A novel HPLC fluorescence method for the quantification of methylphenidate in human plasma

Abstract

A number of analytical methods have been established to quantify methylphenidate (MPH). However, to date no HPLC methods are applicable to human pharmacokinetic studies without the use of mass spectrometry (MS) detection. We developed a sensitive and reliable HPLC-fluorescence method for the determination of MPH in human plasma using 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) as the derivatizing agent. An established GC-MS method was adopted in this study as a comparator assay. MPH was derivatized using DIB-Cl, and separated isocratically on a C18 column using a HPLC system with fluorescence detection (lambda(ex)=330 nm, lambda(em)=460 nm). The lower limit of quantification was found to be 1 ng/mL. A linear calibration curve was obtained over the concentrations ranging from 1 ng/mL to 80 ng/mL (r=0.998). The relative standard deviations of intra-day and inter-day variations were <or=9.10% and <or=7.58%, respectively. The accuracy ranged between 92.59% and 103.06%. The method was successfully applied to the pharmacokinetic study of a subject who received a single oral dose (0.3 mg/kg) of immediate-release MPH and yielded consistent results with that of the GC-MS method. This method is the first HPLC assay with non-MS detection providing sufficient reliability and sensitivity for both pre-clinical and clinical studies of MPH.

Figure 1

The reaction scheme for labeling of MPH with DIB-Cl.

Figure 2

Typical chromatograms of blank human plasma (A), human plasma spiked with 1 ng/mL MPH (B), plasma obtained from a healthy volunteer at the time points of 0 min (C) and 30 min (D) following a single oral dose of 0.3 mg/kg MPH.

Figure 3

Time course of plasma concentrations of MPH in a healthy volunteer receiving a single oral dose of 0.3 mg/kg MPH determined by GC-MS and HPLC-FL.