Desenvolvimento e validação de metodologias analíticas para análise da cefepima e do metronidazol em plasma e microdialisado peritoneal de ratos Wistar

Abstract

Cefepime (CEF) and metronidazole (MTZ) are two antimicrobial agents which can be used to treat secondary peritonitis. Different analytical methodologies have been described for the quantification of these analytes in biological matrices, but there are no reports addressing the determination of concentrations free of these antimicrobials in the peritoneal fluid of rats. Therefore, this study aimed to develop and validate two analytical methodologies to measure concentrations free of these substances in the peritoneal fluid of Wistar rats. Regarding CEF, a quantification method has been developed by using the technique of liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (LC-ESI-QTOF-MS), with a Shimadzu Shim-pack CLC-ODS C18 column (250 mm x 4.6 mm) and a pre-column (5 um C18, 4 mm; Phenomenex). The mobile phase consisted of (A) ultrapure water with a pH of 3.5, and (B) acetonitrile (80:20, v/v). The flow rate was 0.8 mL/min, and the injection volume was 20 uL. Quantification was carried out in the positive mode to monitor ions with m/z of 481.1311 for CEF and with m/z of 171.0283 for IS. Regarding MTZ, quantification has been conducted using high-performance liquid chromatography with ultraviolet detection (HPLC-UV/VIS). Chromatography run was carried out on a C18 column, with a flow rate of 1.6 mL/min at 40ºC, with a mobile phase composed of acetonitrile and an aqueous solution of 50 mM monobasic phosphate buffer, with 0.1% of triethylamine at pH 3.0 (10:90, v/v). Linearity was achieved in both methods with a coefficient of determination > 0.99 for plasma and peritoneal fluid matrices. The results of the analytical performance parameters for the analyzed analytes met the criteria of the Food and Drug Administration's Bioanalytical Method Validation Guidance. The applicability of the methods was demonstrated in a pilot pharmacokinetic study in Wistar rats after intravenous administration (bolus) of 110 mg/kg of CEF and 30 mg/kg of MTZ. To determine the pharmacokinetic parameters, the version 6.4 of the Phoenix software was used. [resumo fornecido pelo autor]