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.