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  • *Correspondence: C.Luchese. Centro de Cincias Qumicas, Farmacuticas e de Alimentos. Universidade Federal de Pelotas. Campus Capo do Leo, 96010-900 - Pelotas - RS, Brazil. E-mail:


    icleBrazilian Journal of

    Pharmaceutical Sciencesvol. 51, n. 4, oct./dec., 2015

    Validation of high performance liquid chromatography method for determination of meloxicam loaded PEGylated nanocapsules

    Francine Rodrigues Ianiski1, Luciane Varini Laporta2, Alexandre Machado Rubim2, Cristiane Luchese3,*

    1Graduate Program in Nanoscience, Franciscan University Center, Santa Maria, RS, Brazil, 2Laboratory of Control of Drug Quality, Franciscan University Center, Santa Maria, RS, Brazil, 3Center for Chemical Sciences, Pharmaceutical and Food,

    Federal University of Pelotas, Pelotas, RS, Brazil

    A method to ensure that an analytical method will produce reliable and interpretable information about the sample must first be validated, making sure that the results can be trusted and traced. In this study, we propose to validate an analytical high performance liquid chromatography (HPLC) method for the quantitation of meloxicam loaded PEGylated nanocapsules(M-PEGNC). We performed a validation study, evaluated parameters including specificity, linearity, quantification limit, detection limit, accuracy, precision and robustness. PEGylated nanocapsules were prepared by interfacial deposition of preformed polymer, and the particle size, polydispersity index, zeta potential, pH value and encapsulation efficiency were characterized. The proposed HPLC method provides selective, linear results in the range of 1.0-40.0 g/mL; quantification and detection limits were 1.78 g/mL and 0.59 g/mL, respectively; relative standard deviation for repeatability was 1.35% and intermediate precision was 0.41% and 0.61% for analyst 1 and analyst 2, respectively; accuracy between 99.23 and 101.79%; robustness between 97.13 and 98.45% for the quantification of M-PEGNC. Mean particle diameters were 261 13 nm and 249 20 nm, polydispersity index was 0.15 0.07 and 0.17 0.06, pH values were 5.0 0.2 and 5.2 0.1, and zeta-potential values were -37.9 3.2 mV e -31.8 2.8 mV for M-PEGNC and placebo(B-PEGNC), respectively. In conclusion, the proposed analytical method is suitable for the quality control of M-PEGNC. Moreover, suspensions showed monomodal size distributions and low polydispersity index indicating high homogeneity of formulations with narrow size distributions, and appropriate pH and zeta potential. The extraction process was efficient for release of meloxicam from nanostructured systems.

    Uniterms: High performance liquid chromatography/quantitative analysis. Meloxicam/determination. PEGylated nanocapsules/quality control. Nanoparticles. Poly(ethylene glycol).

    Para se assegurar que um mtodo analtico produzir informao confivel e interpretvel sobre a amostra este deve ser inicialmente validado, tornando claro que os resultados podem ser confiados e rastreados. Neste estudo, propomos validar um mtodo de cromatografia lquida de alta eficincia (CLAE) para a quantificao do meloxicam encapsulado em nanocpsulas PEGuiladas (M-PEGNC). Efetuamos a validao, avaliando parmetros de especificidade, linearidade, limite de quantificao, limite de deteco, exatido, preciso e robustez. As nanocpsulas PEGuiladas foram preparadas por deposio interfacial do polmero pr-formado e caracterizaram-se o tamanho da partcula, ndice de polidisperso, potencial zeta, pH e eficincia de encapsulao. O mtodo de CLAE proposto fornece resultados seletivos e lineares na faixa de 1,0-40,0 mg/mL; limites de quantificao e deteco de 1,78 mg/mL e 0,59 mg/mL, respectivamente; desvio padro relativo para a repetibilidade de 1,35% e preciso intermediria de 0,41% e 0,61% para o analista 1 e analista 2, respectivamente; exatido entre 99,23 e 101,79%; robustez entre 97,13 e 98,45% para a quantificao de M-PEGNC. Os dimetros mdios das partculas foram 261 13 nm e 249 20 nm; ndice de polidisperso de 0,15 0,07 e 0,17 0,06, valores de pH de 5,0 0,2 e 5,2 0,1 e valores do potencial zeta de -37,9 3,2 mV e -31,8 2,8 mV para o M-PEGNC e o placebo(B-PEGNC), respectivamente. Concluindo, o mtodo analtico proposto adequado para o controle de qualidade do M-PEGNC. Alm disso, suspenses mostraram distribuio

  • F. R. Ianiski, L. V. Laporta, A. M. Rubim, C. Luchese824

    de tamanho monomodal e baixo ndice de polidisperso, indicando alta homogeneidade das formulaes com distribuio estreita de tamanho, pH e potencial zeta apropriados. O processo de extrao foi eficiente para a liberao do meloxicam dos sistemas nanoestruturados.

