Influence of artichoke leaf extract on atorvastatin metabolism and optimization of liquid chromatography methods for determination of atorvastatin and metabolites in biological material

Atorvastatin pripada grupi antihiperlipidemijskih lekova poznatih pod nazivom statini.Osnovni mehanizam delovanja ovih lekova je specifična, kompetitivna i reverzibilnainhibicija HMG-CoA reduktaze, enzima koji je značajan za proces biosinteze holesterola.Osim direktnog dejstva na biosintezu holesterola, atorvastatin pokazuje niz plejotropnihefekata koji imaju povoljno dejstvo na aterosklerotske promene. Atorvastatin metaboliše ujetri pod dejstvom CYP3A4 enzima pri čemu nastaju dva aktivna metabolita: ortohidroksiatorvastatini para-hidroksiatorvastatin i tri neaktivna metabolita: atorvastatin-lakton,orto-hidroksiatorvastatin-lakton i para-hidroksiatorvastatin-lakton.Artičoka (Cynara scolymus L., Asteraceae) je zeljasta, jednogodišnja biljkarasprostranjena širom Mediterana. Terapija hiperlipidemija, pored lekova uključuje inefarmakološke mere (promena ishrane, fizičke vežbe, prestanak pušenja i unošenja alkohola,primena biljnih ekstrakata). Obzirom da su svi delovi artičoke bogati jedinjenjima koja seponašaju kao prirodni antioksidansi, ekstrakt lista artičoke i atorvastatin se često u terapijiprimenjuju zajedno.Cilj doktorske disertacije je da se ispita uticaj vodeno-etanolnog ekstrakta lista artičoke(Tinctura Cynarae) na metabolizam atorvastatina i opravdanost istovremene primeneatorvastatina i navedenog biljnog ekstrakta. Za ispitivanja su korišćene komercijalne biljnekapi na bazi artičoke u kojima je kvantifikovan sadržaj cinarina i hlorogenske kiselineprimenom HPLC metode (određen sadržaj obe komponente je 0,2 %).Eksperimentalni rad je zasnovan na određivanju i praćenju promene koncentracijemetabolita atorvastatina: orto-hidroksiatorvastatina, para-hidroksiatorvastatina i atorvastatinlaktonau uzorcima plazme eksperimentalnih životinja koje su bile na aterogenoj ishrani i kojesu primale atorvastatin kao monoterapiju ili zajedno sa ekstraktom lista artičoke. Za izvođenjeeksperimenta korišćeni su pacovi soja Wistar. Životinje su podeljene u 5 grupa koje su bile narazličitoj ishrani i/ili terapiji. Jedna grupa životinja je tokom celog eksperimenta bila nanormalnoj ishrani, dok su preostale četiri grupe do kraja eksperimenta bile na aterogenojishrani. U skladu sa prethodno postavljenim planom eksperimenta, različite grupe životinja naaterogenoj ishrani su, posle određenog vremena, primale atorvastatin, ili atorvastatin i ekstraktlista artičoke, ili samo ekstrakt lista artičoke...Atorvastatin belongs to the group of antihyperlipidemic drugs known as statins. Thebasic mechanism of action of these drugs is specific, competitive and reversible inhibition ofHMG-CoA reductase, an enzyme involved in cholesterol biosynthesis. In addition,atorvastatin shows many beneficial pleiotropic effects. Atorvastatin is metabolized in the liverunder the influence of the CYP3A4 enzyme to two active metabolites: orthohydroxyatorvastatinand para-hydroxyatorvastatin and three inactive metabolites: orthohydroxyatorvastatinlactone, para-hydroxyatorvastatin lactone and atorvastatin lactone.Artichoke (Cynara scolymus L., Asteraceae) is annual plant, distributed throughoutMediterranean. The treatment of hyperlipidemia, in addition to pharmacotherapy, includesnonpharmacological measures (diet changes, physical activity, cessation of smoking andalcohol intake, use of herbal extracts). Since all the parts of artichoke plant are rich in naturalantioxidants, artichoke leaf extract and atorvastatin are often used together.The Ph. D. disertation is focused to investigate the effect of aqueous-ethanolic extractof artichoke (Tinctura Cynarae) on the metabolism of atorvastatin and justification of coadministrationof atorvastatin and herbal extract mentioned above. Experiments wereconducted using commercial artichoke preparation. The contents of chlorogenic acid andcynarin in Tinctura Cynarae were quantified by means of the HPLC method (determinedcontent of both components is 0.2 %).Experimental work is based on determination and monitoring changes in theconcentration of atorvastatin metabolites: ortho-hydroxyatorvastatin, parahydroxyatorvastatinand atorvastatin lactone in the plasma samples of experimental animalsfed with atherogenic diet and treated with atorvastatin (alone or in combination with artichokeleaf extract). Experiments were conducted on experimental animals (Wistar rats). The animalswere divided into 5 groups on different diet and/or therapy. One group of animals was onnormal diet all the time during experiment, and the remaining four groups were on theatherogenic diet. Three of these four groups were treated with: atorvastatin, artichoke leafextract and the combination of atorvastatin and artichoke leaf extract..

