Nonextractive procedure and precolumn derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole for trace determination of trimetazidine in plasma by high-performance liquid chromatography with fluorescence detection

A highly sensitive high-performance liquid chromatographic method with fluorescence detection has been developed and validated in a single laboratory for the trace determination of trimetazidine (TMZ) in human plasma. Fluoxetine (FLX) was used as the internal standard. TMZ and FLX were isolated from plasma by protein precipitation with acetonitrile and derivatized by heating with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole in pH 8 borate buffer at 70 degrees C for 30 min. Separations were performed in the isocratic mode on a Nucleosil CN column with the mobile phase acetonitrile-10 mM sodium acetate buffer (pH 3.5)-methanol (47 + 47 + 6, v/v/v) at a flow rate of 1.0 mL/min. The derivatized samples were excited at 470 nm and monitored at an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with a good correlation coefficient (r = 0.9997, n = 5) was obtained for the peak area ratio of TMZ to FLX and for TMZ concentrations of 1-120 ng/mL. The proposed method has the lowest limits of detection and quantitation reported to date for the determination of TMZ in plasma with values of 0.3 and 0.95 ng/mL, respectively. The values for intra- and interassay precision were satisfactory; the relative standard deviations were < or =4.04%. The accuracy of the method was demonstrated; the recoveries of TMZ from spiked human plasma were 98.13-102.83 +/- 0.2-4.04%. The method has high throughput because of its simple sample preparation procedure and short run time (<10 min). The results demonstrated that the proposed method would have great value when applied in pharmacokinetic studies for TMZ.