Analytical Letters, 38: 1651 1658, 2005 Copyright # Taylor & Francis, Inc. ISSN 0003-2719 print/1532-236x online DOI: 10.1081/AL-200065813 PHARMACEUTICAL ANALYSIS Rapid HPLC-DAD Determination of Furosemide in Tablets Using a Short Home-Made Column Felipe Silva Semaan, Alvaro José dos Santos Neto, Fernando Mauro Lanças, and Éder Tadeu Gomes Cavalheiro Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Paulo, Brazil Abstract: Furosemide is a loop diuretic widely used as an antihypertensive. Its analysis, in pharmaceutical formulations, is often required for quality control and to develop new formulations. The aim of this work is to describe the development of a fast chromatographic procedure to determine furosemide in tablets. Adequate sample masses were prepared by dissolution of furosemide in ethanol with the aid of an ultrasonic bath, followed by filtration. After dilution, an aliquot was injected into the chromatographic system. The separations were obtained in a C18 home-made column (50 4.6 mm, 3 mm) with methanol:phosphate buffer (10 mmol L 21, ph 5.5) (30 : 70) as the mobile phase, at a flow rate of 1.0 ml min 21. A photodiode array detector (DAD) monitored signals between 190 and 380 nm, with special attention to 237 nm. The findings were 1.4% 6.2% intra-assay precision; 1.6% 6.1% interassay precision (day and operator); recoveries of 90.2%, 97.5%, and 109.2% at 90%, 100%, and 110% concentration levels, respectively. The total chromatographic run lasted 3 minutes. By means of peak purity, obtained using the DAD, no interference of concomitants was observed. The residual graphic demonstrates the adequate Received 11 April 2005; accepted 25 April 2005 The authors are gratefull to CNPq, CAPES, and FAPESP (proc n8 04/08550-0, n8 03/02630-0, and n8 04/00407-4) for financial support. Address correspondence to Éder Tadeu Gomes Cavalheiro, Departamento de Química e Física Molecular DQFM, Instituto de Química de São Carlos, Universidade de São Paulo, P.O. Box 780, Avenhido do Trabalhador Sãocarlense, 400, Centro, CEP 13560-970, São Carlos, São Paulo, Brazil. E-mail: cavalheiro@iqsc. usp.br 1651
1652 F. S. Semaan et al. linearity of the studied concentration interval. The proposed method was applied to commercial samples and shown to be less time and solvent consuming than the procedures previously reported in the literature. Keywords: Antihypertensive drug analysis, furosemide determination, home-made column 1. INTRODUCTION Furosemide (4-chloro-N-furfuril-sulphamoylantranilic acid) (Fig. 1) (Merck Index 2001) represents a powerful loop diuretic, which is widely used in the treatment of hypertension and edema. The reference drug is Lasix w, from Aventis. Nowadays, with the advent of generic drugs, the number of pharmaceutical formulations has increased the demand for quality control. For furosemide determinations in aqueous solutions, the adequate solubility in alkaline solutions must be considered (Agatonovic-Kustrin et al. 1990; Garcia et al. 1997). The use of surfactants is an alternative to improve analyte solubility (Kulichenko and Shevchenko 2003; Kulichenko and Fesenko 2002). Some authors determined furosemide by static-or flow-injection procedures based on complexation with different metallic cations and spectrophotometric detection (Agatonovic-Kustrin et al. 1990; Garcia et al. 1997; Zivanovic et al. 1990). Direct simultaneous determinations in samples containing furosemide and amiloride were developed through derivative spectrophotometry, with no interference from the concomitants of the formulation (Toral et al. 2002). Titrimetric procedures, assisted by surfactants, one using KOH solutions, and another using acid-base potentiometry were developed by Kulichencko and coworkers (2003, 2002). Differential pulse and square wave voltammetric procedures have also been proposed (Barroso et al. 1995). Dias et al. (2004) developed ion-selective electrodes for potentiometric determination of Figure 1. Structural formula of furosemide.
