Simultaneous Determination of Methylphenidate, Amphetamine and their Metabolites in Urine using Direct Injection Liquid Chromatography-Tandem Mass Spectrometry

Nonmedical use of prescription stimulants such as methylphenidate (MPH) and amphetamine (AP) by normal persons has been increased to improve cognitive functions. Due to high potential for their abuse, reliable analytical methods were required to detect these prescription stimulants in biological samples. A direct injection liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and implemented for simultaneous determination of MPH, AP and their metabolites ritalinic acid (RA) and 4-hydroxyamphetamine (HAP) in human urine. Urine sample was centrifuged and the upper layer ($100{\mu}L$) was mixed with $800{\mu}L$ of distilled water and $100{\mu}L$ of internal standards ($0.2{\mu}g/mL$ in methanol). The mixture was then directly injected into the LC-MS/MS system. The mobile phase was composed of 0.2% formic acid in distilled water (A) and acetonitrile (B). Chromatographic separation was performed by using a Capcell Pak MG-II C18 ($150mm{\times}2.0mm$ i.d., $5{\mu}m$, Shiseido) column and all analytes were eluted within 5 min. Linear least-squares regression with a 1/x weighting factor was used to generate a calibration curve and the assay was linear from 20 to 1500 ng/mL (HAP), 40-3000 ng/mL (AP and RA) and 2-150 ng/mL (MPH). The intra- and inter-day precisions were within 16.4%. The intra- and inter-day accuracies ranged from -15.6% to 10.8%. The limits of detection for all the analytes were less than 4.7 ng/mL. The suitability of the method was examined by analyzing urine samples from drug abusers.