Exploring the chemical space around Cannabis sativa L. leaves as a source of bioactive compounds of pharmaceutical interest

Abstract

In the field of Cannabis sativa L. derived products, the attention has been usually focused on plant inflorescences as a source of cannabinoids, with stems and seeds also utilized, while leaves are typically discharged. Although previous studies have examined the composition of C. sativa leaves to evaluate their antioxidant potential, their full value remains largely unknown. In this perspective, this study aims to reveal the hidden potential of C. sativa leaves to reposition them from a waste material to an alternative source of bioactive compounds. To this purpose, the leaves of four non-psychotropic C. sativa (hemp) varieties, including a cannabinoid-free one, were comprehensively characterized for their composition for the first time. The most relevant outcome of the present work was the first identification and characterization of squalene in hemp leaves, along with the development of a suitable extraction and analytical method for this bioactive triterpenoid. Among cannabinoids, cannabinoic acids were the most abundant compounds, complemented by the detection of several minor compounds having either the cannabidiol (CBD) or cannabigerol (CBG) scaffold. As for the other chemical constituents, cannflavins were the predominant non-cannabinoid phenolic compounds. The analysis also addressed to policosanols and terpenes, revealing some variety-specific volatile compounds, beside the most common ones. Overall, hemp leaves showed a rich chemical composition to be exploited in the pharmaceutical field, perfectly aligning with a circular economy perspective.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-39088-0.

Cannabis sativa L. (Cannabaceae family) is an annual and mostly dioecious plant with an extraordinary complex chemical profile characterized by secondary metabolites belonging to various chemical classes^1–4. Among these, cannabinoids (CBs) are the lead compounds and their content define five different chemotypes: chemotype I (Δ⁹-tetrahydrocannabinol (Δ⁹-THC)-rich), chemotype II (Δ⁹-THC and cannabidiol (CBD) in similar amount), chemotype III (CBD-rich, with a content of Δ⁹-THC < 0.3%, according to European regulation², chemotype IV (cannabigerol (CBG)-rich) and chemotype V (CB-free)³. To date, few studies have described the newly developed cannabichromene (CBC)-rich strains⁴, whereas major CBs (Δ⁹-THC, CBD and CBG, ) have been extensively assessed for their biological properties, with CBD being the most studied plant constituent in recent years. CBs are predominantly produced in glandular trichomes found in inflorescences and upper leaves^1,5,6, which explain the historical focus on these parts of the plant. In this scenario, leaves are often considered as a by-product, despite previous studies have highlighted their phenolic composition, together with their antioxidant potential⁷. However, the chemical space of secondary metabolites in the leaves of different varieties remains largely uncharacterized.

Following these premises, this study aims to comprehensive characterize the hidden potential of C. sativa (hemp) leaves from four non-psychotropic varieties, which were fully characterized for their quali- and quantitative composition for the first time. A detailed chemical fingerprint of major and minor CBs in hemp leaves was thoroughly characterized. A broad spectrum of non-cannabinoid phenolic compounds, including prenylated flavones (cannflavin A (CFL-A), cannflavin B (CFL-B), and their corresponding demethoxy derivatives (demethoxy cannflavin A (demethoxy CFL-A) and demethoxy cannflavin B (demethoxy CFL-B)), and the dihydrostilbenoid canniprene, known to be specifically biosynthesized in this plant, was also detected^8–10. Besides these, other phenolics have been identified, including phenolic acids, hydroxycinnamic acids, and phenolic amides^11,12.

Moreover, the phytochemical investigation included policosanols (PCs)¹³, a mixture of long-chain (C₂₀ to C₃₆) aliphatic alcohols¹⁴, showing antioxidant and anti-inflammatory activities¹³. According to the literature, PCs are usually present in very low amounts¹⁴, as they predominantly occur in the esterified form, resulting in a difficult extraction process. For this reason, specific methods have been developed for their isolation¹⁵. Another lipophilic compound previously cited in C. sativa is squalene¹⁶, a polyunsaturated triterpene which is currently extracted from animal sources (i.e., shark liver oil) and widely employed in the pharmaceutical industry as an emulsion-based adjuvant for vaccine delivery¹⁷. Due to both environmental and ethical concerns¹⁸, current research is focusing on the identification of plant-based squalene alternative sources. Currently, Amaranthus caudatus L. is known to be the plant with the highest content of squalene¹⁹.

Finally, monoterpenes and sesquiterpenes were analyzed in detail in hemp leaves. The latter metabolites are quite abundant in the inflorescences and less represented in the leaves¹⁶.

Analytical techniques applied in this study included UHPLC-HRMS and GC-MS for compound identification, whereas HPLC-UV/Vis, HPLC-ELSD and GC-FID were employed for quantification purposes. Overall, hemp leaves could represent a rich source of bioactive compounds to be exploited in the pharmaceutical field in a circular economy perspective.

Results

Chemical characterization and quantification of polyphenols in hemp leaves

The extracts from hemp leaves were analyzed by UHPLC-HRMS to obtain the qualitative composition of polyphenols. Compound identification was achieved by comparison of the retention time (t_R), precursor ion, and fragmentation pattern with those of the corresponding analytical standards when available, such as CFL-A, CFL-B and canniprene. The other constituents were putatively determined by comparing the experimental data with those available in the literature^8,20. The list of compounds identified in the leaves, and the corresponding mass spectral data, are shown in Table 1, while their occurrence in the samples is shown in Supplementary Information (SI, Table S1).

Table 1.

UHPLC-HRMS data of polyphenols detected in the extracts from hemp leaves, both in the positive and negative ion mode, together with their fragmentation data.

