Nova bioanalitička kapilarnoelektroforetska metoda za istovremenu analizu pirfenidona i njegovog metabolita

master's thesis

• Title (english): New bioanalytical capillary electroforetic method for simultaneous analysis of pirfenidone and its metabolite
• Author: Juraj Parag
• Mentor: Miranda Sertić (mentor)
• Committee member: Miranda Sertić (predsjednik povjerenstva)
• Committee member: Biljana Nigović (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2019-07-05, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Pirfenidon je relativno nov lijek registriran za terapiju idiopatske plućne fibroze te je jedan od
samo dva koja se trenutačno koriste za ovu dijagnozu. Iako ima službenu farmakopejsku metodu, kao
novi lijek na tržištu koji je uključen u brojna klinička istraživanja, postoji velika potreba za razvojem
osjetljivih, preciznih, brzih i jednostavnih analitičkih metoda za analizu pirfenidona i njegovog
metabolite u složenim biološkim uzorcima.
Cilj ovog istraživanja je bio razviti novu, jednostavnu kapilarnoelektroforetsku metodu za
analizu pirfenidona i njegovog glavnog metabolita, 5-karboksipirfenidona. S obzirom na neutralnu
narav pirfenidona odabrana je micelarna elektrokinetička kromatografija (MEKC), uz dodatak
unutarnjeg standarda – salicilne kiseline.
Analiza je provedena u kapilari unutrašnjeg promjera 50 μm i ukupne duljine 32 cm (23,5 cm)
pri temperaturi od 25°C. Za detekciju je korišten detektor s nizom fotodioda, na valnim duljinama 210
nm i 310 nm. Tijekom optimizacije parametara napon je mijenjan u rasponu od 25 do 30 kV, a na
kraju je kao optimalan napon odabran onaj od 30 kV. Pri analizi standardnih otopina najboljim se
pokazao boratni pufer (pH 9,3) u koncentraciji od 20 mM uz dodatak visoke koncentracije SDS-a (70
mM) kako bi se osigurali bolji izgled i simetrije pikova. Metoda je uspješno primjenjena na
obogaćenom biološkom uzorku. Prilikom analize bioloških uzoraka životinja tretiranih pirfenidonom,
izazov je predstavljala osjetljivost metode, što će biti predmet daljnjih istraživanja.

Abstract (english)

Pirfenidone is a novel drug registered for the treatment of idiopathic pulmonary fibrosis, and is one of
only two drugs currently in clinical use. Although the official analytic method exists in the European
Pharmacopoeia, to this day the methods for its analysis are scarse. Therefore, the development of new
bioanalytical methods for its analysis is a necessity.
The goal of this research was to develop a new and simple capillary electrophoretic method for analysis
of pirfenidone and its main metabolite 5-carboxypirfenidone. Due to pirfenidone being neutral in charge, a
micellar electrokinetic chromatography (MEKC) was chosen. To minimize the chance of errors, salicylic acid
was used as an internal standard.
Analysis was carried out in a fused silica capillary with 50 μm internal diameter and 32 cm total length
(23.5 cm) with an diode array detecor (DAD) at the end. Detection was performed at 210 nm and 310 nm. The
temperature was 25°C. During the analysis applied voltage varied between 25 kV and 30 kV, but 30 kV showed
better results. Borate buffer (pH 9,3, 20 mM) gave best preliminary results providing fast analysis due to high
electroosmotic flow. Sodium dodecil sulphate (70 mM) was added to improve peak symmetry and shape. This
method showed applicable for the analysis of biological samples treated with pirfenidone. Due to low drug and
metabolite concentrations in biological samples, sensitivity of the method was a problem, and will represent a
challenge in further research.

• Keywords: pirfenidon; kapilarna elektroforeza; bioanalitika; biološki uzorak; plućna fibroza
• Keywords (english): pirfenidone; capillary electroforesis; bioanalytics; biological sample; pulmonary fibrosis
• Language: croatian
• URN:NBN: urn:nbn:hr:163:322847
• 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: 2021-09-07 13:49:34