Metabolomic investigation of inflorescences from Cannabis sativa L. variety Earlina 8FC cultivated in southern Italy: an NMR and LC-MS/MS integrated approachDownload PDF Download PDF ArticleOpen accessPublished: 18 September 2026Enrico Serni1,Anatoly Petrovich Sobolev ORCID: orcid.org/0000-0001-8709-76661,Vincenzo Montesano2 &…Giovanni D’Orazio1 Scientific Reports volume 16, Article number: 29061 (2026) Cite this articleSave articleView saved researchAbstractEarlina 8FC is a monoecious hemp variety developed for seed production characterized by early flowering, a short development period, a high seed yield, and good pest resistance. This work, to the best of our knowledge, reports the first metabolomic investigation of Earlina 8FC inflorescences. For comparison, the same analytical protocol was applied to the well-characterized Futura 75 variety. Both highly polar and less polar/nonpolar compounds were extracted simultaneously as distinct hydroalcoholic and organic (chloroform) extracts. The extracts were analyzed by NMR after being dried and reconstituted in four different solvents for better fractionation. In the hydroalcoholic extract 23 key primary metabolism markers, such as sugars, cyclitols, amino acids, and organic acids were identified by NMR. The six most abundant flavonoids in hydroalcoholic extract, including apigenin and luteolin glycosides, orientin and vitexin were identified and quantified by LC-MS/MS. The most abundant components of organic extracts, including cannabinoids, fatty acids, phospholipids, galactolipids, sterols, and chlorophyll degradation products, were identified and quantified by NMR. According to ANOVA, Earlina 8FC had significantly higher levels of acetic acid and formic acid, a similar level of chlorogenic acid, as well as significantly lower levels of flavonoids, cannabidiolic acid, cannflavin A, myo-inositol, and quebrachitol, compared to Futura 75.IntroductionCannabis sativa L., commonly known as cannabis, is a species within the Cannabaceae family. It has been extensively cultivated since ancient times as a source of fibres, food, and herbal medicine across the Middle East, Central and Southern Asia, and the Mediterranean basin. It is a dioecious plant, meaning that male and female flowers are beard from different individuals, and female plants are characterised by a significantly higher presence of trichome’s secretory structures that produce oleoresin mainly located on flowers and small leaves (inflorescences). Monoecious phenotypes have also been observed to occur spontaneously in nature, albeit at a minor percentage1,2,3. Additionally, monoecious phenotypes have arisen from breeding procedures that have demonstrated advantageous features such as compact size, high pest resistance and a strong tendency to self-pollination, resulting in large seed production4.Agricultural practices and selective breeding have been carried out over the centuries to develop varieties with taller growth habits, longer stems and/or higher seed yields, optimized for fiber and food/oil production, respectively. In common language, these varieties are now referred to as “industrial cannabis” or hemp. Besides, cultivars with higher resin production and/or more potent psychotropic and therapeutic effects are commonly called “medicinal cannabis” or marijuana3,5.The oleoresin contains the majority of secondary metabolites produced by cannabis (more than 500 in total), and its medicinal properties have been related to its content in typical alkyl-phenol metabolites known as phytocannabinoids. These include over 120 structurally related chemicals, among which Δ9-tetrahydrocannabinol (Δ9-THC) is probably the most well-known as it is primarily responsible for euphoric and hallucinogenic effects but also accounted for various therapeutic properties of cannabis6,7. However, recent research has unveiled a therapeutic value of other cannabinoids such as cannabidiol (CBD), cannabinol (CBN) and cannabigerol (CBG)8. Nonetheless, the two acidic precursors, THCA and CBDA (tetrahydrocannabinolic acid and cannabidiolic acid), are usually the most abundant cannabinoids in