Razvoj analitičke metode za određivanje enantiomernog sastava leucin enkefalina (osnove i smjernice u razvoju metode za peptidne lijekove)

master's thesis

• Title (english): Development of analytical method for determination of the enantiomeric composition of leucine enkephalin (basics and guidelines in developing a method for peptide drugs)
• Author: Marija Pikutić
• Mentor: Ivone Jakaša (mentor)
• Granter: University of Zagreb; Faculty of Food Technology and Biotechnology; (Department of Chemistry and Biochemistry); (Laboratory for Analytical Chemistry); Zagreb
• Defense date and country: 2017-09-27, Croatia
• Scientific / art field, discipline and subdiscipline: BIOTECHNICAL SCIENCES; Food Technology

Abstract

Analiza optičke čistoće peptida vaţan je korak u karakterizaciji aktivnih farmaceutskih supstancija peptidnih lijekova. Cilj ovog rada bio je razviti pouzdanu i selektivnu analitičku metodu za odreĎivanje enantiomernog sastava leucin enkefalina te definirati smjernice za primjenu razvijene metode kod odreĎivanja optičke čistoće ostalih API-a peptidnih lijekova. U tu svrhu razvijena je i validirana analitička metoda koja se temelji na hidrolizi peptida leucin enkefalina, derivatizaciji osloboĎenih aminokiselina te njihovoj separaciji na kiralnoj stacionarnoj fazi primjenom plinske kromatografije u sprezi s plameno-ionizacijskim detektorom (GC-FID). U usporedbi sa standardnom metodom hidrolize, brzi alternativni postupci koji uključuju upotrebu smjese klorovodične i trifluoroctene kiseline pokazali su jednaku i čak bolju učinkovitost. Za razliku od derivata aminokiselina dobivenih postupkom dvostupanjske derivatizacije, derivati aminokiselina dobiveni postupkom jednostupanjske derivatizacije uz dodatak trifluoroctene kiseline u otapalo bili su stabilni kroz 24 sata. Od pet ispitanih kiralnih stacionarnih faza baziranih na β-ciklodekstrinima, prihvatljiva separacija enantiomernih derivata aminokiselina postignuta je na kiralnoj stacionarnoj fazi MEGA-DEX DMT beta. Sadrţaj leucin enkefalina u standardu koji sadrţi leucin enkefalin acetat hidrat bio je veći od 75 % što je u skladu s deklariranom vrijednošću.

Abstract (english)

Determination of the optical purity of peptides is an important step in the characterization of active pharmaceutical ingredients of peptide drugs. The aim of this study was to develop a reliable and selective analytical method for determination of the enantiomeric composition of leucine enkephalin and define the guidelines for application of the developed method when determining the optical purity of other peptide APIs. For this purpose analytical method based on hydrolysis of peptide leucine enkephalin, derivatization of free amino acids and their separation on a chiral stationary phase by application of gas chromatography and flame-ionisation detector (GC-FID) was developed and validated. In comparison to standard hydrolysis method, fast alternative methods involving the use of a mixture of hydrochloric and trifluoroacetic acid have shown to be more effective. In contrast to two-step derivatization procedure, one-step derivatization procedure with addition of trifluoroacetic acid to solvent resulted in 24 h stability of all derivatives. Of the five investigated chiral stationary phases based on β-cyclodextrins, acceptable separation of the enantiomeric amino acid derivatives was achieved on the chiral stationary phase of the MEGA-DEX DMT beta. Content of leucine enkephalin in the standard containing leucine enkephalin acetate salt hydrate showed to be within the declared range (> 75 %).

• Keywords: optička čistoća peptida; leucin enkefalin; GC-FID; hidroliza peptida; derivatizacija aminokiselina
• Keywords (english): optical peptide purity; leucine enkephalin; GC-FID; peptide hydrolysis; amino acid derivatization
• Language: croatian
• URN:NBN: urn:nbn:hr:159:504803
• Study programme: Title: Food Engineering; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra inženjer/inženjerka prehrambenog inženjerstva (magistar/magistra inženjer/inženjerka prehrambenog inženjerstva)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-16 10:05:27