The furosemide spectrum: Development of a precise analytical method using UV spectroscopy

 Corresponding author: Sakshi Rajesh Gaikwad. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. The furosemide spectrum: Development of a precise analytical method using UV spectroscopy Sakshi Rajesh Gaikwad *, Prachi Nandkumar Padwal, Pranjal Raghunath Jadhav, Shrutika Pralhad Gadge, Tanvi Sopan Dongare and Asmita Anil Hulawale Samarth Institute of Pharmacy, Belhe, Pune, Maharashtra, India. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 Publication history: Received on 20 September 2025; revised on 26 October 2025; accepted on 29 October 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.1.0936 Abstract A UV-Visible spectrophotometer was used to measure the absorbance of standard solutions, and linearity was established within a specified range, exhibiting a high correlation coefficient (R² > 0.999), indicating excellent linearity.The absorption maxima method was found to be 277nm. The results confirmed that the method is suitable for routine quality control of Furosemide API in bulk and pharmaceutical formulations. The proposed method is advantageous due to its simplicity, rapidity, and minimal use of reagents and solvents. The study's goal is to develop and validate a straightforward, accurate, and economical UV spectrophotometric method for the quantitative analysis of Furosemide. Furosemide contains conjugated double bonds and aromatic rings (e.g., a substituted benzene ring and sulfonamide group), which are chromophores. These absorb UV light due to π→π* and n→π* electronic transitions, making UV spectroscopy ideal for detecting them.The method were validated with respect to linearity,precision,and accuracy studies. Keywords: Furosemide; Absorbance; Wavelength; UV-visible spectroscopy; Chromophore 1. Introduction Pharmaceutical analysis is a branch of chemistry focused on identifying, quantifying, purifying, and characterizing substances used in pharmaceuticals. It involves determining the composition, purity, safety, and quality of drug substances and products[9]. This field plays a crucial role in drug development, manufacturing, quality control, and regulatory compliance. 5-(aminosulfonyl)-4-chloro-2-[(2-furanyl methyl)amino)benzoic acid] is the chemical name for furosemide (Fu). Frusemide, Fursemide, Aisemide, Beronald, Desdimin, Lasilix, and more generic names are among them. C12H11ClN2O5S is the empirical formula, and it has a molecular weight of 330.77. The crystalline powder furosemide is white to slightly yellow, odorless, and nearly tasteless. It dissolves somewhat in water, chloroform, and ether and is soluble in acetone, methanol, and dimethyl formamide [1] as well as alkali hydroxide solutions [2]. It has a melting point of 206°C and an aqueous solution pH between 8.9 and 9.3. Using a DMS 90 Varian spectrophotometer, the UV spectra of furosemide (0.01 mg/ml) in 0.1N NaOH was scanned from 190 to 400 nm. At 226 and 272 nm, it displayed two peaks. Numerous techniques have been documented for identifying the constituents of this significant medication (furosemide). Methods of titration [3–7] techniques using potentiometry [8,9] colorimetric and ultraviolet techniques. UV spectrophotometry is always favored in small-scale companies due to its low maintenance requirements and affordability. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 473 According to a review of the literature, numerous UV spectrophotometric techniques have been documented thus far for the measurement of furosemide either by itself or in conjunction with other medications [9]. However, only a small number of them used a single furosemide estimation. Thus, the primary goal of the suggested techniques was to create easy, novel, and affordable UV spectrophotometric techniques for estimating furosemide dosage in bulk form and validating them in accordance with ICH guidelines[6]. 2. Furosemide API :Properties and Analysis Furosemide is a potent loop diuretic medication used to treat fluid build-up due to heart failure, liver scarring, or kidney disease. It is also sometimes used to treat high blood pressure. Figure 1 Structre of Furosemide • Molecular Formula: C₁₂H₁₁ClN₂O₅S • Molecular Weight: 330.74g/mol. • Apperance: white to slightly yellow crystalline powder. • Melting Point: About 206°C Solubility: It is just weakly soluble in alcohol and nearly insoluble in water. It dissolves readily in alkali hydroxide solutions[11]. A key component of its composition and bodily absorption is its pH-dependent solubility. pKa: Furosemide's acidic dissociation constant (pKa) is roughly 3.9. This suggests that the acid is weak. Its acidic character is mostly caused by the presence of the carboxylic acid group[11]. LogP (Octanol/Water Partition Coefficient): A compound's lipophilicity, or capacity to dissolve in fats, oils, and lipids, is determined by the logarithm of the partition coefficient (LogP). The LogP value for furosemide is around 2.03. Its distribution, absorption, and capacity to pass across cell membranes are all impacted by its mild lipophilicity[11]. 