Razvoj metode za analizu adenozin-fosfata iz ljudske krvi

master's thesis

• Title (english): Method development for analysis of adenosine-phosphates in human blood
• Author: Ivana Kero
• Mentor: Mario Cindrić (mentor)
• Mentor: Biljana Balen (sumentor)
• Committee member: Mario Cindrić (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Sofia Ana Blažević (član povjerenstva)
• Committee member: Dubravka Hranilović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-28, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Adenozin-fosfati su organske molekule iz skupine nukleotida, u koje se ubrajaju adenozin-5'-trifosfat (ATP), adenozin-5'-difosfat (ADP) i adenozin-5'-monofosfat (AMP). Kvantitativno određivanje adenozin-fosfata ima izraženu dijagnostičku ulogu jer se utjecaj koncentracije adenozin-fosfata na brojna fiziološka stanja izravno korelira s homeostazom organizma, primjerice energetski naboj. Iz navedenih je razloga cilj ovog istraživanja bio razviti široko primjenjivu, točnu i preciznu metodu ekstrakcije adenozin-fosfata iz malog volumena kapilarne krvi. Za postizanje navedenog cilja, uz originalni znanstveni doprinos, bilo je neophodno ispitati vezanje adenozin-fosfata na aktivni ugljen adsoprcijskom kromatografijom u svrhu pročišćavanja krvnih uzoraka mikro-ekstrakcijom na čvrstoj fazi. Aktivni ugljen se učestalo koristi kao afinitetni adsorbens u cijelom nizu farmaceutskih, medicinskih i bioloških aplikacija zbog svoje široke dostupnosti, velike aktivne površine u odnosu na masu i jedinstvenih bioloških i fizikalno-kemijskih svojstava. Bilo je neophodno razviti i validirati metodu mikro-ekstrakcije putem tekućinske kromatografije obrnutih faza, koja odjeljuje, detektira i kvantificira adenozin-fosfate ekstrahirane iz uzorka kapilarne krvi. Analizirane koncentracije adenozin-fosfata iz krvi odgovaraju teorijskim vrijednostima pronađenim u literaturnim izvorima, što ukazuje na pouzdanost razvijene ekstrakcijske metode. Shodno do sada navedenom, aktivni ugljen selektivno veže adenozin-fosfate te ponovljivo pročišćava uzorak krvi, a pri tome kromatografski parametri linearno, selektivno i precizno odjeljuju navedene analite.

Abstract (english)

Adenosine-phosphates are organic molecules from the group of nucleotides, which include adenosine-5'-triphosphate (ATP), adenosine-5'-diphosphate (ADP) and adenosine-5'-monophosphate (AMP). Quantitative determination of adenosine-phosphates has a pronounced diagnostic role because the adenosine-phosphate concentration has influence on many physiological conditions and is directly correlated with the homeostasis of the organism, e.g. energy charge. For these reasons, the aim of this study was to develop a widely applicable, accurate and precise method of adenosine-phosphate extraction from a small volume of capillary blood. To achieve this goal, in addition to the original scientific contribution, it was necessary to examine the binding of adenosine-phosphates to activated carbon by adsorption chromatography for the purpose of blood samples purification by solid phase micro-extraction. Activated carbon is often used as an affinity adsorbent in a range of pharmaceutical, medical and biological applications due to its wide availability, large active surface area relative to mass and unique biological and physicochemical properties. It was necessary to develop and validate micro-extraction method by reverse-phase liquid chromatography, which separates, detects and quantifies adenosine-phosphates extracted from a capillary blood sample. The analyzed concentrations of adenosine-phosphates from the blood correspond to the theoretical values found in literature, which indicates the reliability of developed extraction method. Thus, activated carbon selectively binds adenosine-phosphates and purifies the blood sample, while the chromatographic parameters linearly, selectively and precisely separate these analytes.

• Keywords: adenozin-fosfati; energetski naboj; aktivni ugljen; μ-SPE; HPLC
• Keywords (english): adenosine-phosphates; energy charge; activated carbon; μ-SPE; HPLC
• Language: croatian
• URN:NBN: urn:nbn:hr:217:033834
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-10-07 10:36:29