Trendovi u dizajniranju antidepresiva i anksiolitika

Depression constitutes an international health problem. It is estimated that 320 million people worldwide are clinically depressed. Effective antidepressant treatments have been available for more than 40 years. Although modern antidepressants lack many of the side-effects and toxicity of the first generation tricyclics and monoamine oxidase inhibitors, their efficiency has not been substantially improved comparing to older agents. Unfortunately, the modern drug discovery technologies of combinatorial chemistry and throughput screening are based on relatively simple in vitro assays and lack pre-clinical model of depression make this research difficult. Most antidepressants require additional time to achieve therapeutic effects and there is a certain population of patients resistant to current therapies which means that etiology of depression is too complex. Coming research should clarify true depression mechanism and identify new targets of the third antidepressants generation. Anxiety is a normal emotion and essential part of response to stressful and threatening stimuli. At present, a numerous drugs with various mechanism of action are available for pharmacoterapy of anxiety disorders. The benzodiazepines represent the most important drugs in the treatment of anxiety. In addition to their anxiolytic properties, benzodiazepines possess sedative, hypnotic, anticonvulsant and other effects but side effects also-upon prolonged administration they produce withdrawal symptoms and dependency. Research has shown that cholecystokinin and corticotrophin-releasing factor are important neurotransmiters and may have role in the mediation of anxiety. Neuropeptide Y has shown an anxiolytic effect in several animal models. CCK, CRF and neuropeptid Y receptor agonists and antagonists are targets of new anxiolitic drugs.Anksioznost je normalna emocionalna reakcija na preteći ili stresni stimulans. U farmakoterapiji ove bolesti koristi se veliki broj lekova različitog mehanizma delovanja od kojih su benzodiazepini najvažniji. Benzodiazepini, pored anksiolitičkog poseduju sedativni, hipnotički, antikonvulzivni i druge efekte ali posle duže upotrebe razvija se tolerancija, dovode do zavisnosti i često su predmet zloupotrebe. Novija istraživanja su pokazala da, pored serotonina i noradrenalina i drugi neurotransmiteri kao što su holecistokinin (CCK) i oslobađajući faktor za kortikotropin (CRF) imaju značajnu ulogu u anksioznosti. Neuropeptid Y (NPY) takođe pokazuje anksiolitički efekat na životinjama. Agonisti i antagonisti receptora holecistokinina, oslobađajućeg faktora za kortikotropin i neuropeptida Y su cilj sinteze novih anksiolitika

