Determination of methylphenidate and its metabolite ritalinic acid in urine by liquid chromatography/tandem mass spectrometry.

Methylphenidate (MPH) is a drug that is licensed for treatment of ADHD and also narcolepsy. Monitoring of the parent drug and its major metabolite ritalinic acid (RA) in urine is considered necessary to ensure compliance with treatment programmes. A rapid, simple and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay was developed for the determination of MPH and its metabolite RA in human urine. After urine was diluted with water, methylphenidate, the major metabolite ritalinic acid, and d₆-amphetamine as the internal standard were resolved on a PFP propyl column using gradient elution of 0.02% ammonium formate and acetonitrile. The total analysis time was 13.5 min. The three compounds were detected using electrospray ionisation in the positive mode. Standard curves were linear over the concentration range 5-5000 μg/L (r>0.997), bias was ≤ ±20%, intra- and inter-day coefficients of variation (imprecision) were <8% and the limit of detection was 5 μg/L. The limit of quantitation was set at 100 μg/L. Matrix effects were up to 140% but these were accounted for by the internal standard. The assay is being used successfully in clinical practice to enhance the safe and effective use of methylphenidate.