Compound	tR (min)	[M + H]+	MS/MS	[M–H]–	MS/MS
N-Feruloyloctopamine	17.8	–	–	328.1127	310.1083 (53), 295.0846 (17), 161.0232 (100), 133.0520 (76)
Dihydroferulic acid	19.5	197.1172	197.1168 (100), 179.1063 (99), 161.0957 (26), 135.1166 (54), 133.1009 (43), 107.0856 (44)	–	–
Hydroxygallic acid	21.8	–	–	187.0965	187.0963 (65), 125.0958 (100), 97.0645 (17)
N-Coumaroyltyramine	22.5	284.1281	284.1272 (16), 147.0437 (100), 121.0646 (40), 119.0489 (10)	–	–
N-cis-Feruloyltyramine	23.3	314.1385	314.1375 (21), 177.0542 (100), 145.0280 (35), 121.0647 (39), 117.0334 (7)	312.1237	312.1238 (71), 297.1003 (21), 190.0499 (30), 178.0499 (52), 148.0517 (100), 135.0439 (39)
N-Coumaroyltyramine	24.1	284.1281	284.1272 (16), 147.0437 (100), 121.0646 (40), 119.0489 (10)	–	–
N-trans-Feruloyltyramine	24.9	314.1385	314.1375 (21), 177.0542 (100), 145.0280 (35), 121.0647 (39), 117.0334 (7)	312.1237	312.1238 (71), 297.1003 (21), 190.0499 (30), 178.0499 (52), 148.0517 (100), 135.0439 (39)
Apigenin	29.4	271.0601	271.0593 (100), 153.0179 (6), 147.0440 (1)	269.0452	269.0452 (100), 151.0025 (17), 149.0232 (12), 117.0332 (68), 107.0124 (13), 65.0018 (14)
Diosmetin/chrysoeriol	30.1	301.0706	301.0698 (100), 286.0463 (33), 258.0515 (12)	299.0557	299.0557 (46), 284.0322 (100), 256.0374 (26), 227.0340 (4), 107.0122 (5), 63.0225 (8)
Cannflavin derivative	34.5	453.1909	453.1895 (12), 435.1791 (4), 313.0697 (100), 298.0463 (12)	451.1761	451.1757 (100), 309.0401 (36), 297.0373 (24), 163.0026 (50), 133.0281 (26)
Cannflavin derivative	34.7	401.1232	367.1165 (47), 325.0697 (100), 313.0696 (91), 310.0463 (35), 297.0749 (42)	–	–
Acacetin	35.9	285.0757	285.0749 (100), 285.1107 (39), 270.0516 (12), 242.0564 (14)	–	–
Isocannflavin B	37.2	369.1333	369.1319 (31), 313.0695 (100), 298.0458 (12)	367.1185	–
Cannflavin derivative	37.6	435.1804	435.1797 (3), 313.0696 (100), 298.0462 (13), 183.0284 (2), 165.0178 (7)	–	–
Cannflavin derivative	38.0	453.1909	453.1894 (5), 314.0730 (22), 313.0697 (100), 298.0462 (12), 165.0178 (7)	451.1761	451.1759 (100), 351.0973 (29), 309.0400 (51), 297.0401 (52), 163.0024 (10), 133.0281 (55)
Cannflavin derivative	38.4	469.1857	–	467.1707	435.1444 (100), 391.1180 (32), 311.0556 (20), 297.0398 (56), 163.0024 (19), 133.0281 (36)
Cannflavin derivative	38.6	469.1858	313.0696 (100), 298.0463 (13), 183.0284 (2), 165.0178 (7)	–	–
Demethoxy CFL-B	38.7	339.1277	–	337.1079	337.1074 (100), 293.0458 (12), 281.0453 (11), 161.0229 (9), 133.0643 (10), 117.0331 (63)
CFL-B	39.0	369.1332	369.1321 (6), 313.0696 (100), 298.1463 (16), 165.0178 (10)	367.1182	367.1180 (91), 352.0952 (43), 309.0399 (100), 297.0400 (32), 269.0451 (13), 133.0282 (50)
Canniprene	39.7	343.1889	343.1891 (4), 287.1268 (100), 255.1007 (44), 227.1059 (24)	341.1758	341.1751 (100), 326.1517 (20), 283.0977 (13), 269.0918 (5)
CFL-C	41.1	437.1960	437.1947 (66), 313.0696 (100), 298.0462 (17)	435.1810	435.1806 (100), 420.1583 (29), 351.0862 (36), 297.0398 (42), 268.0382 (15), 133.0282 (34)
Demethoxy CFL-A	42.8	407.1854	407.1838 (7), 283.0591 (100), 183.0282 (2), 165.0177 (11)	405.1706	405.1702 (100), 293.0451 (32), 281.0452 (77), 163.0025 (26), 161.0232 (33), 117.0332 (33)
CFL-A	43.1	437.1960	437.1947 (66), 313.0696 (100), 298.0462 (17)	435.1808	435.1806 (100), 420.1574 (40), 351.0866 (36), 309.0404 (19), 297.0388 (15), 133.0281 (30)

The UHPLC-HRMS chromatograms of all the samples analyzed showed the presence of dihydroferulic acid, hydroxygallic acid and several phenolic amides. In addition to CFL-B and CFL-A, their corresponding demethoxy derivatives, namely demethoxy CFL-B and demethoxy CFL-A, were found in each sample analyzed. CFL-C and other cannflavin derivatives were only putatively identified on the basis of the data available in the literature⁸.

Quantitative data of hemp polyphenols in the leaves were obtained by HPLC-UV/Vis. Chromatograms were recorded at 342 nm for cannflavins and at 210 nm for canniprene, respectively. A representative chromatogram is shown in Fig. 1. CFL-A and CFL-B were quantified using a calibration curve derived from their corresponding reference standards. Demethoxy CFL-A and demethoxy CFL-B were quantified using the calibration curve of CFL-A and CFL-B, respectively. As shown in Table 2, CFL-A was found in a higher amount than CFL-B across all samples analyzed. Demethoxy CFL-A and CFL-B were most abundant in sample 1. Canniprene was quantifiable in sample 3 only.

Fig. 1.

HPLC-UV/Vis representative chromatogram of polyphenols in a leaf extract from the CBD-type variety (sample 1), acquired at 342 nm.

Table 2.

Quantitative analysis of polyphenols by HPLC–UV/Vis, expressed as µg/g ± standard deviation (SD, n = 4). ^* SD < 0.05

Compound	Sample 1	Sample 2	Sample 3	Sample 4
Demethoxy CFL-B	28.1 ± 0.6	< LOQ	< LOQ	6.9 ± 0.4
Canniprene	–	< LOQ	23.8 ± 0.7	< LOQ
CFL-B	33.6 ± 0.6	11.0 ± 0.5	10.3*	17.6 ± 0.3
Demethoxy CFL-A	41.7 ± 0.5	< LOQ	< LOQ	< LOQ
CFL-A	70.1 ± 1.8	26.3 ± 1.9	20.9 ± 0.5	174.7 ± 2.5

Chemical characterization and quantification of CBs in hemp leaves

The analysis of CBs in the extracts of hemp leaves belonging to different varieties was carried out using UHPLC-HRMS. The acidic and neutral CBs detected in the samples are detailed in Tables 3 and 4, respectively, with their distribution provided in the SI (Tables S2 and S3). Tables 3 and 4 include data from cannabidiolic acid (CBDA) esters and other minor CBs having either the CBD, CBG or cannabinol (CBN) scaffold, which are investigated here for the first time. For these compounds, the MS data refer to the corresponding standards, provided by chemical synthesis.

Table 3.

UHPLC-HRMS data of acidic CBs investigated in the extracts from hemp leaves, both in the positive and negative ion mode, together with their fragmentation data.

