Оптимизација хроматографског раздвајања арипипразола и нечистоћа: приступ квантификовања односа структуре и ретенционог понашања

A new optimization strategy based on the mixed quantitative struc- ture–retention relationship (QSRR) model is proposed for improving the RP- HPLC separation of aripiprazole and its impurities (IMP A-E). Firstly, experi- mental parameters (EPs), namely mobile phase composition and flow rate, were varied according to Box–Behnken design and thereafter, an artificial neural network (ANN) as a QSRR model was built correlating EPs and sel- ected molecular descriptors (ovality, torsion energy and non-1,4-van der Waals energy) with the log-transformed retention times of the analytes. Values of the root mean square error (RMSE) were used for an estimation of the quality of the ANNs (0.0227, 0.0191 and 0.0230 for the training, verification and test set, respectively). The separations of critical peak pairs on chromatogram (IMP A- B and IMP D-C) were optimized using ANNs for which the EPs served as inputs and the log-transformed separation criteria s as the outputs. They were validated by application of leave-one-out cross-validation (RMSE values 0.065 and 0.056, respectively). The obtained ANNs were used for plotting response surfaces upon which the analyses chromatographic conditions resulting in optimal analytes retention behaviour and the optimal values of the separation criteria s were defined. The optimal conditions were 54 % of methanol at the beginning and 79 % of methanol at the end of gradient elution programme with a mobile phase flow rate of 460 μL min-1Нова оптимизациона стратегија заснована на грађењу мешовитих модела за кван- тификовање односа структуре и ретенционог понашања (QSRR) предложена је за уна- пређење RP-HPLC раздвајања арипипразола и његових нечистоћа (IMP А-Е). Експери- ментални параметри (EP), састав мобилне фазе и брзина протока, варирани су најпре у складу са Box–Behnken дизајном, а затим је награђена вештачка неуронска мрежа као QSRR модел који повезује ЕP и одабране молекуларне дескрипторе (овалност, торзиона енергија и не-1,4-ван дер Валсова енергија) са логаритамски трансформисаним ретен- ционим временом аналита. Вредности средње квадратне грешке (RMSE) коришћене су за процену квалитета мреже (0,0227, 0,0191 и 0,0230 за тренинг, верификацију и тест сет, редом). Раздвајање критичних парова пикова на хроматограму (IMP А-B и IMP D-C) оптимизовано је коришћењем мрежа за које су ЕP послужили као улази, а логаритамски трансформисани критеријуми сепарације s као излази. Ове мреже су валидиране при- меном унакрсне валидације изостанка (RMSE вредности, редом, 0,065 и 0,056). На основу награђених мрежа, конструисани су дијаграми површина одговора чијом ана- лизом су дефинисани услови при којима се постиже оптимална ретенција аналита, односно вредности критеријума сепарације s, а који су подразумевали 54 % метанола на почетку и 79 % на крају програма градијентног елуирања са брзином протока мобилне фазе од 460 mL min -

The potential of corona charged aerosol detector for investigation of telmisartan-β-cyclodextrin inclusion complexes

Cyclodextrins (CDs) are widely used in pharmaceutical analysis due to its biodegradability and eco-friendly character. The particular structure of CDs, characterized by hydrophobic cavity, enables the formation of inclusion complexes with variety of organic compounds. As structures lacking chromophores, CDs could not be detected by Photodiode Array (PDA) detector and Corona Charged Aerosol Detector (CAD) was introduced as the most appropriate detector for the formed complexes. The aim of the study was to investigate the degree of complexation between telmisartan, used as a model substance, and β-CD. Moreover, the effect of different β-CD concentrations (5-15 mM) and pH of the aqueous part of mobile phase (3-7) on the degree of complexation was also assessed. The intensity of the formed complex appeared to be the highest when 15 mM β-CD was used for the complexation. Also, it was noticed that the increase in peak areas with an increasing β-CD concentration was more evident at pH 7 in comparison with the same trend at lower pH values. The reproducibility of the measurement was confirmed by low relative standard deviation (RSD) of peak areas within five measurements. These findings support the use of HPLC-CAD methods for studying the process of inclusion complexes formation

