Simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma using high performance liquid chromatography coupled with tandem mass spectrometric detection: Application to a bioequivalence study

A rapid and sensitive method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of codeine, ephedrine, guaiphenesin and chlorpheniramine in beagle dog plasma has been developed and validated. Following liquid–liquid extraction, the analytes were separated on a reversed-phase C18 column (150 mm × 2.0 mm, 3 μm) using formic acid:10 mM ammonium acetate:methanol (0.2:62:38, v/v/v) as mobile phase at a flow rate of 0.2 mL/min and analyzed by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode. The method was linear for all analytes over the following concentration (ng/mL) ranges: codeine 0.08–16; ephedrine 0.8–160; guaiphenesin 80–16,000; chlorpheniramine 0.2–40. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. It is the first time that the validated HPLC–MS/MS method was successfully applied to a bioequivalence study in 6 healthy beagle dogs.