DRUG FORMULATIONS AND CLINICAL METHODS 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-1,3diazole in pH 8 borate buffer at 70 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 intraand interassay precision were satisfactory; the relative standard deviations were 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. T rimetazidine (TMZ), 1-[(2,3,4-trimethoxyphenyl) methyl]piperazine, is a clinically effective antianginal agent (1). In several studies, TMZ was shown to improve the ergometric exercise capacity and total work output of patients with effort angina (2, 3), reduce attack frequency in patients with chronic stable angina (4), and increase effort tolerance in angina patients (5). The antianginal efficacy of TMZ is comparable to that of propranolol, but TMZ does not reduce cardiac rate-pressure product or coronary blood flow (6). In addition, the therapeutic efficacy of TMZ is comparable to that of nifedipine, but TMZ is devoid of vasodilator activity and alteration of hemodynamic parameters (7). In combination with diltiazem, synergestic antianginal effects were demonstrated (8). Modified-release TMZ tablets are used for the treatment of long-term angina pectoris (9). Because of these clinical successes, TMZ has become unique among the antianginal agents, and it has been clinically used throughout many countries worldwide (10, 11). Analytical methods for the determination of TMZ in pharmaceutical formulations include spectrophotometry (12–15), spectrofluorometry (16), adsorptive stripping voltammetry (17), high-performance thin-layer chromatography (18), and high-performance liquid chromatography (HPLC; 19). These methods do not have adequate sensitivity for measuring TMZ in plasma. The first attempt to determine TMZ in plasma was made by using HPLC coupled with fluorescence detection after derivatization with dansyl chloride (20). The calibration range of dansylated TMZ was 10–500 ng/mL plasma. Gas chromatographic determination of TMZ in plasma was achieved after its liquid–liquid extraction and subsequent derivatization with N-(t-butylsilyl)-N-methyltrifluoroacetamide (21). HPLC with UVdetection was successfully used for quantitation of TMZ in blood and urine after its isolation on Toxi-tubes (22). HPLC with electrochemical detection was described as an alternative for sensitive determination of TMZ in plasma (23). A chemiluminometric method was proposed for determination of TMZ in biological fluids, based on its reaction with potassium permanganate (24). LC/mass spectrometry (LC/MS) was reported for quantitation of TMZ in plasma samples (25). The sample preparation procedures involved liquid–liquid extraction with DARWISH ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 91, NO. 5, 2008 1037