Optimizacija predanalitičke obrade biološkog uzorka za određivanje anastrozola i palbocikliba tekućinskom kromatografijom spregnutom sa spektrometrijom masa

master's thesis

• Title (english): Optimization of pre-analytical processing of a biological sample for the determination of anastrozole and palbociclib by liquid chromatography coupled to mass spectrometry
• Author: Martina Papić
• Mentor: Miranda Sertić (mentor)
• Committee member: Miranda Sertić (predsjednik povjerenstva)
• Committee member: Anita Somborac Bačura (član povjerenstva)
• Committee member: Anita Hafner (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2024-07-25, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Cilj ovog rada je odabir optimalne predanalitičke obrade uzorka plazme te razvoj nove metode za
istovremenu kvantifikaciju anastrozola (ANA) i palbocikliba (PAL) tekućinskom kromatografijom visoke
djelotvornosti spregnute sa spektrometrom masa (LC-MS). Uspoređene su metode taloženja proteina,
ekstrakcije čvrstom fazom (SPE), uklanjanja fosfolipida i disperzivne mikroekstrakcije tekuće-tekuće
(DLLME). Kako bi se odredili optimalni uvjeti ekstrakcije čvrstom fazom uspoređene su vrijednosti
ekstrakcijskih učinkovitosti i utjecaja matrice šest različitih sorbensa u kombinaciji s različitim eluensima.
Najbolji rezultati su primjećeni kod ekstrakcije na kolonici sa C18 sorbensom, a eluens je acetonitril (ACN)
potom 5 %-tni (v/v) amonijak u MeOH. Kako bi se odredili optimalni uvjeti disperzivne mikroekstrakcije
uspoređene su vrijednosti ekstrakcijskih učinkovitosti i utjecaja matrice pet DLLME varijanti. Najbolji
rezultati su primjećeni kod DLLME vodenog uzorka gdje je ekstrakcijsko sredstvo kloroform, disperzijsko
sredstvo izopropanol, a ionska jakost otapala je povećana dodatkom citratne soli. Uzorci su analizirani i
kvantificirani novom LC-MS metodom. Provedena je reverzno fazna kromatografija na bifenilnoj
kromatografskoj koloni uz binarnu gradijentnu eluciju. Mobilna faza sastoji se od 0,1 % mravlje kiseline u
vodi i 0,1 % mravlje kiseline u ACN. MS analiza provedena je uz elektrosprej ionizaciju u pozitivnom
načinu snimanja uz praćenje ionskih tranzicija m/z 448,2→380,0 i m/z 448,2→319,5 za PAL, te m/z
294,1→225,1 i m/z 294,1→210,0 za ANA. Analiti su identificirani na temelju spektara masa i vremena
zadržavanja. Kvantitativna analiza provodila se integriranjem kromatografskih pikova.

Abstract (english)

The aim of this work is to select the optimal pre-analytical processing of the plasma samples and the
development of a novel method for the simultaneous quantification of anastrozole (ANA) and palbociclib
(PAL) by high performance liquid chromatography coupled to a mass spectrometer (LC-MS). The methods
of protein precipitation, solid phase extraction (SPE), phospholipid removal and liquid-liquid dispersive
microextraction (DLLME) were compared. In order to determine the optimal conditions for solid phase
extraction, the values of extraction efficiency and matrix influence of six different sorbents in combination
with different eluents were compared. The best results were observed during extraction on a column with
C18 sorbent, the eluent in the extraction was acetonitril (ACN), then 5% (v/v) ammonia in methanol. In
order to determine the optimal conditions of DLLME, the values of extraction efficiency and matrix
influence of five DLLME variants were compared. The best results were observed with the DLLME of the
water sample, where the extraction agent is clorophorm, the dispersion agent is isopropanol, and the ionic
strength of the solvent is increased by the addition of citrate salt. The samples have been analyzed and
quantified by a novel LC-MS method. Reverse phase chromatography was performed on a biphenyl column
with binary gradient elution in which the mobile phase consists of 0.1% formic acid disolved in water and
0.1% formic acid disolved in ACN. MS analysis was performed with electrospray ionization in positive
imaging mode by monitoring of ion transitions m/z 448,2→380,0 and m/z 448,2→319,5 for PAL, and m/z
294,1→225,1 and m/z 294,1→210,0 for ANA. Analytes were identified based on their mass spectra and
retention times. Quantitative analysis was carried out by integrating the chromatogram spikes.

• Keywords: anastrozol; palbociklib; tekućinska kromatografija; spektrometrija masa; taloženje proteina; ekstrakcija čvrstom fazom; uklanjanje fosfolipida; disperzivna mikroekstrakcija tekuće-tekuće; ekstrakcijska učinkovitost; utjecaj matrice.
• Keywords (english): anastrozole; palbociclib; liquid cromatography; mass spectrometry; protein precipitation; solid phase extraction; phospholipid removal; liquid-liquid microextraction
• Language: croatian
• URN:NBN: urn:nbn:hr:163:514737
• Study programme: Title: Medical biochemistry - integrated undergraduate and graduate - university study; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: sveučilišni magistar medicinske biokemije i laboratorijske medicine (sveučilišni magistar medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Embargoed access; Embargo expiration date: 2025-07-25
• Created on: 2024-10-09 13:32:27