Cannabis sativa L. is the most widely cultivated, trafficked, and abused illicit drug. The reliable distinction between drug type and fiber type C. sativa remains a topic of interest for forensic chemists. For the first time, we applied a combination of simple, cost-effective, and reliable High-Performance Thin-Layer Chromatography (HPTLC) with image analysis as well as sophisticated multivariate tools for differentiating C. sativa drug, fiber and intermediate chemotypes. After extraction, major cannabinoids from 43 seized C. sativa were separated using the HPTLC method. Peak areas of Δ⁹-THC, CBN, and CBD were calculated by simple image processing, and Xfactor = [THC + CBN]/CBD was determined as criteria used to discriminate three C. sativa chemotypes. The obtained results were compared with Gas Chromatography with Flame Ionization Detection (GC-FID) as the reference method for the determination of Xfactor.

Principal Component Analysis and Hierarchic Cluster Analysis were applied to classify three C. sativa chemotypes according to their chemical pattern. The proposed approach clearly distinguishes 26 drugs, 13 fiber, and 4 intermediate chemotypes in seized C. sativa, aligning with GC-FID analysis. The results showed that the HPTLC technique in combination with multivariate methods is an accurate and reliable tool for high-throughput and forensic screening of three C. sativa chemotypes. Compounds such as Δ⁹-THC and CBD were marked as the most important cannabinoids responsible for the classification of the seized C. sativa chemotypes.

大麻是种植、贩运和滥用最广泛的非法药物。药物类型和纤维类型苜蓿之间的可靠区别仍然是法医化学家感兴趣的话题。我们首次将简单、经济高效且可靠的高性能薄层色谱 (HPTLC) 与图像分析以及复杂的多变量工具相结合，用于区分苜蓿药物、纤维和中间化学型。提取后，使用 HPTLC 方法从 43 个查获的苜蓿中分离出主要大麻素。通过简单的图像处理和Xfactor计算Δ9-THC、CBN和CBD^的峰面积 = [THC + CBN]/CBD 被确定为用于区分三种苜蓿化学型的标准。将所得结果与火焰离子化检测气相色谱法 (GC-FID) 进行比较，作为Xfactor测定的参考方法。

应用主成分分析和层次聚类分析根据化学模式对三种苜蓿化学型进行分类。所提出的方法清楚地区分了查获的苜蓿中的 26 种药物、13 种纤维和 4 种中间化学型，与 GC-FID 分析一致。结果表明，HPTLC 技术与多变量方法相结合是对三种苜蓿化学型进行高通量法医筛选的准确可靠的工具。Δ ⁹ -THC 和 CBD 等化合物被标记为最重要的大麻素，负责对查获的苜蓿化学型进行分类。