Upravljanje resursima u HPLC: Otkrivanje zelenog lica farmaceutske analize

High-pressure liquid chromatography (HPLC) is a technique of paramount importance in the analysis of pharmaceuticals because of its ability to separate moderately polar to less polar compounds, such as drugs and related substances. The concept of green analytical chemistry (GAC) aims to provide more environmentally friendly and safer analytical methods in terms of reagents, energy, and waste. One of the major challenges of GAC is to find an appropriate approach to evaluate the greenness of analytical methods. An extension of GAC, called white analytical chemistry (WAC), has been introduced to consider not only environmental friendliness, but also other aspects that contribute to the sustainability of methods, such as analytical and economic or practical efficiency. HPLC methods are intrinsically not green, due to the high consumption of toxic organic solvents and the resulting generation of large amounts of toxic waste. Fortunately, there are many approaches to overcome the non-green character of HPLC methods. In this article, various modifications of the HPLC methods that increase its environmental friendliness are presented, as well as the various tools used to evaluate environmental friendliness. In addition, the new concept of white analytical chemistry is presented.Tečna hromatografija pod visokim pritiskom (HPLC) je tehnika od ogromne važnosti u analitici lekova zbog svoje mogućnosti da razdvoji umereno do manje polarna jedinjenja, kao što su aktivne farmaceutske i srodne supstance. Koncept zelene analitičke hemije (GAC) ima za cilj da obezbedi ekološki prihvatljivije i bezbednije metode u pogledu reagenasa, energije i otpada. Jedan od glavnih izazova GAC je da pronađe odgovarajući pristup za procenu "zelenosti" analitičkih metoda. Proširenje GAC, nazvano bela analitička hemija (WAC), uvedeno je kako bi se uzela u obzir ne samo ekološka prihvatljivost, već i drugi aspekti koji doprinose održivosti metoda, kao što su analitička i ekonomska ili praktična efikasnost. HPLC metode suštinski nisu "zelene" zbog obimne potrošnje toksičnih organskih rastvarača i posledičnog stvaranja velikih količina toksičnog otpada. Srećom, postoji mnogo pristupa za prevazilaženje ne-zelene prirode HPLC metoda. U ovom radu su predstavljene različite modifikacije HPLC metode koje povećavaju ekološku prihvatljivost, kao i različiti alati koji se koriste za procenu ekološke prihvatljivosti. Pored toga, predstavljen je novi koncept bele analitičke hemije

Студија молекулског докинга са биомолекулима изолованим из ендофитних гљива

Recently, growing interest has been devoted to the investigation of compounds with antimicrobial activity due to rising cases of resistance of mic-robes to known therapies. A reliable and versatile source of novel drug disco-very was recently found among endophytic fungi. Hitherto, the research usu-ally enclosed the in vitro evaluation of antimicrobial activity and chemical structure elucidation of biomolecules extracted from fungal material. There-fore, this research was designed as an extension to previous investigations of endophytic fungi growing on conifer needles by means of conducting a mole-cular docking study. The in silico methods were used with the main goal to make a contribution to the understanding of the mechanisms underlying the interaction of biomolecules isolated from fungus Phomopsis species and eight different types of receptors that belong to usually multidrug resistant bacterial pathogens. The results revealed valuable interactions with receptors 3G7B (Staphylococcus aureus’s gyrase B), 1F0K (1.9 Å structure of Escherichia coli’s transferase) and 1SHV (Klebsiella pneumoniae’s SHV-1 β-lactamase) thus pointing out the receptors that trigger antibiotic response upon activation by the most potent compounds 325-3, 325-5, phomoenamide and phomol. These findings also recommended further discovery of novel potent and broad-spectrum antibiotics based on the structure of selected molecules.У последње време, као одговорна повећање резистенције микроорганизама на познату терапију, све већа пажња се поклања истраживању једињења са антимикробном активношћу. Ендофитне гљиве су недавно представљене као поуздан и богат извор за развој нових лекова. До сада, истраживања су се углавном ограничавала на in vitro процену антимикробне активности и разоткривање хемијске структуре биомолекула изолованих из материјала гљива. Из тог разлога, ово истраживање је осмишљено као проширење претходно спроведених испитивања ендофита које расту на иглицама четинара путем in silico студије молекулског докинга. Главни циљ употребе in silico метода је био да се направи прилог разумевању механизама који стоје иза интеракције биомолекула изолованих из гљиве Phomopsis species са осам различитих типова рецептора који припадају патогеним бактеријама у обичајеном ултирезистентних на лекове. Резултати су указали на важне интеракције са рецепторима 3G7B (Staphylococcus aureus гиразаБ), 1F0K (структура Escherichia Coli трансферазе величине 1,9 Å) и 1SHV (SHV-1 β-лакта-маза Klebsiella pneumoniae) указујући на тај начин на рецепторе путем којих се започиње антибиотски одговор након активације најпотентнијим једињењима, 325-3, 325-5, фомо-енамидом и фомолом. Овим открићем се такође препоручује будући развој нових моћних антибиотика са широким спектром деловања базиран на структури изабраних молекула

