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A sensitive and reproducible HPLC procedure was developed for the simultaneous determination of the diastereoisomers of the synthetic amino acid L-buthionine-(SR)-sulfoximine (BSO) in human plasma and urine. Plasma samples were prepared for analysis by addition of internal standard (L-norleucine) followed by ultrafiltration using disposable centrifugal filtration units. Urine samples received internal standard followed by solid phase extraction using disposable C18 cartridges. All samples were derivatized with phenylisothiocyanate (PITC). The derivatized amino acids were separated by HPLC on an octyldecyl column (250 mm x 4.6 mm I.D., 5 microns particle size) using a mobile phase of sodium acetate-acetonitrile-triethylamine-ethylaminediaminetetraacetic acid. The column effluent was monitored at 254 nm and quantitation was performed using peak areas. The linear range for each diastereoisomer of L-(SR)-BSO was from 2 to 100 micrograms/ml in plasma and from 10 to 1000 micrograms/ml in urine. The method is reproducible, convenient and sensitive, illustrating its utility for application in pharmacokinetic studies.