Farmaceutyczny. Przegląd Naukowy. Scientific Review in Pharmacy. lat. Cena 24,50 zł

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1 Miesięcznik copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN Farmaceutyczny Cena 24,50 zł Przegląd Naukowy IC Value - Current 4,55 MNiSW 6 ROK VII (XI) Nr 9/2010 (68) Scientific Review in Pharmacy Valdecoxib stability properties under forced degradation conditions Zastosowanie elektroforezy kapilarnej do analizy potencjalnych radiofarmaceutyków do wczesnej diagnostyki choroby Alzheimera Zmienność cech fenotypowych w szczepach Candida albicans wrażliwych i opornych na azole Przydatność technik przesiewowych SSCP i DGGE w identyfikacji mutacji w zespole long-qt ISSN lat Oddziaływanie bakteryjnej endotoksyny 0 z zainfekowanym makroorganizmem0 0 Analiza termiczna wybranych substancji pomocniczych, stosowanych w procesie formulacji produktów leczniczych Ryzyko niepożądanych działań wybranych niesteroidowych leków przeciwzapalnych (aspiryna) 1

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3 Redaktor Naczelny / Editor-in-Chief: Prof. dr hab. Krystyna Olczyk Adres redakcji / Editorial Adress: ul. Jedności 8, Sosnowiec, Polska / Poland Tel , Fax kolegium.redakcyjne@kwiecinski.pl Konsultacyjna Rada Naukowa / Scientific Board Przewodniczący / Head: Prof. dr hab. Krystyna Olczyk - Sosnowiec Członkowie / Members: 6 Czasopismo jest indeksowane w bazie Scopus. Prof. dr hab. Edward Bańkowski - Białystok Prof. dr Karmela Barišić - Zagreb, Chorwacja Prof. dr hab. Jerzy Brandys - Kraków Prof. dr Vitalis Briedis - Kaunas, Litwa Prof. dr hab. Elżbieta Brzezińska - Łódź Prof. dr Benito Del Castillo Garcia - Madrid, Hiszpania Prof. dr. Lionel Buéno - Toulouse, Francja Prof. dr hab. Kazimierz Głowniak - Lublin Prof. dr hab. Edmund Grześkowiak - Poznań Prof. dr Filiz Hincal - Ankara, Turcja Prof. dr. Michael Horowitz - Adelaide, Australia Prof. dr med. Kinga Howorka - AKH, UW, Wien, Austria Sekretarz Naukowy / Scientific Board Secretary: Dr n. med. Robert D. Wojtyczka fpn@kwiecinski.pl Prof. dr hab. Renata Jachowicz - Kraków Prof. dr hab. Ewa Jagiełło-Wójtowicz - Lublin Prof. dr hab. Krzysztof Jonderko - Sosnowiec Prof. dr hab. Marcin Kamiński - Katowice Prof. dr Vesna Kuntić - Belgrade, Serbia Prof. dr hab. Jan Pachecka - Warszawa Prof. dr hab. Jerzy Pałka - Białystok Prof. dr hab. Janusz Pluta - Wrocław Prof. dr hab. Janusz Solski - Lublin Prof. dr Hiroshi Suzuki - Tokyo, Japonia Prof. dr hab. Yanusz Wegrowski - Reims, Francja Prof. dr hab. Marek Wesołowski - Gdańsk Prof. dr Mira Zečević - Belgrade, Serbia Członkowie Kolegium Redakcyjnego / Members of Editorial Board: Dr n. farm. Paweł Olczyk Dr n. biol. Małgorzata Kępa Mgr Anna Szeremeta Dr n. med. Agnieszka Jura Półtorak Dr n. hum. Anna Kierczak Mgr inż. Marcin Chabior Przemysław Jędrusik Wydawca / Publisher: Grupa dr. A. R. Kwiecińskiego 21 lat Adres Wydawcy / Publisher Adress: Grupa dr. A. R. Kwiecińskiego ul. Wiśniowa 25/2, Bielsko-Biała, Polska / Poland tel , fax Prezes / President: dr n. med. Adam Kwieciński (Ph.D. M.D.) Marketing Manager: Opracowanie graficzne / Graphics: Skład / Technical Editor: Agnieszka Romańska Robert Cyganik Jerzy Partyka agnieszka.romanska@kwiecinski.pl Nakład: do egz. / Print run: up to 7000 copies Farmaceutyczny Przegląd Naukowy jest współfinansowany przez Ministerstwo Nauki i Szkolnictwa Wyższego. Scientific Review in Pharmacy is financially supported by Ministry of Science and Higher Education. Wszystkie materiały opublikowane w piśmie objęte są ochroną Prawa autorskiego. Projekty chronione są Ustawą o Prawie autorskim i pokrewnych prawach z 1994 r. (Dz. U. Nr 24, poz. 83). Redakcja zastrzega sobie prawo dostosowania nadesłanych materiałów do potrzeb pisma. Przedruki możliwe jedynie za zgodą wydawcy. Za treść materiałów reklamowych oraz listów od czytelników redakcja nie odpowiada. All published papers in Scientific Review in Pharmacy are protected by copyright laws (according to Law Gazette NO 24, item. 83). Board of Editors reserves the rights to harmonize the papers obtained to journal rules and requirements. Reprints are allowed only after Publisher agreement. Board of Editors are not responsible for advertisements and reader letters.