Doking studije nekih jedinjenja sa pirazolom u aktivnom mestu ciklooksigenaze-2

Whereas nonselective nonsteroidal anti-inflammatory drugs, such as aspirin, ibuprofen and diclofenac, inhibit both cyclooxygenase-1 and cyclooxigenase-2 enzymes, selective inhibitors target cyclooxygenase-2, which is overexpressed in inflammation, but also in cancer, atherosclerosis, Alzheimer's disease, and Parkinson`s disease. Potential cardiovascular and hepatic side effects of cyclooxygenase-2 inhibitors have limited their use. The development of selective and safe cyclooxygenase-2 inhibitors remains a high priority in drug discovery. Based on the structure of previously investigated newly synthesized β-hydroxy-β-arylpropanoic acids, two groups of compounds were designed: analogs in which one of the benzene rings was replaced by a pyrazole, while the carboxyl group was retained, and amides of β-hydroxy-β-arylpropanoic acids with pyrazole. The compounds were docked into the 3D structure of the catalytic site of the enzyme cyclooxygenase-2 using AutoDock Vina 1.2.0. and the obtained interactions were compared with the interactions of celecoxib, a selective inhibitor. The amides had lower binding energies than the designed acids, which makes them attractive target compounds for synthesis and further examination.Neselektivni nesteroidni antiinflamatorni lekovi poput aspirina, ibuprofena i diklofenaka inhibiraju enzime ciklooksigenazu-1 i ciklooksigenazu-2, a selektivni inhibitori ciljaju ciklooksigenazu-2 koja je prekomerno izražena u inflamaciji, ali takođe i kod kancera, ateroskleroze, Parkinsonove i Alchajmerove bolesti. Potencijalni kardiovaskularni i hepatički neželjeni efekti selektivnih inhibitora ciklooksigenaze-2 su ograničili njihovu primenu. Razvoj selektivnih i bezbednih inhibitora ciklooksigenaze-2 ostaje veoma prioritetna oblast u otkrivanju lekova. Na osnovu strukture prethodno istraživanih novosintetisanih β-hidroksi-β-arilpropanskih kiselina dizajnirane su dve grupe jedinjenja: analozi u kojima je jedan od benzenovih prstenova zamenjen pirazolom, uz zadržavanje karboksilne grupe, i amidi β-hidroksi-β-arilpropanskih kiselina sa pirazolom. Program AutoDock Vina 1.2.0 je korišćen za dokovanje dizajniranih jedinjenja u 3D strukturu katalitičkog mesta enzima ciklooksigenaze-2, a ostvarene interakcije su upoređene sa interakcijama koje ostvaruje selektivni inhibitor celekoksib. Amidi su imali nižu energiju vezivanja od kiselina, što ih čini dobrim kandidatima za sintezu

Development and validation of RP-HPLC method for quantification of trace levels of topical corticosteroids in ambiphilic cream

Corticosteroids are anti-inflammatory and immunosuppressant drugs. Topical corticosteroids formulations (ointments, creams, gels) are used in the treatment of different types of dermatitis and urticaria. Considering their therapeutic and whitening effects, they are frequently used for counterfeiting of cosmetic products. Corticosteroids can cause different local and systemic side effects. HPLC method is often chosen for their analysis, because it is selective, sensitive, precise, simple and fast. The aim of this study was optimization and validation of RP-HPLC method with UV detection for determination of trace levels of corticosteroids in ambiphilic creams. This method is used for qualitative and quantitative analysis of evaluated corticosteroids. Mometasone furoate, hydrocortisone acetate, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone dipropionate and triamcinolone acetonide were evaluated. Separation was performed on Inertsil® ODS-3V 250 × 4.6 mm, 5 μm chromatographic column. Mobile phase was mixture of acetonitrile and water 50:50 (v/v) with gradient elution and flow rate 1 mL min-1. Column temperature was held on 40 °C and UV detection was performed at 240 nm. Selectivity, linearity, accuracy, precision and limit of quantification (LOQ) were evaluated. Method is selective because ambiphilic cream base peaks and corticosteroids peaks were not overlapping. Linearity was confirmed since correlation coefficient was 1 for all compounds. Accuracy and precision were evaluated for hydrocortisone acetate and betamethasone dipropionate. Determined Recovery values were in range of 70-130%. Both RSD values (21.46% and 9.59%) were lower than 30%. Method is highly sensitive since LOQ concentrations were in ng mL-1 range. All evaluated parameters of validation were in accordance with regulatory requirements. Validated RP-HPLC method can be used for qualitative and quantitative analysis of selected corticosteroids in ambiphilic creams

