K Basavaiah * & U Chandrashekar

Indian Journal of Chemical Technology Vol. 12, July 2005, pp. 401-406 Sensitive micro analysis of frusemide (furosemide) in bulk drug and formulations by visible spectrophotometry and high performance liquid chromatography (HPLC) K Basavaiah * & U Chandrashekar Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India Received 25 August 2004; revised received 10 January 2005; accepted 3 March 2005 Two rapid and sensitive methods using visible spectrophotometry and HPLC are described for the determination of frusemide (FRU) in bulk drug and formulations. Spectrophotometry is based on a redox reaction involving FRU followed by complexation reaction and uses iron(iii) and ferricyanide(iii) as reagents. The resulting Prussian blue is measured at 760 nm. The HPLC determination was carried out on a reversed phase Accurasil ODS C 18 column (250 4.6 mm, 5 μm) using a mobile phase consisting of acetonitrile 0.1% orthophosphoric acid (ph 3) (60 + 40) at a flow rate of 1.0 ml min -1 with UV detection at 233 nm. Working conditions of both methods have been optimized and the methods validated as per the ICH guidelines. In spectrophotometry, a regression analysis of Beer s law plot showed a good correlation in the concentration range 0.4-4.0 μg ml -1 with an apparent molar absorptivity of 4.03 10 4 L mol -1 cm -1 and a Sandell sensitivity of 7.85 ng cm -2. The limits of detection and quantification were calculated to be 0.09 and 0.28 μg ml -1 respectively. The linear range of determination by HPLC was 1.01-121.8 μg ml -1. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found to be 0.3 and 0.6 μg ml -1, respectively. Within-day accuracies and precisions were 3% and between-days precisions were less than 5% for all the concentrations tested. The methods were applied to the assay of FRU in tablets and injections. The label claim percentages and relative standard deviations were in the 98.28-103.24 and 0.36-2.04% range, respectively. The validity of the methods was further ascertained by parallel determination by a reference method and by recovery studies via standard addition technique. The results showed that the procedures are suitable for routine analysis of the diuretic. Keywords: Frusemide, assay, spectrophotometry, HPLC, prussian blue, formulations IPC Code:B01D15/00; A61K Frusemide (FRU), is chemically benzoic acid-5- (aminosulphonyl)-4-chloro-2-[2-furanylmethyl) amino)-4-cholro-n furfuryl-5-sulfamoyl anthranilic acid. In chemotherapy, it is extensively used for the treatment of edema associated with pulmonary, cardiac, hepatic, and renal disease 1, and hypertension accompanied by fluid retention or impaired renal failure 2. Numerous analytical methods are available for the assay of FRU in pharmaceutical samples. Spectrophotometric methods based on different reaction schemes have been reported for the assay of FRU. The method developed by Kustrin et al. 3 uses palladium(ii) as complexing agent, but lacks sensitivity (linear range, 79-1028 μg ml -1 ). Procedures based on binary complex formation reaction with iron(iii) 4 and ternary complex formation reaction 5 with iron(iii) and thiocyanate are least *For correspondence (E-mail: basavaiahk@yahoo.co.in; Fax: +91-821-2421263) sensitive with the linear range of applicability being 0.2-3.0 mg ml -1. Spectrophotometric methods utilizing redox 6, oxidative coupling 7,8 and diazo coupling 9 reactions also involve a heating step for a quantitative reaction. Ion-pair complex formation 10,11 and derivatisation 12 reactions based procedures involve an extraction step in addition to using organic solvents. Also, the procedures are poorly sensitive ( 10 3 ) and require a heating step 12. The determination of FRU based on charge transfer complex formation reaction, although sensitive, has to be performed in acetonitrile medium 13. Of the various techniques used in pharmaceutical analysis, HPLC is one of the most sensitive and rapid. However, the number of methods based on this technique for the say of FRU in pharmaceutical samples is limited. Rapaka et al. 14,15 were the first to report the HPLC determination of FRU in bulk material and pharmaceutical samples. The determination of FRU upto 75 μg ml -1 has been

