Identification of Cannabis sativa L. (hemp) Retailers by Means of Multivariate Analysis of Cannabinoids

Abstract

In this work, the concentration of nine cannabinoids, six neutral cannabinoids (THC, CBD, CBC, CBG, CBN and CBDV) and three acidic cannabinoids (THCA CBGA and CBDA), was used to identify the Italian retailers of Cannabis sativa L. (hemp), reinforcing the idea that the practice of categorizing hemp samples only using THC and CBD is inadequate. A high-performance liquid chromatography/high-resolution mass spectrometry (HPLC-MS/MS) method was developed for screening and simultaneously analyzing the nine cannabinoids in 161 hemp samples sold by four retailers located in different Italian cities. The hemp samples dataset was analyzed by univariate and multivariate analysis with the aim to identify the hemp retailers without any other information on the hemp samples like Cannabis strains, seeds, soil and cultivation characteristics, geographical origin, product storage, etc. The univariate analysis highlighted that the hemp samples could not be differentiated by using any of the nine cannabinoids analyzed. To evaluate the real efficiency of the discrimination among the four hemp retailers a partial least squares discriminant analysis (PLS-DA) was applied. The PLS-DA results showed a very good discrimination between the four hemp retailers with an explained variance of 100% and low classification errors in both calibration (5%) and cross validation (6%). A total of 92% of the hemp samples were correctly classified by the cannabinoid variables in both fitting and cross validation. This work contributed to show that an analytical method coupled with multivariate analysis can be used as a powerful tool for forensic purposes.

Keywords: Cannabis sativa L.; HPLC-MS/MS analysis; cannabinoids; multivariate analysis; partial least squares discriminant analysis (PLS-DA).

Figure 1

Box and whisker plot of the relative concentrations of the six neutral (THC, CBD, CBC, CBG, CBN and CBDV) and the three acidic cannabinoids (THCA CBGA and CBDA) in the 161 hemp samples. The hemp samples were grouped as sold by the four Italian hemp retailers. Concentration of cannabinoids was reported as % w/w. Y axis title = Concentration (% w/w). X axis Title = Hemp retailers. Median and average were depicted with a flat black line and a red cross, respectively.

Figure 2

Scores plot (A) and loadings plot (B) obtained from the PCA on the matrix data of the 161 hemp samples (in the matrix rows) and the nine cannabinoids analyzed (the matrix columns). Plots of the first three components (explained variance: PC1 = 42.7%; PC2 = 15.8%; PC3 = 14.9%; total = 73.4%). Data have been auto-scaled (zero mean and unitary variance) before PCA. The four Hemp retailers are marked with different colors: Green = A; Red = B; Yellow = C; Blue = D.