Razvoj in validacija metode za ekstrakcijo in kvantifikacijo klorokina in desetilklorokina v krvi, urinu in izbranih tkivih podgan

Majhenič, Eva (author), Trontelj, Jurij (mentor)

Osrednja točka magistrske naloge je analitika klorokina, ki ga uporabljamo kot antimalarik, vrednotijo pa ga tudi kot potencialno učinkovino v boju proti virusu Sars-CoV-2. Nalogo smo izvedli v podporo študiji in vivo, za preučevanje farmakokinetičnih lastnosti nove farmacevtske oblike s klorokinom, ki so jo izvedli na Inštitutu za patologijo, na Medicinski fakulteti Univerze v Ljubljani. Farmakokinetika klorokina in njegovega aktivnega metabolita desetilklorokina še ni povsem razjasnjena, predvsem slabo raziskana je njuna tkivna distribucija. Namen magistrskega dela je bil razviti in validirati bioanalizno metodo za merjenje koncentracije klorokina in njegovega aktivnega metabolita desetilklorokina v krvi, urinu in v nekaterih tkivih podgan in te metode potem tudi uporabiti na vzorcih iz študije. Med razvojem postopka priprave vzorcev smo preizkusili ekstrakcijo na trdnem nosilcu z reverzno-faznim in ionsko-izmenjevalnim mehanizmom retencije ter ekstrakcijo tekoče-tekoče z različnimi organskimi topili. Slednji postopek se je izkazal za najboljšega, kot ekstrakcijsko topilo pa smo uporabili tercbutilmetil eter. Optimizirali smo tudi kromatografske in masno spektrometrične parametre. Razvito metodo smo ovrednotili na osnovi deloma prilagojenih kriterijev smernic Evropske agencije za zdravila za validacijo bioanaliznih metod in jo aplicirali na vzorce, pridobljene pri študiji in vivo. V podganji krvi smo izmerili (1339 ± 280) μg/L klorokina in (419 ± 62) μg/L desetilklorokina, v urinu pa (30820 ± 8955) μg/L klorokina in (7495 ± 2510) μg/L desetilklorokina. Koncentracija klorokina v jetrih je bila (230 ± 217), v ledvicah (121 ± 91), v srcu (15 ± 9), v skeletnih mišicah (3,6 ± 1,1), v možganih (1,9 ± 1,4) in v maščevju (1,9 ± 0,8) μg/g tkiva. Koncentracija desetilklorokina je bila večkrat nižja: v jetrih (54 ± 34), v ledvicah (21 ± 0,5), v srcu (3,7 ± 1,0), v skeletnih mišicah (0,4 ± 0,1), v možganih (0,6 ± 0,4) in v maščevju (0,6 ± 0,2) μg/g tkiva. Pridobljeni rezultati kažejo na uspešnost razvite metode za podporo predklinični študiji, razkrivajo pa tudi zelo neenakomerno distribucijo klorokina in njegovega aktivnega metabolita po krvi, urinu in po organih ter dobro sovpadajo z do sedaj znanimi informacijami.

• Language: Slovenian
• Keywords: klorokin, desetilklorokin, razvoj in validacija metode, ekstrakcija iz krvi, urina in tkiv, UHPLC-MS/MS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2021
• PID: 20.500.12556/RUL-134141
• Publication date in RUL: 24.12.2021

Secondary language

• Language: English
• Title: Development and validation of a method for chloroquine and desethylchloroquine extraction and quantification in blood, urine, and selected rat tissues

Abstract

The main focus of the master's thesis is chloroquine, used as an antimalarial and evaluated as a potential candidate drug against Sars-CoV-2 virus. The work was performed in support of an in vivo study of the pharmacokinetic properties of a new pharmaceutical dosage form with chloroquine, performed at the Institute of Pathology, Faculty of Medicine, University of Ljubljana. The pharmacokinetics of chloroquine and its metabolite desethylchloroquine has not been fully explained, and their tissue distribution is particularly poorly understood. The aim of this master's thesis was to develop and validate bioanalytical methods for measuring chloroquine and its active metabolite desethylchloroquine concentration in blood, urine, and selected rat tissues, and to measure analyte concentrations in study samples. During the development of the sample preparation process, solid phase extraction with a reverse-phase and ion exchange retention mechanism and liquid-liquid extraction with various organic solvents were tested. The latter procedure provided the best results, and tert-butyl methyl ether was used as the extraction solvent. Chromatographic and mass spectrometric parameters were also optimized. The developed method was evaluated on the basis of partially adjusted criteria from European Medicines Agency guidelines for the validation of bioanalytical methods and applied to samples from the in vivo study. In rat blood, chloroquine and desethylchloroquine concentrations were (1339 ± 280) μg/L and (419 ± 62) μg/L, and in urine (30820 ± 8955) μg/L and (7495 ± 2510) μg/L, respecitvely. The mean concentration of chloroquine in the liver was (230 ± 217), in the kidneys (121 ± 91), in the heart (15 ± 9), in skeletal muscle (3.6 ± 1.1), in the brain (1.9 ± 1.4), and in fat (1.9 ± 0.8) μg/g tissue. The mean concentration of desethylchloroquine was several fold lower: in the liver (54 ± 34), in the kidneys (21 ± 0.5), in the heart (3.7 ± 1.0), in skeletal muscle (0.4 ± 0.1), in the brain (0.6 ± 0.4), and in fat tissue (0.6 ± 0.2) μg/g tissue. The results obtained show a successful application of the developed method to the samples from preclinical study. They also reveal highly uneven distribution of chloroquine and its active metabolite in the blood, urine and in different organs; the results are also in good agreement with literature data.

• Keywords: chloroquine, desethylchloroquine, method development and validation, extraction from blood, urine, and tissues, UHPLC-MS/MS