Compound	tR (min)	[M + H]+	MS/MS	[M–H]–	MS/MS
Cannabinodiolic acid (CBNDA)	7.0	–	–	353.1757	309.1859 (100), 279.1388 (29), 171.0805 (32)
Cannabidivarinic acid (CBDVA)	8.1	331.1799	313.1799 (100), 233.1175 (12), 191.0704 (36)	329.1759	311.1653 (71), 285.1859 (20), 217.1230 (60), 151.0755 (33)
Cannabidibutolic acid (CBDBA)	9.6	345.2068	327.1965 (100), 247.1332 (13), 205.0861 (38)	343.1914	343.1914 (100), 325.1809 (67), 299.2016 (27), 231.1388 (61), 165.0913 (33)
Cannabidiolic acid (CBDA)	10.8	359.2213	341.2114 (100), 261.1489 (10), 219.1017 (21), 135.0441 (4)	357.2073	357.2073 (100), 339.1965 (60), 313.2174 (24), 245.1545 (58), 179.1069 (31)
Cannabigerolic acid (CBGA)	11.1	–	–	359.2228	359.2228 (48), 341.2121 (100), 315.2328 (42), 191.1070 (13)
CBDA geraniol ester	11.7	–	–	–	–
Cannabidihexolic acid (CBDHA)	11.9	–	–	371.2233	371.2231 (100), 353.2125 (49), 327.2326 (23), 325.2169 (19), 259.1703 (57), 193.1226 (39)
Cannabidiphorolic acid (CBDPA)	12.8	–	–	385.2388	385.2388 (100), 367.2283 (56), 341.2495 (29), 339.2325 (22), 273.1863 (72), 207.1385 (39)
Tetrahydrocannabivarinic acid (THCVA)	13.0	331.1801	331.1801 (7), 313.1801 (100), 191.0705 (24)	329.1760	329.1760 (100), 285.1865 (65), 217.1228 (31), 163.0755 (28)
Cannabichromenvarinic acid (CBCVA)	13.5	–	–	329.1758	329.1758 (100), 311.1651 (28), 285.1858 (28), 215.1073 (14), 163.0755 (56)
CBDA borneol ester	13.5	495.3470	495.3487 (3), 341.2112 (59), 219.1017 (100)	–	–
Cannabinolic acid (CBNA)	14.1	–	–	353.1761	353.1761 (64), 309.1806 (100), 279.1391 (52), 171.0807 (19)
Tetrahydrocannabinoic acid (Δ9-THCA)	15.4	359.2224	359.2224 (5), 341.2114 (100)	357.2072	357.2072 (100), 313.2174 (54), 245.1545 (24), 191.1070 (22)
CBDA nerol ester	15.4	–	–	–	–
CBDA fenchol ester	15.6	495.3455	495.3455 (70), 341.2120 (92), 81.0706 (100)	–	–
Cannabigerolic acid monomethyl ether (CBGMA)	16.1	–	–	373.2384	373.2385 (57), 329.2486 (60), 245.1546 (43), 191.1069 (100)
Cannabichromenic acid (CBCA)	16.2	359.2213	359.2219 (100), 341.2113 (85)	357.2076	357.2076 (100), 339.1966 (21), 313.2175 (20), 243.1395 (9), 191.1071 (50), 179.1070 (5)

Table 4.

UHPLC-HRMS data of neutral CBs investigated in the extracts from hemp leaves, both in the positive and negative ion mode, together with their fragmentation data.

Compound	tR (min)	[M + H]+	MS/MS	[M–H]–	MS/MS
Cannabidivarin (CBDV)	8.9	287.2007	287.2007 (100), 231.1383 (18), 165.0912 (52), 135.1171 (19), 123.0444 (9)	285.1861	–
Cannabinerovarin (CBNRV)	8.9	289.2161	289.2164 (4), 165.0912 (100), 123.0443 (7), 69.0707 (2)	–	–
Cannabigerovarin (CBGV)	8.9	289.2159	289.2160 (5), 165.0911 (100), 123.0443 (7), 69.0706 (29)	287.2019	287.2019 (100), 163.0758 (16), 151.0751 (7)
Cannabinerobutol (CBNRB)	10.2	303.2320	303.2320 (3), 179.1068 (100), 123.0443 (10)	–	–
Cannabigerobutol (CBGB)	10.3	303.2316	303.2318 (5), 179.1067 (100), 123.0443 (11)	–	–
Cannabidibutol (CBDB)	10.5	301.2163	301.2163 (100), 245.1539 (17), 179.1068 (47), 123.0443 (14), 93.0705 (19)	299.2016	299.2016 (92), 231.1388 (100), 165.0911 (60), 107.0490 (85)
Cannabinerol (CBNR)	11.3	317.2473	317.2475 (4), 193.1226 (100), 137.0599 (2), 123.0444 (14)	–	–
Cannabigerol (CBG)	11.4	317.2477	317.2477 (4), 193.1225 (100), 137.0599 (4), 123.0443 (12)	–	–
Cannabidiol (CBD)	11.6	315.2321	315.2321 (100), 259.1694 (17), 193.1225 (55), 135.1170 (20), 123.0444 (19), 93.0704 (21)	313.2173	313.2173 (79), 245.1545 (100), 179.1069 (49)
Tetrahydrocannabivarin (THCV)	11.8	287.2007	287.2007 (100), 231.1383 (18), 165.0912 (54), 135.1170 (19), 123.0444 (10)	–	–
Cannabidihexol (CBDH)	12.6	329.2475	329.2481 (52), 273.1889 (23), 207.1382 (28), 135.1170 (16), 93.0705 (39)	–	–
Cannabinerophorol (CBNRP)	13.0	345.2787	345.2788 (3), 221.1536 (100), 123.0443 (13)	–	–
Cannabigerophorol (CBGP)	13.1	345.2787	345.2787 (4), 221.1538 (100), 137.0598 (7), 123.0443 (18)	–	–
Cannabinol (CBN)	13.2	311.2007	311.2007 (100), 293.1900 (26), 241.1226 (16), 223.1119 (55)	309.1860	309.1860 (100), 279.1390 (23), 171.0806 (6)
Cannabidiphorol (CBDP)	13.5	343.2632	343.2628 (100), 287.2002 (17), 221.1537 (36), 135.1170 (23), 93.0704 (25)	–	–
Cannabicitran (CBTC)	13.7	315.2319	315.2319 (100), 259.1693 (15), 193.1225 (41), 135.1169 (17), 93.9704 (16)	–	–
Δ9-Tetrahydrocannabinol (Δ9-THC)	14.2	315.2319	315.2319 (100), 259.1695 (17), 193.1225 (43), 135.1169 (21), 123.0443 (16)	–	–
Sesqui-cannabigerol (sesqui-CBG)	14.7	385.3102	385.3102 (3), 193.1225 (100), 123.0443 (12)	383.2960	383.2960 (100), 245.1540 (3), 191.1069 (15), 179.1065 (4)
Cannabichromene (CBC)	15.0	315.2322	315.2322 (34), 259.1696 (32), 193.1226 (100), 123.0444 (18)	–	–
Cannabigerohexol (CBGH)	15.9	331.2626	331.2629 (2), 207.1380 (100), 137.0597 (8)	–	–

Sample 1 exhibited the most extensive profile, notably including several minor CBs belonging to the CBD-type chemical class. Among these compounds, cannabidihexolic acid (CBDHA), cannabidiphorolic acid (CBDPA), and their corresponding neutral counterparts, i.e., cannabidihexol (CBDH) and cannabidiphorol (CBDP), were putatively identified^6,21. Moreover, other minor CBs identified in this sample included cannabinodiolic acid (CBNDA), tetrahydrocannabivarinic acid (THCVA), cannabichromenvarinic acid (CBCVA) and cannabigerolic acid monomethyl ether (CBGMA) among acidic CBs, and cannabigerophorol (CBGP), cannabicitran (CBTC), sesqui-cannabigerol (sesqui-CBG) and cannabigerohexol (CBGH) as neutral CBs. Only CBGP, having a [M + H]⁺ precursor ion at 345.2787 m/z and two product ions at 221.1538 and 137.0598 m/z, and sesqui-CBG, having a [M + H]⁺ precursor ion at 385.3102 m/z and two product ions at 193.1225 and 123.0443 m/z, were confirmed by comparison with reference standards, while the other compounds were putatively identified on the basis of the literature data^6,21. Samples 2 and 3 exhibited a similar CB profile to sample 1, with the exception that they lacked minor CBD-type compounds (CBDHA, CBDPA, CBDH and CBDP). Sample 4 contained both acidic and neutral CBs, specifically CBNDA, cannabidivarinic acid (CBDVA), cannabidibutolic acid (CBDBA), CBDA, cannabigerolic acid (CBGA), cannabinolic acid (CBNA), CBGMA, cannabichromenic acid (CBCA), CBG, CBD and CBTC. However, their corresponding peak intensities were significantly lower compared to the other samples.