marijuana-type and hemp-type cannabis, respectively, so that the THCA/CBDA (or THC/CBD) content ratio serves as an indicator for characterization of phenotypes and varieties9.Furthermore, the presence of cannabis-specific prenyl-flavonoids, known as cannflavins, in leaves and stems, along with reports on their bioactivity, has drawn attention to other cannabis secondary metabolites and their potential significance for human health10,11,12. Nonetheless, several glycosides based on apigenin and luteolin flavonoid structures had already been identified in various cannabis chemotypes13,14,15.Hemp varieties cultivated for industrial purposes naturally produce cannabinoids but must have a total THC+THCA content below 0.3% (dry weight) according to IT/EU legislation16. This requirement has led to breeding and selection efforts to obtain suitable phenotypes. However, these cultivars often produce significant amounts of other cannabinoids like CBD and CBN, together with considerable amounts of interesting phenolic compounds17.Regarding hemp cultivation for industrial purposes, the recovery of biomass from vegetative parts or seed “cakes” after oil extraction and its reuse is nowadays an important aspect for reduction of waste within a circular economy perspective. Proper processing of hemp matter, containing bioactive compounds such as cannabinoids and flavonoids, might be valuable for pharmaceutical and nutraceutical industries. Metabolomic investigations can fit the purpose for comprehensive chemical description of plant material and extracts, and valorization of ”green waste” matter.In this context, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) are nowadays recognized as powerful tools for metabolomic investigation of plant extracts18,19, and have been successfully applied to the chemical description of both monoecious and dioecious cannabis cultivars in the past20,21,22,23,24. Although less sensitive than LC-MS/MS, NMR has proven to be a highly robust technique for both qualitative and quantitative analyses, pursuing both targeted and untargeted approaches. It has been used in the metabolomic investigation of cannabis extracts for several decades25.Recently, more comprehensive assignments for the 1H-NMR spectra of hydroalcoholic and organic extracts from Cannabis sativa L. inflorescences have been reported, enabling the identification of different classes of compounds using 1D and 2D experiments21,22,26,27,28,29,30. However, only a few studies focus on the characterization of water and/or highly polar extracts. Most research reports only the composition of less polar extracts (particularly those obtained with chloroform), likely due to the greater interest in the qualitative and quantitative analysis of cannabinoids.Earlina 8FC is a monoecious hemp variety developed in France for seed production that has gained attention in recent decades due to its various positive features, including early flowering, a short development period, high seed yield, and good pest resistance. In fact, several authors reported seed oil composition from Earlina 8FC31,32. However, with the exception of cannabinoids, quali-quantitative descriptions of primary and secondary metabolites in this cultivar are currently not available.In this work, a metabolomic investigation of inflorescences from the Earlina 8FC cultivar cultivated in southern Italy is reported. Analyses using NMR spectroscopy aimed to provide a comprehensive qualitative and quantitative chemical description, complemented by LC-MS/MS for confirmation and detailed analysis of polyphenolic fraction. To the best of our knowledge, this is the first report offering a comprehensive metabolomic profile of the Earlina 8FC cultivar, especially using both NMR and LC-MS/MS. To ensure the highest data reliability, extracts from the well-characterized monoecious hemp cultivar Futura 75, cultivated under identical conditions, were prepared following same extraction and analytical methodologies.Materials and methodsChemicals and reagentsDeuterated water (D2O, 99.97 atom% deuterium), deuterated methanol (CD3OD, 99.80% deuterium), deuterated chloroform (CDCl3, 99.80% deuterium), and 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) were purchased from Eurisotop (Saclay, France). 