Journal of Pharmacy Research Vol.5 Issue 5.May 2012 M. I. Walash et al. / Journal of Pharmacy Research 2012,5(5),2648-2656 2648-2656 Research Article ISSN: 0974-6943 Available online through http://jprsolutions.info * Corresponding author. N. El-Enany Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. Micellar High Performance Liquid Chromatographic Determination of Furosemide and Spironolactone in Combined Dosage Forms. Application to Human Plasma M. I. Walash, N. El-Enany*, M. I. Eid, M. E. Fathy Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, 35516, Mansoura, Egypt. Received on:09-01-2012; Revised on: 14-02-2012; Accepted on:27-04-2012 ABSTRACT A Simple and rapid micellar liquid chromatographic method was developed and validated for the simultaneous determination of two diuretic drugs, Furosemide (FUR) and Spironolactone (SPL). Good chromatographic separation was achieved using a 250 mm x 4.6 mm i.d., 5 μm particle size Shim-pack VP-ODS C18 column. A mobile phase consisting of a mixture of 0.1 M sodium dodecyl sulphate (SDS), 0.3% triethylamine (TEA), 10% n-propanol all prepared in 0.02 M orthophosphoric acid at pH 4.0 was pumped at a flow rate of 1 mL/min. with UV detection at 238 nm. Indapamide (IND) was used as an internal standard (IS). The method showed good linearity over the ranges of 0.1-10 μg/mL and 0.4-20 μg/mL with limits of detection (LOD) of 0.03, 0.13 μg/mL and limits of quantification (LOQ) of 0.1, 0.4 μg/mL for furosemide and spironolactone, respectively. The proposed method was successfully applied for the simultaneous analysis of the studied drugs in their laboratory prepared mixture and co-formulated tablets. The method was further extended to the determination of furosemide in human plasma without prior extraction procedure. Statistical evaluation and comparison of the data obtained by the proposed and comparison methods revealed good accuracy and precision of the proposed method. Key words: HPLC, Simultaneous Determination, Furosemide (FUR) , Spironolactone (SPL) , Co-formulated tablets , Human plasma. INTRODUCTION Furosemide (Fig.1a); 5-(Aminosulfonyl)-4-chloro-2-[(2-furanylmethyl) amino] benzoic acid; 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid, [1] is a potent loop diuretic with a rapid action. [2] Spironolactone (Fig. 1b); (7.alpha.,17.alpha.)-7-(Acetylthio)-17-hydroxy-3-oxopregn-4-ene-21-car- boxylic acid .gamma.-lactone; 17-hydroxy-7.alpha.-mercapto-3-oxo-17.al- pha.-pregn-4-ene-21-carboxylic acid .gamma.-lactone, acetate, 1 is an aldos- terone antagonist acting as a potassium-sparing diuretic. [2] Both drugs are useful in the treatment of the edema associated with congestive heart failure, cirrhosis of the liver or the nephrotic syndrome and in the treatment of hypertension. [3] Furosemide is used in the treatment of hypertension either alone or with other antihypertensives. [2] Spironolactone is frequently given with thiazides, furosemide, or similar diuretics, where it diminishes their kaliuretic effects. [2] FUR and SPL are official in the united state pharmaco- poeia (USP). [4] and in the british pharmacopoeia (BP). [5] (a) Furosemide (b) Spironolactone Fig. (1): The structural formulae of the studied drugs. The combination between FUR and SPL is not a recent discovery. There are numerous pharmaceutical products approved to be marketed, which take advantage of the loop diuretic effect of furosemide (increases removal of sodium and potassium salts) and aldosterone antagonist effect of spironolac- tone. By combining effects of the two active substances, hypokalaemia is prevented and an efficient decrease in the volume of fluid circulating through blood vessels is obtained. [6] Combination dosage forms of FUR and SPL are indicated in the treatment and management of edema and hypertension. To the best of our knowledge, up till now no micellar high-performance liquid chromatographic methods have been reported for the simultaneous determi- nation of both drugs in pharmaceutical preparations or for FUR in human plasma. Micellar liquid chromatographic methods [19, 20] have been described for simultaneous determination of a group of diuretics, including the studied drugs, in human urine. Bonet-Domingo et al [19] used a mobile phase consist- ing of 0.042 M SDS and 4.0% propanol at pH 4.5 with U.V. detection at 254 nm. It separated SPL in about 37 min. So, by virtue of the reported method, [19] the proposed method is more time saving. and more sensitive. The other published method [20] separated FUR, SPL and other 12 diuretics using 0.04 M SDS and 4% tetrahydrofuran at pH 3.2 as the mobile phase. Since Fur and SPL were determined at two different wavelengths (220, 245 nm, respec- Reviewing the literature revealed that numerous analytical methods were developed for the assay of FUR and SPL individually or in combination with other drugs. A good guide to the work published for both drugs is presented as the comprehensive monographs in the series of Analytical Profiles of Drug Substances. [7,8] A variety of analytical methods relating to the FUR assay in biological fluids and pharmaceutical samples individually or in combination with other drugs (such as SPL) has been recently reviewed. [9] A comprehen- sive review describing the analysis of hypotensive complex agents including FUR and SPL and many other drugs in pharmaceutical and biological matri ces was established basing on representative publications that were pub- lished between 1995 and 2009. [10] Few spectrophotometric methods were reported for the simultaneous determination of FUR and SPL in pharmaceu- tical preparations. [11-13] Few Liquid chromatographic methods have been re- ported for simultaneous determination of both drugs in pharmaceutical prepa- rations. [14-17] Rapid Ultra HPLC method was described for the simultaneous analysis of FUR, SPL and other two drugs along with their metabolites in human urine and plasma. [18] Also, HPLC- tandem mass spectrometry (HPLC/ MS/MS) has been reported for the simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples. [6]