None of the minor CBN-type cannabinoids, such as cannabinerovarin (CBNRV), cannabinerobutol (CBNRB), cannabinerol (CBNR) and cannabinerophorol (CBNRP), were detected in any of the four samples. CBN was detected only in samples 1 and 2. This is consistent with the fact that CBN is an oxidation product of Δ⁹-THC²², which was detected at very low levels in the samples analyzed.

Several CBDA esters were also investigated in the leaves, including CBDA geraniol ester, CBDA borneol ester, CBDA nerol ester and CBDA fenchol ester. Among this group, CBDA borneol ester, characterized by a [M + H]⁺ at 495.3470 m/z and two product ions at 341.2112 and 219.1017 m/z, was the only one identified in samples 1 and 2.

Following the initial characterization, the main CBs were quantified by HPLC-UV/Vis. A representative chromatogram from the HPLC-UV/Vis analysis of sample 1 is shown in Fig. 2. Table 5 summarizes the amounts of the quantified acidic and neutral CBs. Aligned with their declared plant origin, sample 1 was particularly rich in CBDA, while samples 2 and 3 contained CBGA and CBCA, respectively, as the dominant compounds. CBDA was quantified in sample 4, though its concentration was notably low compared to the other samples. Neutral CBs were present in lower content than their corresponding acidic forms. This result is consistent with the applied sample preparation procedure, which was designed to preserve the native composition of cannabinoids in the plant material.

Fig. 2.

HPLC-UV/Vis representative chromatogram of CBs in a hemp leaf extract from the CBD-type variety (sample 1), acquired at 220 nm.

Table 5.

Quantitative analysis of acidic and neutral CBs by HPLC-UV/Vis, expressed as mg/g ± standard deviation (SD, n = 4). ^* SD < 0.05

Compound	Sample 1	Sample 2	Sample 3	Sample 4
CBDVA	< LOD	< LOD	–	< LOD
CBDV	< LOD	–	–	–
CBDBA	< LOQ	< LOD	< LOD	< LOD
CBDB	< LOQ	–	–	–
CBDA	43.0 ± 1.8	< LOQ	< LOQ	3.8 ± 0.8
CBGA	< LOQ	13.1 ± 0.3	4.4 ± 0.2	< LOD
CBG	< LOQ	1.8*	< LOQ	< LOD
CBD	1.2*	< LOQ	< LOQ	< LOQ
THCV	–	< LOD	< LOD	–
THCVA	< LOD	< LOD	–	–
CBN	< LOD	< LOD	–	–
CBNA	< LOD	< LOD	< LOD	< LOD
Δ9-THC	< LOD	< LOD	< LOD	–
CBC	< LOQ	< LOQ	< LOQ	–
Δ9-THCA	< LOQ	1.7*	5.4 ± 0.2	–
CBCA	< LOQ	< LOQ	12.8 ± 0.8	< LOD

Chemical characterization and quantification of lipophilic compounds in hemp leaves. The samples of hemp leaves were analyzed by means of a validated HPLC-ELSD method for both the identification and quantification of PCs. The specific PCs quantified included docosanol (C₂₂OH), tetracosanol (C₂₄OH), hexacosanol (C₂₆OH), octacosanol (C₂₈OH), triacontanol (C₃₀OH) and dotriacontanol (C₃₂OH)¹³. C₃₀OH was quantified using a calibration prepared from its corresponding reference standard, while C₃₂OH was quantified by using the calibration curve established for C₃₀OH. A representative HPLC-ELSD chromatogram showing PCs in a hemp leaf extract is provided in Fig. 3.

Fig. 3.

HPLC-ELSD representative chromatogram of PCs in a hemp leaf extract from the CBD-type variety (sample 1).

Qualitative data related to the distribution of PCs in the samples are described in the SI (Table S4). Sample 4, notably the CB-free one, was found to contain all the PCs taken into consideration. Only C₃₀OH and C₃₂OH were quantified in the samples analyzed. Samples 1 and 2 exhibited a similar amount of C₃₀OH (19.5 ± 0.4 and 19.0 ± 0.6 µg/g, respectively), whereas in samples 3 and 4 the content of this compound was below the LOQ. As for C₃₂OH, only sample 1 exhibited a quantifiable amount (20.1 ± 0.9 µg/g), while the amount was below the LOQ in samples 2, 3, and 4.

The triterpenoid squalene was initially isolated from a hemp leaf sample. The purification involved several preparative chromatography steps to remove other classes of compounds. The procedure yielded an enriched fraction of squalene (SI, Fig. S1), which was identified by comparison with the literature data²³. The H NMR analysis of the isolated compound confirmed the structure: six olefinic protons (6 H, δ_H 5.14, m), ten methylene moieties (20 H, δ_H 1.98–2.07, m) and eight triterpenyl methyl groups (24 H, δ_H 1.62–1.76, s).

Optimal extraction conditions for squalene were determined by the application of different methods (data not shown). Qualitative and quantitative analyses of squalene were carried out by GC-MS and GC-FID, respectively. Peak assignment was confirmed by comparing the t_R of the pure standard, by analyzing samples spiked with the reference compound and by using a MS spectral library search. The GC-MS analysis of squalene in a hemp leaf extract demonstrated comparable spectral purity and the identical fragmentation pattern of the reference standard (SI, Figs. S2 and S3). A representative GC-FID chromatogram, highlighting the squalene elution window, is shown in Fig. 4. The content of squalene was relatively constant among the samples (12.9 ± 1.1 µg/g for sample 1, 11.0 ± 1.0 µg/g for sample 2, 14.7 ± 0.2 µg/g for sample 4), with a higher amount in sample 3 (29.2 ± 1.4 µg/g).

Fig. 4.

GC-FID representative chromatogram of squalene in a leaf extract from the CBC-type variety (sample 3).

Chemical characterization and quantification of terpenes in hemp leaves

 The analysis of the terpenes in hemp leaves revealed a distinctive profile for each sample. A representative chromatogram from the GC-MS analysis of sample 1 is shown in Fig. 5. The terpenes detected in the samples are detailed in Table 6 and their distribution is available in the SI (Table S5).

Fig. 5.

GC-MS representative chromatogram (TIC) of terpenes in a leaf extract from the CBC-type variety (sample 3).

Table 6.

Quantitative analysis of terpenes by GC-MS, expressed as µg/g ± standard deviation (SD, n = 3).