Determination of neutron energy spectra presence in gamma spectroscopic measurements using Ge-detectors

Određivanje prisustva neutrona tokom niskofonskih gama spektrometrijskih merenja je od posebne važnosti. Zbog toga je u ovom radu analiziran način određivanja energetskog spektra neutrona prisutnog u merenjima sa HPGe detektorima. Ovaj metod se zasniva na korišćenju metoda dekonvolucije. Za to je neophodno poznavati gama aktivnost indukovanu neutronskim reakcijama sa različitim izotopima germanijuma kao i efikasne preseke za date neutronske reakcije. Ovaj pristup je testiran merenjima sa fisionim izvorom neutrona 252Cf koji je bio postavljen u blizini HPGe detektora. Dobijeni rezultati pokazuju da ovaj metod moze pružiti pouzdane podatke o obliku energetskog spektra neutrona tokom gama spektrometrijskih merenja.Determination of neutron spectra in the Ge-detector during low-level gamma spectroscopy measurements is of great importance. Thus, in this paper we analyzed the method for the determination of neutron energy spectra present during measurements with HPGe detectors. This method is based on using the deconvolution procedure. It requires the knowledge of neutron induced gamma activities of Ge isotopes and the cross section data for the neutron reactions of interest. This approach was tested with measurements that used the fission neutron source 252Cf placed in proximity of the HPGe detector. Results show that this method can provide reliable data about the shape of neutron energy spectrum during gamma spectroscopy measurements.Зборник радова : XXIX симпозијум ДЗЗСЦГ : Сребрно језеро, 27-29. септембар 2017. годин

Determination of neutron energy spectra presence in gamma spectroscopic measurements using Ge-detectors

Određivanje prisustva neutrona tokom niskofonskih gama spektrometrijskih merenja je od posebne važnosti. Zbog toga je u ovom radu analiziran način određivanja energetskog spektra neutrona prisutnog u merenjima sa HPGe detektorima. Ovaj metod se zasniva na korišćenju metoda dekonvolucije. Za to je neophodno poznavati gama aktivnost indukovanu neutronskim reakcijama sa različitim izotopima germanijuma kao i efikasne preseke za date neutronske reakcije. Ovaj pristup je testiran merenjima sa fisionim izvorom neutrona 252Cf koji je bio postavljen u blizini HPGe detektora. Dobijeni rezultati pokazuju da ovaj metod moze pružiti pouzdane podatke o obliku energetskog spektra neutrona tokom gama spektrometrijskih merenja.Determination of neutron spectra in the Ge-detector during low-level gamma spectroscopy measurements is of great importance. Thus, in this paper we analyzed the method for the determination of neutron energy spectra present during measurements with HPGe detectors. This method is based on using the deconvolution procedure. It requires the knowledge of neutron induced gamma activities of Ge isotopes and the cross section data for the neutron reactions of interest. This approach was tested with measurements that used the fission neutron source 252Cf placed in proximity of the HPGe detector. Results show that this method can provide reliable data about the shape of neutron energy spectrum during gamma spectroscopy measurements.Зборник радова : XXIX симпозијум ДЗЗСЦГ : Сребрно језеро, 27-29. септембар 2017. годин

Robustan razvoj metode multimodalne hromatografije primenom principa ugrađivanja kvaliteta kroz dizajn za analizu odabarnih lekova

