Determination of Doxapram Hydrochloride in Rabbit Plasma by LC-MS-MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS-MS) method for determination of doxapram hydrochloride in rabbit plasma was developed. After addition of urapidil hydrochloride as internal standard (IS), protein precipitation by 10% trichloroacetic acid in methanol (w/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm x 50 mm, 3.5 mu m) column with acetonitrile-water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; multiple reaction monitoring (MRM) mode was used for quantification using target fragment ions m/z 378.9 -> 291.8 for doxapram hydrochloride and m/z 387.9 -> 204.6 for the IS. Calibration plots were linear over the range of 2-1000 ng mL(-1) for doxapram hydrochloride in plasma. Lower limit of quantitation (LLOQ) for doxapram hydrochloride was 2 ng mL(-1). Mean recovery of doxapram hydrochloride from plasma was in the range 83.7-91.5%. RSD of intra-day and inter-day precision were less than 9%, respectively. This method is simple and sensitive enough to be used in pharmacokinetic research for determination of doxapram hydrochloride in rabbit plasma.