4 Zdj. Zygmunt Wieczorek Szanowni Państwo, Koleżanki i Koledzy, Drodzy Czytelnicy Z radością informuję, iż Farmaceutyczny Przegląd Naukowy znalazł się w naukowej bazie danych SCOPUS, prowadzonej przez wydawnictwo Elsevier, i zawierającej streszczenia (abstrakty) artykułów z ponad czasopism naukowych, m.in. z zakresu nauk medycznych czy przyrodniczych. Nasza sprzed miesięcy aplikacja do Scopus Content Selection & Advisory Board (CSAB) została rozpatrzona pozytywnie. Fakt ten spowoduje podniesienie punktacji czasopisma w rankingu Index Copernicus, lecz przede wszystkim umiejscowi FPN w światowej bazie danych. Długa natomiast jeszcze droga do podniesienia punktacji czasopisma w rankingu MNiSW. Aby wzrosła punktacja ministerialna, artykuły publikowane w Farmaceutycznym Przeglądzie Naukowym muszą być cytowane w innych czasopismach. To ostatni a zarazem jedyny sposób by przekroczyć pułap sześciu punktów MNiSW. Zależy więc od nas samych czy piąć się będziemy w górę. Publikowanie prac w języku angielskim pozwoliłoby natomiast pomyśleć o drodze do uzyskania IF. Jest to przecież możliwe, tak jak stało się to przypadku Acta Poloniae Pharmaceutica Drug Research. W jaki jednak sposób przekonać naszych Autorów, by przygotowywali manuskrypty anglojęzyczne? A przed nami nowy rok akademicki, mnóstwo obowiązków dydaktycznych, i jak zawsze konieczność realizowania badań naukowych. Polecam zatem Farmaceutyczny Przegląd Naukowy jako przyjazne i szybko publikujące nadesłane manuskrypty czasopismo. Zachęcamy Autorów do korzystania z systemu elektronicznej rejestracji i przesyłania prac. Jest to łatwa i wygodna forma komunikowania się, oszczędzająca czas i dająca możliwość śledzenia on-line losów swojej publikacji. A teraz zapraszam do lektury bieżącego numeru. Zawiera on publikacje o zróżnicowanej tematyce, pochodzące z różnych ośrodków naukowych. Jak zawsze, zapowiadamy nadchodzącą konferencję. Tym razem będzie to konferencja poświęcona doskonaleniu jakości szkolnictwa wyższego, mobilności studentów i młodych pracowników nauki, relacjom między efektami kształcenia a potrzebami rynku pracy, która odbywać się będzie pod auspicjami Komisji Europejskiej, w Brukseli, w dniu 9 listopada br. Za nami natomiast liczne zjazdy, krajowe i międzynarodowe. Wielu z nas uczestniczyło w Zjeździe Polskiego Towarzystwa Farmaceutycznego w Gdańsku, w Zjeździe Polskiego Towarzystwa Diagnostyki Laboratoryjnej w Wiśle, czy w Zjeździe Polskiego Towarzystwa Biochemicznego, także w Wiśle. Wcześniej, na przełomie sierpnia i września braliśmy udział w Światowym Kongresie Farmacji i Nauk Farmaceutycznych (World Congress of Pharmacy and Pharmaceutical Sciences, FIP), który odbył się w Lizbonie. Zapowiadając kolejny numer naszego czasopisma, którego skład został już zaakceptowany przez Kolegium Redakcyjne, a i następne numery FPN są już szczęśliwie zapełnione (a tak martwiłam się kiedyś, że zabraknie nam prac), życzę wolnej chwili na przejrzenie tego co przed Państwem. Polecając życzliwej uwadze nasze czasopismo, załączam z wielką przyjemnością moc pozdrowień, Redaktor Naczelny Prof. dr hab. Krystyna Olczyk

5 6 Nr 9 / 2010 Spis treści Valdecoxib stability properties 0 under forced degradation conditions0 11 Zastosowanie elektroforezy kapilarnej do analizy potencjalnych radiofarmaceutyków do wczesnej diagnostyki choroby Alzheimera0 18 Capillary zone electrophoretic analysis of radiopharmaceuticals used in early diagnosis of Alzheimer s disease Zmienność cech fenotypowych w szczepach Candida albicans wrażliwych i opornych na azole 24 Phenotypic variability of Candida albicans 0 strains sensitive and resistant to azoles Przydatność technik przesiewowych SSCP i DGGE w identyfikacji mutacji w zespole long-qt 29 Utility of molecular screening techniques SSCP and DGGE in the detection of long-qt syndrome mutations Oddziaływanie bakteryjnej endotoksyny 0 z zainfekowanym makroorganizmem0 38 Interaction of bacterial endotoxin with infected macroorganism0 Analiza termiczna wybranych substancji pomocniczych, 0 stosowanych w procesie formulacji produktów leczniczych0 45 Thermal analysis of selected excipients 0 used in the formulation process of medicinal products Ryzyko niepożądanych działań wybranych niesteroidowych leków przeciwzapalnych (aspiryna) 0 51 Risk of adverse drug reaction of non steroidal anti inflammatory drugs (acetylsalicylic acid)0

6 An International Symposium for gathering knowledge, discussing the latest challenges and sharing best practices in youth policy and education quality and standards in Europe

7 According to latest EU figures, Five million young Europeans are looking for a job. Many of them will miss opportunities because they lack the right qualifications or experience. In response, the European Commission recently launched Youth on the Move, a new flagship initiative aimed at helping young people to gain the knowledge, skills and experience they need to make their first job a reality. Over the years, despite some notable developments at EU levels and the introduction of effective mechanisms such as the open method for coordination, there still remain many challenges ahead to reduce the high numbers of young people not participating in education, training or employment. The economic downturn in 2008 also exerted an additional negative impact on young people, the long term impact of which will be difficult to reverse. Recognising that education is by far the most important determinant of social and economic mobility, in order to prevent early school leaving, further active investment in education and training systems at EU and national levels is needed to encourage young people to be more actively engaged in the whole education and training cycle. Key to this process is enhancing the openness and relevance of the educational system in Europe in a way that not only appeals to young people but also meets the needs of the European labour market furthermore, bringing young people into the labour market will require an integrated and holistic approach through guidance, counselling and apprenticeships. As the EU seeks to deliver its Europe 2020 Strategy, this International Symposium will look at how we can build on current EU actions and existing educational cooperation initiatives, such as the Erasmus programme, through a wider framework that encourages more young people to participate in the full education cycle. The achievement of such a goal will empower young people to grasp better training and employment opportunities, encourage greater social cohesion and help to safeguard the future economic well-being within the European Union. The Centre for Parliamentary Studies welcomes the participation of all key partners, responsible authorities and stakeholders. The Symposium will support the exchange of ideas and encourage delegates to engage in thought-provoking topical debate.