1.1. Action Mechanism : Inhibiting the Na-K-Cl-2Cl cotransporter in the kidneys' thick ascending limb of the loop of Henle is how furosemide functions. By preventing the reabsorption of potassium, sodium, and chloride ions, this action increases the excretion of these electrolytes and, as a result, water[29]. This diuretic action lowers blood pressure and lessens edema 1.2. Important Analytical Difficulties in the Analysis of Furosemide API : Furosemide's examination as an Active Pharmaceutical Ingredient (API) is complicated by a number of important physicochemical characteristics. Its solubility, stability, and the existence of related chemicals are the main issues here. • Solubility: Poor Solubility in Water Due to its near-insoluble nature in water, furosemide presents significant difficulties for the development of analytical techniques, especially for dissolution testing and HPLC (HighPerformance Liquid Chromatography) analysis[13]. This low solubility may result in: Sample preparation challenges: It can be difficult to completely dissolve the API, which could produce imprecise and erroneous results. Dissolution testing problems: It is challenging to create dissolution techniques that can accurately distinguish between various formulations and forecast in-vivo performance due to the low solubility. To overcome this challenges, analyte often need to use of co-solvent, surfactants, or adjust the pH of analytical solution. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 474 • Analytical variability: The concentration of furosemide may drop if samples are not sufficiently shielded from light during analysis, which could result in erroneously low assay values and an overestimation of contaminants. Analytical techniques must be "stability-indicating," which means they must be able to distinguish and precisely measure furosemide from the byproducts of its breakdown. This calls for meticulous method development and verification[6]. For accurate and trustworthy results, furosemide handling and analysis must always be done in a light-protected environment. All Furosemide API can contain impurities from the manufacturing process .thus it must be carefully monitored and controlled. • Furosemide Impurity : 2,4-dichloro-5-sulfamoylbenzoic acid is its chemical name. This substance is frequently created as a byproduct of synthesis or as a breakdown product[17]. Despite having a chloro group in place of the furfurylamino group, which can cause co-elution issues in chromatography, it shares structural similarities with the furosemide core. Since the aromatic rings in furosemide and its contaminants absorb ultraviolet light, UV detection is utilized. Usually, the detecting wavelength is set at about 272 nm. Existing Analytical method for furosemide API: Chromatographic Techniques For the examination of furosemide • High-performance liquid chromatography (HPLC):- is the gold standard. The most widely utilized mode of HPLC is reversed-phase (RP-HPLC). A polar mobile phase (often a solution of water, acetonitrile, or methanol with a pH-adjusting buffer) is employed with a non-polar stationary phase (such as C18)[14,15]. Usually, a UV detector is used for detection at a certain wavelength (about 270–280 nm) where furosemide has the highest absorption. • Ultra-High-Performance Liquid Chromatography (UPLC): This more sophisticated type of HPLC uses columns with smaller particle sizes, which improves resolution and speeds up analysis times. Because of their effectiveness, UPLC techniques are being used more and more. • Thin-Layer Chromatography (TLC): A less complicated chromatographic method that can be applied to semiquantitative impurity quantification and identification. Methods of Spectroscopy • UV Visible Sprctroscopy : A straightforward and affordable technique for determining the amount of furosemide in a bulk medication is UV-visible spectrophotometry[22]. The idea is founded on Beer-Lambert's law, which states that analyte concentration is directly correlated with a solution's absorbance. The absorbance of a furosemide solution is measured at its wavelength of maximum absorption (λmax)[25]. One of the most effective methods for identifying furosemide is infrared (IR) spectroscopy: To verify the identification of the API, its infrared spectrum is compared to a reference spectrum. It uses the vibrations of the molecule's chemical bonds to create a distinct "fingerprint" of the molecule[27]. • Nuclear Magnetic Resonance (NMR) Spectroscopy: ¹H NMR and ¹³C NMR spectroscopy are used to elucidate the chemical structure of Furosemide and to identify any structural isomers or impurities. Titrimetric Techniques: A straightforward and traditional technique for the testing of • furosemide is acid-base titration: Due to its acidic carboxylic group, furosemide can be titrated using an appropriate indicator with a standardized solution of a strong base, such as sodium hydroxide[15]. • Hyphenated Methods LC-MS: This method, which combines the identification capabilities of mass spectrometry with the separation capacity of HPLC, is extremely sensitive and selective. It is especially helpful for identifying and measuring degradation products and contaminants at the trace level. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 475 3. UV Spectroscopy: Principle and Instrumentation. Basic principle of UV Visible spectroscopy: A potent analytical method that makes use of the way visible and ultraviolet light interact with materials is UV (ultraviolet) spectroscopy. It is extensively utilized for both qualitative and quantitative investigation of substances in many scientific domains, such as chemistry, biology, and materials science[16]. The basic idea behind UV spectroscopy is that certain UV or visible light wavelengths are absorbed by molecules, which elevates electrons from a lower energy ground state to a higher energy excited state. The Law of Beer-Lambert The Beer-Lambert Law governs UV spectroscopy's quantitative component. The absorbance of light, the concentration of the absorbing species, and the length of the light's journey through the sample are all linearly related according to this law[8]. The Beer-Lambert Law can be expressed mathematically as follows: A = εbc Where: • A = Absorbance (unitless) • ε = Molar absorptivity (L mol⁻¹ cm⁻¹) • b = Path length of the light through the sample (cm) • c = Concentration of the absorbing species (mol L⁻¹) This equation describes the relationship between the absorbance of light by a sample and the concentration of the absorbing