Determination of free-form amphetamine in rat brain by ion-pair liquid chromatography–electrospray mass spectrometry with in vivo microdialysis

An ion-pair liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) method with in vivo microdialysis for the determination of free-form amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC–ESI-MS. A reversed phase C18 column was employed for LC separation. Trifluoroacetic acid (TFA) was added in the mobile phase (acetonitrile–water, 10:90, v/v) as an ion-pair reagent. The ion-pair process disguises the protonated amphetamine cations from the ESI-MS electric field as neutral molecules. Post-column addition of volatile organic acid was utilized to minimize TFA signal suppression effect on ESI-MS detection. More than six-fold enhancement of ESI-MS response was achieved by the post-column addition of propionic acid. Good linearity (0.01–1.00 μg/ml, r2=0.99) and detection limit (0.002 μg/ml) were determined. Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain after a single 3.0 mg/kg i.p. administration.