Methylphenidate, a psychostimulant used for the treatment of attention deficit hyperactivity disorder and narcolepsy, is administered as a 50:50 racemic mixture, despite the fact that d-methylphenidate has been shown to have greater pharmacologic activity. This paper presents a validated LC-MS/MS approach to separation and quantification of methylphenidate enantiomers using a vancomycin column and triethylammonium acetate to enhance the chiral separation. The method is applicable to the monitoring of these enantiomers in mouse brain, with a limit of detection of 0.5 ng/mL and a lower limit of quantification of 7.5 ng/mL.
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