HPLC method validation and quantification of flupirtine in canine plasma

INTRODUCTION Flupirtine (FLU) is a non-opioid analgesic drug belonging to the unique class of the ‘Selective Neuronal Potassium Channel Openers’ without antipyretic or antiphlogistic properties [1]. The pharmacological properties of FLU contribute to its therapeutic benefits, without undesirable adverse effects typical of classic analgesic drugs [2] and that might potentially be useful in veterinary medicine. No analytical method to detect FLU in canine plasma samples through a fluorimetric detector has been published to date. The aim of the study was to develop an analytical method providing a selective and accurate quantification of FLU. MATERIAL AND METHODS 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. Typical retention times for FLU and IS were 4.6 0.2 and 5.8 0.2 min, respectively. The extraction method was performed with 500 ll of plasma sample added to 100 ll of IS (100 lg ml 1) and vortexed for 60 s. Four ml of AcOEt:CH2Cl2 (7:3 v/v). Three ml of the organic phase was collected and evaporated under a gentle stream of nitrogen at 40°C and reconstituted with 500 ll of the mobile phase. The described method was validated according to EMA guidelines on the bioanalytical method validation. RESULTS AND CONCLUSION The recoveries of FLU and IS (trazodone) were about 89% and 77%. Limits of quantification and detection were 1 and 0.3 ng ml 1, respectively. The applicability of this method has been verified by determining FLU in canine plasma after single oral treatment with 5 mg kg 1 of Efiret. HPLC analysis of the plasma confirmed the presence of FLU in time related amounts. The average FLU concentration in canine plasma ranged between 4.3 and 760 ng ml 1. The selectivity of the method was also confirmed by HPLC-MS analysis of plasma samples collected in treated dogs. No compounds co-eluting with the analyte of interest were detected by full scan acquisitions in positive ion mode. This is particularly demonstrated from the HPLC–MS extracted ion chromatogram (m/z 305 and 372 Da for FLU and IS, respectively) that exhibited a good correspondence with the HPLC–FL chromatogram. The low LOQ shows that the method could be useful for drug measurement even when administered in sub-clinical doses. As FLU is a drug recently considered for the veterinary medicine application, this method is the most suitable to be used for pharmacokinetic investigations in different animal species. REFERENCES 1. Kornhuber J., M. Maler, J. Wiltfang, S. Bleich, D. Degner and E. R€uther, (1999) Neuronal potassium cannel opening with flupirtine. Fortschr Neurol Psychiatr, 67: 466–475. 2. Devulder J, (2010) Flupirtine in pain management: pharmacological properties and clinical use. CNS Drugs, 24: 867– 881.