2,6-di-tert-butyl-4-methylphenol (DTBMF) was purchased from Sigma-Aldrich (Merck Life Science, Milan, Italy). Methanol (MeOH), chloroform, and formic acid (HPLC grade) were from Carlo Erba Reagenti (Milan, Italy); acetonitrile (ACN) and water (LC-MS grade) were from Honeywell (Thermo Fisher - Milan, Italy).Ultrapure water (filtered through 0.2 μm and packaged under nitrogen) was sourced from VWR (International PBI S.r.l. - Milan, Italy). Standard reference compounds apigenin-7-O-glucoside, apigenin-7-O-glucuronide, apigenin-7-O-rutinoside, apigenin-8-C-glucoside, luteolin-7-O-glucuronide, luteolin-8-C-glucoside, quercetin-3-O-glucuronide, quercetin-3-O-rutinoside, and quercetin-3-O-glucoside were supplied by Medchem Express (DBA - Milan, Italy), while luteolin-7-O-glucoside and cannflavin A were purchased from PhytoLab (Merck Life Science - Milan, Italy).Field trial and experimental designPlants of the two cannabis varieties Earlina 8FC and Futura 75 were cultivated under field conditions in 2023 at the “Alsia-Azienda Pantanello” site in Metaponto di Bernalda, Basilicata, Southern Italy (40°23′031.4″ N, 16°47′010.9″ E), within the local unit of the Institute for Sustainable Plant Protection of the National Council for Research (CNR-IPSP).The experiment was performed from April 14th to October 10th, 2023, using a split-plot design with three replicate plots for each cultivar. Industrial monoecious hemp (Cannabis sativa L.) cultivars Earlina 8FC and Futura 75 (HEMPit Coop - Beaufort en Anjou, France) were directly seeded in the field using a mechanized precision seeder (Model SP94S, Bassi Seminatrici srl, Italy). Earlina 8FC is an early-ripening cultivar, characterized by its short growth cycle. In contrast, Futura 75 is characterized by a longer growth cycle. Plots measuring 10 × 10 m were established with a plant density of 35 kg of seeds per hectare, with 0.2 m of spacing between rows and 0.06 m of spacing between plants within rows. All plots received adequate irrigation, supplying 100% of the estimated crop evapotranspiration until June 30th.The harvest took place on July 20th. The apical buds were combined to create a single sample (about 100 g) representing each cultivar. After harvesting, the fresh plant material was immediately frozen in aluminium-folded packs and stored at − 80 °C. Samples were provided as biological triplicates for both cultivars (n = 3).Sample pre-processingPlant samples (apical buds deprived of major photosynthetic stemmed leaves) were freeze-dried in their original aluminium-folded packages until reaching a constant weight. They were then divided into different plant tissues (stems, seeds, and inflorescence). Briefly, the main stems were first manually separated from all the other parts, and the remaining material was sieved through a 10-mesh screen to separate side stems.The mixture of inflorescences (minor leaves + flowers), seeds, and minor stem residues was then sieved through a 40-mesh screen to separate seeds and minor stems from the inflorescences. Finally, seeds and minor stems were separated by sliding them over a lab-made vibratory bed, constructed from a paper tube on a plastic pipe.The four main fractions (main stems, side-minor stems, seeds, and semi-ground inflorescences) were each collected and separated. The sole inflorescences fraction was further processed and analyzed, being ground into a fine powder using a commercial home grinder and stored at −20 °C until analysis.ExtractionThe dried and powdered inflorescences were subjected to the Bligh-Dyer extraction33. Tree milliliters of a mixture of methanol/chloroform (2:1 v/v), 1.0 mL of chloroform, and 1.8 mL of ultrapure water were sequentially added to 50.0 mg of the powdered sample in 15 mL polypropylene screw-cap centrifuge tubes (Greiner Bio-One S.r.l., 20060 Cassina de’ Pecchi, Italy). The resulting emulsion was vortexed (30 s) and stored at + 4 °C for 40 min. The sample was then vortexed again (30 s) and centrifuged at 4,200 × g