Validation of a New 96-Well Plate Spectrophotometric Method for the Quantification of Compound 48/80 Associated with Par
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Research Article Validation of a New 96-Well Plate Spectrophotometric Method for the Quantification of Compound 48/80 Associated with Particles Dulce Bento,1,2 Gerrit Borchard,3 Teresa Gonçalves,1,4 and Olga Borges1,2,5
Received 3 December 2012; accepted 25 February 2013; published online 7 March 2013 Abstract. A new, simple, inexpensive, and rapid 96-well plate UV spectrophotometric method was developed and validated for the quantification of compound 48/80 (C48/80) associated with particles. C48/80 was quantified at 570 nm after reaction with acetaldehyde and sodium nitroprusside in an alkaline solution (pH 9.6). The method was validated according to the recommendations of the ICH Guidelines for specificity, linearity, range, accuracy, precision, and detection and quantification limits (DL and QL). All the validation parameters were assessed in three different solvents, i.e., deionized water, blank matrix of chitosan nanoparticles, and blank matrix of chitosan/alginate nanoparticles. The method was found to be linear in the concentration range of 5 to 160 μg/ml (R2 >0.9994). Intraday and interday precision was adequate, with relative standard deviation lower than those given by the Horwitz equation. The mean recoveries of C48/80 from spiked samples ranged between 98.1% and 105.9% for calibration curves done with the blank matrices and between 89.3% and 103.3% for calibration curves done with water, respectively. The DL were lower than 1.01 μg/ml and the QL were lower than 3.30 μg/ml. The results showed that the developed method is sensitive, linear, precise, and accurate for its intended use, with the additional advantages of being cost-effective and time-effective, allowing the use of small-volume samples, and the simultaneous analysis of a large number of samples. The proposed method was already successfully applied to evaluate the loading efficacy of C48/80 chitosan-based nanoparticles and can be easily applied during the development of other C48/80-based formulations. KEY WORDS: C48/80; chitosan nanoparticles; mast cell activator; method validation; p-Methoxy-Nmethylphenethylamine.
INTRODUCTION Compound 48/80 (C48/80; polymer formed from pMethoxy-N-methylphenethylamine monomers) is a mast cell activator that has been widely used in allergies-related studies due to its ability to induce the release of histamine (1). More recently, it was demonstrated that C48/80 can also act as a vaccine adjuvant by inducing dendritic cell migration to draining lymph nodes via a mast cell-dependent mechanism (2). In fact, different studies showed that the coadministration of C48/80 with an antigen improves the immunogenicity of the antigen, resulting in higher titers of specific antibodies compared to the antigen alone (2–5). The vectorization of C48/80 to targeted cells could result in an improvement of the adjuvant effect. This can be achieved by the incorporation of the mast cell activator in 1
Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004-504 Coimbra, Portugal. 2 Faculty of Pharmacy, U
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