Mixed-Mode Liquid Chromatography (MMLC) includes several separation mechanisms in a single column, which is why MMLC can analyze compounds in a broad range of polarities and ionization potentials in a single run (1). Acclaim Mixed-Mode WCX-1 column with the ability to expose hydrophobic and weak cation exchange interactions was thus selected to analyse a challenging mixture of neutral and cationic forms: ergotamine, mecloxamine, camylofin, caffeine and propyphenazone, used as a fixed combination. MMLC method was developed in line with Analytical Quality by Design (AQbD) approach implying the scientifically-based understanding of process properties and risk-based management of the method life cycle. AQbD refers to pre-definition of the method’s Analytical Target Profile (ATP) by means of baseline separations within the shortest possible time, as well as definition of Critical Method Attributes (CMAs) as a measure of method quality and Critical Method Parameters (CMPs) affecting CMAs (2). Acetonitrile content, pH and acetate buffer concentration were selected as CMPs since retention mechanism expression in MMLC strongly depends on the mobile phase characteristics. The dependence of CMAs on CMPs was revealed following a face-centred central composition design plan of experiments and accompanying mathematical models, coefficients and standard error values. Design Space in which ATP is achieved with a high level of reliability (π = 90%), was determined by Monte Carlo simulations taking error distribution into account. Its margins pointed out to the working point that assures proper method robustness (pH 5.2, 90 mM acetate buffer solution and 48% (v/v) of acetonitrile).Multimodalna tečna hromatografija (Mixed‐Mode Liquid Chromatography – MMLC) uključuje nekoliko mehanizama razdvajanja u jednoj koloni, zbog čega se ova tehnika može koristiti za simultanu analizu jedinjenja širokog opsega polarnosti i jonizacionog potencijala (1). Acclaim Mixed‐Mode WCX‐1 kolona sa sposobnošću ekspresije hidrofobnih i interakcija slabe katjonske izmene, je odabrana za analizu izazovne smeše neutralnih i katjonskih oblika analita: ergotamina, mekloksamina, kamilofina, kofeina i propifenazona, koji se primenjuju u fiksnoj kombinaciji. MMLC metoda je razvijena u skladu sa pristupom ugradnje kvaliteta kroz dizajn (Analytical Quality by Design – AQbD) koji podrazumeva naučno zasnovano razumevanje svojstava procesa i upravljanje životnim ciklusom metode prema riziku. AQbD se odnosi na unapred definisanje analitičkog ciljanog profila metode (Analytical Target Profile - ATP) odnosno razdvajanje na baznoj liniji za što kraće vreme, kao i na definisanje kritičnih osobina metode (Critical Method Attributes - CMA) kao mere kvaliteta metode i kritičnih parametara metode (Critical Method Parameters - CMP) koji utiču na CMA (2). Sadržaj acetonitrila, pH i koncentracija acetatnog pufera izabrani su kao CMP, pošto ekspresija MMLC retencionih mehanizma zavisi od karakteristika mobilne faze. Zavisnost CMA od CMP definisana je pomoću plana eksperimenata usklađenim sa centralnim kompozicionim dizajnom, ka centru orijentisanim i pratećim matematičkim modelima, koeficijentima i vrednostima standardne greške. Prostor dizajna u kome se ATP postiže sa visokim nivoom pouzdanosti (π = 90%) određen je Monte Karlo simulacijama uzimajuć i u obzir distribuciju grešaka. Njegov okvir ukazuje na radnu tačku koja obezbeđuje odgovarajuć u robusnost metode (pH 5,2, 90 mM rastvor acetatnog pufera i 48% (v/v) acetonitrila).VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Hemometrijski podržan razvoj UHPLC metode: strategija za postizanje ekološke prihvatljivosti