for 15 min at 4 °C. The hydroalcoholic and organic phases were carefully separated. The pellets were re-extracted using half of the original solvent volumes under the same conditions. After careful filtration of the organic phase through cotton wadding, both extract samples were dried under a stream of dry nitrogen at 37 °C using a DC150-2 Nitrogen Sample Concentrator (Yooning, Hangzhou, China). They were weighed and stored at − 20 °C until analysis.Sample preparation for NMR and LC-MS/MS analysesThe dried Bligh-Dyer hydroalcoholic and organic extracts from each sample were dissolved in 0.75 mL of either 400 mM phosphate buffer with D2O or deuterated methanol (CD3OD), with 1.0 mM TSP as an internal standard, and in 0.70 mL of CDCl3 or a 2:1 v/v mixture of CDCl3 and CD3OD containing 1.0 mM DTBMF as an internal standard. The resulting solutions were centrifuged at room temperature (7,500 × g, 10 min) and transferred into 5 mm NMR glass tubes (Norell, USA). For LC-MS analysis, hydroalcoholic extracts were diluted 1:4 (v/v) in ultrapure water, spiked with a 20.0 µg/mL internal standard solution (200 µL extract + 750 µL water + 50 µL I.S.), centrifuged at cool temperature (12,000 × g, 10 min at 4 °C) and directly injected into HPLC system.NMR analysesNMR spectra were recorded at 28 °C on an AVANCE III HD 600 NMR spectrometer (Bruker - Milan, Italy) operating at a proton frequency of 600.13 MHz and equipped with a Bruker multinuclear z-gradient 5 mm probe head. 1H spectra were referenced to methyl group signals of TSP (δ = 0.00 ppm) in D2O and to methyl group signals of TMS (δ = 0.00 ppm) in chloroform or chloroform/methanol mixed solvent. The 1H spectra of hydroalcoholic extracts were acquired with 256 transients and a recycle delay of 7 s. The residual HDO signal was suppressed using pre-saturation for two seconds before pulse application. The experiment was carried out by using a 90° pulse in the range 11.2–11.4 µs and 32 K data points. For other solvents, the experimental conditions are reported in Supplementary Table 1 (NMR).The two-dimensional (2D) NMR experiments, such as 1H-1H TOCSY, 1H-13C HSQC, and1H-13C HMBC, were carried out under the same experimental conditions previously reported22. For the quantification of metabolites, the integrals of the corresponding selected resonances in ¹H-NMR spectra were measured with respect to internal standards, allowing the molar concentration and corresponding weight to be calculated.LC-MS/MS analysesThe LC-MS/MS analysis was performed using an Acquity ARC HPLC system, which included a photodiode array (PDA) detector and was coupled with a Xevo TQ-S Cronos triple quadrupole mass spectrometer (Waters® - Milford, USA). Analyte separation was carried out on an Xbridge™ Premier BEH C18 HPLC column (150 × 2.1 mm, 2.6 μm particle size) with a filter guard column (Waters® - Milford, USA).The mobile phase consisted of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in ACN (B), with linear gradient elution applied as follows: 0–8 min, 10 to 15% (v/v) (B); 8–12 min, 15 to 20% (v/v) (B); 12–14 min, 20 to 100% (v/v) (B); 14–17 min, 100% (v/v) (B); 17–19 min, 100 to 10% (v/v) (B); and 19–24 min, 10% (v/v) (B). The flow rate was set to 0.40 mL/min, and the column was maintained at 45 °C. UV–VIS on-line detection was performed at 330 and 350 nm (spectra were acquired between 200 and 400 nm). The injection volume was two µL. Ion source settings included a spray voltage of 2.60 kV (negative polarity), cone voltage of 20 V, desolvation temperature of 600 °C, desolvation gas flow of 1000 L/h, and cone gas flow of 30 L/h. Collision-induced fragmentation was used with argon at a pressure of 1.5 mTorr. Compound identification and separation of structural isomers (same parent and product ions) were achieved using retention time (Rt) and ion ratio in multiple reaction monitoring (MRM). Analytical parameters for each compound are summarized in Supplementary Table 2.Results and discussionIn this study, NMR spectroscopy was employed to conduct an untargeted analysis of both hydroalcoholic and organic extracts, yielding comprehensive metabolomic profiles