Optimizacija metode za določanje bleomicina v bioloških vzorcih

Golčar, Špela (author), Trontelj, Jurij (mentor), Kosjek, Tina (comentor)

Bleomicin je zmes glikopeptidnih antibiotikov izoliranih iz Streptomyces verticillus, ki so učinkoviti pri zdravljenju številnih vrst raka. Ker ima tudi bleomicin neželene učinke, zlasti toksično delovanje na pljuča, je med terapijo pomembno spremljanje njegove koncentracije v krvi. Zaradi kompleksnosti njegove strukture, polarnosti in sposobnosti keliranja kovin pa je metodo za določanje bleomicina v bioloških vzorcih izjemno zahtevno razviti. V magistrski nalogi smo skušali optimizirati metodo, ki so jo razvili Kosjek s sodelavci, in sicer predvsem s ciljem prenosa ekstrakcije na trdnem nosilcu iz vakuumskega sistema s kolonami na nadtlačni sistem z mikrotitrskimi ploščami, ki je primernejši za ekstrakcijo večjega števila bioloških vzorcev. Na ta način smo želeli poenostaviti pripravo vzorca, skrajšati čas priprave in izboljšati izkoristek ekstrakcije bleomicina iz vzorcev. Izkazalo se je, da je izkoristek ekstrakcije BLM po prenosu metode na mikrotitrske plošče sicer malenkost nižji, ampak ta razlika ni pomembna, če upoštevamo poenostavitev metode. Na mikrotitrskih ploščah smo optimizirali tudi sestavo elucijskega topila. Nadalje smo poskušali postopek skrajšati tako, da smo plazemske oz. serumske vzorce injicirali direktno po denaturaciji in centrifugiranju, torej brez ekstrakcije na SPE ploščah. Rezultati so bili obetavni zgolj pri serumskih vzorcih, pri čemer je bil izkoristek boljši pri nižjem volumnu seruma kot pri višjem. Matriks vzorca namreč inhibira signal našega analita v masnem spektrometru, kar je intenzivneje pri večjem volumnu seruma. Inhibicija je pri plazmi še izrazitejša, zato smo odločili, da ta metoda ni primerna za direktno injiciranje. Metodo za ekstrakcijo BLM iz seruma in tumorskih tkiv smo optimizirali ter ovrednotili vpliv mase tkiva oziroma volumna biološkega vzorca na odziv masnega detektorja. Pri validaciji metode smo sledili smernicam Ameriškega urada za hrano in zdravila (FDA), kjer je bilo to smiselno in mogoče. Zaradi odkrite slabše robustnosti kromatografije internega standarda epirubicina smo se odločili, da ni primeren za to nalogo. Uporabno območje razvite metode za serum je od 100 do 5000 ng/mL, za tumorsko tkivo pa od 50 do 2500 ng/g.

• Language: Slovenian
• Keywords: ekstrakcija na trdni fazi, priprava vzorcev, tekočinska kromatografija, bleomicin
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-135601
• Publication date in RUL: 22.03.2022

Secondary language

• Language: English
• Title: Optimization of a method for determination of bleomycin in biological samples

Abstract

Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. It is efficient in treatment of many neoplastic tissues. Nevertheless, bleomycin has side effects, mostly on pulmonary tissue. Consequently, bleomycin blood level should be monitored. Because of structure complexity, polarity and ability to chelate metals it is very difficult to set sensitive and selective analytical method for its determination in biological tissues. In our work, we tried to optimize the method which was developed in following research Kosjek et al. Mostly with goal to transfer solid phase extraction from cartridge where we used low pressure to microplates under high pressure, which are more appropriate for extraction of multiple biological samples. The goal was to simplify sample preparation, reduce time and increase efficiency of extraction of bleomycin from samples. The method was successfully transferred although efficiency of bleomycin extraction on microplate was lower. Within microplate we optimized composition of the elution solvent. Additionally, we tried to shorten the process with direct injection of blood samples after denaturation and centrifugation without the extraction using SPE microplates step. Results were relevant only for serum samples. Better results were with lower volume samples than higher volume samples. In mass spectrometer bleomycin detection is suppressed by matrix, especially in higher volume serum samples. Suppression is even greater in plasma samples, which is why we decided that it is not appropriate for direct injection. We optimised the method for extraction of bleomycin from serum and tumor tissue and evaluated how mass of tissue and volume of biological sample influence to the mass detector response. In validation process we followed U.S Food and drug administration guidelines where this was reasonable and possible. Due to the poor robustness of internal standard in chromatography we decided that it is not suitable for this task. The working range of analytical method for serum is from 100 ng/mL to 5000 ng/mL and for tumor tissue from 50 to 2500 ng/g.

• Keywords: solid phase extraction, sample preparation, liquid chromatography, bleomycin