Spremljanje oksidacije polisorbatov 20 in 80 s tekočinsko kromatografijo visoke ločljivosti

Strle, Lucija (author), Mravljak, Janez (mentor), Škerlavaj, Tinakara (co-mentor)

V farmaciji so v zadnjih letih vse bolj v uporabi biološka zdravila, ki kot zdravilno učinkovino vsebujejo proteine. Za stabilizacijo proteinov v tekočih parenteralnih farmacevtskih oblikah moramo v formulacijo dodati več pomožnih snovi, med katere spadajo tudi polisorbati. Polisorbati se v farmaciji pogosto uporabljajo kot dobro preizkušene površinsko aktivne snovi, ki izboljšajo fizikalno-kemijske lastnosti v milih, kozmetiki, kapljicah za oči, v hrani. Kljub vsem svojim dobrim lastnostim pa so nagnjeni k razpadu na dva načina: s hidrolizo in avtooksidacijo. Tak razpad močno vpliva na kakovost oziroma stabilnost zdravila saj je to ob razgradnji polisorbata neustrezno. Da bi se temu izognili, je potrebno dobro vrednotenje in karakterizacija polisorbatov, torej je treba določiti vsebnost in sestavo le-teh, kar pa največkrat počnemo z uporabo analiznih tehnik tekočinske kromatografije in jedrske magnetne resonance, pa tudi z uporabo masne spektrometrije. V okviru magistrske naloge smo raziskovali učinkovitost oksidacije polisorbata 20 in polisorbata 80 z različnimi oksidanti, pri različnih temperaturah in časih izpostavljenosti. Namerna oksidacija nam omogoča boljše razumevanje različnih mehanizmov razpada polisorbatov v bioloških zdravilih. Za vrednotenje vsebnosti in čistosti polisorbatov smo uporabili metodo tekočinske kromatografije visoke ločljivosti, sklopljene z detektorjem nabitih aerosolov. Ugotovili smo, da je od testiranih oksidantov najučinkovitejši 2,2'-azobis-(2-metil-propionamidin)-dihidroklorid. Oksidacija je hitreje potekala pri povišani temperaturi in je po daljšem času potekla v večjem obsegu. Omenjeni oksidant smo nato uporabili za namerno razgradnjo polisorbatov v prisotnosti različnih antioksidantov in ugotavljali, kateri antioksidant najbolje zavre oksidacijo. Ugotovili smo, da ob dodatku antioksidanta askorbata oksidacija polisorbata še vedno poteka. Dodan manitol je oksidacijo polisorbata le delno zavrl, dodan metionin pa je oksidacijo popolnoma zavrl. Oksidacijo polisorbatov smo potrdili še s komplementarno metodo – jedrsko magnetno resonančno spektroskopijo.

• Language: Slovenian
• Keywords: polisorbat 20, polisorbat 80, oksidacija, tekočinska kromatografija visoke ločljivosti, antioksidant
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-147524
• Publication date in RUL: 07.07.2023

Secondary language

• Language: English
• Title: Monitoring the oxidation of polysorbates 20 and 80 by high-performance liquid chromatography

Abstract

In recent years, biological drugs containing proteins have been increasingly used in pharmacy. In order to stabilize proteins in liquid parenteral pharmaceutical forms, several excipients must be added to the formulation, including polysorbates. Polysorbates are often used in pharmacy as well-tested surfactants that improve the physico-chemical properties of soaps, cosmetics, eye drops, and food. Despite all their good properties, they tend to break down in two ways: by hydrolysis and auto-oxidation. Such decomposition has a strong impact on the quality or stability of the medicine, as this alone is inadequate when the polysorbate is decomposed. In order to avoid this, a good evaluation and characterization of polysorbates is necessary, i.e., the content and composition of only these should be determined, which is mostly done using analytical techniques of liquid chromatography and nuclear magnetic resonance, as well as using mass spectrometry. As part of the master's thesis, we investigated the effectiveness of the oxidation of polysorbate 20 and polysorbate 80 with different oxidants, at different temperatures and exposure times. Intentional oxidation allows us to better understand the various mechanisms of polysorbate degradation in biological drugs. To evaluate the content and purity of polysorbates, we used the method of high-resolution liquid chromatography coupled with a charged aerosol detector. We found that 2,2'-azobis-(2-methyl-propionamidine)-dihydrochloride is the most effective of the tested oxidants. Oxidation took place faster at elevated temperature and took place to a greater extent after a longer time. Saidoxidant was then used for the intentional degradation of polysorbates in the presence of various antioxidants and we determined which antioxidant inhibits oxidation best. We found that with the addition of the antioxidant ascorbate, the oxidation of polysorbate still takes place. Added mannitol only partially inhibited polysorbate oxidation, while added methionine completely inhibited oxidation. The oxidation of polysorbates was also confirmed by a complementary method - nuclear magnetic resonance spectroscopy.

• Keywords: polysorbate 20, polysorbate 80, oxidation, high-performance liquid chromatography, antioxidant