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11 Farm Przegl Nauk, 2010,9, copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN Valdecoxib stability properties under forced degradation conditions Gordana Pejović 1, Biljana Otašević 2, Mira Zečević 2, Vesna Kuntić 2, Zorica Vujić 2 1 Medicines and Medical Devices Agency of Serbia, Vojvode Stepe 458, Belgrade 2 Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, Belgrade Abstract The present work describes forced degradation study of valdecoxib, under different ICH recommended stress conditions photolytic degradation, oxidation, hydrolysis in alkaline and acidic conditions as well as its thermal degradation. Stress studies were performed at valdecoxib drug substance and the final dosage form. Chromatographic separations of drug and the degradation products formed under various experimental conditions was successfully achieved on RP-18e Chromolith performance monolithic column (100 mm x 4.6 mm, macropore size 2 μm, mesopore size 13 nm) with an eluent containing the mixture of methanol and water solution of TEA (1 %, ph 7.4) in the ratio 40:60 (v/v) while the column temperature was adjusted to 30 C. Detection was performed using photodiode array detector on several wavelengths in order to ensure the proper insight into drug degradation pathway in case the formed products differ in absorption characteristics. Key words: valdecoxib, forced degradation studies, stability-indicating method Abbreviations: ICH International Conference for Harmonisation, HPLC High Presure Liquid Chromatography, RP- HPLC Reversed Phase High Presure Liquid Chromatography, US-FDA United States Food and Drug Administration Introduction In the past several decades a great attention has been given to the stability evaluation, both for active pharmaceutical ingredients (API) and drug products. Information on the stability of the drug substance is an integral part of the systematic approach to stability evaluation. This aspect is regulated by ICH guideline [1] which gives recommendations specifically for intermediate and long term stability studies. Stability-indicating methods are, therefore, carefully developed and validated for the purpose of stability samples analysis. The first definition for stability-indicating methods has been provided by US-FDA stability guideline [2]. In accordance with this guideline, stability-indicating method is quantitative analytical method that is based on the characteristic structural, chemical or biological properties of each active ingredient of a drug product and that will distinguish each active ingredient from its degradation product, so that the active ingredient content can be accurately measured. In the following draft guideline [3] this definition is amended as stability-indicating method is validated quantitative method that can detect the changes with time in the chemical, physical, or microbiological properties of the drug substance and drug products, and that are specific so that the contents of active ingredient, degradation products, and other components of interest can be accurately measured without interference. By the introduction of the ICH guideline, stabilityindicating methods have become closely connected to the stability stress studies and mandatory for the pharmaceutical industry. Forced degradation or stress studies have to be an integral part of stability-indicating method development and should be included in the early stages. They are undertaken to demonstrate specificity when developing stabilityindicating methods, particularly when little information is available about potential degradation products. To be more precise, stress studies should be the first step in method development, which will ensure that method development and the first degradation products identification processes can be carried out in parallel. Apart from this, forced degradation studies should examine the degradation pathways of the drug substance, as well as identify degradation products that may form during storage. The special attention should be paid to the drug stability evaluation considering the available drug pharmaceutical formulations since it is well known that the excipients can sometimes interact with the active drug and the formed reaction products, considered as impurities, can influence the drug pharmacological properties. The important aspect on forced degradation studies is that they may help facilitate complete pharmaceutical development in areas such as drug formulation development, manufacturing and packaging, in which knowledge of chemical behavior can be used to improve a drug product [4]. Following the forced degradation studies, the appropriate stability-indicating method should be properly optimized and validated with the special emphasis to the drug substance and drug degradation product separation. Apart from this, stability-indicating method must provide drug substance quantification without interferences coming from degradation products, process-related impurities, excipients or any drug related products. Considering the variability of 11

12 Farm Przegl Nauk, 2010, 9 The following chemicals were used for forced degradation experiments: sodium hydroxide (NaOH) (Zorka Pharma, Sabac, Serbia), hydrochloric acid (HCl) (J.T. Baker, Deventer, Holland) and hydrogen peroxide (H 2 O 2 ) (Centrohem, Stara Pazova, Serbia). Chromatographic conditions An Agilent 1200 Series (Agilent Technologies, Waldbronn, Germany) chromatographic system was used, equipped with Rheodyne manual injector, 1200 binary pump, UV/VIS photodiode-array detector and Agilent ChemStation integrator. The detection was performed at 220 nm, 235 nm, 254 nm and 280 nm. RP-HPLC analyses were performed using the mobile phase consisting of water solution of TEA (1 %, ph 7.4) mixed with methanol in ratio 60:40 (v/v) pumped at the flow rate of 4 ml/min and the column temperature was 30 C. Separations were performed on a Chromolith Performance RP-18e column (100 mm x 4.6 mm, macropore size 2 μm, mesopore size 13 nm) (Merck, Darmstadt, Germany). Fig. 1. a) Valdecoxib b) SC commercially available pharmaceutical formulations, the developed stability-indicating method may even be subjected to revalidation to ensure the method specificity/selectivity, accuracy and precision. Valdecoxib (4-(5-methyl-3-phenyl-4-isoxazolyl)benzensulfonamide), presented on Fig. 1a, is a non-steroidal anti-inflammatory drug (NSAID), reported to be a selective inhibitor of enzyme cyclo-oxygenase-2 (COX-2). Having in mind the importance of evaluation of drug stability properties through stress testing as well as the fact that there are no valuable records in the literature of such kind for the mentioned drug, the principal aim of this study was postulated to be the detail investigation of intrinsic stability of valdecoxib under the forced degradation conditions and the proposition of following stability-indicating method based on RP-HPLC. Materials and methods Materials and reagents Valdecoxib reference substance, valdecoxib impurity SC reference substance, as well as Bextra filmtablets, containing 40 mg of valdecoxib were obtained from Pharmacia-Pfizer EEIG, Sandwich, Kent, UK. All reagents were of analytical grade. Methanol (Sigma- Aldrich Chemie GmbH, Taufkirchen, Germany) and triethylamine (TEA) (Fisher Scientific, Leicestershire, UK) were used to prepare the mobile phase. Water HPLC grade was obtained using purification system Simplicity 185 (Millipore, Billerica, Massachusetts, USA) and 85 % orthophosphoric acid (J. T. Baker, Deventer, Holland) was used for ph adjustment of the mobile phase. The mobile phase and the solutions to be injected were degassed and vacuum filtered prior to use. Solutions Valdecoxib stock solution was prepared by dissolving 25 mg of valdecoxib standard substance in methanol and diluting to 25 ml in volumetric flask with the same solvent. Concentration of valdecoxib in stock solution was 1 mg ml -1. Valdecoxib degradation product (SC-77852) stock solution was prepared by dissolving 10 mg of SC standard substance in methanol and diluting to 50 ml in volumetric flask with methanol. 0.5 ml of this solution were transferred into 10 ml volumetric flask and diluted to volume with methanol. The concentration of this SC stock solution was 10 μg ml -1. Sample preparation Ten Bextra film-coated tablets were finely powdered. Equivalent to 10 mg of valdecoxib was accurately weighed and transferred to a 10 ml volumetric flask. About 5 ml of methanol were added to dissolve the drug. After sonicating and shaking the mixture for 30 minutes, it was completed to volume with the same solvent. This mixture was centrifuged for 10 minutes at 4000 rpm and filtrated. The concentration of valdecoxib was 1 mg ml -1. Methodology of stress testing Stress studies were carried out under the conditions of photolysis, dry heat, acidic and alkaline hydrolysis, oxidation as mentioned in ICH guidelines. Photolysis was carried out with valdecoxib active substance, Bextra film-tablets powder, as well as with stock solutions of both active substance and the sample from tablets. Each of these four samples was exposed to day light for 72 hours. 10 mg of valdecoxib active substance powder was dissolved with methanol in 10 ml volumetric flask after the exposure to the day light. 1 ml of this solution were transferred to 10 ml volumetric flask and diluted to volume with the same solvent. Ten Bextra film-tablets were finely powdered, left exposed to the day light and the powder was afterwards used for preparation of the sample solution in the same manner as previously described. For the photostability investigations with drug methanol solution, 1 ml of val- 12