Compound	Sample 1	Sample 2	Sample 3	Sample 4
α-Pinene	31.0 ± 4.0	< LOD	< LOD	6.0 ± 1.0
β-Myrcene	46.0 ± 6.0	6.0 ± 1.0	< LOD	7.0 ± 1.0
Camphene	< LOD	< LOD	< LOD	4.0 ± 0.5
Terpinolene	< LOD	< LOD	< LOD	3.1 ± 0.5
Limonene	25.0 ± 3.0	10.0 ± 2.0	< LOQ	5.3 ± 0.7
Eucaliptol	27.0 ± 3.0	< LOD	< LOD	< LOD
Ocimene	< LOD	< LOD	70.0 ± 8.0	< LOD
Fenchyl alcohol	29.0 ± 5.0	< LOD	< LOD	< LOQ
Borneol	< LOD	< LOD	< LOD	3.0 ± 0.4
β-Caryophyllene	420.0 ± 12.0	170.0 ± 10.0	700.0 ± 10.0	120.0 ± 5.0
α-Humulene	103.0 ± 9.0	46.0 ± 6.0	250.0 ± 8.0	20.0 ± 2.0
Valencene	309.0 ± 10.0	< LOD	110.0 ± 1.0	< LOD
cis-Nerolidol	405.0 ± 11.0	101.0 ± 10.0	90.0 ± 10.0	< LOD
Farnesene	1035.0 ± 30.0	442.0 ± 12.0	< LOD	< LOD
trans-Nerolidol	< LOD	< LOD	20.0 ± 5.0	< LOD
Caryophyllene oxide	52.0 ± 4.0	50.0 ± 4.0	130.0 ± 9.0	60.0 ± 6.0
α-Bisabolol	895.0 ± 17.0	645.0 ± 13.0	30.0 ± 4.0	10.0 ± 2.0
Guaiol	< LOD	173.0 ± 10.0	< LOD	< LOD
Cedrol	392.0 ± 12.0	198.0 ± 9.0	< LOD	< LOD

In particular, sample 4 exhibited the lowest total terpene content, while samples 1, 2 and 3 all had a total terpene content approximately 1000 µg/g. In all samples, the most abundant terpenes were α-humulene, β-caryophyllene, α-bisabolol and caryophyllene oxide. Valencene was found in significant amounts in samples 1 and 3, while farnesene was the most abundant terpene in samples 1 and 2. Other terpenes, such as camphene, limonene and β-myrcene, were generally present at concentrations in the tenths of µg/g amounts. Eucalyptol was characteristic for sample 1, while camphene and borneol for sample 4. Ocimene was found only in sample 3, while sample 2 did not exhibit typical terpenes.

Discussion

A comprehensive chemical characterization of bioactive compounds was performed for the first time in non-psychotropic C. sativa leaves from different varieties. The analysis focused on polyphenols, CBs, unsaponifiable lipids (PCs and squalene) and terpenes.

The data confirmed the ubiquitous presence of CFL-A and B, along with the corresponding demethoxy derivatives, highlighting their role as relevant markers of this chemical class in hemp leaves^8,10. Several studies have investigated these flavones, particularly in CBD-rich varieties in both leaves and inflorescences^12,24,25. The CFL-A amount in the CBD-type varieties (sample 1, 70.1 ± 1.8 µg/g) was consistent with literature reports for leaves of the same variety (70.0–76.1 µg/g)²⁵ and also for inflorescences (19.6–130.0 µg/g, with a mean value of 61.8 µg/g)¹². Interestingly, a significantly higher content of CFL-A was found in the CB-free variety (sample 4, 174.7 ± 2.5 µg/g), suggesting that this variety is a promising source of this compound. Leaves from the CBG- and CBC-type varieties (samples 2 and 3) exhibited lower levels of CFL-A. Conflicting quantitative data exist in the literature with one study reporting a substantially higher amount (1369.1 µg/g) of CFL-A in a C. sativa leaf extract obtained under reflux extraction with methanol (MeOH) ²⁴.

The CB analysis provided a detailed chemical fingerprint of C. sativa leaves. In addition to the main CBs, the profile included minor and type-specific compounds. Leaves from the CBD-type variety (sample 1) contained minor compounds related to the CBD-type scaffold (CBDHA and CBDPA), and their corresponding neutral counterparts (CBDH and CBDP). CBGP and CBGH were identified in both CBD-type and CBG-type leaves (samples 1 and 2). Sesqui-CBG was ubiquitous across all leaf samples. Notably, the content of CBDA in sample 1 (43.0 ± 1.8 mg/g) was on average comparable to the levels observed in the inflorescences from a CBD-chemotype (3.7 to 36.4 mg/g)²⁶. The highest content of CBGA was in sample 2 (13.1 ± 0.3 mg/g), in agreement with previous results on CBG-type inflorescences (9.8 mg/g)²⁷. CBCA levels were higher in sample 3 (12.8 ± 0.8 mg/g), resulting slightly lower than those described in the literature for a CBC-type variety biomass (22.0–28.4 mg/g) ⁴. Very few CBs were detected in the CB-free leaves (sample 4), with CBDA being the only quantifiable compound. In general, hemp leaves were demonstrated to be a viable source of CBs, suggesting their potential for recovery as an alternative to commonly used inflorescences.

Concerning PCs, a wide range of these compounds was identified, thought only triacontanol (C₃₀OH) and dotriacontanol (C₃₂OH) were present in a quantifiable amount. In general, hemp leaves exhibited a lower PCs content compared to inflorescences (C₃₀OH and C₃₂OH range: 21.6–141.2 µg/g and 23.1–96.2 µg/g)¹³, probably due to a lower content of waxy material. Noteworthily, C. sativa leaves were investigated for the first time as a potential “green” source of squalene. An appropriate extraction procedure and analytical method were newly developed and optimized, identifying ethyl acetate (EtOAc) as the most efficient solvent using dynamic maceration for 4 h. The content of squalene determined in hemp leaves (from 11.0 ± 1.0 µg/g for sample 2 to 29.2±1.4 µg/g for sample 3) was notably higher than that found in inflorescences from sample 3 itself (2.4±0.3 µg/g). Even if the content of squalene was lower than that achieved via Soxhlet extraction with heptane (60–160 µg/g, depending on the harvest stage)²⁸, the developed method followed a more “eco-friendly” procedure. With the development of appropriate enrichment and fractionation procedures, C. sativa leaves could really represent a new source of this pharmaceutically relevant compound.

The volatile profile revealed β-caryophyllene, α-humulene, α-bisabolol and caryophyllene oxide commonly present across the samples, together with some variety-specific terpenes. Although some differences among the subvarieties analyzed were observed, the overall chemical similarity prevented a clear differentiation, as previously described in the literature²⁹. As expected, sesquiterpenes were predominant over monoterpenes³⁰.

Conclusions

Overall, C. sativa leaves, often considered a waste product, were demonstrated to be a diverse and rich source of bioactive compounds belonging to different chemical classes, paving the way to their possible exploitation in the pharmaceutical field in a concrete circular economy perspective.

Materials and methods

Hemp plant material

This study did not involve wild-collected material or species at risk of extinction (IUCN/CITES). The plant material was provided by certified companies operating under official licenses in compliance with Italian law No. 242/2016. Specifically, hemp leaves were purchased in 2024 from Canvasalus s.r.l. (Monselice, Padua, Italy). A reference sample of each variety is stored in in the laboratory of phytochemistry at the Department of Pharmaceutical Sciences, University of Eastern Piedmont, Novara, Italy, with the following specifications: CBD-type (reference standard CBD-Hl/12/24), CBG-type (reference standard CBG-Hl/12/24), CBC-type (reference standard CBC-Hl/12/24) and CB-free (reference standard CB-free-Hl/12/24). In the text, the samples were labelled as 1, 2, 3 and 4, corresponding to the CBD-type, CBG-type, CBC-type and CB-free variety, respectively. For each sample, the leaves were manually sieved before the extraction procedure. Hemp leaves (Carmagnola) used for the isolation of squalene were provided by Dimensione Canapa (Cuccaro Monferrato, Alessandria, Italy) and collected in September 2024 (44°59’39.1"N 8°26’29.1"E). A reference sample named CarmgLeaves-DC/09/24 is stored in the laboratory of phytochemistry in Novara.