Burden of potentially harmful effects of analytical techniques used in pharmaceutical analysis on human health and environment has recently shown a dramatic increase (1). Therefore, analytical methods able to minimize the production of hazardous waste and use less energy are developing. Reversed-phase high performance liquid chromatography (RP-HPLC) is the most frequently used technique in determination of various compounds, due to its representative analytical features (2). However, RP-HPLC is associated with a substantial amount of waste. Simultaneously, pharmaceutical industry seeks for technologies that produce high resolution within a reduced analysis time without loss of separation efficiency (3). Using fully porous sub-2-μm stationary phase particles provides better resolution in comparison to traditional HPLC stationary phases. However, it causes high column backpressure and thus initiated development of ultra high performance liquid chromatography (UHPLC) instruments. Along with reduced particle size, UHPLC uses shorter columns, which leads to diminished analysis time and minimized production of waste comparing to HPLC. In that respect, UHPLC method for separation of dronedarone hydrochloride and its degradation products was developed utilizing experimental design methodology. Model mixture was selected to cover diversity in physicochemical characteristics. Compounds’ varying polarity represents a challenge from the analytical point of view. Dronedarone hydrochloride is lipophilic, thus requires substantial amounts of organic solvent for its elution. In that respect, there is a recognized need for developing a chromatographic method able to separate the aforementioned compounds with reduced organic solvent consumption and/or analysis time. Preliminary experiments determined factors with significant influence on retention. These factors, namely the initial and final content of acetonitrile and time of gradient were varied according to central composite design. The goal was to achieve satisfying separation between critical peak pairs. Firstly, mathematical models for the time of start and end of peak of each critical pair were obtained. Afterwards, the separation factor (S) standing for the distance between peaks in one critical pair was indirectly modelled. An appropriate separation is achieved if S is higher than zero, which was accomplished with the initial content of acetonitrile of 6% (v/v), the final content of acetonitrile of 50% (v/v), and the gradient duration of 5 min. Ecological acceptability of UHPLC method was evaluated with green analytical procedure index (GAPI), which confirmed eco- friendliness. GAPI showed that the most critical segment is the sample collection and that the advantage should be given to UHPLC over HPLC in terms of energy saving.Opterećenje štetnim uticajem analitičkih tehnika koje se koriste u farmaceutskoj analizi na zdravlje ljudi i životnu sredinu je doživelo dramatičan porast (1). Iz tog razloga, razvijaju se metode koje generišu manje štetnog otpada i koriste manje energije. Reverzno- fazna tečna hromatografija pod visokim pritiskom (RP-HPLC) je najčešće korišćena tehnika u određivanju različitih jedinjenja, zahvaljujući svojim reprezentativnim analitičkim osobinama (2). Sa druge strane, primenom RP-HPLC proizvode se značajne količine otpada. U isto vreme, farmaceutska industrija je u potrazi za tehnikama, koje će obezbediti visoku rezoluciju u redukovanom vremenu trajanja analize, bez gubitka efikasnosti razdvajanja (3). Korišćenjem stacionarnih faze sa potpuno poroznim česticama veličine ispod 2 μm obezbeđuje bolju rezoluciju u poređenju sa tradicionalnim HPLC stacionarnim fazama. Međutim, one dovode do visokih pritisaka, koji su kompatibilni sa instrumentima za izvođenje hromatografije pod veoma visokim pritiskom (UHPLC). UHPLC instrumenti koriste više pritiske u kraćim kolona sa smanjenom veličinom čestica, što dovodi do kraćeg trajanja analize i manje produkcije otpada nego kod HPLC. UHPLC metoda za razdvajanje dronedaron hidrohlorida i degradacionih proizvoda razvijena je korišćenjem metodologije eksperimentalnog dizajna. Model smeša je odbrana kako bi se pokrile raznovrsne fizičko- hemijske osobine. Različita polarnost ispitivanih jedinjenja predstavljala izazov sa aspekta analitike. Dronedaron hidrohlorid je lipofilan, pa je za njegovo eluiranje potrebna značajna količina organskog rastvarača. Prema tome, postoji potreba za razvojem hromatografske metode za razdvajanje ovih jedinjenja sa redukovanom upotrebom organskog rastvarača i/ili smanjenim trajanjem analize. Preliminarni eksperimenti su odredili faktore sa značajnim uticajem na retenciju. Ovi faktori, početni i krajnji udeo acetonitrila i vreme trajanja gradijenta su varirani prema centralnom kompozicionom dizajnu. Cilj je bio da se postigne zadovoljavajuće razdvajanje između kritičnih parova. Prvo su dobijeni matematički modeli za vreme početka i kraja pika svakog kritičnog para. Nakon toga, indirektno je modelovan separacioni faktor (S), koji predstavlja razdaljinu između dva pika jednog kritičnog para. Do odgovarajućeg razdvajanja dolazi ukoliko je S veći od 0, što se postiže sa početnim udelom acetonitrila od 6% (v/v), krajnjim udelom acetonitrila od 50% (v/v) i trajanjem gradijenta od 5 minuta. Ekološka prihvatljivost UHPLC metode je potvrđena određivanjem indeksa ekološke prihvatljivosti analitičke procedure (GAPI). GAPI je pokazao da je najkritičniji segment prikupljanje uzorka, kao i da u pogledu energetske uštede prednost nad HPLC treba dati UHPLC metodi.Drugi naučni simpozijum Saveza farmaceutskih udruženja Srbije sa međunarodnim učešćem, 28. 10. 2021. Beogra

