Bioanalytical Method Validation and Quantification of Flupirtine in Canine Plasma by HPLC with Spectrofluorimetric Detection

Flupirtine (FLU) is a non-opioid analgesic drug belongs to the class of N-Methyl-D-Aspartate (NMDA) antagonist without antipyretic or antiphlogistic properties. No analytical method to detect FLU in canine plasma samples through a fluorimetric detector has been published to date. The analytical method described in this work provides a selective and accurate quantification of FLU. The mobile phase consisted of ACN:AcONH₄ (20 mM) pH 6.8 (60:40, v/v) at a flow rate of 1 mL min^-1 in isocratic mode. Excitation and emission wavelengths were set at 323 and 370 nm, respectively. The recoveries of FLU and IS (trazodone) were about 89 and 77%. Typical retention times for FLU and IS was 4.6±0.2 and 5.8±0.2 min, respectively. Limits of quantification and detection were 1 and 0.3 ng/mL, respectively. The described method was validated according to international guidelines on the bioanalytical method validation. The applicability of this method was verified by determining FLU in canine plasma after single oral treatment with 5 mg kg^-1 of Efiret^®. The low LOQ showed that the present method could be useful for the FLU measurement even when administered in sub-clinical doses.