Razvoj i validacija RP-HPLC metode za određivanje trijodtironina i tiroksina u biološkom uzorku

master's thesis

• Title (english): Development and validation of the RP-HPLC method for the determination of triiodthyronine and thyroxins in biological sample
• Author: Mihaela Jovanović
• Mentor: Ruža Frkanec (mentor)
• Mentor: Davor Šakić (mentor)
• Committee member: Ruža Frkanec (predsjednik povjerenstva)
• Committee member: Davor Šakić (član povjerenstva)
• Committee member: Lidija Bach-Rojecky (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of analytical chemistry); Zagreb
• Defense date and country: 2022, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

Hipotireoza je patološko stanje koje se javlja zbog smanjenja stvaranja i lučenja hormona štitnjače. Terapija hipotireoze oslanja se na primjenu levotiroksina, no identificirana je subgrupa pacijenata s ostatnim simptomima unatoč toj terapiji. Zbog toga u zadnja dva desetljeća sve veći broj pacijenata poseže za tiroidnim pripravcima koji osim tiroksina sadrže i trijodtironin. Prirodni tiroidni pripravci su po sastavu suhi prah štitne žlijezde. Problem njihovog korištenja su neriješena pitanja sadržaja koji se razlikuje između proizvođača, ali i između pojedinih serija istog proizvođača. Do sada mali se broj istraživanja bavio utvrđivanja u tiroidnim pripravcima i stabilnosti T3 i T4 u njima. Stoga je cilj ovog rada razviti i validirati brzu, specifičnu i pouzdanu metodu tekućinske kromatografije visoke djelotvornosti na reverznoj fazi s UV-Vis detekcijom za istovremeno kvantificiranje T3 i T4 u takvom biološkom uzorku. Antrakinon je korišten kao unutarnji standard za kvantifikaciju T3 i T4. Metoda je validirana ispitivanjem točnosti, preciznosti, specifičnosti, linearnosti te granice detekcije i granice kvantifikacije. Razvijena metoda je visoko specifična (RSD 0,41170 % za T3 i 0,35716 % za T4) i točna (analitički prinos 103,29 % – 116,81 %). Metoda je linearna u ispitivanom koncentracijskom području (0,125 – 2,5 μg/mL). Intra i inter-testna preciznost je visoka (RSD za T3 i T4 te unutarnjeg standarda bio niži od 5 %). Limit detekcije i limit kvantifikacije niski su za oba analita (LOD 80 ng/mL, LOQ 243 ng/mL). Metoda je primijenjena za kvantitativnu analizu T3 i T4 u komercijalnom tiroidnom pripravku unutar i izvan roka trajanja. Rezultati kvantitativne analize tiroidnog pripravka koji je bio unutar roka valjanosti slažu se s deklariranim vrijednostima sadržaja dok je analiza uzorka izvan roka trajanja pokazala smanjenu količinu T3 i T4.

Abstract (english)

Hypothyroidism is a pathological condition that occurs due to a decrease in the formation and secretion of thyroid hormones. Hypothyroidism therapy relies on the use of levothyroxine, but a subgroup of patients with residual symptoms has been identified despite this therapy. That is why in the last two decades an increasing number of patients are resorting to thyroid preparations that contain triiodothyronine in addition to thyroxine. Natural thyroid preparations are by composition dry powder of the thyroid gland. The problem of their use is unresolved issues of content that differs between manufacturers but also between individual series of the same manufacturer. A small number of studies have been done so far with the aim of solving this issue, so the aim of this paper is to develop and validate a fast, specific and reliable method of High-performance liquid chromatography on the reverse phase with UV-Vis detection for simultaneous quantification of T3 and T4 in such biological sample. Anthraquinone was used as an internal standard for quantifying T3 and T4. The method is validated by examining the accuracy, precision, specificity, linearity and limit of detection limit of quantification. The developed method is highly specific (RSD 0.41170 % for T3 and 0.35716 % for T4) and accurate (analytical yield 103.29 % - 116.81 %). The method is linear in the concentration area studied (0.125-2.5 μg/mL). Intra and inter-test accuracy is high (RSD for T3 and T4 and internal standard was lower than 5%). Limit of detection and limit od quatification are low for both analytes (LOD 80 ng/mL, LOQ 243 ng/mL). The method was applied for quantitative analysis of T3 and T4 in a commercial thyroid preparation inside and outside the shelf life. The results of quantitative analysis of the thyroid preparation that was within the shelf life correspond to the declared content values, while the sample analysis outside the shelf life showed a decreased amount of T3 and T4.

• Keywords: tiroidni prah; T3; T4; RP-HPLC; validacija
• Keywords (english): thyroid powder; T3; T4; RP-HPLC; validation
• Language: croatian
• URN:NBN: urn:nbn:hr:163:854785
• 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; Embargo expiration date: 2023-07-04
• Created on: 2022-07-12 12:35:32