HPLC-DAD Determination of Furosemide 1653 furosemide, such sensors used tricaprilmetilamonium as ionic par. Other reports have also demonstrated the applicability of lanthanides in the determination of derivatives of anthranilic acid, like furosemide, by using its complexation reaction and exploration of the luminescence properties of the complexes with higher sensibility when compared with other spectral techniques. The lanthanides most used were cerium (Rao et al. 1999), europium (Beltyukova et al. 1998), and terbium (Ioannon et al. 1998; Georges 2003). Chromatographic procedures have also been applied in the determination of furosemide in tissues, serum, and urine, using UV or amperometric detection. One method was previously described to determine this analyte in tablets, and this method uses amperometric detection (Mills et al. 1997; Barroso et al. 1996; Guzmán et al. 2003). To achieve significantly lower analysis times, a current trend in conventional high pressure liquid chromatography (HPLC) separations is to use smaller particles (3 mm) packed into shorter columns (20 50 mm) (Majors 2003). Column efficiency (H) is proportional to the d p x (where d p is the particle diameter and x a factor between 1.6 and 1.9), while resolution is proportional to (L/H) 1/2 (where L is the column length). So, with the use of smaller particles packed into shorter columns of the same internal diameter, the resolution decreases much less than the efficiency improves (Majors 2003). The present work proposes a simple, fast, and cheap method for furosemide determination by HPLC-DAD using a short home-made column and its application in commercially available tablets. 2. EXPERIMENTAL 2.1. Apparatus and Column The system consisted of a Shimadzu 10A series HPLC equipped with two pumps LC-10AD, a degasser DGU-2A, a diode array detector SPD-M10A VP controlled by the unit CBM-10A and the software Class-LC10, and with a Rheodyne injection valve. The C18 column (50 mm 4.6 mm, 3 mm) used in the chromatographic system was home-packed by slurry packing. 2.2. Reagents and Standard Solutions Furosemide was obtained from Natural Pharma (Brazil) and it was characterized as standard by nuclear magnetic resonance (NMR), Fourier transformed-infrared spectrometry (FT-IR), elemental analysis (EA), and differential scanning calorimetry (DSC). Solvents were J.T. Baker (USA) HPLC grade, and the water was obtained from a Milli-Q Waters system (USA). All the salts were from Mallinckrodt (USA). Furosemide stock solution was prepared by direct dilution of the analyte in absolute ethanol in order to achieve a 10 22 mol L 21 solution. Intending to
1654 F. S. Semaan et al. prepare the calibration solutions (8 to 12 mg L 21 ), aliquots from the stock solution were transferred to 10 ml volumetric flasks and volumes were made up with mobile phase. 2.3. Chromatographic Conditions The mobile phase was a mixture of phosphate buffer (10 mmol L 21, ph 5.5) : methanol (30 : 70), at a flow rate of 1.0 ml min 21. Separations were carried out after the injection of 20 ml sample aliquots, and the analytical signals were obtained using a diode array detector, monitoring wavelengths between 190 and 380 nm, special attention was deserved to 237 nm. 2.4. Sample Preparation The pharmaceutical formulations analyzed in this work were presented as tablets, from three different commercial brands (Lasix, from Aventis; Neosemid w, from Neoquimica; and Furosemida w from Biosintetica). In order to perform the determinations, tablets were weighed and triturated in a mortar until the achievement of a fine and homogeneous powder. Suitable amounts of these powders were weighed out and solubilized in 30 ml absolute ethanol, the systems were sonicated during 10 minutes and the solutions were then filtered. The volumes were made up with absolute ethanol to 50 ml in volumetric flasks. Adequate aliquots from these solutions were transferred to 10 ml volumetric flasks and the volumes were adjusted with mobile phase. 3. RESULTS AND DISCUSSIONS 3.1. Figures of Merits and Statistical Analysis The proposed method showed adequate linearity, precision, and accuracy in the range from 8 to 12 mg L 21 (Fig. 2). Samples were prepared to achieve 10 mg L 21 (the central point of the analytical curve) (Fig. 3). Applying the described chromatographic conditions, the furosemide retention time was 2 minutes, with a total time of 3 minutes, which allows a high analytical frequency (20 measurements per hour). In order to evaluate the significance of the obtained data an analysis of variance (ANOVA) was developed (Burke 2004). The R-squared was 0.998 with a lack of fit 0.133 (F crit ¼ 5.41). The response was significant, which can be seen by a regression significance of 4077.7 against a F crit of 5.32. The linearity was evaluated by residual graphic, and the value calculated by ANOVA was 0.002 (F crit ¼ 5.41). 1.4 6.2% Intra-assay precision of 1.4% 6.2% and interassay precision of 1.6% 6.1% (day and operator) were found.
HPLC-DAD Determination of Furosemide 1655 Figure 2. Chromatograms of the standard solutions (A: 8 mg L 21, B: 9 mg L 21,C: 10 mg L 21,D:11mgL 21, and E: 12 mg L 21 ). Figure 3. Sample chromatograms.
1656 F. S. Semaan et al. Figure 4. Furosemide spectrum by DAD. In the commercial samples and standard analysis, no interferences were observed, as could be seen by the resolution of the peaks in the chromatograms (Fig. 4). Recovery tests were performed at three different levels. Recoveries of 90.2%, 97.5% and 109.2% for 90%, 100%, and 110% concentration levels were found, respectively. 3.2. Analytical Application Three commercial brands of tablet, each containing 40 mg of furosemide per unit were analyzed by applying the described chromatographic procedure. The results and respective standard deviations are presented in Table 1. Table 1. Results from sample determinations and its standard deviations Sample Labeled a Found a SD b Lasix w 40 38.18 0.72 Neosemid w 40 39.34 0.42 Biosintetica w 40 39.59 0.37 a mg furosemide per tablet. b SD means standard deviation, n ¼ 5.
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