of cannabis inflorescences using a single analytical method. To offer a more detailed characterization of phenolic and flavonoid compounds, hydroalcoholic extracts were analysed in both phosphate buffer and methanol solutions. Similarly, organic extracts were examined in chloroform and a chloroform/methanol mixture for a more thorough screening of cannabinoids and lipid profiles. Finally, LC-MS/MS analysis was also used for more detailed analysis of polyphenols in methanol solutions.Hydroalcoholic extract analysis in different solventsBuffered aqueous solutions–NMR analysisAssignment of signals to single molecular structures in the hydroalcoholic fraction from Bligh-Dyer extraction, dissolved in phosphate buffer/D2O, follows previous works on selected cultivars and is performed with a similar methodology21,22,26,30. Twenty-three metabolites, mainly amino acids, sugars, and organic acids, were identified through this process (Table 1).Table 1 List of metabolites detected in hydroalcoholic extracts dissolved in aqueous phosphate buffer solution and in methanol, 1H-NMR signals used for quantitation (δ = chemical shift) and their content in the cultivars examined (mg/g dry weight).Full size tableMetabolites were quantified using their characteristic 1H-NMR signals. Raffinose and leucine were not quantified due to significant signal overlap. The low-field part of the spectra (7.5–6.5 ppm) containing aromatic signals was also characterized by extensive overlap (Supplementary Fig. 1), impeding the differentiation of individual phenolic compounds and other key metabolites, such as aromatic amino acids.Тo the best of our knowledge, no literature data on the composition of hydroalcoholic extracts of Earlina 8FC or Futura 75 inflorescences are available. A limited comparison of our data can be made with NMR metabolite profiling study of four monoecious hemp cultivars22 and the Cannabis Compound Database34. Unfortunately, the first reference reports only the relative percentage of metabolites, whereas the second relates to Cannabis sativa and Cannabis indica hybrids with high THC content.Asparagine was the most abundant among nine quantified amino acids, followed by glutamic acid, glutamine and proline. The prevalence of asparagine aligns with the aforementioned studies22,34, though the relative abundances of less expressed amino acids vary considerably. In terms of absolute quantification, the levels of asparagine, proline and glutamine were lower than in Cannabis sativa and Cannabis indica hybrids34, while the level of glutamic acid was significantly higher. These differences can be explained by differences in genotype, cultivation and harvesting conditions. In fact, substantial changes in the content of amino acids, sugars and organic acids during plant development, as well as metabolite variations due to genetic background, were demonstrated when the metabolite compositions of four monoecious hemp cultivars were compared22.As in the case of other monoecious cultivars, citric acid was the most abundant organic acid, followed by malic acid22,34. Acetic, succinic and formic acids were much less abundant (Table 1). According to ANOVA, the levels of acetic and formic acids were significantly higher in Futura 75 than in Earlina 8FC.Carbohydrate fraction includes sugars (glucose, fructose and sucrose) and cyclitols (myo-inositol and quebrachitol). Sucrose was the predominant sugar, accounting for approximately 50% of soluble carbohydrates, while the amounts of the other less abundant components were comparable. Earlina 8FC cultivar has a significantly lower quantity of myo-inositol and quebrachitol than the Futura 75 cultivar. Due to their common biosynthetic pathway, it seems that quebrachitol and myo-inositol levels in hemp are correlated. In fact, quebrachitol is a product of myo-inositol methylation, followed by epimerization35. Quebrachitol exhibits potential as an anti-diabetic agent by improving glucose tolerance and has been shown to protect stomach cells from damage, likely due to its antioxidant and free-radical scavenging capabilities36. Although the presence of quebrachitol