Izgradnja QSRR modela za razvoj RP/WCX HPLC metode

Reverse-phase high-performance liquid chromatography (RP-HPLC) is most common in pharmaceutical analysis and requires significant consumption of toxic mobile phase. Therefore, more eco-friendly solutions are preferred (1). When it comes to complex samples, RP-HPLC, due to inability to separate highly polar and charged analytes, often requires multiple unimodal or two-dimensional HPLC analyzes. The development of mixed-mode liquid chromatography (MMLC), where multiple separation modalities are incorporated into a single stationary phase, allows the separation of complex samples in a single run. Numerous factors affect MMLC separation, which makes method development demanding and limits their practical application (2). Building predictive mathematical models, such as Quantitative structure-retention relationship (QSRR), could improve method development. QSRR links the molecules’ retention behavior with their physicochemical properties (molecular descriptors (MD)), which allows retention behavior prediction of untested analytes. Including experimental parameter values in the QSRR extends the predictability of the model to entire experimental space (3). For model development purposes, experiments were performed on Thermo’s Acclaim Mixed-Mode WCX-1 (3 μm, 2.1 x 150 mm) column which combines hydrophobic and weak cation exchange (WCX) interactions. Small diameter column agrees with low mobile phase flow rate (400 μl/min). Mobile phase composition (acetonitrile content (30 – 50 % (v/v)), pH (3.8 - 5.6) and ionic strength (20 - 40 mM) of acetic buffer) and column temperature (30 – 38 °C) were varied according to face-centered central composite design. Retention factor of 33 pharmaceuticals of different pharmacological and ionization properties were monitored. MDs were calculated using AlvaDesc software. RapidMiner software was used for obtaining QSRR models. Several machine learning algorithms were considered and the most informative (gradient boosted trees (GBT) and bagging neural network (BNN)) were selected. Models were built upon data of 30 analytes, and the remaining three (anion, cation, neutral) were used as a test set. The most influential MDs for BNN were chosen by forward selection, contrary to GBT which did not require preselection. For internal model evaluation 10-fold cross-validation was applied, while external was performed with a test set. Models were compared based on the relative mean square error (RMSE) of the test set. The BNN (RMSE = 0.104; R2 = 0.976) model outperformed GBT (RMSE = 0.122; R2 = 0.963). The obtained QSRR models showed good potential to predict the retention behavior of molecules of different ionization abilities in the RP/WCX system. This could improve the development of MMLC methods and make them more accessible for practical use.U farmaceutskim analizama najzastupljenija je reverzno-fazna tečna hromatografija visokih performansi (reverse‐phase high‐performance