Assay and Physicochemical Characterization of the Antiparasitic Albendazole
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Brazilian Journal of Pharmaceutical Sciences vol. 48, n. 2, apr./jun., 2012 Article Assay and physicochemical characterization of the antiparasitic albendazole Noely Camila Tavares Cavalcanti1*, Giovana Damasceno Sousa2, Maria Alice Maciel Tabosa1, José Lamartine Soares Sobrinho3, Leila Bastos Leal1, Davi Pereira de Santana1 1Pharmaceutical and Cosmetics Development Center, NUDFAC, Federal University of Pernambuco, 2Pernambuco’s Health College, FPS, Recife, 3Center of Pharmaceutical Technology, Federal University of Piauí The aim of this study was to characterize three batches of albendazole by pharmacopeial and complementary analytical techniques in order to establish more detailed specifications for the development of pharmaceutical forms. The ABZ01, ABZ02, and ABZ03 batches had melting points of 208 °C, 208 °C, and 209 °C, respectively. X-ray diffraction revealed that all three batches showed crystalline behavior and the absence of polymorphism. Scanning electron microscopy showed that all the samples were crystals of different sizes with a strong tendency to aggregate. The samples were insoluble in water (5.07, 4.27, and 4.52 mg mL-1, respectively) and very slightly soluble in 0.1 M HCl (55.10, 56.90, and 61.70 mg mL-1, respectively) and additionally showed purities within the range specified by the Brazilian Pharmacopoeia 5th edition (F. Bras. V; 98% to 102%). The pharmacopeial assay method was not reproducible and some changes were necessary. The method was validated and showed to be selective, specific, linear, robust, precise, and accurate. From this characterization, we concluded that pharmacopeial techniques alone are not able to detect subtle differences in active pharmaceutical ingredients; therefore, the use of other complementary techniques is required to ensure strict quality control in the pharmaceutical industry. Uniterms: Albendazole/characterization. Antiparasitics/quality control. O objetivo do trabalho foi caracterizar três lotes de albendazol com técnicas analíticas farmacopéicas e complementares a fim de estabelecer especificações mais detalhadas para o desenvolvimento de formas farmacêuticas. Os lotes ABZ01, ABZ02 e ABZ03 apresentaram fusão em 208 °C, 208 °C e 209 °C. Foi possível evidenciar, por difração de raios X, que os três lotes apresentaram comportamento cristalino e ausência de polimorfismo. Através da microscopia eletrônica de varredura verificou-se que todas as amostras apresentaram cristais com diferentes tamanhos e forte tendência de agregação. As amostras foram insolúveis em água (5,07; 4,27 e 4,52 µg mL-1) e muito pouco solúveis em HCl 0,1M (55,10; 56,90 e 61,70 µg mL-1) e, ainda, apresentaram pureza dentro da faixa especificada pela F.Bras.V (98% a 102%). O método farmacopéico de doseamento não foi reprodutível, e algumas mudanças foram necessárias. O método foi validado e demonstrou ser seletivo, específico, linear, robusto, preciso e exato. A partir dessa caracterização, pode-se concluir que apenas técnicas farmacopéicas não são capazes de detectar diferenças sutis entre os ingredientes farmacêuticos ativos, necessitando, portanto, de uso de outras técnicas complementares para garantir um rígido controle de qualidade na indústria farmacêutica. Unitermos: Albendazol/caracterização. Antiparasitários/controle de qualidade. INTRODUCTION ment of pharmaceutical forms and is the first step in the drug design process. Changes in the active pharmaceutical Characterization of the physical and physico- ingredient (API) may impair productivity, affect product chemical properties of drugs is essential for the develop- quality, and even cause the loss of a production batch (Nery et al., 2008; Soares Sobrinho et al., 2010). *Correspondence: N.C.T. Cavalcanti. Núcleo de Desenvolvimento Farmacêu- Albendazole (ABZ; methyl-5-propylthio-2-benz- tico e Cosmético - NUDFAC, Universidade Federal de Pernambuco - UFPE. imidazolecarbamate; Figure 1), a member of the benz- Av. Professor Artur de Sá, s/n, Cidade Universitária, 50740-521 - Recife - PE, imidazole family of compounds, is a crystalline white Brazil. E-mail: [email protected] 282 N. C. T. Cavalcanti, G. D. Sousa, M. A. M. Tabosa, J. L. Soares Sobrinho, L. B. Leal, D. P. Santana odorless powder that melts at 207 °C–209 °C. It has a Brazil), batches 1005024006, 1005022006, and molecular weight of 265.34 g mol-1 and the molecular 1002008006, which for the proposed study were identi- formula is C12H15N3O2S. In the Biopharmaceutical Clas- fied as ABZ01, ABZ02, and ABZ03, respectively. Pharma- sification System (BCS), it is a Class II substance, with copeial Standard Batch 1034, content (99.80%) INCQS, low solubility and high permeability (Merck Index, 1996; Rio de