402 INDIAN J. CHEM. TECHNOL., JULY 2005 achieved by Rao and Raghuveer 16 on a micro Bondapak NH 2 column with UV-detection at 254 nm and methanolic 0.01 M sodium acetate of ph 7.8 as mobile phase. The limit of detection was 0.5 μg ml -1. One of the most sensitive HPLC methods for FRU has been accomplished with an electrochemical detection 17. The method employed ODS material as the analytical column with methanol 0.04 M KH 2 PO 4 of ph 2.0 (1:1) as the mobile phase and detection at + 1.30 V versus Ag-AgCl electrode. The calibration graph was rectilinear from 1 to 10 μg ml -1 and the detection limit was 0.5 μg ml -1 which is a 10-fold improvement compared to UV-detection. This paper describes two methods based on visible spectrophotometry and reversed phase HPLC techniques for the assay of FRU in pharmaceutical formulations containing FRU alone. Spectrophotometry is based on the formation of Prussian blue when the drug is treated with iron(iii) chloride and ferricyanide (III) and measurement of the resulting blue colour at 760 nm. The HPLC analysis was carried out by injecting the drug solution onto an Accurasil ODS column with the elution being effected by a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid of ph 3 (60+40) and UV detection at 233 nm. The spectrophotometric method is the most sensitive ever reported and the HPLC procedure is the most sensitive with UV detection for FRU in single dosage forms. Both methods were found to be selective, accurate and precise. Experimental Procedure Apparatus A systronics model 106 digital spectrophotometer with 1 cm matched quartz cells was used for absorbance measurements. The chromatographic system consisted of an Agilent 1100 series chromatograph equipped with an inbuilt solvent degasser, quaternary pump, photodiode assay detector with variable injector and autoanalyzer. A steel column (250 4.6 mm) of Accurasil ODS C 18 (5 μm) was used. Reagents and materials All reagents used were of analytical reagent grade and distilled water was used to prepare solutions for spectrophotometric work. For HPLC work, distilled water filtered through 0.45 μm filter was used to prepare solutions. A 0.2% aqueous solution of iron(iii) chloride was prepared afresh every time before use by dissolving 0.2 g of the chemical in 100 ml of water and stored in an amber coloured bottle. A 0.4% aqueous solution of potassium ferricyanide was prepared by dissolving 0.4 g of the reagent in 100 ml of water. Sulphuric acid (10 M) was prepared by adding 55 ml of concentrated acid to 45 ml of water with cooling. Orthophosphoric acid (0.1%) was prepared by diluting 1 ml of the acid to one litre with distilled water and filtered through 0.45 μm filter. The mobile phase used in HPLC consisted of HPLC grade acetonitril and 0.1% H 3 PO 4 of ph 3 in the ratio 60:40. The dilutent solution was prepared by mixing acetonitrile and water in the ratio 60:40. Pharmaceutical grade frusemide was received as gift from Geno Pharmaceuticals Ltd., Mumbai, India. Formulations containing FRU were purchased from local commercial sources. A stock standard solution equivalent to 200 μg ml -1 FRU was prepared by dissolving 20 mg of pure drug in 20 ml of methanol and diluting to the mark with water in a 100 ml calibrated flask. This was diluted 10-fold with water and used in spectrophotometric analysis. For assay by HPLC, a stock standard solution containing 203 μg ml -1 FRU was prepared by dissolving 20.3 mg of pure drug in diluent solution and diluting to the mark in a 100 ml calibrated flask. Methods Spectrophotometric method Varying aliquots (0.2-2.0 ml) of 20 μg ml -1 FRU solution were transferred into a series of 10 ml calibrated flasks by means of a microburette and the volume was brought to 2 ml by adding water. To each flask was then added 2 ml of 0.2% iron(iii) chloride solution followed by 1 ml of 0.4% ferricyanide solution. The contents were mixed well and let stand