liquid chromatography (RP-HPLC)) koja iziskuje značajnu potrošnju mobilne faze toksične prirode. Iz tog razloga, teži se ekološki prihvatljivijim rešenjima (1). Kada su u pitanju kompleksne smeše uzorka, RP-HPLC zbog nemogućnosti sparacije visoko polarnih i naelektrisanih analita, često zahteva više unimodalnih ili dvodimenzionalne HPLC analize. Razvoj multimodalne tečne hromatografije (mixed‐mode liquid chromatography (MMLC)) koja podrazumeva više separacionih modaliteta inkorporiranih u jednu stacionarnu fazu, omogućava razdvajanje složenih uzorka jedinstvenom analizom. Brojni faktori utiču na MMLC separaciju, što razvoj metoda čini zahtevnim i ograničava im praktičnu primenu (2). Izgradnja prediktivnih matematičkih modela, kao što su modeli kvantitativnog odnos strukture i retencionog ponašanja (Quantitative structure‐retention relationship (QSRR)), može ubrzati razvoj metode. QSRR povezuje fizičko-hemijska svojstva (molekulski deskriptori (MD)) sa retencionim ponašanjem molekula, što omogućava predviđanje retencionog ponašanja neispitanih analita. Uključivanje vrednosti eksperimentalnih parametara u QSRR, proširuje prediktivnost modela na ceo eksperimentalni prostor (3). Podaci o retencionom ponašanju za potrebe razvoja QSRR modela, dobijeni su upotrebom Thermo Acclaim Mixed‐Mode WCX‐1 (3 μm; 2,1x150 mm) kolone koja uključuje hirdrofobne i interakcije slabe katjonske izmene (weak cation exchange (WCX)). Malim prečnikom kolone omoguć en je nizak protok i utrošak mobilne faze (400 μl/min). Sastav mobilne faze (udeo ACN (30 – 50 % (v/v)), pH (3,8 – 5,6) i jonska jačina (20 – 40 mM) acetatnog pufera) i temperatura kolone (30 – 38 °C) menjani su u skladu sa centralnim kompozicionim dizajnom – ka centru orijentisanim. Praćen je retencioni faktor 33 farmaceutska jedinjenja različitih farmakoloških i jonizacionih osobina. MD su računati AlvaDesc softverom. Za izgradnju QSRR modela RapidMiner softverom razmatrana je nekolicina algoritama mašinskog učenja, a odabrani su najinformativniji (gradient boosted trees (GBT) i bagging neural networks (BNN)). Modeli su građeni na osnovu podataka za 30 analita, dok su preostala tri (anjon, katjon, neutralni) odabrani za test set. Selekcijom unapred odabrani su najznačajniji MD za izgradnju BNN, za razliku od GBT koji ne zahteva preselekciju MD. Interna procena modela vršena je desetostrukom unakrsnom validacijom (10‐fold cross‐validation), dok je eksterna vršena test setom podataka. Modeli su upoređeni na osnovu relativne srednje kvadratne greške (RMSE) test seta. BNN (RMSE = 0,104; R 2 = 0,976) se pokazao boljim u poređenju sa GBT (RMSE = 0,112; R 2 = 0,963). Dobijeni QSRR modeli pokazali su dobru sposobnost predviđanja retencionog ponašanja molekula različitih jonizacionih sposobnosti u RP/WCX sistemu. Tako bi mogao da se unapredi razvoj MMLC metoda i učini ih pristupačnijim za praktičnu upotrebu.Drugi naučni simpozijum Saveza farmaceutskih udruženja Srbije sa međunarodnim učešćem, 28. 10. 2021. Beogra

