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Uspešno formulisanje stabilnog i delotvornog farmaceutskog preparata zahteva pažljiv odabir pomoćnih supstanci, s obzirom da one mogu da stupe u interakciju sa aktivnim supstancama. Interakcije između lekovitih i pomoćnih supstanci mogu dovesti do fizičke i hemijske nestabilnosti farmaceutskog proizvoda. Za potrebe razvoja olanzapin film tableta sprovedeno je ispitivanje kompatibilnosti olanzapina sa pomoćnim supstancama predloženim za razvoj formulacije. U izvedenim studijama su detaljnije ispitani oni ekscipijensi za koje je pokazano da utiču na stabilnost aktivne supstance. Međusobne interakcije su ispitane praćenjem sadržaja nečistoća primenom visoko efikasne tečne hromatografije (engl. High Pressure Liquid Chromatography, HPLC) metode, zatim primenom termalnih tehnika kao što su diferencijalno skenirajuća kalorimetrija (engl. Differential Scanning Calorimetry, DSC) i termogravimetrijska analiza (engl. Thermogravimetric Analysis, TGA). Dodatno, izvedena je procena fizičke kompatibilnosti primenom difrakcije x-zraka iz supstanci u čvrstom stanju (engl. X-Ray Powder Diffraction, XRPD). Prema ICH (engl. International Conference on Harmonization) smernicama, sve nečistoće prisutne u farmaceutskim preparatima u količini većoj od 0,1% moraju da se kvantifikuju i identifikuju. U slučaju nečistoća sa potencijalno jakim ili toksičnim dejstvom kvantifikacija i identifikacija se izvode ispod ovog nivoa. Degradacioni proizvodi olanzapina, detektovani pod stres uslovima u studijama kompatibilnosti olanzapina i pomoćnih supstanci, kao i u studiji preliminarne stabilnosti olanzapin film tableta, su strukturno definisani primenom HPLC metode spregnute sa masenom spektrometrijom (engl. Mass Spectrometry, MS), preparativne HPLC metode, kao i IR (engl. Infrared Spectroscopy) i NMR spektroskopije (engl. Nuclear Magnetic Resonance Spectroscopy)... Successful formulation of stabile and effective pharmaceutical products requires careful selection of excipients, due to their ability to interact with active pharmaceutical ingredients (APIs). API-excipient interactions could lead to physical or chemical instability of drug products. The compatibility testing of olanzapine and excipients proposed for formulation development was conducted to support development of olanzapine film-coated tablets. Excipients that were shown to influence the stability of API were more profoundly examined. Interactions were tested by monitoring the content of impurities. High pressure liquid chromatography (HPLC), as well as thermal methods, such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA), were used for the evaluation of chemical compatibility. In addition, assessment of physical compatibility was performed using X-ray powder diffraction (XRPD). According to relevant International conference on harmonization (ICH) guidelines all impurities present in new pharmaceutical products in the level greater than 0.1% must be quantified and identified. For impurities known to be unusually potent or to produce toxic pharmacological effects quantification and identification are performed below this level. Degradation products of olanzapine were detected under the stress experimental conditions in API-excipient compatibility study and preliminary stability study of olanzapine film-coated tablets. Their structural characterization was performed using HPLC coupled with mass spectrometry (MS), preparative HPLC, infrared (IR) and nuclear magnetic resonance (NMR) spectroscopy. Analysis of the content of impurities is very important test parameter during the stability testing of pharmaceutical products. It enables us to appropriately monitor quality of drug products during the defined shelf-life. The importance of this testing is especially emphasised for drug product where increase in the level of impurities represents critical stability attribute that could influence chemical, pharmacological, and toxicological drug product profile...

Retenciono ponašanje i retencioni mehanizmi lekova različitih kiselo–baznih osobina na polarnim i umereno polarnim stacionarnim fazama detaljno su ispitani pomoću teorijskih i eksperimentalnih modela u tečnoj hromatografiji hidrofilnih interakcija (eng. Hydrophilic Interaction Liquid Chromatography – HILIC). Teorijskim modelima prevashodno je omogućeno razjašnjavanje retencionih mehanizama u ispitivanim HILIC sistemima, dok su eksperimentalni modeli bili od značaja u analizi retencionog ponašanja u zavisnosti od većeg broja ispitivanih faktora i kvantifikaciji faktorskih uticaja, kao i njihovih interakcija. Literaturni podaci pokazali su da je do sada najveći broj analiza u HILIC sistemima izveden u silika kolonama, pri čemu su najčešće bile razdvajane bazne supstance zbog svog velikog afiniteta prema ovom tipu stacionarnih faza. U ovom delu disertacije, primenom hemometrijskog pristupa, ispitano je retenciono ponašanje kiselih i amfoternih farmaceutski aktivnih supstanci na primeru model smeše β–laktamskih antibiotika u silika koloni. Kvantifikacija faktorskih uticaja omogućena je građenjem eksperimentalnih modela u formi polinoma drugog reda, a faktorske interakcije grafički su prikazane u vidu trodimenzionih površina odgovora. Verifikacija dobijenih modela urađena je poređenjem simuliranih i eksperimentalno dobijenih hromatograma u proizvoljno odabranim tačkama eksperimentalnog prostora. Separacioni mehanizam u HILIC sistemima izuzetno je složen i do sada nedovoljno proučen. Smatra se da mogu biti uključeni procesi raspodele, adsorpcije i/ili jonske izmene, a da relativan udeo svakog od njih zavisi od tri grupe faktora: strukturnih karakteristika analita, sastava mobilne faze i vrste kolone. Zato su u nastavku istraživanja ispitani retencioni mehanizmi i retenciono ponašanje baznih analita na primeru model smeše odabranih farmaceutski aktivnih supstanci u dve silika, dve cijano i jednoj diolnoj stacionarnoj fazi korišćenjem teorijskih i eksperimentalnih retencionih modela. Fitovanjem retencionih podataka u katjonsko–izmenjivačke retencione modele kvantifikovan je doprinos procesa katjonske izmene u ukupnom HILIC retencionom mehanizmu ovih analita, dok je udeo procesa raspodele i adsorpcije ispitan na osnovu kreiranih particionih i adsorpcionih retencionih modela... The assessment of the retention behavior and retention mechanisms of drugs with different acido–basic properties on various polar and moderately polar stationary phases was carried out with the aid of theoretical and empirical models in hydrophilic interaction liquid chromatography (HILIC). Theoretical models primarily enabled the elucidation of the retention mechanisms in the examined HILIC systems, whereas empirical models were useful in the analysis of the retention behavior as a function of several examined factors and in the quantification of factor influences, as well as their interactions. Literature survey has shown that the greatest number of separations in HILIC mode was performed on bare silica columns. Since basic analytes have high affinity towards this type of stationary phases, they were most commonly analyzed on bare silica columns. In this part of dissertation the retention behavior of acidic and amphoteric analytes on the example of a model mixture of β–lactam antibiotics on bare silica column utilizing chemometric approach was investigated. Quantification of factor influences was enabled through the creation of empirical models in the form of second–order polynomial and factor interactions were graphically represented as 3D response surface plots. Verification of the created models was conducted by comparing simulated and experimentally obtained chromatograms at random points of experimental space. Separation mechanism in HILIC systems is complex and insufficiently investigated so far. It is believed that it involves partition, adsorption and/or ion–exchange processes, and that the dominance of each process depends on three groups of factors: structural characteristics of the analytes, mobile phase composition and column type. Thus, further on, the retention mechanisms and retention behavior of basic analytes on the example of a model mixture of selected basic drugs on two bare silica, two cyano and one diol stationary phase using theoretical and empirical retention models were assessed. By fitting the retention data in cation–exchange models the involvement of cation–exchange process in the overall HILIC retention mechanism was quantified...