for 20 min with occasional shaking. Finally, 1 ml of 10 mol L -1 H 2 SO 4 was added and diluted to the mark with water, and absorbance was measured at 760 nm against a reagent blank. A calibration graph was prepared by plotting the absorbance versus concentration of drug. The concentration of the unknown was read from the calibration graph or computed from the regression equation derived from the Beer s law data. HPLC Method Chromatographic conditions The chromatographic separation was achieved at ambient temperature on a reversed phase Accurasil ODS 5 μm C 18 column using a mobile phase consisting of acetonitrile-0.1% H 3 PO 4 of ph 3

BASAVAIAH & CHANDRASHEKAR: ANALYSIS OF FRUSEMIDE BY SPECTROPHOTOMETRY & HPLC 403 (60+40) at a flow rate of 1.0 ml min -1. The detector wavelength was set at 233 nm with a sensitivity of 0.2 a.u.f.s. Preparation of calibration graph Working standard solutions containing 1.02-101.5 μg ml -1 FRU were prepared by appropriate dilution of the stock standard solution with the diluent solution. Twenty μl aliquot of each solution was injected automatically onto the column in duplicate and the chromatograms were recorded. Calibration graph was constructed by plotting the mean peak area versus concentration of FRU. The concentration of the unknown was read from the calibration graph or calculated with the help of regression equation derived from the peak areaconcentration data. Assay procedure for formulations Twenty tablets were weighed and ground into a fine powder with agate pestle and mortar. The contents of ten ampoules were emptied into a beaker and mixed. An accurately weighed amount of the tablet powder equivalent to 20 mg of FRU was transferred into a 100 ml calibrated flask, 20 ml of methanol was added and shaken for 20 min. The mixture was diluted to the mark with water and mixed well, and filtered using a Whatman No. 42 filter paper. First 10 ml portion of the filtrate was discarded and subsequent 10 ml portion was diluted to 100 ml with water in a calibrated flask. A suitable aliquot of this solution (20 μg ml -1 FRU) was subjected to analysis by spectrophotometry. An accurately measured aliquot of the injectable product equivalent to 20 mg of FRU was transferred into a 100 ml calibrated flask, 20 ml of methanol added and diluted to the mark with water. This solution was diluted 10-fold and the convenient aliquot of the diluted solution was then analysed spectrophotometrically as described previously. An amount of tablet powder equivalent to 20 mg of FRU was accurately transferred into a 100 ml calibrated flask, 60 ml diluent solution added and shaken for 20 min. The mixture was diluted to the mark with the diluent and mixed well. A small portion of this (~ 10 ml) was withdrawn and filtered through a 0.2 μm filter to ensure the absence of particulate matter. The filtered solution was appropriately diluted to a convenient working concentration and 20 μl aliquot was then injected to get the chromatogram. In the case of injection, a measured aliquot equivalent to 20 mg of FRU was diluted to 100 ml with the diluent and mixed well. A 10 ml aliquot of this solution was filtered through a 0.45 μm filter. A suitable aliquot of the filtrate was appropriately diluted before analysis as described for tablets. Results and Discussion Spectrophotometry Primary and secondary amines reduce ferric ferricyanide to Prussian blue 18 and this reaction has been successfully used for the spectrophotometric determination of several pharmaceuticals 19-25. FRU which contains both primary and secondary amino groups reduces ferric ferricyanide to the blue colour which could be measured at 760 nm and related to the drug concentration. The optimum experimental conditions were established by varying one parameter at a time and observing its effect on the absorbance of the coloured species. Two ml of 0.2% iron(iii) chloride and 1 ml of 0.4% ferricyanide solutions in a total volume of 10 ml were found to produce maximum sensitivity in the concentration range investigated. The blue colour formed following the reduction of ferric ferricyanide was found to flocculate within 30-40 min. However, addition of 1 ml of 10 mol L -1 H 2 SO 4 after full colour development but before diluting to the mark was found to delay flocculation and increase the stability of colour. The reduction reaction was found to be slow at room temperature (30 ± 2 C) but the absorbance increased with time and reached maximum in 20 min. The developed colour was stable for at least 90 min. After fixing all other parameters, experiments were performed to study the effect of order of addition reactants. The order, FRU: iron(iii): ferricyanide followed by sulphuric acid after full colour development gave maximum sensitivity and stability of coloured species and hence followed throughout the investigation. HPLC method FRU was determined by HPLC by injecting the solution onto an Accurasil ODS 5 μm column with UV detector set at 233 nm. No internal standard was used. The composition and ph of the mobile phase and its flow rate were varied to optimize the chromatographic conditions. A mobile phase consisting of acetonitrile and 0:1% H 3 PO 4 of ph 3 was selected after several preliminary experiments with acetonitrile-water and methanol-water. Acetonitrile and phosphoric acid increase the

404 INDIAN J. CHEM. TECHNOL., JULY 2005 solubility of FRU and prevent its adherence to the packing material in the column. At a flow rate of 1.0 ml min -1, the retention time for FRU was 3.05 min. Under the described experimental conditions the analyte peak was well defined and free from tailing. Method validation Spectrophotometric method The increasing absorbance values at 760 nm were plotted against the drug concentration to obtain the calibration graph. Beer s law is obeyed over the concentration range 0.4-4.0 μg ml -1 the equation of the line being, A = - 0.0117+0.1352 C where A is the absorbance and C concentration in μg ml -1. The correlation coefficient of the calibration graph was calculated to be 0.9985 (n=8) confirming a linear increase in absorbance with increasing concentration of FRU. The calculated molar absorptivity was 4.03 10 4 L mol -1 cm -1 and the Sandell sensitivity was 7.85 ng cm -2. The limits of detection (LOD) and quantification (LOQ) were calculated from the standard deviation of the absorbance measurement obtained from a series of seven blank solutions. The LOD and LOQ established according to ICH guidelines 26 were 0.09 and 0.28 μg ml -1, respectively. HPLC Method The concentration of the unknown was determined by measuring the peak area. A plot of mean peak area versus concentration gave a linear relationship (r= 0.9999) over the concentration range 1.01-121.8 μg ml -1. Using the method of least squares, the linear regression equation obtained was Y = 93.21+ 115.5 X where Y is the mean peak area and X concentration in μg ml -1. The LOD was established at a signal to noise ratio (S/N) of 3 and LOQ was established at a S/N ratio of 9. The LOD was calculated to be 0.3 μg ml -1 and the LOQ was calculated to be 0.6 μg ml -1. Precision and accuracy of the methods The within-day precision of the methods was determined by replicate analyses of the standard solution containing FRU at three different concentration levels and the results are presented in Tables 1 and 2. For HPLC method, the relative standard deviation (RSD) which is a measure of precision was evaluated for both retention time and peak area. The inter-day precision was established by performing analysis over a 5 days period with solutions prepared freshly each day. The RSD values were not more than 4 and 5% for spectrophotometric and HPLC methods, respectively. The peak area based and retention time based inter-day RSD values were 0.8 and 4.5%, respectively. In order to demonstrate the accuracy of the proposed methods, seven replicate analyses were performed on solutions containing FRU at three different levels. The results obtained are compiled in Tables 1 and 2 and the accuracy expressed as percent relative error was found satisfactory. Application Commercially available tablets and injections containing FRU