Objective: To establish a gas chromatography-mass spectrometry (GC-MS)-isotope internal standard method for the simultaneous determination of three active components [cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabinol (CBN)] in hemp, and to explore their content characteristics and chemical phenotypes in hemp from different habitats. Methods: The target component was extracted by ultrasonic extraction with methanol, and the sample solution was prepared after centrifugation. GC-MS was used to determine the contents of THC, CBN and CBD in hemp by using isotope internal standard method under reasonable chromatographic and mass spectrometry conditions. The selected qualitative and quantitative ion pairs were as follows: CBD, m/z 231→238/314; THC-D3, m/z 302-234/317; THC, m/z 299-231/314; CBN, m/z 295-238/310. Results: The three components had a good linear relationship, and the correlation coefficients (r) were 0.999 6~0.999 9 in the range of 0.2-32.0 μg·mL-1, the recoveries were 93.2%-112.7%, the lower limit of detections (LODs) were 0.002 3-0.006 2 μg·mL-1, the lower limit of quantitation (LOQs) were 0.007 6-0.02 1 μg·mL-1, the precision and reproducibility were good, and the stability was good within 48 h. The results of 18 batches of samples showed that the CBD contents of Kunming in Yunnan Province and Longxi in Gansu Province were higher (1.24%-3.43%), the THC contents of Jining in Inner Mongolia were higher (0.20%-0.29%), close to the critical value of drug cannabis (THC content>0.3%), and the hemp samples from Heilongjiang Cyclophanthus had the lowest contents of the three components. Conclusion: The method used in this study could simultaneously determine the contents of three active components in hemp. It was easy to operate, specific, sensitive and efficient, and the results were accurate and reliable. By analyzing and discussing the content distribution characteristics and chemical phenotypes of the three components in different areas. It was concluded that the content characteristics were significantly different, and 18 batches of samples were preliminarily judged to be fibrous hemp.
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