in hemp inflorescences has been documented26, there is no quantitative data available for comparison.Most compounds identified play essential roles in various primary metabolic pathways, such as glycolysis and the Krebs cycle. Therefore, the fingerprint obtained from hydroalcoholic extracts in buffer solution serves as a valuable tool for screening the primary metabolism of cannabis.Solutions in methanol–NMRAfter Bligh-Dyer extraction, most polyphenolic compounds, like flavonoids, are found in the hydroalcoholic fraction, as also confirmed by LC-MS/MS analyses of hydroalcoholic extracts in this work. However, a strong signal overlapping, line broadening, and unpredictable shifts of signals in the aromatic region of ¹H spectra (δ = 6.5–7.5 ppm) (Supplementary Fig. 1) impede the determination of single phenolic structures in aqueous solutions without further sample processing and clean-up. Moreover, literature data with 1H chemical shift values relative to aromatic protons of polyphenols, including flavonoids, are primarily reported in methanol. For these reasons, the fraction of hydroalcoholic extracts soluble in deuterated methanol (CD3OD) was subjected to NMR analysis to identify the major flavonoids present in the extracts.A set of isolated signals between δ = 6.50 and 7.90 ppm matched protons from the two aromatic rings of flavonoid structures and the H3 signal of flavones (Fig. 1). As widely documented in the literature, glycoside derivatives with flavone structures such as apigenin and luteolin, especially as −7-O-glucuronides and − 7-O-glucosides, are major flavonoids in various cannabis varieties, both monoecious and dioecious. Therefore, confirming their presence in the extracts was initially attempted by spiking the samples with aliquots of specific reference standards directly in the NMR tubes. Spiking with apigenin-7-O-glucuronide increased the intensity of the doublets at δ = 7.90 and 6.93 ppm (J = 8.8 Hz), corresponding to H2’, H6’ and H3’, H5’, respectively, and the singlet at δ = 6.65 ppm corresponding to H3. Conversely, spiking with luteolin-7-O-glucuronide increased the intensity of the multiplet at δ = 7.42 ppm, corresponding to H2’ and H6’, doublet at 6.90 ppm from H5’, and the singlet at δ = 6.60 ppm for H3. For both compounds, the spiking gave rise to the increment of a doublet at 6.50 ppm due to H-6 signal and a doublet at 6.85 or 6.84 due to H-8 in apigenin or luteolin aromatic ring, respectively. As shown in Table 1, the quantification results of apigenin-7-O-glucuronide and luteolin-7-O-glucuronide clearly indicate that the levels of both flavonoids were approximately twice as low in the Earlina 8FC cultivar as in the Futura 75 cultivar.Fig. 1Full size image1H-NMR spectrum of hydroalcoholic extract dissolved in CD3OD. The signals of apigenin-7-O-glucuronide: 1, CH-2′6′; 3, CH-3′,5′; 5, CH-8; 7, CH-3; 9, CH-6. The signals of luteolin-7-O-glucuronide: 2, CH-2′ and CH-6′; 4, CH-5′; 6, CH-8; 8, CH-3; 9, CH-6.Similar to the discussion about buffer solutions and markers of primary metabolism, analysis of the methanolic fraction of hydroalcoholic extracts can be a powerful method for identifying major flavonoids in cannabis extract using NMR analyses without requiring additional purification steps.The methanolic fractions obtained from the hydroalcoholic extracts were fatherly spiked with standard reference compounds corresponding to less abundant flavonoids, as suggested by the results of the LC-MS/MS analysis. Spiking with apigenin-7-O-glucoside, luteolin-7-O-glucoside, vitexin, and orientin proved that their content in non-spiked solutions was below the limit of quantitation (LOQ) of the proposed NMR method.Solutions in methanol - LC-MS/MS analysisConsidering that LC-MS/MS analysis is more sensitive and usually more selective than proton NMR spectroscopy, especially for highly complex mixtures, it has been successfully used in the metabolomic analysis of plant extracts, including cannabis, employing both targeted and non-targeted approaches.In this regard, recent reports indicated that untargeted computational LC-Q/ToF analysis of two medicinal