Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study.

Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gamma-aminobutyric acid (GABA). A simple and sensitive liquid chromatography mass spectrometry method for determination of bicuculline in rat plasma was developed over the range of 5-500ng/mL. After addition of midazolam as internal standard, protein precipitation with acetonitrile-methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1mm×150mm, 5µm) column with acetonitrile -0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 368 for bicuculline and m/z 326 for the IS. Linear calibration was obtained with correlation coefficients r>0.99. The CV of the precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. Mean recoveries of bicuculline in plasma were in the range of 80.5-91.8%. The method was successfully applied to the pharmacokinetic study after gavage administration of 15mg/kg bicuculline in rats.

GABA antagonism reverses hypoxic respiratory depression in the cat.

We assessed the role of gamma-aminobutyric acid (GABA) as a potential causative agent of hypoxic respiratory depression by monitoring the response of the phrenic neurogram to systemic infusion of the GABA antagonist bicuculline (0.01 mg.kg-1.min-1) under control conditions and during isocapnic brain hypoxia produced by CO inhalation in separate groups of anesthetized, glomectomized, vagotomized, paralyzed, and ventilated cats with blood pressure held constant. The maximum effect of bicuculline in subseizure doses in control cats was to increase minute phrenic activity to 151 +/- 14% of preinfusion values. Infusion was continued until seizure activity was seen in the electroencephalogram. A 53% decrease of arterial O2 content resulted in a marked reduction of both peak phrenic amplitude and phrenic firing frequency to 16 and 64% of control values, respectively. Infusion of bicuculline while the level of hypoxia was maintained constant restored both peak phrenic amplitude and phrenic firing frequency to prehypoxic levels. The maximum effect of bicuculline was to increase minute phrenic activity to 123 +/- 13% of the prehypoxic value. These results suggest that although GABA has only a modest role in determining the output of the control phrenic neurogram, a significant portion of the phrenic depression that occurs during hypoxia can be attributed to inhibition of respiratory neurons by GABA.