species, path length, and molar absorptivity. The Beer-Lambert Law basically says that the concentration of the absorbing molecules and the distance that light must travel through them determine how much light is absorbed. The foundation of quantitative analysis with UV spectroscopy is this relationship[8]. Transitions in Electronics Electrons are excited from their ground electronic state to a higher energy electronic state when a molecule absorbs UV or visible light. Electrons are promoted from bonding (σ and π) or non-bonding (n) molecular orbitals to anti-bonding (σ* and π*) molecular orbitals during these transitions. The energy of the incident light and the molecule structure determine the kind of electronic transition that takes place. Electronic transitions of the following frequent forms include σ ▶ σ* (sigma to sigma star) transitions: Since these transitions demand a lot of energy, they usually take place at shorter wavelengths, usually in the far ultraviolet (below 200 nm) range[12]. Saturated hydrocarbons and other compounds with only sigma bonds exhibit them. Transitions known as n ▶ σ* (n to sigma star) occur when an electron is excited from a non-bonding orbital to an antibonding sigma orbital. They are seen in saturated compounds with atoms having lone pairs of electrons and need less energy than σ ▶ σ* transitions. Alkenes, alkynes, carbonyls, and other compounds with unsaturated centres, including double or triple bonds, undergo π ▶ π* (pi to pi star) transitions. Their energy requirements are typically lower than those of n ▶ σ* transitions. The promotion of an electron from a non-bonding orbital to an anti-bonding pi orbital is known as a π* (n to pi star) transition. They are typical of unsaturated compounds with atoms with lone pairs and have the lowest energy requirements of all the common electronic transitions[22]. In conclusion, the fundamental idea behind UV spectroscopy is that molecules absorb visible or ultraviolet light, which causes electronic changes. By connecting absorbance to concentration, the Beer-Lambert equation serves as the foundation for quantitative analysis, and the ideas of chromophores and auxochromes aid in comprehending the structural characteristics that affect a molecule's absorption spectrum[28]. 4. Instrumentation A UV-Visible spectrophotometer's equipment is made to measure how much light at different wavelengths is absorbed by a sample. The fundamental elements cooperate to transmit a light beam through a sample and measure the transmitted light's intensity[31]. Important Elements and Their Roles The key components of a UV-visible spectrophotometer and their functions are broken down as follows: World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 476 • Source of Light The visible and ultraviolet light that passes through the sample is supplied by the light source. Two lamps are usually utilised because one does not give enough light intensity over the whole UV-visible spectrum: Deuterium Lamp and Tungsten Halogen Lamp. • Monochromator: The function of the monochromator is to choose a particular light wavelength from the continuous spectrum that the light source produces. It is an essential element that makes it possible to quantify absorbance at specific wavelengths. The components of the monochromator are: Entrance Slit, Dispersing element (Prism,Diffraction Grating ),Exit Slit. • Sample or Reference cell: Cuvettes, which are tiny, transparent containers that are positioned in the line of the laser beam, are used to hold the sample and reference solutions[33]. Detector: The light that has flowed through the sample must be transformed into an electrical signal by the detector. The intensity of the light that reaches the detector determines how strong this signal is. Typical detector types include: Photomultiplier tube (PMT) ,Photodiode, Charge-Coupled Device (CCD). There are two main configurations for UV-Visible spectrophotometers: • Single-Beam Spectrophotometer: In this simpler design, all the light passes through the sample. To obtain the absorbance of the sample, a measurement of the reference (the blank) is taken first, and the instrument is set to zero absorbance. Then, the sample is placed in the light path, and its absorbance is measured[32]. Advantages: Lower cost and simpler mechanics. Disadvantages: Any fluctuations in the light source intensity between the reference and sample measurements can lead to errors. • 2. Double Beam Spectrophotometer: The light beam from the monochromator is divided into two distinct beams in this more complex configuration. At the same time, one beam travels through the sample and the other through the reference cuvette. The ratio of the two beams' intensities is then measured by the detector. Benefits: It automatically accounts for variations in the strength of the light source and the solvent's absorbance, resulting in measurements that are more precise and reliable. Cons: Costlier and more complicated. The electrical signal from the detector is then processed by the instrument's electronics and software to generate the final absorbance value or the complete UV-Visible spectrum, which is a plot of absorbance versus wavelength. 1.3. Method development and optimization A crucial initial step in creating a strong and trustworthy UV spectroscopy analysis method for furosemide is choosing the right solvent and analytical wavelength. Choosing a Solvent Effective dissolution of furosemide and transparency in the UV spectrum, where furosemide absorbs light, are the two main requirements for the perfect solvent for this application[25]. Although furosemide is soluble in acetone, methanol, and alkali hydroxide solutions, it is nearly insoluble in water and chloroform. A comparison of appropriate solvents is shown below: 0.1 M NaOH (Sodium Hydroxide): