Razvoj metode za merjenje koncentracij kanabidiola in tetrahidrokanabinola ter nekaterih njunih metabolitov v človeški krvni plazmi

Kočevar, Marko (author), Trontelj, Jurij (mentor)

Kanabidiol (CBD) in ?9-tetrahidrokanabinol (THC) sta dva kanabinoida iz konoplje, ki pri strokovni javnosti vzbujata vse več zanimanja za njuno terapevtsko uporabo. Glavni namen magistrske naloge je bil razviti metodo za merjenje koncentracij CBD, THC, 11-hidroksi-THC (THC-OH) in 11-karboksi-THC (THC-COOH) v človeški krvni plazmi, ki bo ustrezala namenom klinične študije zdravljenja refraktarne epilepsije pri otrocih. Med razvojem priprave vzorca smo preizkusili 5 različnih tekočinskih ekstrakcij, 18 vrst kartuš za ekstrakcijo na trdnem nosilcu, 1 filter in 2 kartuši za podprto tekočinsko ekstrakcijo. Med vsemi se je za najustreznejšo izkazala ekstrakcija tekoče-tekoče z zmesjo topil heksana in etilacetata v razmerju 9 : 1. LC-MS/MS metodo smo razvili s tekočinskim kromatografom sklopljenim s tandemskim kvadrupolnim masnim spektrometrom. Analite smo kromatografsko ločili z gradientnim programom z 0,2 mM amonijevim fluoridom in acetonitrilom na analitski koloni Kinetex C18 (50 mm × 2,1 mm, 2,6 µm). Umeritvene krivulje smo pripravili z osmimi kalibratorji v območju 1–100 µg/L za THC in 6–100 µg/L za THC-OH ter z desetimi kalibratorji v območju 1–1000 µg/L za CBD in 2,5–1000 µg/L za THC-COOH. Kot interne standarde smo uporabljali izotopno označene analoge analitov: CBD-d3, THC-d3 in THC-COOH-d3. Z namenom preverjanja selektivnosti analizne metode smo poskušali z jetrno S9 frakcijo in vitro pripraviti metabolite CBD, predvsem 7-karboksi-CBD, ki komercialno ni bil dostopen. Analizno metodo smo vrednotili po smernicah za validacijo bioanaliznih metod. Točnost in ponovljivost sta bili pri CBD, THC in THC-COOH v območju 88–110 % ter 1,1–8,1 %, pri THC-OH pa v območju 97–117 % ter 2,0–15,8 %. Vzorce lahko z redčenjem točno in ponovljivo analiziramo do 5-kratnika zgornje meje kvantifikacije. Stabilnost analitov v plazmi smo dokazali za 17 dni pri –80 °C, tri cikle zamrzovanja in odtajanja, štiri ure pri sobni temperaturi in 36 ur v avtomatskem vzorčevalniku pri 10 °C. Pri CBD, THC in THC-COOH smo dokazali odsotnost relativnega učinka matrice. Razvita metoda za namene klinične študije omogoča merjenje CBD v plazmi v koncentracijskem območju 1–5000 µg/L, THC 1–500 µg/L, THC-COOH 2,5–5000 µg/L in THC-OH 6–500 µg/L.

• Language: Slovenian
• Keywords: epilepsija, kanabinoidi, S9 frakcija, tekočinska kromatografija, validacija metode, LC-MS/MS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-110998
• Publication date in RUL: 21.09.2019

Secondary language

• Language: English
• Title: Development of a method for quantitation of cannabidiol, tetrahydrocannabinol and their selected metabolites in human blood plasma

Abstract

Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) are two cannabinoids from cannabis that are of increasing medical interest to professional public. The main aim of master’s thesis was to develop a method for quantitation of CBD, THC, 11-hydroxy-THC (THC-OH) and 11-carboxy-THC (THC-COOH) in human blood plasma, which would be fit for purpose of a clinical study studying treatment of refractory epilepsy in children. During method development we tested 5 different liquid-liquid extractions, 18 kinds of solid phase extractions, 1 filter and 2 supported liquid extractions. The most optimal was liquid-liquid extraction with hexane and ethyl acetate in 9 to 1 ratio. LC-MS/MS method was developed with liquid chromatograph with tandem triple quadrupole mass spectrometer. Chromatographic separation was achieved on Kinetex C18 column (50 mm × 2.1 mm, 2.6 μm) using a gradient comprising of 0.2 mM ammonium fluoride and acetonitrile. Calibration curves were prepared with eight calibrators 1–100 μg/L for THC and 6–100 μg/L for THC-OH and with ten calibrators 1–1000 μg/L for CBD and 2.5–1000 μg/L for THC-COOH. Isotopically labeled analogs were used as internal standards: CBD d3, THC-d3 and THC-COOH-d3. To prove method selectivity we tried to prepare in vitro CBD metabolites, especially commercially unavailable 7-carboxy-CBD, with liver S9 fraction. Method was evaluated according to the guidelines for validation of bioanalytical methods. Accuracy and precision for CBD, THC in THC-COOH were between 88–110 % and 1.1–8.1 % and for THC-OH between 97–117 % and 2.0–15.8 %. Accurate and precise dilution integrity was up to 5 times the upper limit of quantification. Sample stability was proven for 17 days at –80 °C, three freeze and thaw cycles, four hours at room temperature and 36 hours in autosampler at 10 °C. Absence of relative matrix effect was proven for CBD, THC and THC-COOH. The method developed for the purpose of the clinical study allows the measurements of plasma CBD from 1 to 5000 μg/L, THC from 1 to 500 μg/L, THC-COOH from 2.5 to 5000 μg/L and THC-OH from 6 to 500 μg/L.

• Keywords: epilepsy, cannabinoids, S9 fraction, liquid chromatography, method validation, LC-MS/MS