Antiaritmički efekti novosintetisanih derivata propafenona

It is well known that the presence of different chemical groups in drug molecules influences their pharmacological properties. The aim of our study is to investigate whether newly synthesized derivatives of propafenone, with changes in benzyl moiety, have a different effect upon arrhythmia, compared to propafenone. 5OCl-PF and 5OF-PF are derivatives of propafenone with-Cl or –F substituent on the ortho position of the benzyl moiety. For verification of their antiarrhythmic effect, we used an in vivo rat model of aconitine-induced arrhythmia. 5OCl-PF speeded the appearance of supraventricular premature beats (SVPB) and death more than aconitine. All animals treated with 5OCl-PF developed ventricular premature beats in salvos (VPBS), bigeminies (VPBB) and paroxysmal ventricular tachycardia (PVT). 5OF-PF had a negative chronotropic effect and potentiated atrial excitability (more SVPB). It had a positive effect on the occurrence and onset time of supraventricular tachycardia, VPBS, and PVT. Based on the obtained results, it can be concluded that newly synthesized propafenone derivatives have no better antiarrhythmic effect than the parent compound. In the future, our research will be focused on the synthesis of different derivatives and examining their antiarrhythmic effects.Dobro je poznato da prisustvo različitih hemijskih grupa u molekulima leka utiče na njegova farmakološka svojstva. Cilj našeg istraživanja je ispitati da li novosintetisani derivati propafenona, s promenama u benzilnoj grupi, imaju drugačiji efekat na aritmiju u odnosu na propafenon. 5OCl-PF i 5OF-PF su derivati propafenona sa -Cl ili –F supstituentom na orto položaju benzilnog dela. Za proveru njihovog antiaritmičnog efekta koristili smo in vivo model na pacovima sa aritmijom izazvanom akonitinom. 5OCl-PF je ubrzao pojavu supraventrikularnih prevremenih otkucaja (SVPB) i smrt više nego akonitin. Sve životinje lečene sa 5OCl-PF razvile su ventrikularne prevremene otkucaje (VPBS i VPBB) i paroksizmalnu ventrikularnu tahikardiju (PVT). 5OF-PF je imao negativan hronotropni efekat i potencirao atrijalnu ekscitabilnost (više SVPB). Pozitivno je uticao na pojavu i vreme početka supraventrikularne tahikardije, VPBS i PVT. Na osnovu dobijenih rezultata se može zaključiti da novosintetisani derivati propafenona nemaju bolji antiaritmijski efekat od polaznog jedinjenja. U budućnosti, istraživanje će biti usmereno ka sintezi hemijski drugačijih derivata i ispitivanju njihovog antiaritmijskog efekta