were analysed by the described spectrophotometric and HPLC methods. The results obtained are summarized in Table 3. As can be seen, the results are in agreement with the labelled amounts. For comparison, a conventional UV-absorption method ( λ = 275 nm) due to Moustafa and Abdel Moety 27 was used to analyse the same batch tablets Table 1 Evaluation of accuracy and precision (spectrophotometry) FRU taken μg ml -1 FRU found* μg ml -1 Range μg ml -1 RE RSD, % ROE 1.0 0.98 0.05 2.0 1.28 ±1.18 2.0 2.06 0.10 3.0 1.75 ±1.62 3.0 2.97 0.20 1.0 2.87 ±2.65 * Mean value of seven determinations, RE: Relative Error, RSD: Relative standard deviation, ROE: Range of error at 95% confidence level Table 2 Evaluation of accuracy and precision (HPLC) FRU taken μg ml -1 FRU found μg ml -1 Range, μg ml -1 RE, RSD % ROE % RSD** ROE** 20.3 20.77 0.35 2.66 0.65 0.60 4.23 3.90 40.6 42.32 0.37 4.06 0.31 0.29 3.35 3.09 81.2 82.69 0.62 1.80 0.49 0.45 4.24 3.92 *Based on peak area, ** Based on retention time

BASAVAIAH & CHANDRASHEKAR: ANALYSIS OF FRUSEMIDE BY SPECTROPHOTOMETRY & HPLC 405 Formulation and Brand name * Frusenex a tablets Lari b Injections Table 3 Results of analysis of formulations containing frusemide Nominal amount mg/tablet or mg/ml Found ** (% of nominal amount ± SD) Reference method Spectrophotometric method HPLC method 40 99.64±0.85 100.32±1.62 t= 0.86 F= 3.63 100 97.86±1.12 98.28±2.05 t= 0.42 F=3.31 10 102.58±0.76 101.76±1.85 t= 1.00 F=5.92 * Marketed by : a. Geno Pharm Ltd, : b. Indian Drugs and Pharmaceuticals Ltd. ** Mean value of five determinations Tabulated t-value at 95% confidence level in 2.77 Tabulated t-value at 95% confidence level in 6.39 Table 4 Results of recovery experiments Formulation studied Frusenex tablets (40 mg) Drug in formulation μg ml -1 Spectrophotometric method Pure drug added, μg ml -1 Total found μg ml -1 Recovery* of pure drug added, % Drug in formulation μg ml -1 HPLC method Pure drug added, μg ml -1 Total found μgml -1 100.65±0.44 t= 2.45 F=3.73 98.96±0.62 t= 2.00 F=3.26 103.24±0.36 t= 2.31 F=4.45 Recovery*of pure drug added, % 0.50 1.0 1.50 98.56 5.03 20.0 24.58 97.68 0.50 1.5 2.01 100.28 5.03 40.0 45.58 101.35 0.50 2.0 2.56 102.67 5.03 60.0 64.10 98.44 Lasix injections (10 mg /ml) 0.51 1.0 1.53 101.56 5.16 20.0 25.34 100.88 0.51 2.0 2.58 103.67 5.16 40.0 44.22 97.64 0.51 3.0 3.48 98.92 5.16 60.0 64.77 99.35 *Mean value of three determinations and injections. The results, compiled in Table 3 were statistically compared by Student s t-test and F-test. As shown, the calculated t - and F - values were less than the tabulated values inferring that the proposed methods have the same accuracy and precision as that of the reference method at the 95% confidence level. The accuracy and reliability of the methods were further established by performing recovery experiments. The pre-analysed tablets and injections were spiked with pure FRU at three different levels and the total was found by the proposed methods. Each experiment was repeated three times. The recoveries of the added pure drug were in the range 97.64-103.67% (Table 4) indicating that coformulated substances such as talc, starch, gumacacia, lactose, magnesium stearate, sodium alginate, calcium gluconate, and calcium dihydrogenorthophosphate did not interfere in the determination. Conclusions A simple and highly sensitive spectrophotometric method based on a well known reduction - complexation reaction is reported. The procedure does not involve either a heating or extraction step unlike many reported previously. The present HPLC method is superior to many reported earlier in terms of sensitivity, linear range of response, and analysis time (6 min). In terms of limit of detection, the proposed method is next only to the procedure employing electrochemical detector 17. Acknowledgements The authors gratefully acknowledge the receipt of pure frusemide from Geno Pharmaceuticals Ltd., Mumbai as gift. One of the authors (UC) thanks the authorities of the University of Mysore, Mysore for facilities.

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