Primena hemometrijskih tehnika u razvoju metoda tečne hromatografije omogućava da se iz minimalanog broja inicijalnih eksperimenata modeluje ponašanje sistema u funkciji različitih faktora i da se zatim, kreirajući odgovarajuće algoritme, generiše veliki broj simuliranih hromatograma koji će omogućiti automatizovano pretraživanje eksperimentalnog prostora s ciljem pronalaženja optimalnih rešenja. Ovaj proces je u potpunosti zavistan od adekvatnosti definisanog matematičkog modela kao i od odabira funkcije cilja. U ovoj disertaciji predlaže se nova funkcija hromatografskog odgovora kao funkcija cilja u optimizacionim procedurama. Funkcija je dizajnirana tako da procenjuje, istovremeno, kvalitet razdvajanja i ukupnu dužinu trajanja hromatografske analize. Individualni članovi funkcije definisani su tako da omoguće maksimalnu pouzdanost merenja kvaliteta hromatograma, a težinski koeficijenti koji su dodati u dizajn funkcije omogućavaju postavljanje željene ravnoteže između individualnih ciljeva. Funkcija je testirana poređenjem sa šest prethodno razvijenih funkcija hromatografskog odgovora na seriji simuliranih hromatograma i eksperimentalno dobijenih u sistemu reverzno–fazne tečne hromatografije (RP–LC) i tečne hromatografije hidrofilnih interakcija (HILIC). Nakon verifikacije adekvatnosti funkcije cilja da karakteriše hromatogram u celini, novorazvijena funkcija inkorporirana je u metodologiju eksperimentalnog dizajna za optimizaciju metoda za analizu smeše raloksifen hidrohlorida i njegovih nečistoća u RP–LC sistemu, kao i smeše agonista i antagonista beta receptora u HILIC sistemu. Za optimizaciju u oba slučaja primenjen je centralni kompozicioni dizajn. Novorazvijena funkcija hromatografskog odgovora modelovana je i praćena kao jedini odgovor sistema, a optimum je definisan primenom metodologije površine odgovora. U nastavku istraživanja funkcija je unapređena tako, da osim kvaliteta razdvajanja i dužine trajanja analize, ima sposobnost da proceni oblik dobijenih pikova što može biti od velikog značaja u farmaceutskoj analizi jer je otežana kvantifikacija pikova neadekvatnog oblika... The application of chemometrical techniques in liquid chromatographic method development enables modeling of system behavior dependence on several factors after performing minimal number of experiments. Further on, by creating the appropriate algorithms, the large number of simulated chromatograms is constructed providing automatized experimental space search and identification of optimal solutions. This process is completely dependent on the accuracy of defined mathematical model and chosen objective function. This dissertation proposes a new chromatographic response function for optimization strategies. The function is designed to simultaneously estimate the quality of separation and the total analysis duration. Individual function terms are defined to provide maximal reliability in chromatograms quality evaluation and weighting factors are added to enable the proper adjustment of balance between individual goals. The function is compared with six previously developed chromatographic response functions on set of simulated chromatograms and experimentally obtained chromatograms in reversed–phased liquid chromatography (RP–LC) and hydrophilic interaction liquid chromatography (HILIC). After the function ability to estimate the chromatograms adequately had been confirmed, it was incorporated in Design of experiments methodology for the optimization of method for the analysis of raloxifene hydrochloride and its impurities in RP–LC system, as well as mixture of agonists and antagonists of beta receptors in HILIC system. The optimization in both cases is performed applying central composite design. The newly developed function was the only output of the system and the optimum is located by response surface methodology. Further on, the function is improved to estimate the peak shape along with separation quality and analysis duration. This is very important in pharmaceutical analysis since the quantification of peaks of inadequate shape is very difficult. Improved function is verified on a set of experimentally obtained chromatograms acquired in the analysis of antidepressants in HILIC system and subsequently applied for method optimization...