Chemicals and reagents

Acetone, acetonitrile (ACN), chloroform (CHCl₃), cyclohexane, EtOAc, ethanol (EtOH), MeOH, isopropanol, methyl tert-butyl ether (MTBE), n-hexane, petroleum ether (PE), formic acid (HCOOH) and potassium hydroxide (KOH) were purchased from Sigma-Aldrich (Milan, Italy), while ammonium formate was provided from Fluka (Charlotte, NC, USA). Water (H₂O) was purified using a Milli–Q Advantage 10 system from Millipore (Milan, Italy). Basic alumina (50–75 μm), silica gel 60 (60–200 μm), silica gel for flash chromatography (50 μm), reversed-phase (RP) C₁₈ silica gel (25 μm), and Celite 545 (particle size 0.02–0.1 mm), used for low-pressure chromatography (LPC), flash chromatography, and vacuum chromatography, were purchased from Macherey-Nagel (Düren, Germany). Purifications were monitored by TLC on 60 F254 (0.25 mm) plates purchased from Merck (Darmstadt, Germany) and visualized by staining with 5% H₂SO₄ in EtOH and heating. Cannflavin reference standards (CFL-A and CFL-B) were purchased from LGC standards (Milan, Italy), while canniprene (≥ 97%), isolated according to a procedure described in the literature⁹, was kindly provided by Prof. Federica Pollastro. The standard solution of cannabidivarin (CBDV), cannabidibutolic acid (CBDBA), cannabidibutol (CBDB), cannabigerol (CBG), cannabidiol (CBD), tetrahydrocannabivarin (THCV), cannabinol (CBN), Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and cannabichromene (CBC) were purchased from LGC standards (Milan, Italy). A cannabinoic acids mixture, composed of cannabidivarinic acid (CBDVA), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), tetrahydrocannabivarinic acid (THCVA), cannabinolic acid (CBNA), Δ⁹-tetrahydrocannabinoic acid (THCA) and cannabichromenic acid (CBCA), was purchased from Merck Life Science s.r.l. (Milan, Italy). CBDA esters (≥ 97%), including CBDA geraniol ester, CBDA borneol ester, CBDA nerol ester and CBDA fenchol ester, obtained through a CBDA esterification procedure described in the literature³¹, as well as minor CBG-type compounds (≥ 97%), namely cannabigerovarin (CBGV), cannabigerobutol (CBGB), cannabigerophorol (CBGP) and sesqui-CBG and CBN-type compounds (≥ 97%), namely cannabinerovarin (CBNRV), cannabinerobutol (CBNRB), cannabinerol (CBNR) and cannabinerophorol (CBNRP), were provided by Prof. Federica Pollastro. Reference policosanols, including docosanol (C₂₂OH, ≥ 97%), tetracosanol (C₂₄OH, ≥ 97%), hexacosanol (C₂₆OH, ≥ 95%), octacosanol (C₂₈OH, ≥ 95%) and triacontanol (C₃₀OH, ≥ 90%), were purchased from LGC standards (Milan, Italy). Squalene standard was purchased from TCI (Japan). Spex CertiPrep CAN-TERP-KIT-H Can-Terp Kit (42 components, 1.000 µg/mL) Kit was purchased from Cole-Parmer, England.

Extraction of polyphenols from hemp leaves

According to a previous work, 0.25 g of dried leaves was weighed and treated with 10 mL of n-hexane for 15 min under magnetic stirring to remove CBs. The washing procedure was repeated twice with 10 mL and 5 mL of n-hexane, respectively. After filtration with a paper filter, the filtrates were sent to waste, and the dried residue was extracted three times with 10 mL, 10 mL and 5 mL of acetone. The filtrates were collected and brought to dryness under vacuum with a rotary evaporator (Laborota 4000 Heidolph, Schwabach, Germany) and adjusted to a final volume of 1 mL with acetone. The solution was filtered through a 0.45 μm PTFE filter into an HPLC vial. The sample preparation was performed in duplicate for all the samples considered in this study. The samples were stored at − 20 °C until analysis was performed²⁶.

Extraction of CBs from hemp leaves

A portion of 0.25 g of dried leaves was weighed and submitted to a dynamic maceration under magnetic stirring with 10 mL of EtOH for 15 min. The extract was filtered with a paper filter. The procedure was repeated twice with 10 mL and 5 mL of the extraction solvent, respectively. The extracts were pooled and adjusted to a final volume of 25 mL with EtOH. The solution was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) filter into an HPLC vial. The sample preparation was performed in duplicate for all the samples considered in this study. The samples were stored at 4 °C until analysis was performed²⁶.

Extraction of PCs from hemp leaves

The extraction of PCs was performed following a previously developed method, slightly modified¹³. A portion of 5 g of leaves was weighed and submitted to extraction with 60 mL of n-hexane in an ultrasonic bath for 30 min (AU–32, Argolab, Italy). The extraction was repeated with the same amount of n-hexane. After vacuum filtration, the extracts were pooled and brought to dryness by using a rotary evaporator (Laborota 4000 Heidolph, Schwabach, Germany). The oily residue was dissolved in 15 mL of hot EtOH (75 °C) and then placed at − 20 °C for 24 h to winterize. The waxy precipitate was centrifugated at 8000 rpm at 2 °C for 30 min. After vacuum filtration, the solid residue was washed with 2.5 mL of EtOH and dried overnight in a desiccator. The waxy material was then placed in a closed glass vial with 2.5 mL of EtOH and 50 mg of KOH and subjected to a microwave-assisted extraction (MAE) with a microwave apparatus (Biotage Initiator Sixty, Biotage, Sweden) to promote the trans-esterification and hydrolysis reactions of long-chain esters and fatty acids. The MAE conditions were set as follows: 80 °C for 30 min under medium speed magnetic stirring. The resulting greyish suspension underwent vacuum filtration, washed with 5 mL of H₂O, and finally dried in a desiccator overnight. Finally, 5 mg of the residue was dissolved in 720 µL of CHCl₃ and filtered through a 0.45 μm PTFE filter prior to the HPLC-ELSD of PCs. The extraction procedure was performed in duplicate for all the samples considered in this study. Samples were stored at − 20 °C until analysis was performed.

Isolation and characterization of squalene from hemp leaves

CBD-type hemp leaves were arranged in a thin layer on stainless-steel trays and dried at room temperature. After 4 days, the dried vegetable material (520 g) was pulverized, extracted with acetone (1:10, w/v, 2 × 12 h) in a vertical stainless-steel percolator at room temperature, filtered in sintered funnel to avoid vegetal material residue, evaporated at reduced pressure (556 mbar, 40 °C) to finally obtain a dark green syrup (29 g, 5.7% yield).

