Liquid-liquid extraction using 96-well plate format in conjunction with liquid chromatography/tandem mass...

Methylphenidate (MPH; Ritalin: methyl-alpha-phenyl-2-piperidinacetate hydrochloride) is utilized for the treatment of attention deficit hyperactivity disorder (ADHD) and narcolepsy. Recently, we described a rapid enantioselective liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of the enantiomers of MPH (Rapid Commun. Mass Spectrom. 1999; 13: 2054). A lower limit of quantification (LLOQ) of 87 pg/mL was attained for the human plasma assay. The present paper describes a high-throughput sample preparation procedure in conjunction with racemic LC/MS/MS analysis for MPH with a LLOQ of 50 pg/mL. A semi-automated robotics method using liquid-liquid extraction (LLE) in a 96-well plate format was developed and validated. The correlation coefficients were > or =0.998 for MPH indicating good fits of the regression models over the range of the calibration curve. The accuracy and precision of the semi-automated approach were comparable to those obtained using the manual sample preparation technique reported previously (vide supra). The current method can easily be adapted to the enantioselective LC/MS/MS assay of MPH. The assay was simple, fast, specific, and exhibited excellent ruggedness.