Identification and quantification of cannabinol as a biomarker for local hemp retting in an ancient sedimentary record by HPTLC-ESI-MS

Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-020-02492-0 RESEARCH PAPER Identification and quantification of cannabinol as a biomarker for local hemp retting in an ancient sedimentary record by HPTLC-ESI-MS Theresa Schmidt 1 & Annemarie Elisabeth Kramell 1 & Florian Oehler 2 & Ralph Kluge 1 & Dieter Demske 3 & Pavel E Tarasov 3 & René Csuk 1 Received: 28 November 2019 / Revised: 22 January 2020 / Accepted: 5 February 2020 # The Author(s) 2020 Abstract Cannabis products have been used in various fields of everyday life for many centuries, and applications in folk medicine and textile production have been well-known for many centuries. For traditional textile production, hemp fibers were extracted from the stems by water retting in stagnant or slow-moving waters. During this procedure, parts of the plant material‚ among them phytocannabinoids‚ are released into the water. Cannabinol (CBN) is an important degradation product of the predominant phytocannabinoids found in Cannabis species. Thus, it is an excellent indicator for present as well as ancient hemp water retting. In this study, we developed and validated a simple and fast method for the determination of CBN in sediment samples using highperformance thin-layer chromatography (HPTLC) combined with electrospray ionization mass spectrometry (ESI-MS), thereby testing different extraction and cleanup procedures‚ as well as various sorbents and solvents for planar chromatography. This method shows a satisfactory overall analytical performance with an average recovery rate of 73%. Our protocol enabled qualitative and quantitative analyses of CBN in samples of a bottom sediment core‚ having been obtained from a small lake in Northern India, where intense local retting of hemp was suggested in the past. The analyses showed a maximum CBN content in pollen zone 4 covering a depth range of 262–209 cm, dating from approximately 480 BCE to 1050 CE. These findings correlate with existing records of Cannabis-type pollen. Thus, the method we propose is a helpful tool to track ancient hemp retting activities. Keywords HPTLC . Cannabinol . Sediment . Biomarker . Cannabis . Hemp retting Introduction Cannabis has been used by humans for many centuries and is probably one of the oldest cultivated plants [1]. It is widely distributed around the world, and archaeological finds indicate Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02492-0) contains supplementary material, which is available to authorized users. * Annemarie Elisabeth Kramell 1 Department of Organic Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 2, 06120 Halle, Germany 2 Department of Inorganic Chemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 2, 06120 Halle, Germany 3 Section Paleontology, Institute of Geological Sciences, Freie Universität Berlin, Malteserstr. 74-100, 12249 Berlin, Germany its usage for more than 2500 years. For instance, almost complete ancient Cannabis plants as well as parts of it have been excavated from different tombs in the Jiayi and Yanghai cemetery located in Northwestern China dating back to the first millennium BCE [2, 3]. Cannabis is a versatile plant and has been used as medicine, food source (seeds and oil), fuel, and psychedelic drug and also as a construction material or for the production of textiles and paper. The production of hemp fibers, e.g., for the manufacturing of robes, requires the separation of the fibers from the stems through microbiological and physical processes occurring during retting. Traditionally, the extraction of the fibers from stems is performed in stagnant or slow-moving waters, thereby submerging the stems in water for several days. During this treatment plant material and among other substances, phytocannabinoids are released into the waters. Phytocannabinoids are unique to the Cannabis species. The predominant phytocannabinoids in drug- and fiber-type Cannabis are (-)-Δ9-trans-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) that are Schmidt T. et al. storage, e.g., the transformation to Δ9-THC by an acidcatalyzed cyclization, followed by the decay of Δ9-THC to CBN (Fig. 1) [8, 9]. Therefore, it is not surprising that CBN has been detected as the major degradation product of cannabinoids in dried Cannabis flowers dating from around 1896–1905 [10]. Studies concerning the CBN content in sedimentary records, however, are rare. Thus, Lavrieux et al. reported about the detection of CBN, preserved in sediment samples from lake Aydat in the French Massif Central, covering the past 1800 years [11], thereby relating the presence of CBN to the retting of locally grown Cannabis plants for fiber production. This finding was supported by the analysis of pollen and historical data. Thus, determination of CBN contents in sedimentary cores seems to be an excellent possibility to trace ancient water retting activities. transformed by a non-enzymatically decarboxylation upon heating after harvesting and during storage into their corresponding neutral forms, namely (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). Phytocannabinoids accumulate in female flowers and in most aerial parts of the plant. In contrast, Cannabis pollen, seeds, and roots contain only low concentrations of these compounds [4]. The concentration of cannabinoids and the ratio Δ9-THC:CBD depend on different parameters such as growth conditions, variety, age, harvest time, and storage conditions [4, 5]. Especially during storage, Δ9-THC is relatively unstable whenever Cannabis products such as flowering tops, oils, and resins are exposed to air, light, heat, or acidic conditions [6]. Eventually, cannabinol (CBN) is one of the most important products of degradation [7, 8]. CBD also undergoes changes during long-term Fig. 1 Conversion of CBDA and THCA to CBD and THC as well as the formation of the main degradation product CBN OH OH COOH COOH HO O C5H11 CBDA C5H11 THCA T - CO2 T - CO2 OH HO OH cyclization C5H11 O CBD C5H11 -THC oxidation OH O C5H11 CBN Identification and quantification of cannabinol as a biomarker for local hemp retting in an ancient... In a previous study, sediment samples from lake Aydat were investigated, and the detection of CBN was performed by gas chromatography mass spectrometry (GC-MS). However, this approach required a time-consuming derivatization with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). In general, different methods, e.g., GC-MS, liquid chromatography tandem mass spectrometry (LC-MS/MS), or (high-performance) thin-layer chromatography MS ((HP)TLC-MS) technique, are available for the quantification of cannabinoids in various matrices such as human blood, plasma, hair, urine, rodent tissues, or plant material [12–14]. Here, HPTLC combined with MS detection is a versatile and useful tool for the analysis of complex compounds (...truncated)