Chiral separation and quantification of d,l-methylphenidate, d,l-ethylphenidate and ritalinic acid in blood by LC-MS/MS

Abstract

Drugs such as methylphenidate (MPH) are commonly prescribed for Attention-Deficit/Hyperactivity Disorder but may be abused recreationally. Threo-MPH is associated with pharmacological effects and is present as dextro (d) or levo (l) configuration, with the d configuration being more potent. However, many medications are sold as a racemic mixture and thus analytic separation of isomers is essential. Additionally, enantiomeric separation can be used to potentially determine illegal production. MPH metabolizes into ritalinic acid (RA) as well as ethylphenidate (EPH) in the presence of ethanol. Chiral analysis poses challenges to researchers. Due to limited assays, this project aimed to develop a method that separates and quantifies the enantiomers of MPH and EPH as well as RA in blood. Methods such as this are critical to understanding the pharmacokinetics of chiral cognitive stimulants. This method uses solid phase extraction and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was fully validated. The linear range for MPH and EPH was 0.5–200 ng/mL and 0.5–500 ng/mL for RA. Limit of detection was 0.1 ng/mL (with the exception of RA with a LOD of 0.5 ng/mL) and the limit of quantification was 0.5 ng/mL, despite significant matrix effects. Extraction recovery was > 79%. Bias was −4.8 to −12.7% and maximum within-run precision was ± 12.5% for all analytes. The optimized and validated technique offers chiral separation of the threo-enantiomers of d,l-MPH, d,l-EPH and RA and quantification in blood utilizing LC-MS/MS.