Determination of enantiomeric drugs in physiological fluids using on-line solid phase derivatizations and...

A new analytical approach has been developed for the determination of d,l-amphetamine in urine using on-line solid phase derivatizations in HPLC-UV/FL. Several other enantiomeric drugs were also investigated using the same method. The method was validated by several experiments, including: (1) kinetic studies for the reaction of each enantiomeric drug with the solid phase chiral reagent; (2) "single blind spiking" experiments; and (3) polarimetry for confirmation of the enantiomeric composition determined by the solid phase diastereomer formation-HPLC-UV/FL method. The resulting diastereomers were well resolved (Rs = 1.05-1.40) under typical reversed-phase HPLC conditions. Enantiomeric contamination at the 1.1% level could be detected. The lowest amount of d,l-amphetamine that could be simultaneously derivatized and detected was about 50 ng ml-1. The linearity of the overall measurement was 3-4 orders of magnitude. d,l-Amphetamine spiked into urine at different concentration levels and different d,l-ratios, only followed by filtration, were directly injected into the on-line solid phase derivatization-HPLC-UV/FL system for quantitation with relative standard deviations 1.8-6.4% and relative errors 0.6-9.8%.