13 copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN decoxib stock solution was transferred to 10 ml volumetric flask, diluted to volume with methanol and exposed to the day light. Also, 1 ml of Bextra film-tablets sample solution was transferred to 10 ml volumetric flask, diluted to volume with methanol and exposed to the day light. The predicted concentration of valdecoxib in all these solutions used for photostability testing was expected to be 100 μg ml -1 if no drug degradation occurs. Thermal degradation was also carried out with valdecoxib active substance and Bextra film-tablets powder, which were heated at 75 C for 10 hours in 1 mm layer in a Petriplate, as well as with stock solutions of both active substance and sample prepared with Bextra film-tablets. After exposure, the same methodology as for photolysis investigation is used for preparation of solutions, so the predicted concentration of valdecoxib in each case, if no drug degradation occurs, was expected to be 100 μg ml -1. Alkaline hydrolysis was performed with valdecoxib stock solution. Two sets of experiments were carried out in the first set 1 ml of valdecoxib stock solution was transferred into three 10 ml volumetric flask and 1 ml of NaOH in concentration 0.5 mol L -1, 1 mol L -1 and 2 mol L -1 was added, respectively; these solutions were kept in dark place and at room temperature for 5 hours. The second set of experiment was carried out with the valdecoxib stock solution to which 1 ml of 2 mol L -1 and 5 mol L -1 NaOH was added, respectively and then the samples were exposed to these conditions for 30 hours. After the exposure, 1 ml of HCl of appropriate concentration (0.5 mol L -1, 1 mol L -1, 2 mol L -1, 5 mol L -1 ) was added to each sample to neutralize the solutions and all solutions were diluted to volume with methanol. The predicted concentration of valdecoxib in these sample solutions, if no drug degradation occurs, was expected to be 100 μg ml -1. Acidic hydrolysis was performed with valdecoxib stock solution. Two sets of experiments were carried out in the first set 1 ml of valdecoxib stock solution was transferred into three 10 ml volumetric flask and 1 ml of HCl in concentration 0.5 mol L -1, 1 mol L -1 and 2 mol L -1 was added, respectively; these solutions were kept in dark place and at room temperature for 5 hours. The second set of experiments was carried out with the valdecoxib stock solution to which 1 ml of 2 mol L -1 and 5 mol L -1 HCl was added, respectively and then the solutions were exposed to the these conditions for 30 hours. After the exposure, the samples were neutralized with addition of 1 ml NaOH of appropriate concentration (0.5 mol L -1, 1 mol L -1, 2 mol L -1, 5 mol L -1 ) to the solutions. The predicted concentration of valdecoxib in these sample solutions, if no drug degradation occurs, was expected to be 100 μg ml -1. Oxidative stress was performed with valdecoxib stock solution to which H 2 O 2 was added. Two sets of experiments were carried out in the first, 1 ml of valdecoxib stock solution was transferred into three 10 ml volumetric flask and 1 ml of H 2 O 2 in concentration 10 %, 15 % and 30 % (v/v) was added respectively; these solutions were kept in dark place and at room temperature for 5 hours. The second set of experiments was carried out with the valdecoxib stock solution which was mixed with 30 % H 2 O 2 (v/v) and then was left for 30 hours. The predicted concentration of valdecoxib in each prepared sample solution, if no drug degradation occurs, was expected to be 100 μg ml -1. Research results and discussion The ICH guideline [2] gives only few details on conducting of stress studies. It is recommended that testing should include investigation of temperature, humidity (where appropriate), oxidation effects, as well as photolysis and susceptibility to hydrolysis across a wide range of ph values. Apart from the recommendation that the effect of temperature should be investigated in 10 C increments above the accelerated temperature test conditions (e.g. 50 C, 60 C, etc.), no other detail has been given, which leaves the possibility to conduct stress studies in relation to specific physical-chemical characteristics of drug substance tested. There are several literature references, outlining the recommended forced degradation conditions [5-11]. In order to select proper conditions for forced degradation studies, a special attention should be paid to the chemical structure of active substance analyzed, investigating the properties of its functional groups. For example, it is known that amides, esters, lactams and lactones are very susceptible to hydrolysis; thiols and thioeters to oxidation, whereas chemical compounds with aromatic nitro group, N-oxids, olefins and aryl-halo derivatives are susceptible to photodegradation. In general, there are several important principles to follow in conducting forced degradation studies. The main goal is to achieve degradation level of about 5 % to 20 % in order to enable chromatograms to be relatively easy to interpret (readily visible peaks in case of incomplete separations) and sufficient to give photo-diode array software a good chance of detecting co-elutions. Further degradation is not recommended, as it could lead to very complex and difficult chromatograms interpretations and judgment of results may be influenced by secondary, tertiary or higher order degradations that are probably very unlikely in real time stability studies. At the same time, based on regulatory requirements, only 5 20 % degradation of initial drug amount is considered to be significant at all. Any forced degradation that results in less than 5 % is not relevant to method development since that level of degradation is not likely ever to occur in long term stability studies. On the other hand, it is not justified to overstress a drug molecule, in order to achieve degradation over 20 %, since these extreme stress conditions would probably not ever occur in long term stability studies and are not representative of real storage conditions. Mass balance correlates the measured loss of a parent drug to the measured increase in the amount of degradation products. It is a good quality control check on analytical methods to show that all degradation products are adequately detected and do not interfere with quantification of the parent drug. Regulatory agencies use mass balance to assess the appropriateness of the analytical method and determine whether all degradation products have been accounted for. The fundamental approach for determining mass balance is to measure the degradation peaks and then reconcile the measured loss in the parent drug with the amount of degradation products. In some cases it is not appropriate to estimate mass balance directly from the amount of degradation products formed, but rather the percent parent drug lost due to unknown analytical precision and differences in response factor [12]. 13

