Razvoj i validacija LC-MS/MS metode za određivanje N-nitrozamina u farmaceutskim proizvodima

N-nitrosamines are a class of organic compounds with a chemical structure that contains a nitroso (-NO) functional group attached to the amine nitrogen. Such an amine can have one or two alkyl groups attached to the nitrogen atom. According to various studies, most nitrosamines have been confirmed to be carcinogenic in various animal studies but potentially also in humans. For example, a higher incidence of gastric and oesophageal cancer has been described in people who were more exposed to nitrosamines in food. Additionally, another study reported that workers in the rubber industry who were exposed to increased levels of nitrosamines also had an increased incidence of mouth, oesophagus, and throat cancers. This mechanism is associated with cytochrome P450, which mediates the oxidation of nitrosamine compounds that then produce an alkylating agent that can react with genetic material. Since June 2018, the US Food and Drug Administration (FDA), as well as the European Medicines Agency (EMA), have monitored the presence of nitrosamine contaminants in various drugs and introduced rules on maximum permissible levels. The aim of this work was to develop LC- MS/MS method(s) for the qualitative and quantitative determination of six nitrosamine contaminants in six different drugs on the market. The final forms of the drug (Finished Dosage From – FDF) and the standards of their active pharmaceutical ingredient (API) will be used during the development of the method. A review of the literature found several different methods regarding the technique of separation and analysis, as well as some methods that are used for several different pollutants. In this paper, the goal is to develop a method(s) adapted to as many potential nitrosamines as possible impurities in active substances. N-nitrosamines, which are the subject of study in this work, must be well chromatographically separated from each other as well as from the main peak of the active substance being analysed. The N-nitrosamines of interest that will be studied are N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitroso-N-methyl-4-aminobutyric acid, N-nitrosoethylisopropylamine, N-nitroso-diisopropylamine and N-nitrosodibutylamine. Medicines and standards of active substances used in this work are Azithromycin, Betahistine, Metformin, Metronidazole, Simvastatin, Sitagliptin and Vildagliptin in the form of various salts or free bases. Two chromatographic methods were developed using three different chromatographic columns, and calibration curves were created for all combinations of methods and columns. Also, detection limits and quantification limits are determined for each nitrosamine contamination and for each method and type of column. For N-nitroso-N-methyl-4-aminobutyric acid, satisfactory detector sensitivity was achieved using only one method and one column.N-nitrozamini su klasa organskih spojeva s kemijskom strukturom koja sadrži nitrozo (-NO) funkcionalnu skupinu vezanu na dušik amina. Ovakav amin može imati jednu ili dvije alkilne skupine vezane na dušikov atom. Prema različitim provedenim studijama na životinjama, potvrđeno je da je većina nitrozamina kancerogena, a potencijalno je to slučaj i kod ljudi. Opisana je veća učestalost karcinoma želudca i jednjaka kod ljudi koji su više bili izloženi nitrozaminima u hrani. Osim toga, jedno drugo istraživanje navodi da radnici u gumarskoj industriji, koji su bili izloženi povećanim razinama nitrozamina, su također imali povećanu incidenciju karcinoma usne šupljine, jednjaka i grla. Ovaj mehanizam je povezan s citokromom P450 koji je posrednik oksidacije spoja nitrozamina koji potom proizvodi alkilirajuće sredstvo koje može reagirati s genetskim materijalom. Od lipnja 2018. Američka agencija za hranu i lijekove (Food and Drug Administration, kao i Europska agencija za lijekove (European Medicines Agency) prate prisutnost nitrozaminskih onečišćenja u raznim lijekovima i uvode pravila o maksimalnim dopuštenim razinama. Cilj ovog rada bio je razviti kromatografsku, LC-MS/MS metodu(e) za kvalitativno i kvantitativno određivanje šest nitrozaminskih onečišćenja u šest različitih lijekova na tržištu. Kao materijal za analizu koristiti će se konačni oblici lijekova (gotove formulacije), a standardi njihovog aktivnog farmaceutskog sastojka (Active Pharmaceutical Ingredient - API) će se koristiti prilikom razvoja metode. Pregledom literature nađeno je više različitih metoda s obzirom na tehniku razdvajanja i analize, kao i neke metode koje se koriste za više različitih onečišćenja. U ovom radu cilj je razviti metodu(e) prilagođenu što većem broju potencijalnih nitrozaminskih onečišćenja u aktivnim supstancama. N-nitrozamini, koji su predmet izučavanja u ovom radu, moraju se dobro kromatografski odvojiti međusobno, kao i od glavnog vrha aktivne supstance koja se analizira. N-nitrozamini od interesa koji će biti izučavani su N-nitrozodimetilamin, N-nitrozodietilamin, N-nitrozo-N-metil-4-aminomaslačna kiselina, N-nitrozoetilizopropilamin, N-nitrozo-diizopropilamin i N-nitrozodibutilamin. Lijekovi i standardi aktivnih supstanci korištene u ovom radu su azitromicin, betahistin, metformin, metronidazol, simvastatin, sitagliptin i vildagliptin u obliku različitih soli ili slobodnih baza. Razvijene su dvije kromatografske metode korištenjem tri različite kromatografske kolone, te su izrađene kalibracijske krivulje za sve kombinacije metoda i kolona. Također, za svako nitrozaminsko onečišćenje te za svaku metodu i vrstu kolone određene su granice detekcije i granice kvantifikacije. Za N-nitrozo-N-metil-4-aminomaslačnu kiselinu zadovoljavajuća osjetljivost detektora je postignuta korištenjem samo jedne metode i jedne kolone