Sensitive determination of oxybutynin and desethyloxybutynin in dog plasma by LC-ESI/MS/MS

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) was developed for the quantification of oxybutynin and desethyloxybutynin in dog plasma. Diazepam was used as internal standard, with plasma sample extracted using n-hexane and back-extracted using hydrochloric acid. A centrifuged lower layer (aqueous layer) was injected into a C(18) XTerra MS column (2.1 x 30 mm(2)) with 3.5 microm particle size. The analytical column lasted for at least 500 injections. The mobile phase was composed of 90% methanol, with flow rate at 200 microl/min. The mass spectrometer was operated in positive ion mode using electrospray ionization. Nitrogen was used as the nebulizer gas and argon was used as the collision gas. Using MS/MS with multiple reaction monitoring (MRM) mode, oxybutynin and desethyloxybutynin were detected without severe interferences from plasma matrix. Oxybutynin produced a protonated precursor ion ([M+H](+)) at m/z 358 and a corresponding product ion at m/z 142. Desethyloxybutynin produced a protonated precursor ion ([M+H](+)) at m/z 330 and a corresponding product ion at m/z 96. And internal standard (diazepam) produced a protonated precursor ion ([M+H](+)) at m/z 285 and a corresponding product ion at m/z 193. Detection of oxybutynin and desethyloxybutynin in dog plasma were accurate and precise, with detection limit at 0.1 ng/ml. This method has been successfully applied to a study of oxybutynin and desethyloxybutynin in dog plasma.