Short- and Long-Term Stability of Methylphenidate and Its Metabolites in Blood

Methylphenidate (MPH) is a medication used to combat attention-deficit/hyperactivity disorder by speeding up brain activity. MPH has two chiral centers; however, d-threo-MPH is responsible for its effects. Few studies have analyzed MPH and its metabolites, ritalinic acid (RA) and ethylphenidate (EPH), in blood. Stability studies are crucial in a forensic setting to provide insight on ideal storage conditions and analysis time. In this study, d,l-MPH, d,l-EPH and RA were analyzed at two concentrations (15 and 150 ng/mL) over 5 months at room temperature (∼25°C), refrigerated (4°C), frozen (-20°C) and elevated (35°C) temperatures. Analytes were analyzed using a validated liquid chromatography--mass spectrometry method. RA concentrations increased 53% at 25°C after 24 h, while d- and l-MPH concentrations dropped 18.1 and 20.6%, respectively. Additionally, d- and l-EPH concentrations decreased 22.3 and 28.8%, respectively. All analytes were stable at 4°C for 1 week (±17% change). At -20°C, all analytes were stable for 5 months. At 35°C, l-EPH remained stable for 24 h (14.4% loss) at the high concentration, while RA increased 244%. Losses of 64.1, 68.7 and 27.2% were observed for d- MPH, l-MPH and d-EPH, respectively. Due to this, a follow-up study was designed to assess the breakdown of MPH. The short-term experiment assessed d,l-MPH at two concentrations for 1 month in the same conditions. As MPH decreased, RA concentrations rose. At 25°C, it took 2 weeks for MPH to metabolize completely into RA. In refrigerated and frozen temperatures, MPH did not completely metabolize to RA. In elevated temperatures, MPH broke down to RA within 2 weeks. Due to this, it was concluded that d,l-MPH breaks down in the blood to its metabolite RA and may make data interpretation difficult if samples are not properly stored. The optimal storage for these analytes is recommended at -20°C.