cannabis varieties could be a helpful approach for cultivar discrimination. Flavonoid glycosides of luteolin and isorhamnetin can be used as markers to differentiate Amnesia Haze and Royal Dutch Cheese varieties20. Previous analyses of cannabis polar extracts using LC-MS/MS methods have also been conducted for targeted detection of phenols and polyphenols. A targeted investigation on samples from the Kompolti, Tiborszallasi, Antal and Carmagnola varieties cultivated in Italy returned over 20 analytes, including glycosides of apigenin, luteolin, and quercetin23.The LC-MS system proved to be a reliable tool for compound identification even without using fragmentation of precursor ion. In this context, Lewis and colleagues obtained an interesting chemical profile of medicinal cannabis extracts using UHPLC coupled with single quadrupole MS24.In this study, methanol solutions from hydroalcoholic extracts of both Earlina 8FC and Futura 75 cultivars were first screened using MRM (multiple reaction mode) to identify the most common flavonoids among reference compounds at our disposal (Fig. 2, Supplementary Figs. 2, 3). These primarily included derivatives of luteolin and apigenin. To ensure comprehensive screening of flavonoids, selected quercetin and kaempferol derivatives were also included in the study together with chlorogenic acid. A list of standard flavonoid analyses is provided in the Supplementary Table 2.Fig. 2Full size imageUV and TIC of the MRM chromatographic profile of the hydroalcoholic fraction of the Earlina 8FC cultivar.It must be emphasized that, in attempting to fulfill both fastness and selectivity for the chromatographic method, the MRM time window relative to elution of luteolin-7-O-glucuronide showed two peaks, that is one from the corresponding to the standard plus another probably due to occurrence of a structural isomer (likely luteolin-5-O- or −3′-O-glucuronide), meaning that separation of the two compounds was not fully achievable in the conditions adopted. Besides, the authors decided that a semi-quantitative determination of flavonoids indicated was sufficient for a comparison of the cultivars, so that analyte/internal standard area ratio was evaluated and compared for the purpose.The most abundant flavonoids measured with the method proposed hereby showed a similar order of abundance in both cultivars examined but with different relative content (see Table 2).Table 2 Polyphenolic content after LC-MS/MS analysis in the cultivars examined.Full size tableGlucuronides as −7-O- derivatives of luteolin and apigenin were the two most abundant flavonols, followed in order by luteolin-7-O-glucoside, orientin (luteolin-8-C-glucoside), vitexin (apigenin-8-C-glucoside) and apigenin-7-O-glucoside in Earlina 8FC. In the Futura 75 the pattern is the same except for orientin content being slightly higher than luteolin-7-O-glucoside.Regarding relative abundance of each single analyte between the cultivars, Futura 75 showed higher content of all the compounds considered respect to Earlina by a factor between ca. 2 (apigenin-7-O-glucuronide) and ca. 6 (vitexin). These results are perfectly in line with those emerging from our NMR analyses (Table 1).It is interesting to note that the additional peak corresponding to hypothetic luteolin-7-O-glucuronide isomer is clearly occurring in MRM chromatograms from Futura 75, while almost absent in those referring to Earlina 8FC, thus being a putative candidate as a marker for cultivar discrimination.For a more comprehensive description of flavonoid composition, scans of precursors ion (precursor ion scan, PIS) of each aglycone after characteristic fragmentation of mono- and di-glycosides were performed within the appropriate mass range. This indicated the potential presence of −3-O-glucuronide and −3-O-rhamnoside of quercetin, along with − 3-O-glucuronide and −3-O-glucoside of kaempferol (data not shown).Additionally, screening for the precursors of isorhamnetin revealed no significant peaks in the chromatogram; therefore, isorhamnetin-3-glucoside was selected as the internal standard (IS) for semi-quantitative analyses.The