Sinteza in vrednotenje fluorogenih reagentov za zaznavanje nitritov

Jelenko, Tit (author), Pajk, Stane (mentor), Mravljak, Janez (comentor)

Nitrozamini so skupina organskih spojin, ki zaradi svojega mutagenega in rakotvornega potenciala predstavljajo pomembno tveganje za zdravje ljudi. Njihova prisotnost je bila ugotovljena v različnih okoljskih vzorcih, živilih in farmacevtskih izdelkih, zato je razvoj občutljivih in zanesljivih metod za njihovo zaznavanje pomemben predvsem v farmacevtski industriji. Namen magistrske naloge je bil razviti in ovrednotiti novo molekulsko sondo za določanje nitritov ter na njeni osnovi razviti analitsko metodo tekočinske kromatografije ultra visoke ločljivosti, skopljene z masno spektrometrijo (UHPLC-MS). Izhodišče raziskave je predstavljala že opisana molekulska sonda, pri kateri pri reakciji z nitritom nastaneta dva regioizomera. Nastanek nefluorescentnega izomera otežuje čiščenje produkta in lahko vpliva na zanesljivost analitskega določanja. Z uvedbo dodatne metilne skupine smo želeli usmeriti reakcijo v nastanek enega samega produkta in s tem poenostaviti analitski postopek. Novo molekulsko sondo smo pripravili z večstopenjsko sintezo. Identiteto sintetiziranih spojin smo preverili z jedrsko magnetno resonanco in UHPLC-MS analizo. Za določanje nitritov smo razvili in optimizirali metodo UHPLC-MS ter ovrednotili njeno linearnost, natančnost in točnost. Umeritvena krivulja je pokazala dobro linearnost v območju koncentracij od 0,7 do 10,0 µmol/L, pri čemer je koeficient determinacije znašal 0,9984. Relativni standardni odklon je znašal 0,74 % pri koncentraciji 5,0 µmol/L, medtem ko je relativna napaka znašala 9,30 %. Rezultati kažejo na zadovoljivo natančnost in točnost metode v preučevanem koncentracijskem območju. Preverjali smo tudi možnost zaznavanja nitritov v različnih organskih topilih po izpostavitvi zraku in izpušnim plinom dizelskega avtomobila. V izvedenih vzorcih nitriti niso bili zaznani oziroma so bile njihove koncentracije pod mejo detekcije uporabljene metode. Za zanesljivejšo oceno tega vpliva bi bilo potrebno povečati čas izpostavitve, površino stika med topilom in zrakom ali izboljšati občutljivost analitske metode. Rezultati kažejo, da ima nova molekulska sonda potencial za uporabo pri analitskem določanju nitritov, pri čemer je njena nadaljnja uporabnost povezana predvsem z izboljšanjem občutljivosti, topnosti in optimizacijo analitskih pogojev.

• Language: Slovenian
• Keywords: nitrozamini, NDMA, molekulska sonda, UHPLC-MS, Griessov reagent
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FFA - Faculty of Pharmacy
• Publisher: [T. Jelenko]
• Year: 2026
• PID: 20.500.12556/RUL-189559
• UDC: 543.544:543.51(043.2)
• COBISS.SI-ID: 294817027
• Publication date in RUL: 09.10.2026

Secondary language

• Language: English
• Title: Synthesis and evaluation of fluorogenic reagents for nitrite detection

Abstract

Nitrosamines are a group of organic compounds that pose a significant health risk due to their mutagenic and carcinogenic potential. Their presence has been detected in various environmental samples, food products, and pharmaceutical products, making the development of sensitive and reliable methods for their detection particularly important in the pharmaceutical industry. The aim of this master’s thesis was to develop and evaluate a novel molecular probe for nitrite determination and based on this probe, to develop a UHPLC-MS analytical method. The starting point of the study was a previously described molecular probe, which reacts with nitrite to form two regioisomers. The formation of a non-fluorescent isomer complicates product purification and may affect the reliability of analytical determination. By introducing an additional methyl group, we aimed to direct the reaction towards the formation of a single product, thereby simplifying the analytical procedure. The novel molecular probe was prepared through a multistep synthesis. The identities of the synthesized compounds were confirmed by NMR and UHPLC-MS analyses. A UHPLC-MS method for nitrite determination was developed and optimized, and its linearity, precision, and accuracy were evaluated. The calibration curve demonstrated good linearity over the concentration range of 0,7 to 10,0 µmol/L, with a coefficient of determination of 0,9984. The relative standard deviation was 0,74 % at 5,0 µmol/L, while the relative error was 9,30 %. The results indicate satisfactory precision and accuracy of the method within the investigated concentration range. We also investigated the possibility of detecting nitrites in various organic solvents following exposure to air and to the exhaust gases of a diesel-powered vehicle. Nitrites were not detected in the samples analyzed, or their concentrations were below the detection limit of the applied method. A more reliable assessment of this effect would require increasing the exposure time, the contact surface area between the solvent and air, or the sensitivity of the analytical method. The results indicate that the novel molecular probe has potential for use in the analytical determination of nitrites. Its further applicability will primarily depend on improving its sensitivity and solubility and on further optimization of the analytical conditions.

• Keywords: nitrosamines, NDMA, molecular probe, UHPLC-MS, Griess reagent