14 Farm Przegl Nauk, 2010, 9 Fig. 2. HPLC chromatograms demonstrating photostability (after exposure to day light for 72 hours): a) valdecoxib active substance, b) Bextra film-tablets powder, c) valdecoxib active substance solution after exposure to day light, d) Bextra solution after film-tablets exposure to day light. From the literature records [13] it was noticed that the manufacturer reported the presence of the potential degradation product of valdecoxib denoted as SC and chemically described as 4-(2,3-dihydro-5-methyl- 3-phenyl-4-isoxazolyl)-N-(4-o-β-D-galactopyranosyl-dglucopyranosyl)benzenesulfonamide. SC represents a mixture of α- and β- anomer (Fig. 1b.) and it is the result of the interaction between lactose from the excipient and valdecoxib. Also, it was noticed that stereo chemical aspect of SC has a great importance, since only β- anomer can interact with lactose. Due to this reaction, the sulfonamide groups of valdecoxib become blocked which finally results in depression of its pharmacological effect. Therefore, according to the regulatory requirements, β- anomer is needed to be identified and quantified in the final product. As mentioned, this interaction is only expected in final drug and therefore it was considered necessary to fulfill the present forced degradation studies with the sample of commercially available valdecoxib pharmaceutical preparation, Bextra film-tablets, both as powder and methanol solution. Stock solutions of valdecoxib and final drug product in methanol have been used in stress studied to investigate the possible interference of methanol in degradation process. SC was also chromatographed on the same conditions as used for all other samples in order to be able to extract appropriate conclusions concerning the selectivity of the proposed stability-indicating method. Chromatographic behavior of prepared solutions was investigated on various wavelengths of detection, in order to be able to notice those degradation products which have different absorption characteristics in comparison to initial drug substance. Also, additional spectral analysis of obtained peaks has been conducted, since peak purity depends on the amount of variations in valdecoxib absorption spectrum. Having all this in mind, the proper procedure of stress testing was selected and the following stability properties of valdecoxib were noticed. It was demonstrated that valdecoxib drug substance is very stable in dry heat and light exposure conditions, both in solid state and methanol solution. Similar stability is obtained in experiment with Bextra film-tablets, both as powder and methanol solution. After these stress conditions, solutions prepared were subject to chromatographic analysis, which showed no additional peaks. Namely, it was not possible to detect any degradation product with different polarity and therefore different retention time than valdecoxib, although the separation was performed two times longer than valdecoxib retention time (Fig. 2 and Fig. 3). For practical purposes only chromatograms performed at 220 nm were presented on figures, due to high detection ability at this wavelength. Degradation peaks were not observed at any wavelength used. Peaks purity factors were in the interval , so the possibility of peaks co-elution can be excluded. This is due to the fact that degradation products in small increments can be identified as peak purity factor variation. Also, mass balance was determined by comparing the valdecoxib peak from the control sample, which was prepared in the same valdecoxib concentration as in the stressed sample. Since the total amount of potential peak areas in investigated stress samples was 100 % comparing to the control sample, this indicated that under applied stress conditions no degradation of valdecoxib occurred. No degradation product was detectable using UV/VIS detector, which can be due to either undetectable amount of degradation products or their lack of chromophores in the molecule. 14

15 copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN Fig. 3. HPLC chromatograms demonstrating thermal stability (after exposure to 75 C for 10 hours): a) valdecoxib active substance, b) Bextra film-tablets powder, c) valdecoxib active substance solution after exposure to high temperature, d) Bextra solution after film-tablets exposure to high temperature. When it comes to other stress conditions (hydrolysis, oxidation), some level of instability was noticed. In order to better detect all possible changes in the appearance of the chromatograms and to extract the proper conclusions about Fig. 4. HPLC chromatograms of valdecoxib stock solution (c = 100 μg ml -1 ) after alkaline hydrolysis due to exposure to a) 1 mol L -1 NaOH, 5 hours, b) 2 mol L -1 NaOH, 5 hours, c) 2 mol L -1 NaOH, 30 hours, d) 5 mol L -1 NaOH, 30 hours. the drug behavior, additional control samples were prepared blank samples containing only the stress agents mixed with methanol and zero time samples containing the valdecoxib and stress agent but analyzed directly after the preparation. 15