discussion of the results obtained for Earlina 8FC hydroalcoholic extracts regarding its composition in phenolics cannot rely on a strict comparison with previously released data since these latter seem to be totally lacking.As for Futura 75, being one of the most widespread cultivars in Europe, reports about chemical composition of plants grown in Italy are available. Nevertheless, most papers describe terpene and cannabinoid fractions, while comprehensive quali-quantitative analyses of flavonoid fractions are limited, especially when comparison between monoecious cultivars is concerned. In this regard, André et al. used reversed phase (RP) C18 HPLC-MS/MS to evaluate the polyphenol composition of methanol/water (70:30) extracts of inflorescences from six monoecious varieties, including Futura 75, and two dioecious varieties cultivated in Switzerland and harvested at different developmental stages37. They indicated that at the end of flowering for high-sowing density field cultivation the main compound was luteolin-7-O-glucuronide, followed by apigenin-7-O-glucuronide by a factor of ca. 2.5., This is in perfect agreement with our data, although in their case the content of luteolin-C-hexoside (not specified if corresponding to orientin) was lower than the content of luteolin-7-O-glucuronide by a much higher ratio than in our case when orientin was considered (ca. 60 vs. 5 respectively). With a similar methodology (extraction of flowers and leaves with methanol/water 3:1 and analysis with C18 HPLC/UV/MS-MS), Vanhoenacker et al. had previously reported that major flavonoids in flowers and leaves from Futura 75 cultivated in Holland were, in order, luteolin-7-O-glucuronide, apigenin-7-O-glucuronide, vitexin and orientin, thus being highly consistent with our results38.When acidified methanol/water extracts from monoecious and dioecious cultivars grown in Tuscany were analyzed by HPLC/UV for few selected flavonoids, orientin and rutin resulted to be the most abundant, representing 63.8–72.3% of the total flavonoids; in Futura 75 their amount was comparable (3741.4–3227.7 µg/g dry weight resp.) and followed by vitexin by ca. 1:4 ratio (856.9 µg/g dry weight)39. Orientin and vitexin as major compounds in methanol/water (75:25) extracts of Futura 75 grown in Denmark were also described40.NMR metabolite profiling of chloroform extractsChloroform solutionOrganic solvents, such as chloroform and hexane, are commonly used for extracting less polar and more lipophilic compounds from the cannabis plant. As described in several studies, these solvents are particularly effective in extracting cannabinoids and other hydrophobic constituents from both hemp and marijuana matrices. Among them, chloroform has emerged as the preferred solvent for quantifying major cannabinoids, largely due to its ability to produce well-resolved and easily identifiable signals in ¹H NMR spectra26,27,28,41.In this work, the extracts from both Earlina 8FC and Futura 75 cultivars were expected to contain very small quantities of THC compared to other neutral cannabinoids like CBD or CBG, as typical for industrial hemp cultivars.Among cannabinoids only the signals of CBD and CBDA were detected and quantified in the 1H NMR spectra of chloroform solution. According to the literature, the characteristic signals at 4.64 and 4.72 ppm were ascribed to the geminal vinylic protons (CH2−9) of CBD, whereas the more intense couple of signals at 4.39 and 4.53 ppm were ascribed to the corresponding CH2−9 group of CBDA27. The CBDA content was ten to twenty times more abundant than that of CBD, as shown in Table 3. Earlina 8FC showed a significantly lower content of CBDA compared to Futura 75. Considering that CBD derives from CBDA, the sum of CBD and CBDA content by weight% was about 1.49% for Futura 75 and 0.99% for Earlina 8FC.Table 3 List of metabolites detected in organic extracts (chloroform and chloroform-methanol solutions), 1H-NMR signals used for quantitation (δ = chemical shift) and their content in the cultivars examined (µmol/g dry weight). Statistically different amounts between the two cultivars are indicated (p