Depigmentation occurred on the extracts by solid-phase filtration under vacuum on C₁₈ silica gel. To this purpose, the extract was dissolved in EtOH (1:2, w/v) at 40 °C and charged on C₁₈ (raw extract/stationary phase ratio of 1:3, w/w), protected by a layer of Celite, packed with EtOH in a sintered funnel (9 × 15 cm) with a side arm for vacuum and eluted with EtOH to obtain the ethanolic fraction (18.6 g) after evaporation at reduced pressure (337 mbar, 40 °C).

The ethanolic fraction was vacuum filtered on basic alumina (90 g, PE-EtOAc gradient from 95:5 to 60:40) to obtain four fractions (I-IV). Fraction II (4.8 g) was further purified with flash chromatography (Isolera One with UV/Vis detection) on silica (50 g, PE-isopropanol gradient from PE 100:0 to 98:2, detection at 205 nm) to obtain, after solvent evaporation at reduced pressure (137 mbar, 40 °C), 57 mg of a fraction concentrated in squalene as a viscous yellow oil, which was identified according with H NMR data previously described in scientific literature²³. ¹H (400 MHz) and C (100 MHz) NMR spectra were recorded with a Bruker 400 spectrometer (Billerica, MA, USA). Chemical shifts were referenced to the residual solvent signal (CDCl₃: δ_H = 7.25). ¹H NMR data are shown in Fig. S1 in the SI.

Squalene extraction method from hemp leaves

The extraction procedure of squalene was performed following the method developed and optimized in this study. A weighed amount of sample (1 g) was extracted with 10 mL of EtOAc for 4 h by means of dynamic maceration. The extract was filtered through a paper filter and adjusted to the final volume of 10 mL. Then, the solution was filtered through a 0.45 μm PTFE before the injection into GC-MS and GC-FID instruments. The extraction procedure was performed in duplicate for all the samples considered in this study. Samples were stored at − 20 °C until analysis was performed.

Extraction of terpenes from hemp leaves

A portion of 0.5 g of finely ground leaves was treated with 5 mL of EtOAc at room temperature for 15 min on an orbiting shaker. The extract is filtered on a 0.45 μm Nylon syringe filter, dodecane was added to a final concentration of 50 µg/mL as the internal standard and the resulting solution was analyzed^32–34.

Polyphenol profiling and quantification

The qualitative analysis of polyphenols in hemp leaves was performed on a Thermo Scientific (Waltham, MA, USA) UHPLC Ultimate 3000 equipped with a vacuum degasser, a binary pump, a thermostatted autosampler, a thermostatted column compartment and a Q-Exactive Orbitrap mass spectrometer with a heated electro-spray ionization (HESI) source (Thermo Scientific, Waltham, MA, USA). An Ascentis Express C₁₈ column (150 mm × 3.0 mm I.D., 2.7 μm, Supelco, Bellefonte, PA, USA) was used for the analysis of the target compounds. The separation was achieved using a gradient elution with a mobile phase composed of 0.1% HCOOH in H₂O (solvent A) and 0.1% HCOOH in ACN (solvent B). The gradient elution was modified as follows: 0–20 min from 2 to 25% B, 20–30 min from 25 to 40% B, 30–40 min from 40 to 80% B, which was kept for 5 min, 45–55 min from 80 to 90% B, which was kept for 5 min. The post-running time was 10 min for re-equilibration of the system. The flow rate and injection volume were set at 0.3 mL/min and 3 µL, respectively. The column temperature was set at 30 °C. MS acquisition was carried out with a heated electro-spray ionization source (HESI) operated in both the positive and in the negative ion mode. For what concerns the MS detector, the source parameters were set as follows: sheath gas (N₂) 37, auxiliary gas (N₂) 28, electrospray voltage 3.4 kV (+) and 2.9 kV (−). The auxiliary gas temperature and the capillary temperature were set at 290 and 320 °C, respectively. The analysis was acquired at a resolving power of 35.000 full width at half maximum (FWHM). The other mass analyzer parameters were set as follows: scan range 100–1000 m/z, automatic gain control (AGC) target 1 × 10⁶ ions, maximum ion injection time (IT) of 243 ms. Data acquisition in data dependent MS/MS (dd–MS/MS) mode was performed at 17.500 resolution and an isolation window for the filtration of the precursor ions of 1 m/z. The fragmentation of precursors ions was performed at 20, 50 and 75 as normalized collision energies (NCE)⁸.

The HPLC-UV/Vis analysis for the quantification of polyphenols was performed on an Agilent Technologies (Waldbronn, Germany) modular model 1260 Infinity II system, consisting of a quaternary pump, an autosampler injection and a UV variable wavelength detector under the same chromatographic conditions applied for the UHPLC-HRMS analysis. Chromatograms were recorded using an Agilent OpenLab (3.7 version). Chromatograms were acquired at 210 nm and 342 nm for canniprene and cannflavins, respectively⁸. The standard solutions of CFL-A and CFL-B were prepared by dissolving 1 mg in 1 mL EtOH. The calibration curve of cannflavins was built with five points in the range of 5–100 µg/mL. Canniprene calibration curve covered a range of 1–25 µg/mL. Three injections were performed for each standard solution and two injections for each sample. Full validation parameters of the method are shown in Table S6 in the SI.

CB profiling and quantification

The qualitative analysis of CBs was performed by using the same instrument as described in the previous paragraph. An Ascentis Express C₁₈ column (150 mm × 3.0 mm I.D., 2.7 μm, Supelco, Bellefonte, PA, USA) was used for the separation of the target compounds. According to a previously reported method, slightly modified³⁵, the chromatographic conditions for the qualitative analysis of CBs consisted of a binary gradient elution by using 0.1% HCOOH in H₂O (solvent A) and 0.1% HCOOH in ACN (solvent B) as the mobile phase. The gradient elution program was set up as follows: the initial conditions were 50% B then raised to 67% B in 2 min, held at 67% B for 4 min and then raised to 90% B until 10 min, kept at 90% B until 14 min, decreased to 50% B over the next min, and held at 50% B until 20 min for re-equilibration of the system. A flow rate of 0.3 mL/min was used. The injection volume was 3 µL. The column temperature was set at 30 °C. As for MS acquisition, the HESI source was operated both in the positive and the negative ion mode. The MS source parameters were set as follows: sheath gas flow rate (N₂) 37, auxiliary gas flow rate (N₂) 28, auxiliary gas temperature 290 °C, capillary temperature 320 °C, electrospray voltage 3.4 kV (+) and 2.9 kV (–). MS was operated in the full-scan mode, followed by data-dependent MS/MS mode. Data acquisition in full MS mode was performed at 70.000 resolution, and the AGC target was set to 1 × 10⁶ with a maximum ion IT of 243 ms. The scan range was 100–1000 m/z for all acquisition. Data acquisition in dd–MS/MS mode was performed at 17.500 resolution and an isolation window of 3 m/z. To study the response of the major product ions of selected CBs with energy, the samples were analyzed at 20, 30, 50 NCE levels³⁵.

To improve the identification of CBDA esters, the aforementioned method was optimized for MS parameters. Conversely to the previous approach, the acquisition was operated exclusively in positive ion mode, and the scan range explored was 300–1000 m/z.