16 Farm Przegl Nauk, 2010, 9 Fig. 5. HPLC chromatograms of valdecoxib stock solution (c = 100 μg ml -1 ) after acidic hydrolysis due to exposure to a)1 mol L -1 HCl, 5 hours, b) 2 mol L -1 HCl, 5 hours, c) 2 mol L -1 HCl, 30 hours, d) 5 mol L -1 HCl, 30 hours. Fig. 6. HPLC chromatograms of valdecoxib stock solution (c = 100 μg ml -1 ) after oxidation with a) 10 % H 2 O 2, 5 hours, b) 15 % H 2 O 2, 5 hours, c) 30 % H 2 O 2, 5 hours, d) 30 % H 2 O 2, 30 hours. 16

17 copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN In case of alkaline hydrolysis, in all concentrations of NaOH, less than 0.5 % of valdecoxib has been degraded (Fig. 4 a, b). Also, when acid hydrolysis was applied, only 3.5 % of valdecoxib degradation was observed (Fig. 5 a, b). Similar stability is demonstrated with oxidation stress conditions, where 2.5 % of valdecoxib has been degraded (Fig. 6 a, b, c). Because valdecoxib has demonstrated significant stability on these conditions, in order to confirm such conclusion, even more severe stress conditions were selected in the second set of experiments. Alkaline hydrolysis was performed using higher concentrations of NaOH with longer exposure, where 4 % degradation of valdecoxib is obtained (Fig. 4 c, d). Spectral analysis of chromatograms showed appropriate peak purity in each case as well as the evidence of mass balance. This led to conclusion that valdecoxib is very stable in terms of alkaline hydrolysis. On the other hand, valdecoxib has shown some susceptibility to acid hydrolysis and oxidative stress conditions. Namely, 8.2 % of valdecoxib has degraded with HCl in 30 hours (Fig. 5 c, d), whereas 5.3 % valdecoxib degradation occurred with oxidative stress agents in 30 hours (Fig. 6 d). Peak purity test results also confirmed that valdecoxib peak was homogeneous and pure in all the analyzed stress samples. Although some degradation was obtained in acid hydrolysis and oxidative stress conditions, not any individual peak is statistically relevant, and can not be identified as main i.e. typical degradation product. Second set of experiment showed that mass balance was less than 100 %, which indicates the possibility that the formed degradation products may not have the chromophores in their structure. Therefore, it was finally concluded that no additional qualification and quantification of degradation products is needed and the previously published method [13] could be used in further investigations as a stabilityindicating method. Conclusions A thorough forced degradation studies have been performed in order to examine valdecoxib behavior. Valdecoxib has shown a significant stability in thermal and photolytic stress conditions, where no degradation product was identified. In case of mild hydrolysis and oxidative stress conditions less than 5 % valdecoxib degradation occurred, which has no significance in terms of long term stability studies. Higher degree of degradation was obtained in more severe stress conditions (higher concentration of stress agents, longer time of exposure), but individual degradation products detected under described conditions have no statistical relevance and therefore there is no need for their further evaluation or structure elucidation. Acknowledgements: These results are part of the project Synthesis, Quantitative Structure/Properties and Activity Relationship, Physical Chemical Characterization and Analysis of Pharmacologically Active Substances No B, financed by the Ministry of Science and Technology of the Republic of Serbia. References 1. International conference for harmonization of technical requirements for registration of pharmaceuticals for human use. Q1A(R2): Stability testing of new drug substances and products. Fed Regist 2003; 68: Food and Drug Administration. Drugs/GuidanceComplianceRegulatoryInformation/ Guidances/default.htm. 3. Food and drug Administration. downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm htm. 4. Reynolds DW et al. Available guidance and best practices for conducting forced degradation studies. Pharm Technol 2002; 26: Alsante KM et al. The role of degradant profiling in active pharmaceutical ingredients and drug products. Adv Drug Deliv Rev 2007; 59: Bedse G, Kumar V, Singh S. Study of forced decomposition behavior of lamivudine using LC, LC MS/TOF and MS n. J Pharm Biomed Anal 2009; 49: Mohan A et al. Identification and characterization of a principal oxidation impurity in clopidogrel drug substance and drug product. J Pharm Biomed Anal 2008; 47: Breier R et al. Isolation and structure elucidation of photodegradation products of fexofenadine. J Pharm Biomed Anal 2008; 46: Bhardwaj SP, Singh S. Study of forced degradation behavior of enalapril maleate by LC and LC MS and development of a validated stability-indicating assay method. J Pharm Biomed Anal 2008; 46: Bakshi M, Ojha T, Singh S. Validated specific HPLC methods for determination of prazosin, terazosin and doxazosin in the presence of degradation products formed under ICH-recommended stress conditions. J Pharm Biomed Anal 2004; 34: Bakshi M, Singh S. Development of validated stabilityindicating assay methods-critical review. J Pharm Biomed Anal 2002; 28: Nussbaum MA, Jansen PJ, Baertschi SW. Pharmaceutical stress testing: predicting drug degradation, Taylor & Francis group, New York, USA, G. Savić et al. Validation of an HPLC method for the determination of valdecoxib and its degradation product: a mixture of α- and β-n-lactosyl sulfonamide anomers. Chromatographia 2007; 66: data otrzymania pracy: r. data akceptacji do druku: r. Corresponding author: Prof dr Mira L Zečević Faculty of Pharmacy, Department of drug analyses Vojvode Stepe 450, Belgrade tel: , fax: mzecevic@pharmacy.bg.ac.rs 17