Primena LC‐MS/MS metoda u ispitivanju parametara oksidativnog stresa

Oxidative stress is a phenomenon that occurs due to the disturbance in the balance between the production of reactive oxygen species and the ability of biological systems to remove the resulting compounds. Oxidative stress is involved in the pathogenesis of many disorders in the human organism. This indicates the importance of quantification of oxidative stress parameters in biological samples. Traditionally, these parameters are determined by biochemical tests. Although these tests are routinely performed, they have many drawbacks. To determine the exact concentration of selected compounds, more sensitive analytical methods are becoming more important. In the modern scientific literature, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly mentioned (1). This method, with adequate selection of stationary and mobile phases, enables quantification of very low concentrations of selected parameters. In addition, these methods can simultaneously determine the concentration of many selected components. However, it is necessary to take into account that LC-MS/MS methods require a very purified biological sample from which the proteins have been maximally removed. In this work, we will present the results of determination of cysteine, cystine, reduced and oxidized glutathione in patients with microcellular lung cancer. The use of LC-MS/MS methods is becoming increasingly common for the analysis of oxidative stress markers in biological fluids. In the future, we are expected to move to even more modern, fully automated methods, which simultaneously purify and analyze samples.Oksidativni stres je pojava koja nastaje usled narušavanja ravnoteže između proizvodnje i nagomilavanja reaktivnih jedinjenja kiseonika u organizmu i sposobnosti bioloških sistema da ukloni nastala jedinjenja. Oksidativni stres učestvuje u patogenezi mnogih poremećaja kao što su kardiovaskularne bolesti, dijabetes i bolesti bubrega. Ovo ukazuje na značaj određivanja odabranih parametara oksidativnog stresa u uzorcima biološkog materijala. Tradicionalno, najveći broj parametara se određuje biohemijskim testovima. Iako se ovi testovi rutinski izvode, oni imaju mnoge nedostatke. Da bi se odredila tačna koncentracija izabranih parametara, koji se u uzorcima nalaze u veoma niskim koncentracijama, sve veću prednost imaju osetljivije analitičke metode. U savremenoj naučnoj literaturi se u te svrhe sve više spominje tečna hromatografija spregnuta sa masenim detektorom (LC-MS/MS) (1). Ova metoda, uz adekvatan izbor stacionarne i mobilne faze, omogućava kvantifikaciju veoma niskih koncentracija odabranih jedinjenja. Sem toga, ovim metodama se istovremeno, u jednom uzorku, može odrediti koncentracija većeg broja odabranih komponenti. Ipak, neophodno je voditi računa o tome da tečna hromatografija spregnuta sa masenim detektorom zahteva dobro prečišćen uzorak biološkog materijala iz kog su maksimalno uklonjeni proteini. U ovom radu biće prikazani rezultati određivanja cisteina, cistina, redukovanog i oksidovanog glutationa kod pacijenata sa mikrocelularnim karcinomom pluća. Upotreba LC-MS/MS metoda je sve uobičajenija za analizu markera oksidativnog stresa u biološkim tečnostima zbog svoje osetljivosti. U budućnosti se očekuje razvoj ka još savremenijim, potpuno automatizovanim metodama, kojima se istovremeno uzorci prečišćavaju i analiziraju.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

Hemijska stabilnost lekova - uticaj svetlosti i temperature na stabilnost montelukasta u rastvoru

In this paper, influence of temperature and light on stability of montelukast in solutions (standard substance, chewable tablets and film coated tablets) which were prepared during routine analysis of related substances was investigated. The content of montelukast and degradation products was determined by HPLC method at defined time intervals (influence of temperature was tested at the beginning, after 24 hours, and after 48 hours, and influence of light was tested at the beginning of the experiment and after 12 hours). Stability of montelukast in tested solutions under influence of temperature was acceptable. The content of montelukast ranged from 98 % to100 %, which meets the pharmacopoeia requirements and specifications. Individual and total impurities were within acceptable limits. Presence of photosensitive functional groups in structure of montelukast led to significant photodegradation. Individual and total impurities in samples which were under influence of light were above acceptable limits. Photodegradation occured during first 12 hours. Content of MOK-3 sulphoxide and unknown impurity on RRT 0.76 was above limits in all examined samples. Over a period of 12 h the content of those impurities grew 8-40 fold compared to time zero values.U radu je dat prikaz ispitivanja uticaja temperature i svetlosti na stabilnost montelukasta u rastvorima (standardne supstance, tabletama za žvakanje i filmom obloženih tableta) koji se pripremaju u rutinskoj analizi u toku postupka ispitivanja prisustva srodnih supstanci. Sadržaj montelukasta i nastalih degradacionih proizvoda praćen je primenom RP-HPLC metode u definisanim vremenskim intervalima (0, 24 i 48h kada je u pitanju temperatura, odnosno 0 i 12h kada je u pitanju svetlost). Rezultati ispitivanja uticaja temperature, kao spoljašnjeg faktora nestabilnosti, ukazuju da su analizirani rastvori standarda montelukasta, tableta za žvakanje i filmom obloženih tableta stabilni u prihvatljivim granicama. Sadržaj montelukasta se kreće od 98 % - 100 % što zadovoljava farmakopejske zahteve, kao i zahteve specifikacije proizvoda. Pojedinačne i ukupne nečistoće su u dozvoljenim granicama. Prisustvo fotoreaktivnih funkcionalnih grupa u strukturi montelukasta uslovilo je nastanak fotodegradacionih proizvoda u količinama koje su izvan granica definisanih u monografiji montelukasta, kao i granica definisanih u specifikaciji gotovog proizvoda. Do fotodegradacije dolazi već u toku prvih 12 h. Porast sadržaja MOK-3 sulfoksida i nečistoće sa relativnim retencionim vremenom oko 0,76 zabeležen je u sva tri ispitivana uzorka. U vremenskom intervalu od 12 h uočen je porast sadržaja navedenih nečistoća 8-40 puta u poređenju sa vrednostima u početnom vremenu ispitivanja

