Ethano Pharmocology, 28(1): Heroudkova, K Slozeni a uziti listove casti odrud konopi seteho. Diploma Thesis, Vysoká škola chemicko-technologická v Praze. Holler, J.M., Bosy, T.Z., Dunkley, C.S., Levine, B., Past, M.R., Jacobs, A Delta9- tetrahydrocannabinol content of commercially available hemp products. Journal of Analytical Toxicology, 32(6): Lachenmeier, D.W., & Walch, S.G Current Status of THC in German Hemp Food Products. Journal of Industrial Hemp, 10(2): Leson, G., Pless, P., Grotenhermen, F., Kalant, H., Elsohly, M.A Evaluating the impact of hemp food consumption on workplace drug tests. Journal of Analytical Toxicology, 25(8): Manniche, L An ancient Egyptian herbal. Third University of Texas Press Printing. Mechtler, K., Bailer, J., de Hueber, K Variations of D9-THC content in single plants of hemp varieties. Industrial Crops and Products, 19: Ross, S.A., Mehmedic, Z., Murphy, T. P., Elsohly, M.A GC-MS analysis of the total Δ9-THC content of both drug-and fiber-type cannabis seeds. Journal of Analytical Toxicology, 24(8): Russo, E.B History of cannabis and its preparations in saga, science, and sobriquet. Chemistry and Biodiversity, 4(8): Small, E., Marcus, D Tetrahydrocannabinol levels in hemp (Cannabis sativa) germplasm resources. Economic Botany, 57: 545. Turner, C.E., Elsohly, M.A., Boeren, E. G Constituents of Cannabis sativa L. XVII. A review of the natural constituents. Journal of Natural Products, 43(2): P age

Analysis of Cannabinoids in Cannabis by UHPLC Using PDA Detection

APPLICATION NOTE Liquid Chromatography Author: Wilhad M. Reuter PerkinElmer, Inc. Shelton, CT Analysis of Cannabinoids in Cannabis by UHPLC Using PDA Detection Introduction Cannabis sativa, from which