Characterization of hydrophilic interaction liquid chromatography system with UV and MS/MS detection in analytics and bioanalytics of olopatadine

Cilj doktorske disertacije bio je da se kroz sveobuhvatnu karakterizaciju sistema tečne hromatografije hidrofilnih interakcija (eng. Hydrophilic Interaction Liquid Chromatography – HILIC) sa UV (eng. Ultraviolet) i MS/MS (eng. Mass Spectrometry/Mass Spectrometry) detekcijom unapredi analitika i bioanalitika olopatadina. U dosadašnjoj naučnoj literaturi HILIC sistem je nedovoljno ispitivan primenom metodologije eksperimentalnog dizajna (eng. Design of Experiments – DoE). Takođe, mogućnost analize amfoternih jedinjenja sa cviterjonskom strukturom, kao i geometrijskih izomera pomoću HILIC tehnike još uvek je nedovoljno proučena. U prvom delu doktorske disertacije predstavljen je razvoj nove HILIC metode sa UV detekcijom za istovremeno određivanje olopatadina, njegove nečistoće E izomera i benzalkonijum-hlorida u kapima za oči uz podršku savremene hemometrijske strategije. Na osnovu podataka iz preliminarnih studija odabrana je cijanopropil silikagel kolona i definisani su kvantitativni faktori sa najvećim uticajem na hromatografsku analizu pomenutih jedinjenja. Zatim je, u cilju kompletne karakterizacije retencionog ponašanja ispitivanih jedinjenja u složenom HILIC sistemu i postavljanja robusnog optimuma metode, uspostavljena adekvatna matematička zavisnost kritičnih faktora od razmatranih odgovora sistema primenom Box–Benhken dizajna. Optimalni hromatografski uslovi razdvajanja identifikovani su korišćenjem unapređenog postupka pretrage čvorova mreže. Na kraju je predložena metoda uspešno validirana, čime je pokazana njena pogodnost za rutinsku kvantitativnu analizu geometrijskih izomera olopatadina i konzervansa u farmaceutskoj formulaciji...The goal of the doctoral dissertation was to improve analytics and bioanalytics of olopatadine through comprehensive characterization of hydrophilic interaction liquid chromatography (HILIC) system with UV (ultraviolet) and MS/MS (mass spectrometry/mass spectrometry) detection. In the previous scientific literature, HILIC system has been poorly examined by the application of Design of Experiments (DoE) methodology. Also, the possibility of analysis of amphoteric compounds with zwitterionic structure and geometric isomers by HILIC technique has been still insufficiently studied. In the first part of the doctoral thesis, it was presented the development of a new HILIC method with UV detection for the simultaneous determination of olopatadine, its impurity E isomer and benzalkonium chloride in the eye drops with the support of contemporary chemometrical strategy. On the basis of the data from the preliminary studies, cyanopropyl silica column was selected and quantitative factors with the greatest influence on chromatographic analysis of the mentioned compounds were defined. Then, it was established the adequate mathematical dependence of the critical factors on considered system responses by using Box–Benhken design in order to fully characterize retention behavior of the investigated compounds in complex HILIC system and set robust optimum of the method. The optimal chromatographic separation conditions were identified by employing an improved grid point search procedure. At the end, the proposed method was successfully validated thereby demonstrating its suitability for routine quantitative analysis of olopatadine geometric isomers and preservative in the pharmaceutical formulation..