Janeiro, Brazil. All solvents and reagents used Lindenberg et al., 2004). in the trials were of analytical grade, the glassware was calibrated, and the tests described below were performed in 3 replicates. To improve understanding, the analyses were clas- sified as pharmacopeial and non-pharmacopeial (comple- mentary), owing to some discriminative characteristic FIGURE 1 - Chemical structure of albendazole. methods cited in the Brazilian Pharmacopoeia 5th edition (F. Bras. V, 2010). The molecule was patented in 1975 but there were no reports available regarding its solid-state characteriza- PHARMACOPEIAL TESTS tion or possible polymorphisms. In 2010, Pranzo et al. showed that the re-crystallization of ABZ from methanol Organoleptic characterization and N,N-dimethylformamide affords a new stable poly- morphic form (Form II), enantiotropically related to the Organoleptic characteristics were compared to those commercially available ABZ. described in the F. Bras. V (2010) and Drug Master File ABZ is the API most widely used for controlling in- (DMF) for the API provided by Formil Química®. testinal parasites, because it has broad-spectrum activity, is well tolerated, and has low cost (Moriwaki et al., 2008). Ac- Infrared spectroscopy cording to the National Health Surveillance Agency (AN- VISA), the reference drug is Zentel® (GlaxoSmithKline), Infrared spectroscopy with a KBr tablet was verified and currently, there are 11 types of generic medications from using a Fourier Transformed Infrared Spectrometer (model several pharmaceutical laboratories in Brazil. ABZ is avail- IFS66, Bruker, Madison, WI, USA). The spectrum of the able in the form of 200 mg tablets, 400 mg chewable tablets, API was drawn in the range of 4000 cm-1 to 400 cm-1 ac- and a 400 mg10 mL-1 oral suspension (ANVISA, 2010). cording to the method described in the F. Bras. V (2010). Knowing that ABZ is a class II (BCS) substance and considering that the presence of polymorphic forms could Melting point cause changes in the melting point, density, solubility, dis- solution, physical or chemical stability, and consequently The melting point was evaluated using a Mars´ drug bioavailability, it is extremely important to obtain Fusiometer, model III, in accordance with the general in-depth knowledge of the drug, if the required pharma- method of F. Bras. V (2010) and by differential thermal copeial tests are not enough to identify subtle differences analysis (DTA). among the raw materials, especially in pre-formulation studies, thereby threatening the quality of the product. Assay Thus, it is very important to use different parameters, e.g., melting point, and techniques, e.g., infrared spectroscopy Initially, 40 mg of ABZ was weighed into a 50 mL and X-ray diffraction, for characterization (Nery et al., round-bottomed volumetric flask; 37% HCl was added to 2008; Storpirtis et al., 2009; Maximiano, 2010). 10% of the flask volume; and then the volume was made Therefore, the aim of this study was to evaluate up with methanol from a stock solution (concentration, the physical and physicochemical characteristics of ABZ 800 μg mL-1). The solution was shaken with a magnetic bar through different identification and characterization tests, for 5 min and subjected to dispersion in an ultrasonic bath as a way to establish quality standards and aid technologi- for a further 5 min. To evaluate the content, an analytical cal improvements in this antiparasitic drug. curve was used, in triplicate, with concentrations of 60, 80, 120, 180, 320 μg mL-1, generated by dilution of the stock MATERIAL AND METHODS solution in monobasic sodium phosphate/methanol (40:60) buffer and analyzed by high-performance liquid chroma- ABZ was provided by Formil Química® (Barueri, tography coupled with an ultraviolet detector (HPLC-UV) Assay and physicochemical characterization of the antiparasitic albendazole 283 set at 308 nm. The concentration of 120 μg mL-1 was materials, nuclear magnetic resonance (NMR) analyses considered 100%. were performed. Proton (1H) and Carbon-13 (13C) NMR The chromatographic conditions were as fol- experiments were performed on a Varian® spectrometer, lows: stationary phase: Luna 5 μm C18 column, 100 Å, model Unity plus-300 MHz (Palo Alto, CA), using di- 250 × 4.6 mm; mobile phase: monobasic sodium phos- methyl sulfoxide as the solvent. phate/methanol buffer (40:60), pH 4.8, isocratic; flow rate: 2.0 mL min-1; detector: UV (308 nm), Temperature: 40 ºC, Flow properties and injection volume: 20 mL. The validation process was performed according to Density was determined by testing 20 g of each the present guidelines contained in Resolution RE-899 sample using an automatic compactor (Tapped Density passed on May 29, 2003 (ANVISA, 2003). To evaluate Tester Varian 50-100) equipped with a calibrated cylin- selectivity and specificity,