Estimation of lipophilicity of newly synthesized potential COX-2 and 5-LOX inhibitors using RP-HPLC analysis

The chronic inflammatory process is associated with the development and progression of many diseases such as cancer, arthritis, autoimmune diseases, etc. Dual COX-2 and 5-LOX inhibitors have been developed to provide more potent anti-inflammatory agents with a better safety profile [1]. Nineteen newly synthesised potential dual COX-2 and 5-LOX inhibitors [2] were selected and their retention properties were tested in different RP-HPLC systems. RP-HPLC analysis was performed using a C18, a cyano and an amino column. The mobile phases were binary combinations of acetonitrile and phosphate buffer (pH 5.5 and pH 7.4) as well as methanol and phosphate buffer (pH 5.5 and pH 7.4) in different ratios. The amino column proved to be inadequate as the tested compounds were not retained on it. According to preliminary results, methanol was not suitable as an organic modifier in the mobile phase as the retention time of the compounds was increased several times compared to acetonitrile. Acetonitrile was chosen as the organic modifier. The logarithmic values of the retention factors (logk) were calculated for each sample and plotted with the percentage of organic modifier in the mobile phase. The chromatography parameters logkw, a and ϕ0 were determined according to equation (1) and equation (2) (S-percentage of organic modifier in the mobile phase). Logk = logkw + aS (1) ϕ0 = - logkw/a (2) On C18 and cyano columns, seven compounds (1A, 1G, 1ME, 1PE, and 2D) had no retention properties, which is consistent with their very low logD values, while three (1F, 1H, and QSAR17) compounds were outlayers. Systems consisting of C18 and cyano columns (on both pH 5.5 and 7.4) showed good correlation coefficients between the chromatography parameters logkw, a, and ϕ0 and the logD values determined with MarvinSketch. The most suitable system consisted of a C18 column, acetonitrile, and phosphate buffer pH 7.4, as it had the highest correlation coefficient between the chromatography parameter logkw and the logD value (R2=0.9023) (3 compounds were discarded)

Razvoj i validacija metode tečne hromatografije spregnute sa masenom spektrometrijom za određivanje sadržaja rivaroksabana u uzorcima plazme