18 Farm Przegl Nauk, 2010,9, Zastosowanie elektroforezy kapilarnej do analizy potencjalnych radiofarmaceutyków do wczesnej diagnostyki choroby Alzheimera Capillary zone electrophoretic analysis of radiopharmaceuticals used in early diagnosis of Alzheimer s disease Kamila Padkowska 1,2, Paweł Szymański 2, Elżbieta Mikiciuk-Olasik 2 1 Nycomed Pharma Sp. z o.o. 2 Zakład Chemii Farmaceutycznej i Analizy Leków Katedry Chemii Farmaceutycznej i Biochemii, Uniwersytet Medyczny w Łodzi Abstract A new capillary zone electrophoresis (CZE) method for determination of compounds that are likely to become radiopharmaceuticals used for early diagnosis of Alzheimer s disease was developed. These compounds are tacrine and hynic analogs. Agilent Technologies CE, equipped with a diode array detection (DAD) system, automatic injector, along with ChemStation software were used for tacrine analogs analysis. Studies were carried out in order to optimize measurement conditions. The method of compound determination was optimized. The best results were obtained when: fused-silica capillary tubing was 48.5 cm long (40 cm to the detector) with i.d. of 50 μm, the background electrolyte (BGE) was phosphate buffer (ph 2.6, 50 mm), the separation voltage was 20 kv, the capillary temperature was 25 C, samples were injected hydro-dynamically for 5 s (with injection pressure 25 mbar) and detection was carried out at 240 nm (tacrine hydrochloride) and 248 nm (tacrine and hynic analogs). In aqueous solutions, the detection limits (S/N=3) were 5.7 μg/ml, 6.9 μg/ml, 3.7 μg/ml, and 3.8 μg/ml for 6a, 6b, 6c, and 6d, respectively. The quantification limit (S/N=3:10) obtained in aqueous solutions was 16.1 μg/ml for 6a, 16.8 μg/ml for 6b, 11.0 μg/ml for 6c and 11.2 μg/ml for 6d. The method is suitable for a routine pharmaceutical quality control analysis of the potential radiopharmaceuticals for early diagnosis of Alzheimer's disease. Keywords: capillary zone electrophoresis, CZE, Alzheimer s disease, tacrine analog, hynic analog, radiopharmaceutical Streszczenie Opracowano i zoptymalizowano nową metodę analityczną z wykorzystaniem strefowej elektroforezy kapilarnej (CZE) do oznaczania jakościowego i ilościowego czterech radiofarmaceutyków będących potencjalnymi substancjami diagnostycznymi do wczesnego wykrywania choroby Alzheimera. Związki te są analogami takryny i hynica. Do optymalizacji i walidacji metody wykorzystano zestaw elektroforezy kapilarnej CE zaopatrzony w detektor diodowy (DAD) i automatyczny system nastrzyku próby. Metoda oznaczania związków została zoptymalizowana. Najlepsze rezultaty otrzymano stosując następujące warunki: kapilara ze stopionej krzemionki pokrytej otoczką poliamidową, długość całkowita 48,5 cm (długość do detektora 40 cm), średnica wewnętrzna 50 μm. Detekcja oznaczanych analogów oraz chlorowodorku takryny miała miejsce przy 240 nm (chlorowodorek takryny) i 248 nm (analogi takryny). Ostatecznie do oznaczeń analitycznych wybrano 50 mm bufor fosforanowy o ph 2,6. Podczas analiz temperatura kapilary wynosiła 25ºC a wartości przykładanego napięcia 20 kv. W trakcie prowadzonych badań stosowano iniekcję hydrodynamiczną (czas nastrzyku 5 s, wartość przykładanego ciśnienia 25 mbar). Określono liniowość uzyskując informację, że metoda jest liniowa w zakresie stężeń: 0,01-0,05 mg/ml dla związków 6a, 6b, 0,01 0,06 mg/ml dla związku 6c, 0,02 0,08 mg/ ml dla związku 6d. Dla badanych związków otrzymano następujące wartości stężeń odpowiadające granicy wykrywalności: Związek 6a 5,7 μg/ml, Związek 6b 6,9 μg/ml, Związek 6c 3,7 μg/ml, Związek 6d 3,8 μg/ml. Otrzymano następujące wartości stężeń odpowiadające granicy oznaczalności dla badanych substancji: Związek 6a 16,1 μg/ml, Związek 6b 16,8 μg/ml, Związek 6c 11,0 μg/ml, Związek 6d 11,2 μg/ml. Udowodniono, że metoda ta może być z powodzeniem wykorzystywana do rutynowych analiz jakościowych potencjalnych radiofarmaceutyków będących analogami takryny. Słowa kluczowe: strefowa elektorforeza kapilarna, CZE, choroba Alzheimera, analog takryny, analog hynic a, radiofarmaceutyki 18

