Accelerated quantification of amphetamine enantiomers in human urine using chiral liquid chromatography and on-line column-switching coupled with tandem mass spectrometry

Amphetamine (AM) is a powerful psychostimulant existing in two enantiomeric forms. Stereoselective analysis of AM in biosamples can assist clinicians and forensic experts in differentiating between abuse of illicitly synthesized racemic AM and ingestion of pharmaceutical AM formulations containing either S-AM or different proportions of the S- and R-enantiomers. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying AM enantiomers in urine was newly developed. The method comprised dilution with water, followed by injection of the diluted sample onto an achiral C18 trapping column for purification and subsequent backflush elution to a chiral Lux 3 mu m AMP LC column by means of a switching valve. An isocratic mobile phase of 25 % acetonitrile in 0.1 M aqueous ammonia was used for enantiomeric separation. Injection, cleanup, and backflush of the next sample were performed before the previous sample had eluted from the analytical column, thus enabling simultaneous enantioseparation of up to three samples within the analytical column. This novel chromatographic concept allowed for increased sample throughput by accelerating both the sample preparation and the LC analysis. Analyte detection was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was successfully validated through assessment of its linearity, lower limit of quantification, accuracy and precision, selectivity, matrix effect, carry-over, dilution integrity, and re-injection reproducibility. Linearity ranged from 0.05 to 25 mg/L for both enantiomers. Proof of the method included analysis of urine samples obtained from drug abusers and patients receiving an S-AM prodrug.