Razvoj in validacija občutljive metode za merjenje koncentracij idarubicina in idarubicinola v plazemskih vzorcih bolnikov na transarterijski kemoembolizacijski terapiji

Lorbek, Maja (author), Trontelj, Jurij (mentor)

Idarubicin je antraciklinski antibiotik s širokim spektrom protirakavega delovanja. Idarubicin se interkalira v DNA in tako vpliva na delovanje encima DNA-topoizomeraza II, kar zavre podvojevanje DNA. Glavni presnovni produkt je idarubicinol, ki prav tako izkazuje citotoksično delovanje in zaradi svoje daljše razpolovne dobe podaljšuje učinkovito delovanje idarubicina. V podporo in vivo klinični študiji v Univerzitetnem kliničnem centru Ljubljana, v kateri so raziskovali povezanost izpostavljenosti idarubicinu in neželenimi učinki ter terapevtskim izidom pri bolnikih z jetrnim rakom na transarterijski kemoembolizacijski terapiji (TACE), smo želeli razviti občutljivo, točno, natančno in selektivno metodo za merjenje koncentracij idarubicina in idarubicinola v realnih plazemskih vzorcih bolnikov. Do sedaj objavljene metode so imele limito kvantifikacije pri 0,1 μg/L, s čimer pa je možno spremljati plazemske koncentracije po terapiji TACE le v obdobju 48 ur, kar pa je premalo, posebej ob upoštevanju razpolovne dobe idarubicinola, ki znaša od 40 do 60 ur. Zato smo želeli razviti občutljivejšo metodo, s katero bi lahko ovrednotili celovit plazemski profil obeh citotoksičnih spojin. Preskusili smo več ekstrakcij na trdnem nosilcu in več tekočinskih ekstrakcij z željo po čim večjem in ponovljivem izkoristku. Najbolje se je izkazala tekočinska ekstrakcija s terc-butil metil etrom in izopropanolom v razmerju 9:1. Po kromatografski separaciji smo preskusili dva načina detekcije: s fluorescenčnim detektorjem in z masnim detektorjem, pri čemer se je bolje izkazal slednji. Razvito in optimizirano metodo smo poskusili ovrednotiti skladno s smernicami FDA in EMA za validacijo bioanaliznih metod, vendar smo nekatere smernice prilagodili namenu našega raziskovanja. Selektivnost, točnost in natančnost metode smo potrdili na koncentracijskem območju med 0,025 μg/L in 50 μg/L, s čimer smo lahko kvantificirali idarubicinol celo do 14 dni po aplikaciji, kar bo omogočilo celovito farmakokinetično analizo in boljšo oceno izpostavljenosti citostatiku. Metodo smo uporabili na velikem številu vzorcev iz klinične študije in tako potrdili njeno ustreznost v realnem kliničnem okolju.

• Language: Slovenian
• Keywords: idarubicin, idarubicinol, tekočinska ekstrakcija, LC-MS/MS
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-147089
• Publication date in RUL: 23.06.2023

Secondary language

• Language: English
• Title: Development and validation of a sensitive method for determination of idarubicin and idarubicinol concentrations in human blood plasma samples of patients on transarterial chemoembolization therapy

Abstract

Idarubicin is an anthracycline antibiotic with a broad spectrum of anti-cancer activity. Idarubicin intercalates into DNA and interferes with the activity of enzyme DNA topoisomerase II, which inhibits DNA replication. The main metabolyte is idarubicinol, which also exhibits cytotoxic activity and, due to its longer half-life, prolongs efficacy of idarubicin. In support of an in vivo clinical study at the University Clinical Center Ljubljana, which studied the relationship between exposure to idarubicin and adverse effects and therapeutic outcome in patients with hepatocellular carcinoma undergoing transarterial chemoembolization (TACE), we aimed to develop a sensitive, accurate, precise and selective method for measuring the concentrations of idarubicin and idarubicinol in patients' plasma samples. The methods published so far had a quantification limit of 0,1 μg/L, which makes it possible to monitor plasma concentrations after TACE therapy only for a period of 48 hours, which is insufficient, especially considering the half-life of idarubicinol, which ranges from 40 to 60 hours. Therefore, we aimed to develop a more sensitive method that could be used to evaluate the complete plasma profile of both cytotoxic compounds. We tested several solid-phase extraction methods and several liquid-liquid extractions with the desire for the highest possible and repeatable recovery. A liquid-liquid extraction procedure using a 9:1 mixture of tert-butyl methyl ether and isopropanol proved to be the most efficient. After chromatographic separation, we tested two detection methods: the fluorescence and the mass spectrometry detection; out of which the latter proved to be superior. We tried to validate the developed and optimized method in accordance with the FDA and EMA guidelines for bioanalytical method validation, with some parameters adapted to the aims of our study. The selectivity, accuracy and precision were confirmed in the concentration range between 0,025 μg/L and 50 μg/L, which allowed us to quantify idarubicinol even up to 14 days after the application, which enables a comprehensive pharmacokinetic analysis and a better assessment of cytostatic exposure. We tested the method on a large batch of samples from a clinical study and confirmed its suitability and usefulness in an actual clinical environment.

• Keywords: idarubicin, idarubicinol, liquid-liquid extraction, LC-MS/MS