Methylphenidate (MPH) is a phenethylamine derivative used in the treatment of childhood attention-deficit hyperactivity disorder. MPH is biotransformed in the body by the hydrolysis of the methyl ester linkage to its metabolite, ritalinic acid. Whereas both compounds are usually measured in plasma and urine, preliminary observations show that only the parent compound is present in hair from treated individuals. Since in children hair samples can be easily collected without the need for special skills and exposing a patient to discomfort, hair testing of MPH should be an alternative to check compliance in a wider time-window than if using blood. A procedure based on liquid chromatography-mass spectrometry (LC-MS) has been developed for the determination of MPH in hair of treated children. After addition of 3,4-methylenedioxypropylamphetamine as internal standard, hair samples were overnight digested with 0.1M HCl at 37 degrees C. Then, after pH adjustment to 6 using 1N NaOH, and 0.1M phosphate buffer, the analyte was extracted with Bond-Elut Certify columns. Chromatographic separation was achieved at ambient temperature using a reverse phase column and a mobile phase of 80% 10mM ammonium acetate-20% acetonitrile with a 20 min gradient program. The mass spectrometer was operated in positive electrospray ionization and selected ion monitoring acquisition mode. The method was validated in the range 0.15-50 ng MPH/mg hair, using 20mg hair per assay. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 73.2 and 77.1%. First results show MPH hair concentration varying from 0.15 to 4.17 ng/mg hair, with decreasing drug concentration in distal hair segments, even in children treated with the same MPH dose during the period corresponding to different segments. This fact could be either attributed to sebum or sweat shunt with the most proximal hair segment or drug degradation by cosmetic treatments in more distal segments.