ELSEVIER Journal of Chromatography B, 666 (1995) 241-250 JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL APPLICATIONS Determination of d-amphetamine in biological samples using high-performance liquid chromatography after precolumn derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid John F. Bowyer*, Peter Clausing, Glenn D. Newport Division of Neurotoxicology, National Center for Toxicological Research, 3900 NCTR Dr, Jefferson, AR 72079-9502, USA First received 7 September 1994; revised manuscript received 13 December 1994; accepted 13 December 1994 Abstract An HPLC method is described for the determination of amphetamine using fluorometric detection after derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid. This procedure is more sensitive (detection limit 370 fmol in microdialysate buffer standards, 1.5 pmol in extracted plasma and tissue samples) than most of the previous methods described for the determination of amphetamine with HPLC-fluorescence detection. Due to the stability of the derivative it is also suitable for autosampling after manual derivatization. Investigators currently using o-phthaldialdehyde derivatization and fluorometric detection for amino acid determination should be able to rapidly implement this method. I. Introduction Chromatographic methods for the quantitation of amphetamine (AMPH) in tissues and body fluids are known for more than two decades and include gas chromatography [1], gas-liquid chro- matography [2] and combined gas chromatog- raphy-mass spectrometry (GC-MS) [3]. Today, with GC-MS minimum AMPH concentrations as low as 0.1 ng/ml can be quantitated [4]. However, HPLC often is an alternative, when mass fragmentation is not an analytical require- ment, which is less expensive and simpler yet sensitive enough for many applications in bio- medical research. The determination of AMPH * Corresponding author. 0378-4347/95/$09.50 (~) 1995 Elsevier Science B.V. All rights SSDI 0378-4347(94)00573-7 by HPLC has been utilized for 20 years [5]. AMPH was quantitated after derivatization by UV [6-8], fluorescent [9-11], chemilumines- cence [12], and more recently by diode-array UV [13] detection. For laboratories without access to a chemiluminescent or photodiode-array detec- tor, fluorescent detection frequently is the meth- od of choice for the quantitation of primary amines. In this context, o-phthaldialdehyde (OPA) is the most widely reported derivatizing reagent for HPLC quantitation of primary amines, with either electrochemical, UV, or fluorescence detection [14]. Over decades, AMPH and related compounds have attracted attention in pharmacology and neuroscience because of their neurotoxic poten- tial and use as a neuropharmacological tool in reserved