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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. 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.

Reducirani glutation (GSH) tripeptid je cisteina, glutaminske kiseline i glicina te predstavlja najvažniji neenzimatski antioksidans u organizmu. Cilj ovoga istraživanja bio je optimizirati metodu mjerenja GSH u ljudskoj krvi i plazmi te ispitati stabilnost GSH prilikom pohranjivanja uzoraka. Uzorci krvi i plazme pribavljeni su od dobrovoljnog davatelja krvi. Ispitivanje stabilnosti GSH provedeno je tako da je jedan uzorak podijeljen u četiri epruvete. U prvom uzorku plazme ili krvi koncentracija GSH izmjerena je odmah. Dio je uzoraka pohranjen u inkubatoru (na 37 °C u vlažnoj atmosferi s 5% CO2), a preostala po dva uzorka nakon sakupljanja pospremljeni su u zamrzivač na -20 °C na 8 i 30 dana. Koncentracija GSH izmjerena je pomoću Ellmanovog reagensa (5,5'-ditiol-(2-nitrobenzojeva kiselina), DTNB), spektrofotometrijski pri valnoj duljini  = 412 nm i sobnoj temperaturi. Reakcija tiolne skupine GSH s DTNB-om odvija se na sobnoj temperaturi i pH = 7,4 pri čemu nastaje žuti produkt 5'-tio-2-nitrobenzojeva kiselina (TNB). Mjerenja su provedena na UV-Vis spektrofotometru, a koncentracija GSH određena je pomoću molarnog apsorpcijskog koeficijenta (ε) koji iznosi 14,15 x 10-3 M-1 cm-1. Ispitivanja su pokazala da je centrifugiranje plazme, prije spektrofotometrijskog određivanja GSH optimalno kako bi se uklonio dio interferencija koje bi mogle reagirati sa DTNB reagensom i odredila koncentracija GSH. Za određivanje koncentracije GSH u krvi potrebno je krv tretirati s 5 % klorovodičnom kiselinom kako bi se uklonile interferencije i omogućilo određivanje GSH. Nakon pohranjivanja uzorka krvi na 37 °C u vlažnoj atmosferi tijekom 24 sata uočen je statistički značajan porast koncentracije GSH i u plazmi i u krvi te se može zaključiti da su ovi uvjeti potakli sintezu GSH u krvnim stanicama. U uzorcima plazme i krvi pohranjenima -20°C tijekom 8 i 30 dana nisu uočene statistički značajne razlike u koncentraciji GSH. Stoga se može zaključiti da prilikom pohranjivanja uzoraka plazme i krvi na -20 °C do 30 dana ne dolazi do razgradnje GSH. Reduced glutathione (GSH) is cysteine, glutamic acid and glycine tripeptide. It is the most important non-enzymatic antioxidant in the body. The aim of this study was to optimize the GSH measurement method in human blood and plasma samples, and to investigate GSH stability when storing samples. Blood and plasma samples were obtained from volunteer blood donor. To test GSH stability blood or plasma sample was divided into four tubes. In the first sample, concentration of GSH was measured immediately. Part of the samples were stored in the incubator (at 37 ° C in a humid atmosphere with 5% CO2) and the remaining samples were frozen at -20 ° C and kept for 8 or 30 days. GSH concentration was measured using an Ellman reagent (5,5'-dithiobis- (2-nitrobenzoic acid), DTNB spectrophotometrically at wavelength λ = 412 nm on room temperature. The reaction of thiol group GSH with DTNB was carried out at room temperature and pH 7.4, resulting in a yellow product of 5'-thio-2-nitrobenzoic acid (TNB). The measurements were performed on UV-Vis spectrophotometer and the GSH concentration was determined by a molar absorption coefficient (ε) which is 14.15 x 10-3 M-1 cm-1. Tests have shown that centrifugation of plasma prior to spectrophotometric determination of GSH is optimal in order to remove interfering molecules and to determine the actual GSH concentration. Blood sample needs to be treated with 5% chlorodhydrogen acid, to remove possible interfering molecules with the reagents, and to reliably measure GSH. Storing plasma and blood sample at 37 ° C and the humid atmosphere after 24 hours showed statistically significant increase in GSH level in blood and plasma, probably due to GSH synthesis in blood cells. Storing blood and plasma samples at -20 ° C for 8 or 30 days did not affect the level of the GSH. Therefore can be concluded that during storage of plasma and blood samples at -20 °C for 30 days GSH was not degraded.