Izazovi razvoja MEKC metode za određivanje pirfenidona i 5-karboksipirfenidona u biološkom uzorku

master's thesis

• Title (english): Challenges of development of MEKC method for determination of pirfenidone and 5-carboxypirfenidone in biological sample
• Author: Laura Serdar
• Mentor: Miranda Sertić (mentor)
• Committee member: Miranda Sertić (predsjednik povjerenstva)
• Committee member: Dubravka Vitali Čepo (član povjerenstva)
• Committee member: Kristina Pavić (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2020-07-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Idiopatska plućna fibroza (IPF) je kronična bolest pluća kojoj se ne zna točan uzrok, a i patofiziologija nije u potpunosti razumljiva. Lijek pirfenidon se pokazao vrlo korisnim u terapiji bolesti IPF. On smanjuje propadanje funkcije pluća, a time i progresiju bolesti i posljedičnu smrtnost. Pirfenidon je analiziran istovremeno sa svojim glavnim metabolitom, 5-karboksipirfenidonom i unutarnjim standardom salicilnom kiselinom s ciljem uspostavljanja optimalnih radnih uvjeta na CE. Budući da se radi o neutralnoj molekuli, korištena je micelarna elektrokinetička kapilarna kromatografija koja je omogućila analizu pirfenidona, ali i 5-karboksipirfenidona te salicilne kiseline u složenom biološkom matriksu. Kako bi utvrdili vremena migracije, najprije su napravljene analize na standardnim otopinama pirfenidona, 5-karboksipirfenidona i salicilnoj kiselini, pri čemu se najbolja analiza pokazala kad su standardi bili razrijeđeni u čistoj vodi. Slijedeće su se analize provodile na plazmi i serumu, pri čemu je veliki naglasak bio na predobradi uzorka zbog složenosti biološkog uzorka. Osim toga, svaki je put uz „obogaćeni“ uzorak napravljen i kontrolni uzorak da bi se utvrdila vremena migracije ispitivanih analita te dostatno odjeljivanje samih pikova. Prilikom analize „obogaćenih“ uzoraka, pravi je izazov predstavljalo osiguravanje osjetljivosti s obzirom na vrlo niske koncentracije ispitivanih analita u biološkim uzorcima. U većini analiza uočena je nepravilna simetrija pika metabolita 5-karboksipirfenidona te je stoga njemu bilo posvećeno više pažnje tokom ovog diplomskog rada. Međutim, bez obzira na to, optimalni uvjeti analize dobiveni su korištenjem 20 mM boratnog pufera (pH 9,3) uz dodatak SDS-a koncentracije 65 mM. Temperatura od 25°C bila je konstanta dok je napon bio ili 20 ili 25 kV ovisno o analizi. Odabrana valna duljina na kojoj su proučavani ispitivani analiti je 210 nm. Na kraju je kao otapalo za uzorke ispitan i acetonitril, što je pokazalo zanimljive rezultate poput skraćenja vremena analize, dobru simetriju i odjeljivanje pikova. Ovim diplomskim radom utvrđeni su i ustanovljeni optimalni uvjeti odvajanja pikova analita od interesa, primjenom na biološkim uzorcima, metodom kapilarne elektroforeze.

Abstract (english)

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease whose exact cause is not known, and the pathophysiology is not fully understood. The drug pirfenidone has been shown to be very useful in therapy of IPF. It reduces the deterioration of lung function and thus disease progression and consequent mortality. Pirfenidone was analyzed simultaneously with its major metabolite, 5-carboxypyrfenidone, and the internal standard salicylic acid in order to establish optimal assay conditions in CE. Since it is a neutral molecule, micellar electrokinetic capillary chromatography was used, which enabled the analysis of pirfenidone, but also 5-carboxypyrfenidone and salicylic acid in a complex biological matrix. To determine migration times, analyses were first performed on standard solutions of pirfenidone, 5-carboxypyrfenidone, and salicylic acid, with the best analysis being shown when the standards were diluted in pure water. The following analyses were performed in plasma and serum, with great emphasis on sample pretreatment due to the complexity of the biological sample. Furthermore, a control sample was made each time in addition to the spiked sample in order to determine the migration times of the tested analytes and to provide sufficient separation of the peaks themselves. When analyzing spiked samples, the challenge was to ensure sufficient sensitivity given the very low concentrations of test analytes in the biological samples. In most analyses, an irregular symmetry of the peak of the metabolite 5-carboxypyrfenidone was observed and therefore brought more attention to itself. Nevertheless, optimal assay conditions were obtained using 20 mM borate buffer (pH 9.3) with the addition of 65 mM SDS. The temperature of 25 ° C was constant while the voltage was either 20 or 25 kV depending on the analysis. The selected wavelength at which the test analytes were studied was 210 nm. Finally, acetonitrile, was tested as the sample solvent, which showed interesting results such as shortening of the analysis time, good symmetry and peak separation. This diploma thesis determines and establishes the optimal assay conditions for separation of the tested analytes in biological samples by capillary electrophoresis.

• Keywords: pirfenidon; kapilarna elektroforeza; 5-karboksipirfenidon; salicilna kiselina; acetonitril
• Keywords (english): pirfenidone; capillary chromatography; 5-carboxypyrfenidone; salicylic acid; acetonitrile
• Language: croatian
• URN:NBN: urn:nbn:hr:163:779002
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2022-09-02 12:24:19