Rivaroxaban belongs to the group of direct oral anticoagulants (DOAC), drugs used to prevent and treat venous thrombosis and venous thromboembolism. Drugs from this group are considered safer than vitamin K antagonists. In case of overdose, their most significant side effect is bleeding. Given the great toxicological significance, it is very important to develop an analytical method for quantification of this drug in biological samples. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of rivaroxaban content in plasma samples was optimized and validated. Plasma samples were prepared by protein precipitation with cold acetonitrile. Carbamazepine was used as an internal standard. The analysis was performed on an Infinity Lab Poroshell 120 EC-C18, 4.6 ×100 mm, 2.7 μm chromatographic column. The mobile phase consisted of acetonitrile and 0.1% formic acid (50:50, v/v), at a flow rate of 400 μL/min and a column temperature set at 30°C. The autosampler temperature was 4C. The injection volume was 10 μL. Detection of analytes and internal standard was performed in multireaction monitoring mode (MRM), at the following ion transitions: 437>145 (m/z) for rivaroxaban, and 237>194 (m/z) for the internal standard. The optimized method was validated and the obtained parameters indicate that the method is sensitive, specific, selective, precise and accurate. The samples were stable under the tested conditions. A validated method has been used to determine the concentration of rivaroxaban in plasma samples of patients with atrial fibrillation who were hospitalized under strict medical supervision. Obtained concentrations were in the expected range.Rivaroksaban pripada grupi direktnih oralnih antikoagulanasa (DOAK), lekova koji se koriste za prevenciju i lečenje venske tromboze i venske tromboembolije. Lekovi iz ove grupe se smatraju bezbednijim od antagonista vitamina K. U slučaju predoziranja, njihov najznačajniji neželjeni efekat je krvarenje. S obzirom na veliki toksikološki značaj, veoma je važno postojanje analitičke metode za određivanje sadržaja ovog leka u uzorcima biološkog materijala. Optimizovana je i validirana metoda tečne hromatografije spregnute sa masenom spektrometrijom (LC-MS/MS) za određivanje sadržaja rivaroksabana u uzorcima plazme. Uzorci plazme su pripremani metodom precipitacije proteina koja je vršena hladnim acetonitrilom. Kao interni standard korišćen je karbamazepin. Analiza je izvršena na Infinity Lab Poroshell 120 EC-C18, 4,6×100 mm, 2,7 μm hromatografskoj koloni. Mobilna faza se sastojala od acetonitrila i 0,1% mravlje kiseline (50:50, v/v), pri protoku od 400 μL/min i temperaturi kolone podešenoj na 30°C. Temperatura autosemplera je bila 4C. Injekciona zapremina je bila 10 μL. Detekcija analita i internog standarda je izvršena u multireakcionom monitoring modu (MRM), pri sledećim jonskim prelazima: 437>145 (m/z) za rivaroksaban, odnosno 237>194 (m/z) za interni standard. Optimizovana metoda je validirana i dobijeni parametri ukazuju da je metoda osetljiva, specifična, selektivna, precizna i tačna. Uzorci su stabilni pri ispitivanim uslovima. Validirana metoda je primenjena za određivanje koncentracije rivaroksabana u uzorcima plazme pacijenata sa atrijalnom fibrilacijom, koji su bili hospitalizovani, pod strogim medicinskim nadzorom. Određene koncentracije su bile u očekivanom opsegu.VIII Kongres farmaceuta Srbije sa međunarodnim učešćem, 12-15.10.2022. Beogra

ex vivo–in vivo comparison of drug penetration analysis by confocal Raman microspectroscopy and tape stripping

When it comes to skin penetration analysis of a topically applied formulation, the number of suitable methods is limited, and they often lack in spatial resolution. In vivo studies are pivotal, especially in the approval of a new product, but high costs and ethical difficulties are limiting factors. For that reason, good ex vivo models for testing skin penetration are crucial. In this study, caffeine was used as a hydrophilic model drug, applied as a 2% (w/w) hydrogel, to compare different techniques for skin penetration analysis. Confocal Raman microspectroscopy (CRM) and tape stripping with subsequent HPLC analysis were used to quantify caffeine. Experiments were performed ex vivo and in vivo. Furthermore, the effect of 5% (w/w) 1,2-pentanediol on caffeine skin penetration was tested, to compare those methods regarding their effectiveness in detecting differences between both formulations