Validation of isocratic reverse-phase HPLC method for benznidazole determination in human plasma

GARCÍA, MC; MARTINEZ, R; MANZO, RH; JIMENEZ-KAIRUZ, AF

Córdoba

Congreso; 3 rd International Meeting on Pharmaceutical Sciences; 2014

Facultad de Ciencias Químicas, Universidad Nacional de Córdoba y Coorganizada por la Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario

Benznidazole (BZN) is the first choice medicine for Chagas disease treatment denoting a clinical efficacy higher than 80%. However, few studies of bioavailability/bioequivalence (BD/BE) have been published despite high incidence of toxic and adverse events, more often in young and adult patients, reported. The aim of this work was the validation of a simple HPLC methodology for assay BZN in human plasma, for be used in Phase-4 Clinical Trials of new BNZ delivery systems in the framework of the BD/BE Program of UNC (1,2). The BNZ was assayed using reverse-phase isocratic HPLC method. Chromatographic analysis was performed on a 250x4.6-mm (5-µm) C18-column. Acetonitrile:water (6:4) was used in the mobile phase at flow rate of 0.9-µL.min-1. The UV-detector was set at 324-nm. The procedure was fully validated to meet the requirements of FDA and GLP Guidelines for Industry (3) in terms of: calibration curve, selectivity, accuracy, precision and recovery, sensitivity, reproducibility and stability. Benzocaine and metronidazole were used as internal standard and analogue structura (4). In assayed conditions, BZN and benzocaine showed tR between 6.3-7.0 and 11.0-12.0 min, respectively. Adequate selectivity was demonstrated using metronidazole. Calibration curve showed linearity (r2=0.9987, N=3) in the BNZ concentration range between 1.6-100.0µg/mL (LLOQ-High) with acceptable sensitivity. Accuracy and precision showed main values of 98% (CV≥10%) and 94% (CV≥5%) for inter-/intra-day experiments, respectively (N=3). Reinjection (N=5) denoted acceptable reproducibility. Recovery of analyte was close 100%. Analyte stability in mobile phase (stored for 24-h) and after freeze and thaw cycles (N=3) comply with requirements (CV≥10%). A simple, robust and selective HPLC-method was validated for benznidazole determination in human plasma meeting the bioanalytical method validation requirements and may be used in clinical trials. References. 1. Programa BD/BE de la UNC para la determinación de la Biodisponibilidad y Bioequivalencia de medicamentos. 2. Olivera ME. (directora). Estudios Clínicos de Fase 4 en voluntarios sanos. Evaluación farmacocinética de formulaciones optimizadas para el tratamiento de infecciones re-emergentes y endémicas. Proyecto de I+D orientado, Clase "C". Res. SECyT-UNC 373/2012. Período 11/2012-11/2014. 3. FDA 2013. Guidance for industry: Bioanalytical method validation. Center for Drug Evaluation and Research. Revision 1, september 2013, Biopharmaceutics. URL: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM368107.pdf) [last accessed: 06/23/2014] 4. Guerrero L. et al. A high-performance liquid chromatographic method for benznidazole quantitation in plasma of patients with Chagas disease. Clin Chem Lab Med 2011;49(1):77?82.