Razvoj in optimizacija analitske metode za določitev nitritov v pomožnih snoveh za namen ocene tveganja za tvorbo nitrozaminov

Logar, Maša (author), Pompe, Matevž (mentor), Trdan Lušin, Tina (co-mentor)

Aktualna tematika v farmacevtski industriji je še dandanes zelo povezana s področjem nitrozaminov. V zadnjem času je prišlo do odpoklica določenih zdravil, v katerih je bilo ugotovljeno, da je vsebnost nitrozaminov v zdravilu presegala dovoljene meje, zato so regulatorne oblasti uvedle zahtevo po oceni tveganja za nitrozaminske nečistoče, ki mora biti priložena vlogi za pridobitev marketinškega dovoljenja. Nitrozamine je namreč IARC uvrstil v potencialno kancerogene snovi za človeka, FDA pa v razred visoko potentnih mutagenih kancerogenih snovi, za katere je potrebna dosledna kontrola nad vsebnostjo. Največkrat nitrozamini s funkcionalno skupino nitrozo (N=O) nastanejo z reakcijo med sekundarnimi amini in nitriti v kislem. Nitriti so močna nitrozirajoča sredstva in jih lahko v zdravilih najdemo v zdravilni učinkovini in pomožnih snoveh, ki so ključne komponente v farmacevtski obliki. V pomožnih snoveh lahko nastopajo tudi reaktivne nečistoče v sledovih, ki lahko povzročijo reakcijo z učinkovino, kar lahko vodi v zmanjšano ali celo izgubljeno učinkovitost zdravila ter nastanek potencialno nevarnih razpadnih produktov. Težava v primeru prisotnosti nitrita kot nečistoče nastane ob interakciji z dušik vsebujočimi farmacevtskimi spojinami, saj to lahko vodi v nastanek N-nitrozo spojin (nitrozaminov). Da bi že vnaprej poskusili preprečiti nastanek nitrozaminov, lahko že v zgodnji fazi razvoja izdelka testiramo pomožne snovi na vsebnost nitritov in ocenimo tveganje za nastanek nitrozaminov. Z uporabo pomožnih snovi z nizko vsebnostjo nitritov, bi lahko zmanjšali tveganje za tvorbo nitrozaminov. V okviru magistrske naloge smo razvili, optimizirali in preverili HPLC metodo za določanje sledov nitritov v pomožnih snoveh. Za določanje smo razvili HPLC metodo z uporabo derivatizacije z reagentom, ki omogoča detekcijo s fluorescenčnim detektorjem. Razvita metoda je točna, natančna in ponovljiva. V sklopu magistrskega dela smo analizirali pomožne snovi in jih na osnovi določenih vsebnosti nitrita razvrstili glede na nizko in visoko rizične za nastanek nitrozaminov. Analizirali smo tudi zdravilno učinkovino, intermediate in končni izdelek farmacevtske oblike modelnega farmacevtskega izdelka ter preko določitve vsebnosti nitrita ocenili prispevek vstopnih materialov in procesa izdelave farmacevtskega izdelka k nivoju nitritov in posledično k povišanju tveganja za nastanek nitrozaminov.

• Language: Slovenian
• Keywords: nitriti v pomožnih snoveh, ocena tveganja nitrozaminov, tekočinska kromatografija, fluorescenčna detekcija, derivatizacija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-142258
• COBISS.SI-ID: 135131139
• Publication date in RUL: 28.10.2022

Secondary language

• Language: English
• Title: Development and optimization of analytical method for nitrites determination in excipients for the purpose of nitrosamines formation risk assessment

Abstract

Topical issue in the pharmaceutical industry today is still very much related to nitrosamines. Recently, there has been a recall of certain medicines that were found to contain nitrosamines in excess of permitted limits, which led the regulatory authorities to require a risk assessment for nitrosamine impurities to accompany the marketing authorisation application. Nitrosamines are classified by the IARC as potentially carcinogenic to humans and by the FDA as a highly potent mutagenic carcinogen, requiring strict control of levels. Most nitrosamines with a nitroso functional group (N=O) are formed by reaction between secondary amines and nitrites in acid. Nitrites are strong nitrosating agents and can be found in medicines in excipients, which are the key components in the pharmaceutical form. The excipients may also contain reactive trace impurities that can react with the active substance, leading to reduced or even lost efficiency of the medicinal product and the formation of potentially hazardous degradation products. The problem with nitrite as an impurity arises when it interacts with nitrogen-containing pharmaceutical compounds, as this can lead to the formation of N-nitroso compounds (nitrosamines). To try to prevent nitrosamines from forming in advance, we can test excipients for nitrite content and assess the risk of nitrosamines from an early stage of product development. The use of low-nitrite excipients could also reduce exposure to the risk of nitrosamine formation. In the framework of the MSc thesis, we developed, optimised and validated the HPLC method for the determination of trace nitrites in excipients. We have developed an HPLC method for the determination using derivatization with a reagent that allows detection with a fluorescence detector. The method developed is accurate, precise and reproducible. As part of the Master`s thesis, we analysed the excipients, evaluating them for low- and high-risk for the formation of nitrosamines, based on the nitrite content determination. We also analysed the active substance, intermediates and the final product of the model pharmaceutical manufacturing process to the nitrite levels and, consequently, to the increased risk of nitrosamines through the determination of nitrite content.

• Keywords: nitrites in excipients, nitrosamines risk assessment, liquid chromatography, fluorescence detection, derivatization