Hexahydrocannabinol (HHC) is a phytocannabinoid, classified as a semisynthetic cannabinoid; the HHC found commercially is synthesized directly from cannabidiol (CBD), which is either extracted from Cannabis with a low Δ9-THC content or produced directly from Δ9-THC following the process of hydrogenation of the C9-C10 bond in the cyclohexyl ring.In this study, the first high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous separation and quantitative determination of (9R)- and (9S)- HHC, and their metabolites, in urine, oral fluid, and human blood samples was developed and validated. The developed method showed good selectivity and specificity; moreover, the simple and rapid sample preparation makes the method suitable for routine analysis. The developed and validated method was subsequently applied to real human samples in order to study for the first time the pharmacokinetics of HHC epimers and their metabolites. Real human samples (blood, urine and oral fluid) were taken from 6 healthy volunteers after the administration of 25 mg of HHC in 500 mg of tobacco. The application of the method to human biological samples showed that the maximum concentration of the (9R)-HHC epimer in blood was reached 10 minutes after consumption, while for the (9S)-HHC epimer the maximum concentration was reached 1 hour after administration. The mean maximum concentration was 7.9 ng/mL for (9R)-HHC and 2.3 ng/mL for (9S)-HHC. Analytical discrimination between (9R)-HHC and (9S)-HHC and their metabolites provided insight into possible differences between the two epimers from a pharmacokinetic point of view in humans, differences already observed in animals.The (9R)-HHC showed higher bioavailability and higher peak concentration with slower elimination, while both epimers exhibit rapid onset in the blood. Based on the data obtained, some considerations about commercially available HHC products and the possible secondary consequences of their use are mandatory. Commercially available HHC products do not specify the amounts of the two isomers contained in the different mixtures. It is therefore obvious that products with a higher amount of (9R)-HHC may cause more severe clinical effects with possible acute toxicity. Therefore, further studies regarding pharmacokinetics and possible adverse effects caused by the use of this new semisynthetic substance are essential.
L'esaidrocannabinolo (HHC) è un fitocannabinoide, classificato come un cannabinoide semisintetico; l'HHC che si trova in commercio viene sintetizzato direttamente dal cannabidiolo (CBD), che viene estratto dalla Cannabis con un basso contenuto di Δ9-THC o prodotto direttamente dal Δ9-THC in seguito al processo di idrogenazione del legame C9-C10 nell'anello cicloesilico. L’obiettivo di questa tesi è stato quello di sviluppare e validare il primo metodo di cromatografia liquida ad alte prestazioni con spettrometria di massa tandem (HPLC-MS/MS) per la separazione simultanea e la determinazione quantitativa di (9R)- e (9S)- HHC, e dei loro metaboliti, in campioni di urina, fluido orale e sangue umano. Il metodo sviluppato ha mostrato una buona selettività e specificità; inoltre, la preparazione semplice e rapida del campione rende il metodo adatto all'uso in analisi di routine. Il metodo è stato successivamente applicato a campioni reali al fine di studiare per la prima volta la farmacocinetica dell’HHC e dei suoi metaboliti nell’uomo. I campioni reali (sangue, urina e fluido orale), sono stati prelevati da 6 volontari sani dopo la somministrazione di 25 mg di HHC in 500 mg di tabacco. L'applicazione del metodo a campioni biologici umani ha mostrato che la massima concentrazione degli epimeri 9R-HHC e 9S-HHC in sangue è stata raggiunta rispettivamente 10 e 60 minuti dopo il consumo. La concentrazione massima media era di 7,9 ng/mL per il 9R-HHC e di 2,3 ng/mL per il 9S-HHC. La discriminazione analitica tra 9(R)-HHC e 9(S)-HHC e i loro metaboliti ha permesso di comprendere le differenze tra i due epimeri da un punto di vista farmacocinetico nell'uomo, differenze già osservate negli animali. Il 9(R)-HHC ha mostrato una maggior biodisponibilità e un picco di concentrazione più elevato, con un’eliminazione più lenta, mentre entrambi gli epimeri presentano una rapida insorgenza nel sangue. Sulla base dei dati ottenuti, sono d'obbligo alcune considerazioni sui prodotti HHC disponibili in commercio e sulle possibili conseguenze secondarie del loro utilizzo. I prodotti HHC commercializzati non specificano le quantità dei due isomeri contenuti nelle diverse miscele. È quindi ovvio che i prodotti con una quantità maggiore di 9R-HHC possano provocare effetti clinici più gravi con possibile tossicità acuta. Risultano quindi fondamentali ulteriori studi in merito alla farmacocinetica e ai possibili effetti avversi provocati dall’uso di questa nuova sostanza semisintetica.
Sviluppo e validazione del primo metodo analitico in UHPLC-MS/MS per la quali-quantificazione del 9R- e 9S-HHC e dei principali metaboliti. Applicazione a campioni reali per lo studio della sua farmacocinetica nell'uomo.
RICCIARDELLI, GIORGIA
2022/2023
Abstract
Hexahydrocannabinol (HHC) is a phytocannabinoid, classified as a semisynthetic cannabinoid; the HHC found commercially is synthesized directly from cannabidiol (CBD), which is either extracted from Cannabis with a low Δ9-THC content or produced directly from Δ9-THC following the process of hydrogenation of the C9-C10 bond in the cyclohexyl ring.In this study, the first high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous separation and quantitative determination of (9R)- and (9S)- HHC, and their metabolites, in urine, oral fluid, and human blood samples was developed and validated. The developed method showed good selectivity and specificity; moreover, the simple and rapid sample preparation makes the method suitable for routine analysis. The developed and validated method was subsequently applied to real human samples in order to study for the first time the pharmacokinetics of HHC epimers and their metabolites. Real human samples (blood, urine and oral fluid) were taken from 6 healthy volunteers after the administration of 25 mg of HHC in 500 mg of tobacco. The application of the method to human biological samples showed that the maximum concentration of the (9R)-HHC epimer in blood was reached 10 minutes after consumption, while for the (9S)-HHC epimer the maximum concentration was reached 1 hour after administration. The mean maximum concentration was 7.9 ng/mL for (9R)-HHC and 2.3 ng/mL for (9S)-HHC. Analytical discrimination between (9R)-HHC and (9S)-HHC and their metabolites provided insight into possible differences between the two epimers from a pharmacokinetic point of view in humans, differences already observed in animals.The (9R)-HHC showed higher bioavailability and higher peak concentration with slower elimination, while both epimers exhibit rapid onset in the blood. Based on the data obtained, some considerations about commercially available HHC products and the possible secondary consequences of their use are mandatory. Commercially available HHC products do not specify the amounts of the two isomers contained in the different mixtures. It is therefore obvious that products with a higher amount of (9R)-HHC may cause more severe clinical effects with possible acute toxicity. Therefore, further studies regarding pharmacokinetics and possible adverse effects caused by the use of this new semisynthetic substance are essential.| File | Dimensione | Formato | |
|---|---|---|---|
|
Tesi di Giorgia Ricciardelli matricola s1103342 pdfA.pdf
accesso aperto
Dimensione
3.92 MB
Formato
Adobe PDF
|
3.92 MB | Adobe PDF | Visualizza/Apri |
I documenti in UNITESI sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
https://hdl.handle.net/20.500.12075/16870