    Unitermos: Cromatografia lquida de alta eficincia/anlise quantitativa. Meloxicam/determinao. Nanocpsulas PEGuiladas/controle de qualidade. Nanopartculas. Poli(etileno glicol).


    B i o d e g r a d a b l e p o l y m e r s , s u c h a s p o l y (-caprolactone) (PCL) and their copolymers diblocked or multiblocked with poly(ethylene glycol) (PEG), have been commonly used to form polymeric nanoparticles and to encapsulate a variety of therapeutic compounds (Chan et al., 2010; Plapied et al., 2011). Nanoparticles feature dimensions from one to hundreds of nanometers, and remarkable applications in biomedicine due to the unique way in which they interact with matter (Wagner et al., 2006; Kim, Rutka, Chan, 2010). However, several challenges hamper the use of nanoparticles in clinical practice, such as their uptake by the reticuloendothelial system (Knop et al., 2006). The addition of PEG to the surface of nanoparticles (PEGylation) can reduce many of these challenges. Furthermore, PEG is one of few polymers approved for internal use in humans by the FDA, which is an important consideration in developing new pharmaceutical formulations (USP-NF, 2013).

    PEGylated nanoparticles increase the potential for drug delivery and targeting system, besides crossing the blood-brain barrier interacting with microvascular transport proteins. Also, PEG chains can create a barrier layer with the finality of camouflage the phagocytic cells, thus prolonging the circulation period in the blood (Owens III, Peppas, 2006; Grover, Hirani, Sutariya, 2013; Rabanel, Hildgen, Banquy, 2014). Moreover, PEG provides a system with adequate stability in physiological media, which is useful to target tumoral and inflamed regions (Beduneau et al., 2007; Diaz-Lopez et al., 2010; Hervella et al., 2014). Accordingly, the nanoencapsulation of anti-inflammatory drugs is very promising for use as adjuvant treatment of various diseases. In this context, meloxicam [4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide] is a nonsteroidal anti-inflammatory drug with analgesic and antipyretic effects that preferentially inhibits cyclooxygenase-2 (Megan, 2006).

    However, nanoencapsulation requires considerable quality control. The choice of an appropriate analytical methodology is crucial for efficient quality control of an active substance, as such, or in a certain pharmaceutical

    form (Ermer, 2001). In order to ensure that an analytical method will generate reliable and interpretable information about a sample, this method must first be validated, ensuring the reliability and traceability of results (Ribani et al., 2004). In fact, according to Brazilian legislation, an analytical methodology for analysis of a drug or pharmaceutical form should be validated when it is not described in pharmacopeias or official forms recognized by the National Health Surveillance Agency (ANVISA, 2003). No previous data in the literature demonstrated the development and validation of a quantitative bioanalytical high-performance liquid chromatography (HPLC) method for the quantification of meloxicam-loaded PEGylated nanocapsules(M-PEGNC).

    Moreover, experimental studies should ensure that the method meets the requirements of analytical applications, demonstrating the reliability of the results (ANVISA, 2003). The characterization of nanostructured materials is carried out by different methods. These methods are essential to determine the drug content incorporated into them. Some methods for the quantification of meloxicam in pharmaceutical forms by HPLC method are described in the literature (Zhang, Choi, 2008; Bandarkar, Vavia, 2009; Sahoo et al., 2014). However, there is no specific methodology for nanostructured systems containing meloxicam. Therefore, in this study, we revalidate the analytical HPLC method for quantitation of M-PEGNC prepared according to the interfacial deposition of preformed polymer. This method was validated according to the official guidelines (ANVISA, 2003; ICH, 2005).


    Reagents and chemicals

    Meloxicam (Henrifarma - Brazil). Caprylic/capric triglyceride and polysorbate 80 (Delaware - Brazil). Span 60 (sorbitan monostearate) (Sigma Aldrich - USA). Polyethylene glycol 4000 (PEG) (Galena Brazil). Poly(-caprolactone) (PCL) (70000) (Sigma-Aldrich - USA). HPLC-grade methanol (Sigma-Aldrich - Venezuela). Milli-Q Water (Millipore

  • Validation of high performance liquid chromatography method for determination of meloxicam loaded PEGylated nanocapsules 825

    Corporation). All other chemicals and solvents used were of analytical or pharmaceutical grade. All reagents were used as received.

    Preparation of loaded PEGylated nanocapsules

    M-PEGNC were prepared by interfacial deposition of preformed polymer according to Fessi et al. (1989). Meloxicam was dissolved in acetone with the polymer (PCL), the oil (caprylic/capric triglyceride) and sorbitan monostearate at 40 C. This organic phase was poured into an aqueous phase composed of polysorbate 80 and PEG under stirring, leading to the immediate formation of a milky dispersion. Lastly,


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