Nove strategije u razvoju ekološki prihvatljivih RP‐HPLC metoda primenom detektora naelektrisanja u aerosolu

Qualitative and quantitative assessment of drug substances and drug dosage forms is of utmost importance for the quality control in pharmaceutical industry. Among the analytical techniques available, high performance liquid chromatography (HPLC) stands out due to its high efficiency and robustness. But, HPLC is also characterized by high consumption of toxic organic solvents used both for method development and routine analyses. These solvents could harmfully impact the environment and human health. Therefore, various strategies are developing for attracting eco-friendly character to HPLC. Recently introduced green chromatography concept is based on the 3R rule (Reduce – Replace – Recycle) implying the HPLC method development governed by the decreased use of acetonitrile labeled as the most commonly used HPLC solvent and its replacement with ethanol or acetone (1). However, these greener alternatives are known for their high UV absorbtion cut-off values making them unsuitable for common UV/VIS detection. As appropriate solvent compatible solution, the use of Corona Charged Aerosol Detector (CAD) is proposed. CAD is recognized as a universal detector providing persistent mass sensitive analyte response independent of chemical structure (no need for UV/VIS chromofores or ionization ability). The detection principle involves the nebulization of chromatographic mobile phase, the evaporation of aerosol droplets and finally the production of charged analyte particles (2). Assuming that chromatographic behavior and CAD generated responses of analytes could be affected by various mobile phase related factors and/or detector parameters settings, design of experiments (DoE) supported HPLC-CAD method development was performed for the analysis of antipsychotic drug risperidone in presence of its impurities. By carefully planning the number and the order of performing experiments, DoE methodology enables investigation of significance effects of multiple factor effects together with their factor interactions. Different experimental settings involving variation of organic solvent type and content in range 15-25% (V/V), mobile phase flow rate (0.50-1.00 mL min -1) and column temperature (25-50 °C) were examined in accordance with Box-Behnken design and selectivity factor between adjacent peaks on the chromatogram was measured. The optimal conditions were defined applying mucticriteria decision making approach by means of Derringer desirability function evaluation. Optimal separations were achieved using 20% (V/V) of ethanol in mobile phase flowing at 0.6 mL min -1 under 37.5 °C column temperature, while in case of acetone, optimum comprised 17% (V/V) of organic modifier, 0.8 mL min -1 and 37.5 °C. Finally, eco-friendly character of both methods was rated by Green Analytical Procedure Index, GAPI (3).Kvalitativna i kvantitativna analiza farmaceutskih supstanci i doziranih oblika predstavlja činioce od najvećeg značaja za kontrolu kvaliteta u farmaceutskoj industriji. Među dostupnim analitičkim tehnikama, tečna hromatografija pod visokim pritiskom (HPLC) ističe se svojom visokom efikasnošć u i robusnošću. Ali, HPLC takođe karakteriše velika potrošnja toksičnih organskih rastvarača koji se koriste za razvoj metoda i za rutinsku analizu lekova. Ovi rastvarači mogu štetno uticati i na životnu sredinu i na zdravlje ljudi. Zbog toga se razvijaju različite strategije za obezbeđivanje ekološki prihvatljivog karaktera HPLC metode. Nedavno predstavljeni koncept zelene hromatografije zasnovan je na pravilu 3 R (Smanji - Zameni - Recikliraj), što podrazumeva razvoj HPLC metode koji se rukovodi smanjenom upotrebom acetonitrila kao najčešće korišć enog rastvarača i njegovu zamenu etanolom ili acetonom (1). Međutim, ove zelenije alternative poznate su po visokim vrednostima za UV apsorpciju, što ih čini neprikladnim za uobičajenu UV/VIS detekciju. Kao odgovarajuće rešenje kompatibilno sa pomenutim rastvaračima, predlaže se upotreba detektora naelektrisanja u aerosolu (CAD). CAD je prepoznat kao univerzalni detektor koji pruža postojan odgovor zavistan od mase analita, a nezavisan od hemijske strukture (postojanje UV/VIS hromofora ili sposobnosti jonizacije). Princip CAD detekcije uključuje raspršivanje hromatografske mobilne faze u vidu kapljica aerosola, njihovo isparavanje i konačno naelektrisavanje dobijenih čestica analita (2). Pretpostavljajuć i da na hromatografsko ponašanje i CAD generisane odgovore analita mogu uticati različiti faktori povezani sa mobilnom fazom i/ili parametrima podešavanja detektora, izvršen je razvoj HPLC-CAD metode za analizu antipsihotika risperidona u prisustvu njegovih nečistoća uz podršku metodologije dizajna eksperimenata (DoE). Pažljivim planiranjem broja i redosleda izvođenja eksperimenata, DoE omoguć ava ispitivanje značaja efekata više faktora istovremeno zajedno sa njihovim faktorskim interakcijama. Različite eksperimentalne postavke koje uključuju varijacije tipa i sadržaja organskog rastvarača u opsegu 15- 25% (V/V), protok mobilne faze (0,50-1,00 mL min -1) i temperaturu kolone (25-50 °C) ispitivane su u skladu sa Boks-Behnken-ovim dizajnom i faktor selektivnosti između susednih vrhova na hromatogramu je praćen. Optimalni uslovi definisani su primenom pristupa mutikriterijumskog odlučivanja baziranog na izračunavanju Derringer-ove funkcije poželjnosti. Optimalna separacija je postignuta primenom 20% (V/V) etanola u mobilnoj fazi koja teče pri 0,6 mL min -1 i pri temperaturi kolone od 37,5 °C, dok je u slučaju acetona optimum podrazumevao 17% (V/V) organskog modifikatora, protok 0,8 mL min -1 i temperaturu 37,5 °C. Konačno, ekološki karakter obe metode procenjen je na osnovu indeksa zelenih analitičkih procedura, GAPI (3).Drugi naučni simpozijum Saveza farmaceutskih udruženja Srbije sa međunarodnim učešćem, 28. 10. 2021. Beogra