Determination of amphetamine in dog plasma by gas chromatography with mass selective detection

This paper describes the validation of an analytical method for the determination of amphetamine in beagle dog plasma by gas chromatography coupled to mass spectrometry (GC-MS). d-Amphetamine-d(6) was used as the internal standard. The method consisted of a rapid single-step liquid-liquid extraction and derivatization of amphetamine with 2,2,2-trichloroethyl chloroformate, followed by sensitive GC-MS detection. This is the first report utilizing the combination of trichloroethyl chloroformate as a derivatization reagent and a deuterated amphetamine analog as an IS for the quantification of amphetamine in plasma. The method was validated in terms of specificity, curve fit, precision, accuracy, recovery and stability, and was acceptable according to FDA draft guidelines for validation of bioanalytical methods. The limit of detection was 0.65 ng/mL. The calibration range was 5-150 ng/mL. The validated method was successfully employed for the quantitation of amphetamine in dog plasma samples for pharmacokinetic profiling.