Development and validation of an analytical method for simultaneous quantification of certain nitrosamines in selected medicines by liquid chromatography coupled with tandem mass spectrometry

Leta 2018 sta tako ameriški kot evropski regulatorni organ sprožila odpoklic kontaminiranih serij zdravil s sartani zaradi prisotnih kancerogenih nitrozaminskih nečistot. Posebna pozornost je bila namenjena dvema spojinama z nitrozo skupino: NDMA in NDEA. Nitrozamini predstavljajo tveganje za razvoj raka pri ljudeh, zato je eden ključnih korakov pri preprečevanju razvoj dovolj občutljivih in selektivnih metod. Pri večini objavljenih analiznih metod lahko hkrati spremljamo največ dva nitrozamina oziroma so primerne za analizo posamezne zdravilne učinkovine iz skupine sartanov. Iz tega razloga je bil cilj magistrskega dela razvoj dovolj občutljive analizne metode za sočasno vrednotenje petih reguliranih nitrozaminov (NDMA, NMBA, NDEA, NEIPA in NDIPA) v izbranih zdravilih, ki vsebujejo sartane. Instrumentalna LC-MS/MS metoda za zaznavo nitrozaminov je bila že predhodno razvita. Pri delu smo se osredotočili na optimizacijo postopka priprave vzorca z namenom zmanjšanja učinka ozadja. Preverili smo vpliv ekstrakcijskega topila, zmanjšanja volumna injiciranja in vpliv ionskega izvora (ESI in APCI) na učinek ozadja. Najnižji učinek ozadja (90–110 %) ter najvišji izkoristek (90–110 %) in dobro ponovljivost metode (RSD ˂ 5 %) smo dosegli z ekstrakcijo nitrozaminov z 20 % metanolom v vodi in pri analizi z APCI ionskim izvorom. Poleg zmanjšanja učinka ozadja smo izboljšali tudi občutljivost in ponovljivost analizne metode ter z validacijo potrdili, da je metoda selektivna, linearna, točna in ponovljiva. Meja določitve je pri analitih v koncentracijskem območju od 0,08 µg/L do 0,61 µg/L. Nato smo preverili ali so nitrozamini prisotni v izbranih zdravilih z zdravilno učinkovino iz skupine sartanov, ki se nahajajo na slovenskem trgu. Pri analizi šestih izbranih zdravilih nismo zaznali nitrozaminov, kar pa je pričakovano, saj so morali proizvajalci zdravil na zahtevo regulatornih organov vzpostaviti ustrezne kontrolne strategije za preprečitev ali omejitev prisotnosti nitrozaminskih nečistot na najmanjšo možno mero. Rezultati so pokazali, da je metoda sposobna zaznati vse nitrozamine na regulatornem nivoju. Prav tako lahko kvantitativno ovrednotimo izbrane nitrozamine v testiranih zdravilih, le NDEA in NEIPA sta malenkost pod mejo določitve za posamezne primere zdravil. V primerjavi z večino analiznih metod iz literature lahko vpeljana metoda sočasno vrednoti pet reguliranih nitrozaminov v zdravilih, ki vsebujejo eno zdravilno učinkovino iz skupine sartanov. Pomembna prednost je tudi občutljivost razvite metode, ki omogoča še nadaljnjo nadgradnjo pri pripravi in analizi vzorca.In 2018, both the US and European Regulatory Authorities initiated a recall of contaminated batches of sartan medicines due to the presence of carcinogenic nitrosamine impurities. Two compounds containing a nitroso group have received particular attention: NDMA and NDEA. Nitrosamines pose a risk of cancer in humans, so one of the key steps in prevention has been the development of a sufficiently sensitive and selective analytical method. For most methods found in literature, no more than two nitrosamines are simultaneously detected, or the methods are suitable just for the analysis of a particular sartan substance in medicines. Therefore, the main objective of our thesis was to develop a sufficiently sensitive method for simultaneous quantification of five nitrosamines (NDMA, NMBA, NDEA, NEIPA and NDIPA) in selected sartan medicines. As the method for the detection of nitrosamines by LC-MS/MS has been previously developed we focused on optimizing the sample preparation procedure to reduce the matrix effect. We have examined the influence of the extraction solvent, injection volume and the ion source (ESI and APCI) on the matrix effect. The lowest matrix effect (90–110 %) and highest recovery (90–110 %) with good reproducibility (RSD ˂ 5 %) were obtained by extracting the nitrosamines with 20 % methanol in water and using APCI ion source. Along with reduced matrix effect also improved sensitivity and reproducibility of the method was achieved. The method was validated and proved to be selective, linear, accurate and reproducible. The limit of quantification for the selected nitrosamines is in the concentration range between 0.08 µg/L and 0.61 µg/L. The method was then applied to real samples and tested for the presence of nitrosamine impurities in selected sartan-containing medicines present on the Slovenian market. The analysis of the six selected pharmaceuticals did not detect nitrosamines, which was expected as Authorities demand from the drug manufacturers the use of appropriate control strategies to prevent or minimize the nitrosamine presence. The results showed that our method enables the detection of all tested nitrosamines in selected medicines at the regulatory level. In addition, the method is capable to quantify the selected nitrosamines in medicines, only NDEA and NEIPA are slightly below the limit of quantification in certain cases. Compared to other methods in the literature, the proposed method enables simultaneous quantification of five nitrosamines in medicines containing mono active ingredient from the sartan group. An advantage is also the method sensitivity, which could be further improved by the adjusted sample preparation and LC-MS/MS analysis