19 copyright 2010 Grupa dr. A. R. Kwiecińskiego ISSN Introduction Alzheimer s disease (AD) was first mentioned in 1906, when the German psychiatrist and neuropathologist Alois Alzheimer gave a speech presenting the clinical symptoms and pathology of presenile dementia observed in one of his patients a 51 year old woman. This was the first time AD was described. Later Emil Kraeplin (head of the psychiatric division in Munich University) named that type of dementia after Alzheimer [1]. The disease currently affects nearly 15 million people (mainly elderly). Prognosis shows that in 2025 there will be about 22 million sufferers (which is mostly the effect of prolonged life expectancy) and by 2050, the amount of patients will have quadrupled. It is assumed that AD affects about 5 to 10% of society and is the main dementia type among people above 65 years of age [2]. For the last two decades it has been observed observe significant progress in the effective, but symptomatic treatment of Alzheimer disease [3]. The earliest observable symptoms are often mistakenly thought to be age-related concerns, or manifestations of stress [4]. Therapy is mainly based on drugs that increase the level of acetylcholine, or drugs that improve nerve conductivity [5]. Acetylcholinesterase inhibitors (IAChE) play the main role in AD treatment [6, 7]. Studies on the role acetylcholine plays in the learning and remembering process lead to creation of cholinergic hypothesis of the disease. The hypothesis assumes that loss of cholinergic stimulation of brain neurons is one of the causes of the disease [8]. Thus one of the symptomatic treatment methods is the inhibition of acetylcholinesterase to increase the amount of acetylcholine in the synaptic gap. Early diagnosis is especially important in the treatment of Alzheimer s as an early treatment can, to some extent, slow down the development of the disease, thus, prolonging the mental agility of the patient. Having knowledge about cholinergic theory we can design bifunctional compounds used as drugs for AD. Moreover, those compounds, after the introduction of radioactive technetium, can be used for early diagnosis of this disease. Radiopharmaceuticals that were developed display particles derived from tacrine which is responsible for binding with the enzyme (acetylcholinesterase), and particles derived from hynic (containing radioisotope). Compared to tacrine they are more selective for AChE and less selective for BChE. Specifically compounds 6a and 6d show high AChE affinity in enzymatic studies [9]. Those compounds (i.e. 6a, 6b, 6c and 6d) are radio-labelled with 99m Tc and given to the patient; then scintigraphic methods are used for imaging and early diagnosis. The next stage in compound research is establishing analytical methods that are best suited for qualitative and quantitative studies. For this purpose analysis using capillary electrophoresis has been chosen, and such analytical technique is gaining importance in routine-analysis as well as in R&D laboratories. Further, this technique has proven to be an excellent choice for the determination of compounds 6a, 6b, 6c and 6d. Materials and methods Reagents Tacrine hyrochloride and tacrine and hynic analogs: 6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino) ethyl]nicotinamide hydrochloride later called 6a, 6-hydrazino-N-[8-(1,2,3,4-tetrahydroacridin-9-ylamino)propyl]nicotinamide hydrochloride later called 6b, 6-hydrazino-N-[8- (1,2,3,4-tetrahydroacridin-9-ylamino)heksyl]nicotinamide hydrochloride later called 6c, 6-hydrazino-N-[8-(1,2,3,4- tetrahydroacridin-9-ylamino)octyl]nicotinamide hydrochloride later called 6d. We have synthesized these compounds at the Medical University in Łodz [9]. Tacrine hydrochloride 9-Amino- 1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA) used in the experiment at the University of Łódź was of analytical grade (Aldrich). Other reagents used: sodium hydroxide 0.1 M and sodium hydroxide 1 M, acetic acid, acetone, anhydrous ethanol 99.8% were of analytical grade (Poch Gliwice), fosforic acid (V) 85.7 % was of analytical grade (Fluka). Solutions were prepared with deionized water. Apparatus An Agilent Technologies CZE system DE equipped with a diode array detection (DAD) system, automatic injector, thermostating column cartridge and Chem- Station software was used in all our experiments. Fusedsilica capillary type G , tubing 48.5 cm long (40 cm to the detector) with i.d. of 50 μm was used. Spectrophotometric measurements were done on a UV-Vis Carry 100 spectrometer Bio Varian. A ph-meter Metrohm 744 and Metrohm ph sensitive, combined glass electrode were used for the ph measurements. CE measurements and procedure CZE separations were carried out at 25ºC. The solution of a 50 mm phosphate buffer used throughout this study, was prepared daily, filtered through 0.22 μm nylon filters and submitted to Ultrasonic Cleaner (Ultron). The ph was adjusted with diluted NaOH and/or HCl solutions. The capillary inlet and outlet vials were replenished after every three injections. The capillary was washed daily for 15 min. with 0.1 M NaOH, 15 min. with deionized water and 15 min. with run buffer. In each analysis prewash of 5 min. with 0.1 M NaOH and 2 min. with background electrolyte (BEG) was used. Absorbance was monitored at 220 nm. In the preliminary experiments electrokinetic injection (20 s, 10 kv) was used, then hydrodynamic injection 25 mbar, 5 s was applied in the rest of the experiments. Sample preparation Solutions of examined compounds 6a, 6b, 6c and 6d were prepared using deionized water. Samples were dissolved with the help of an ultrasonic bath and filtered through 0.22 μm nylon filters. Solutions were prepared on the day of the experiments. 19

20 Farm Przegl Nauk, 2010, 9 Fig. 1. CE analytical method. development and optimisation. Results and calculation Capillary electrophoresis (CE) is a dynamically developing analytical technique, which plays a more and more important role in a pharmaceutical laboratory. Fundamental advantages of CE are: very high efficiency, short analysis time, ecological character, ease of automation, requirement of minute amounts of sample. The latter is especially important when the tested compound synthesis is time-consuming and expensive. Before starting analysis that leads to the development of an analytical method that uses CE for a new compound one should know the solubility of the sample (it is most convenient if the sample is water/buffer soluble, or low conductance solution soluble). The next step is the determination of the ionic character of the sample. Once this is established one can start the analysis form using: - phosphate buffer or acetate buffer, concentration about 25 mm, ph about 2.5 for cations - borate buffer, concentration about 25 mm, ph about 9.4 for anions - borate buffer, concentration about 25 mm, ph about 9.4 with addition sodium salt addition for neutral ions Fig. 2. Molecular structure of the drugs for early diagnosis of Alzheimer's disease. For given n equal: n=2 - Compound 6a 6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino) ethyl]nicotinamide hydrochloride n=3 - Compound 6a 6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino) propyl]nicotinamide hydrochloride n=6 - Compound 6a 6-hydrazino-N-[2-(1,2,3,4-tetrahydroacridin-9-ylamino) heksyl]nicotinamide hydrochloride n=8 - Compound 6d 6-hydrazino-N-[8-(1,2,3,4-tetrahydroacridin-9-ylamino) octyl]nicotinamide hydrochloride Fig. 3. Absorption spectra of the radiopharmaceuticals for early diagnosis of Alzheimer's disease. (b) 6a compound 23.5 ppm, (c) 6d compound 24.5 ppm in aqueous solution. After selecting the base buffer, it should be decided what CE type to use. If the compound can be determined with the use of zone CE one should decide what kind, length, i.d., and temperature of capillary to use; other factors left to determine are: voltage and injection type. If peaks are separated correctly (i.e. have good symmetry and sufficient resolution) it can be concluded that parameters are optimal. Otherwise modifications to buffer parameters and optimizations of hardware-related parameters should be performed. Development and optimisation of CE analytical method are shown schematically in [Fig. 1]. The molecular structures of compounds being analysed is shown in [Fig. 2]. The absorption spectra of the compounds studied are presented in [Fig. 3]. Detection at 240 nm for THA and 248 nm for other studied compounds were respectively used as the most sensitive wavelengths for the determination. CE analyses were started by determining environment influence on electrophoretic mobility of examined compounds. Measurement was oriented towards finding optimal ph and concentration of background electrolyte i.e. conditions under which THA gives high, clear signal with correct symmetry. For that purpose two series of electropherograms were prepared one for phosphate and one for acetate buffer (which 20