Validacija analizne metode na osnovi tekočinske kromatografije, sklopljene s tandemsko masno spektrometrijo, za kvantifikacijo analitov v človeških jetrnih mikrosomih

Urbanček, Alja (author), Pajk, Stane (mentor), Rozenski, Jef (comentor)

Namen magistrske naloge je razviti in potrditi analitično metodo z reverzno-fazno tekočinsko kromatografijo visoke ločljivosti (UHPLC), povezano s trojnim kvadrupolnim masnim spektrometrom (MS/MS), za kvantitativno bioanalizo klindamicina (CLI) in njegovih metabolitov, klindamicin sulfoksida (CSO) in N-demetil klindamicina (CDM). Postopek validacije je bil namenjen pridobitvi podatkov iz in vitro modelov za opazovanje metabolizma s humanimi jetrnimi mikrosomi (HLM), potrebnih za razvoj in silico fiziološko temelječega farmakokinetičnega (PBPK) modela za izboljšanje razumevanja klinične farmakologije CLI. Preiskovana učinkovina CLI spada v skupino linkozamidov, ki so razred antibiotikov, ključnih pri zdravljenju in preprečevanju bakterijskih okužb, saj preko vezave na ribosomsko ribonukleinsko kislino večje podenote bakterijskih ribosomov, zavirajo sintezo bakterijskih beljakovin. Kljub dolgoletni uporabi še vedno obstajajo vrzeli v razumevanju nekaterih vidikov povezanih s CLI, vključno z mehanizmi odpornosti, interakcija z drugimi zdravili in posledičnim optimalnim odmerjanjem ter dolgoročnimi učinki. Sklopljeni analizni tehniki UHPLC in MS/MS sta bila uporabljeni zaradi izboljšane učinkovitosti ločevanja, občutljivosti in zmožnosti kvantifikacije. Poleg prve, smo razvili še drugo LC-MS/MS metodo za vrednotenje midazolama (MDZ). Obe validirani metodi sta se izkazali za linearni s koeficientom determinacije (R2) nad 0,990. Čas ene kromatografske meritve je bil približno 6 minut, kar je omogočilo natančne, točne in zanesljive rezultate, ki izpolnjujejo kriterije validacije. Metoda prav tako izkazuje dobro selektivnost, občutljivost in ustrezne meje detekcije (LOD) in kvantifikacije (LOQ). Presnova CLI v HLM, ki posnemajo jetrno in vivo presnovo, je pokazala močno inverzno korelacijo med koncentracijo CLI in tvorbo CSO, kar potrjuje primarni metabolizem CLI prek S-oksidacije, katalizirane preko encimov iz družine citokromov P450 (CYP). Medtem ko v drugih vzorcih, metabolizma MDZ ni bilo opaziti, najverjetneje zaradi neaktivnih hepatocitov ali drugih dejavnikov priprave inkubacij. Validirani metodi LC-MS/MS sta se izkazali za uspešno analitično orodje pri kvantificiranju CLI in MDZ v in vitro modelih s HLM. Pridobljeni pričakovani rezultati dajejo dragocen vpogled v metabolizem CLI, kar bo z razvojem in silico modelov PBPK in terapevtskim spremljanjem zdravil v prihodnosti omogočilo nadaljnje klinične študije.

• Language: Slovenian
• Keywords: klindamicin, metabolizem, LC-MS/MS, validacija, mikrosomske inkubacije
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-150039
• Publication date in RUL: 13.09.2023

Secondary language

• Language: English
• Title: Development and validation of a liquid chromatography coupled with tandem mass spectrometry method for the quantification of analytes in human liver microsomes

Abstract

The aim of this master’s thesis is to develop and validate an analytical method using reversed-phase liquid chromatography coupled with a triple quadrupole mass spectrometer (LC-MS/MS), equipped with electrospray for the quantitative bioanalysis of clindamycin (CLI) and its metabolites; clindamycin sulfoxide (CSO) and N-demethyl clindamycin (CDM). The validation process aims to obtain in vitro data from microsomal incubations, essential for developing an in silico physiologically based pharmacokinetic (PBPK) model to improve the understanding of the clinical pharmacology of CLI. Lincosamides are a class of antibiotics that inhibit bacterial protein synthesis. However, there is a limited understanding of certain aspects related to CLI, including resistance mechanisms, optimal dosing, long-term effects, and drug-drug interactions. UHPLC and MS/MS were used because of their improved separation efficiency, sensitivity, and quantification capabilities. We also developed and evaluated a method for midazolam (MDZ) and 1-OHMDZ, and both validation methods were proven to be linear with a coefficient of determination (R2) surpassing 0.990. The chromatographic run time was approximately 6 minutes, allowing precise, accurate and reliable results meeting the validation criteria. Moreover, the methods demonstrated good selectivity, sensitivity, and suitable limits of detection (LOD) and quantification (LOQ). Multiple reaction monitoring (MRM) mode was employed, and fragment transitions were calculated for precursor ions. The metabolism of CLI and MDZ in human liver microsomes (HLM) incubations, which mimics in vivo hepatic metabolism, showed a strong inverse correlation between CLI concentration and CSO formation, confirming the primary metabolic pathway of CLI through S-oxidation mediated by CYP3A4. However, no metabolism of MDZ was observed, possibly due to inactive hepatocytes or other issues during preparation. In conclusion, the optimised and validated LC-MS/MS methods successfully quantify CLI and MDZ in microsomal incubations. The results provide a clearer understanding of CLI's metabolism, confirming the hypothesis and provide valuable insights for further research. LC-MS/MS is highlighted as a powerful analytical tool in pharmaceutical research, and the optimized methods will contribute to the development of in silico PBPK models and therapeutic drug monitoring.

• Keywords: clindamycin, metabolism, LC-MS/MS, validation, microsomal incubations