The quantitative analysis of CBs was performed with the same HPLC-UV/Vis equipment as described in the previous paragraph. The mobile phase was composed of a 2 mM ammonium formate solution and 0.1% HCOOH in H₂O (solvent A) and 0.1% HCOOH in ACN (solvent B). According to a previous work²¹, the gradient was set as follows: 0–20 min from 70% to 90% B, which was held for 5 min with a 10 min post-running time. Flow rate and injection volume were set at 0.2 mL/min and 3 µL, respectively. Chromatograms were recorded at the wavelength of 210 and 220 nm for the detection of neutral cannabinoids and cannabinoid acids, respectively²¹. Standard solutions for acidic CBs (CBDVA, CBDA, CBGA, THCVA, CBNA, THCA and CBCA) and neutral CBs (CBDV, CBG, CBD, CBN, THCV and CBC) were diluted with EtOH to reach a stock solution of 100 µg/mL. A five-point calibration curve was generated covering the concentration range of 5–100 µg/mL, while the Δ⁹-THC calibration curve was in the concentration range of 2.5–50 µg/mL. The stock standard solutions for CBDBA and CBDB were prepared as follows: 1 mg of the compound was dissolved in 1 mL of MeOH, then diluted to generate a five-points calibration curve in the concentration range of 5–100 µg/mL. Three injections were performed for each standard solution and two injections for each sample. Full validation parameters of the method are shown in Table S6 in the SI.

PC profiling and quantification

The analysis of PCs was performed on an Agilent Technologies (Waldbronn, Germany) modular model 1260 Infinity II system, consisting of a quaternary pump, a thermostatted column compartment, and an evaporative light scattering detector (ELSD). The separation occurred on an Atlantis dC₁₈ column (150 × 3.0 mm, 3 μm, Waters, Milford, MA, USA). A gradient elution with a mobile phase composed of ACN (solvent A) and MTBE-MeOH 90:10 (v/v) (solvent B) was used for the separation, which was set as follows: 0–1 min isocratic elution at 20% B, 1–16 min linear gradient from 20% to 45% B, which was held constant for 4 min, with a post-running time of 5 min. The flow rate was set at 1.5 mL/min, and the injection volume was 10 µL. The ELSD evaporator temperature was set at 35 °C, while the nebulizer temperature was 30 °C. Nitrogen flow rate was set at 1.50 SLM¹³. An accurate amount of C₃₀OH reference standard (10 mg) was weighed and dissolved in CHCl₃ in a 5 mL volumetric flask as a stock solution. A four-point calibration curve was built covering the ranges of 25–100 µg/mL. Three injections were performed for each standard solution and two injections for each sample. Full validation parameters of the method are shown in Table S6 in the SI.

Squalene identification and quantification

Squalene was analyzed by means of GC-MS and GC-FID techniques. GC-MS analyses were carried out on a 7890 B GC system (Agilent Technologies, Waldbronn, Germany), coupled with a 5975 C MS detector (Agilent Technologies). Compounds were separated on an Agilent HP-5MS capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness, Agilent Technologies). The oven temperature was initially set at 150 °C, then increased to 320 °C at a rate of 4 °C/min, this final temperature being kept for 15 min. The injection volume was 1 µL, with a 1:20 split ratio. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injector and the transfer line temperature were set at 330 °C. Electron ionization (EI) at 70 eV was used to perform MS detection, operating in the full-scan acquisition mode in the m/z range 50–600. Peak identification was performed through a search of mass spectra in the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) mass-spectral database (version 2.0d, 2005)¹⁴.

GC-FID analyses were performed by using a Shimadzu GC–2010 system (Shimadzu Corporation, Kyoto, Japan), equipped with a split/splitless injector. The column used was a HP–5MS capillary column (30 m × 0.25 mm I.D., 0.25 m film thickness, Agilent Technologies). The GC-FID conditions were the same as those described in the literature¹⁴, with slight changes: the initial column temperature was set at 150 °C, programmed to increase at a rate of 4 °C/min until 300 °C, and then held constant for 15 min. The injector and detector temperatures were 280 and 300 °C, respectively. The injection volume was 1 µL, with a split ratio of 1:20. The injection was performed by using the three-layer sandwich mode, with 0.5 µL of EtOAc, another 0.5 µL air gap and 1.0 µL of sample. Helium was used as the carrier gas at a flow rate of 1 mL/min¹⁴. A squalene stock standard solution of 1 mg/mL in EtOAc was prepared, obtaining a five-point calibration curve in the concentration range of 1–25 µg/mL. Two injections were performed for each standard solution and sample. Full validation parameters of the method are shown in Table S6 in the SI.

Terpene profiling and quantification

The qualitative analysis of terpenes was performed by GC-MS analyses with an Agilent Technologies 7890 A GC system coupled with a 5975 C MS detector (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed on a HP-5MS capillary column (30 m length × 0.25 mm ID, 0.25 μm film thickness, Restek, Milan, Italy) with helium as the carrier gas at a constant flow-rate of 1.0 mL/min. An injection volume of 1 µL was employed. The injector temperature was set at 250 °C and operated in the splitless mode. The oven temperature was programmed from 60 to 115 °C at the rate of 3 °C/min, followed by a ramp to 250 °C at the rate of 10 °C/min (toral run time: 32 min). Data acquisition started 5 min after the injection. Mass transfer line temperature was set at 300 °C. All mass spectra were acquired with EI at 70 eV and source temperature of 250 °C, with spectral acquisition in the full-scan mode in the m/z range 50–600. Data were analyzed by MSD 5975 VL data analysis software (Agilent Technology).

Diluted standards were prepared in EtOH to obtain five different concentrations in the range 2–100 µg/mL, and dodecane (50.0 µg/mL) was added as the internal standard^32–34. Three injections were performed for each standard solution and sample. Full validation parameters of the method are shown in Table S6 in the SI.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Conceptualization: Daniele Merli, Gianni Sacchetti, Federica Pollastro, Federica Pellati. Methodology: Matilde Marani, Aurora Camola, Caterina Fantino, Virginia Brighenti, Massimo Tacchini, Maria Eleonora Foletti. Investigation and formal analysis: Matilde Marani, Aurora Camola, Caterina Fantino, Virginia Brighenti, Massimo Tacchini, Maria Eleonora Foletti. Writing original draft preparation: Matilde Marani, Aurora Camola. Writing review and editing: Massimo Tacchini, Daniele Merli, Gianni Sacchetti, Federica Pollastro, Federica Pellati. Funding acquisition: Daniele Merli, Gianni Sacchetti, Federica Pollastro, Federica Pellati. Resources: Daniele Merli, Gianni Sacchetti, Federica Pollastro, Federica Pellati. Supervision: Federica Pollastro, Federica Pellati.

Funding

This study was supported by the project “PNRR - Missione 4 “Istruzione e Ricerca” - Componente C2 Investimento 1.1 “Fondo per il Programma Nazionale di Ricerca e Progetti di Rilevante Interesse Nazionale (PRIN)”, “NOrCa - Not Ordinary Cannabis - Exploring the chemical space around hemp (Cannabis sativa L.) waste and by-products from a circular economy perspective”, project code P2022TXJX8, CUP E53D23021050001, financing Decree MUR D.D. n. 1377 dated 1 st September 2023.

Data availability

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Declarations

Competing interests

The authors declare no competing interests.