Determination of methylphenidate and ritalinic acid in blood, plasma and oral fluid from adolescents and adults using protein precipitation and liquid chromatography tandem mass spectrometry--a method applied on clinical and forensic investigations.

A validated, accurate and sensitive LC-MS/MS method for determination of racemic methylphenidate and its metabolite ritalinic acid has been developed. The analytes were quantified by tandem mass spectrometry operating in positive electrospray ionization mode with multiple reaction monitoring. Blood, plasma and oral fluid samples of 100μl were prepared by simple precipitation with 200μl of an aqueous solution of zinc sulphate in methanol. Corresponding deuterated internal standards were used for quantification. Calibrations for methylphenidate and ritalinic acid were linear within the selected range of 0.2-30ng/ml and 10-1500ng/ml in blood or plasma and in the range of 1-500ng/ml and 0.25-125ng/ml in oral fluid, respectively. The method was successfully applied for the analysis of samples from patients treated with methylphenidate in the dose range of 36-72mg/day and some representative ante mortem and post mortem samples from clinical and forensic toxicological investigations. A three to fourfold higher concentration of methylphenidate was found in oral fluid compared with blood while for ritalinic acid the concentrations were about 25-fold lower in oral fluid.