UV-VIS Spectrophotometric Method for Estimating Furosemide in Dosage Form

Furosemide, chemically known as 4-chloro-2-[(furan-2-ylmethyl)amino]-5-sulfamoylbenzoic acid, is a potent loop diuretic widely used in the management of edema associated with conditions such as congestive heart failure, liver cirrhosis, and renal disorders.¹ It exerts its pharmacological effect by inhibiting the Na?/K?/2Cl? co-transporter in the thick ascending limb of the loop of Henle, resulting in increased excretion of sodium, potassium, chloride, calcium, magnesium, and bicarbonate ions.²?³ Furosemide is a crystalline powder that is practically insoluble in water but soluble in acetone, methanol, and alkaline hydroxide solutions.? Furosemide is commercially available in the following forms: tablets (20 mg, 40 mg, and 80 mg per dose), oral solutions (10 mg/mL and 40 mg/5 mL), and injectable solutions (10 mg/mL).

Figure 1 Structure of furosemide

Due to its therapeutic significance, the accurate and reliable quantification of furosemide is essential for ensuring drug quality and efficacy. Among various analytical techniques, UV-Vis spectrophotometry is widely preferred because of its simplicity, sensitivity, and cost-effectiveness. Although several UV spectrophotometric methods have been developed for furosemide estimation, many exhibit limitations. Some studies utilized both absorbance maxima and area under the curve (AUC) approaches for bulk and tablet analysis but lacked comprehensive method validation.? In degradation studies, UV-based techniques were applied without the development or validation of a specific spectrophotometric protocol.? A kinetic UV spectrophotometric method involving Co(II) salts and acidic ethanol was reported, but it employed reagents that are environmentally hazardous and unsuitable for routine quality control.? Another method used methanol as the solvent without assessing alternative solvents or buffer systems.? A different approach employed a pH 9.2 buffer capsule and reported maximum absorbance at 276?nm, but did not account for possible interference from excipients or degradation products.? Additionally, a validated method using 0.1?N NaOH demonstrated a narrow linearity range (1–6?µg/mL) with absorbance at 229?nm, limiting its broader application.¹⁰  However, a method using water has not been reported. Therefore, in the present study, an attempt was made to develop a simple and economical UV-vis spectrophotometric method for the analysis of furosemide in water.

MATERIALS AND METHODS

Materials

Furosemide was obtained as gift samples from Hetero Drugs Ltd. Tablet formulation (Lasix 40mg Tab, Sanofi India Ltd) containing 40 mg was purchased from retail outlets. Analytical-grade methanol used in this study was obtained from Merck Life Science. For analysis UV-vis spectrophotometer Shimadzu (UV-1800) paired with 1-cm quartz cells. Samples were weighed with a Shimadzu (AY-220) digital balance, and an ultrasonicator (Microclean-103) was employed to sonicate the sample solution.

Methods

Preparation of standard solutions

Precisely, 10?mg of furosemide was weighed, transferred into a 10?ml volumetric flask, dissolved and diluted to 10 ml with methanol, to obtain a stock solution of 1000?μg/ml. From this stock solution, 1?ml was pipetted out and diluted to 20?ml with distilled water to obtain a standard solution of 50?μg/ml.

Determination of maximum absorption

To identify the optimal analytical wavelength, 10?μg/ml concentration solution was scanned in range of 400-200nm using UV-vis spectrophotometer.

Linearity Study

1, 2, 3, 4 and 5 ml of standard solutions was diluted up to 10?ml with distilled water in volumetric flasks. This resulted in solutions with concentrations of 5, 10, 15, 20, and 25?μg/ml, respectively. The absorbance of each solution was then measured at 277?nm, distilled water as the blank. Then absorbance vs concentration graph was plotted.

Precision Study

A solution of furosemide (15 µg/ml) was analyzed for precision. Absorbance values were recorded six times at 277 nm using a UV-Vis spectrophotometer. The relative standard deviation (%RSD) was calculated to evaluate the repeatability and consistency of the method.

Preparation for Accuracy study

The accuracy study demonstrates that the analytical method was reliable and produces consistent results across different levels of recovery.

Test solution: Accurately weighed 20 tablets of furosemide and average weight of tablets recorded. The tablets were triturated in glass mortar and pestle to get fine powder. From this tablet powder weight equivalent to 5 mg of furosemide powder was weighed, dissolved and diluted up to 10 ml with methanol in a volumetric flask resulting concentration of (500 µg/ml). From this (500 µg/ml) 1 ml was pipetted out and diluted to 10 ml with distilled water to get 50 µg/ml concentration.

For the recovery study, 1?mL of test solution (50?μg/mL) was kept constant. To this, 0.8, 1.0, and 1.2?mL of standard solution (50?μg/mL) were added to achieve 80%, 100%, and 120% recovery levels, respectively. Each mixture was then diluted with distilled water to 10?mL in a volumetric flask. The absorbance of these solutions was measured at 277 nm. The drug content was quantified using the standard calibration curve equation.

Limit of Detection

The Limit of Detection is the smallest amount of a substance that can be clearly detected. It was often calculated using the formula:

LOD=3.3×SD÷Slope

Here: SD: Standard deviation of the response, Slope: Slope of the calibration curve

Limit of Quantitation

This value indicates the smallest concentration of furosemide that can be quantified. To calculate the Limit of Quantitation following formula was used:

LOQ=10×SD÷Slope

Here: SD: Standard deviation of the response, Slope: Slope of the calibration curve

Preparation for furosemide tablet assay

2ml of test solution (50 µg/ml) was diluted to 10 ml with distilled water in a volumetric flask to get concentration of (approx10 µg/ml). The absorbance of this (approx10 µg/ml) was recorded at 277nm with distilled water serving as the blank. Percent assay was calculated from regression equation (Y= 0.0651x + 0.0718).

RESULTS AND DISCUSSION

1. 1. Method development

The absorption spectrum revealed a peak at 277?nm, which was selected as the analytical wavelength for whole study.

Figure 2 Absorption Spectra of Furosemide

Method Validation

The validation of this method was done by as per International Council for Harmonization.^11,12

Linearity

When absorbance was plotted against concentration, the resulting graph exhibited a linear relationship with a strong correlation (R²=0.999), confirming the method's reliability and adherence to Beer-Lambert’s law.

Table 1 Absorbance of standard solution

Figure 3 Linearity response of furosemide standard solution

Figure 4 Overlay of UV Spectra of Standard Solutions of Furosemide

Precision

The precision study demonstrated that the UV-Vis spectrophotometric method for furosemide exhibited high reliability and consistency. As shown in Table 2, the absorbance values across six replicates showed minimal variation, with a relative standard deviation (RSD) of only 0.123%. This low RSD value indicates excellent repeatability.

Table 2 Precision Study

Accuracy

The accuracy of the method was evaluated through recovery studies at three concentration levels: 80%, 100%, and 120%. As shown in Table 3, the recovery percentages ranged from 98% to 101%, which fall well within the acceptable limits set by ICH and IP guidelines. These results confirm that the UV-Vis spectrophotometric method provides accurate quantification of furosemide across the tested concentration levels.

Table 3 Accuracy Study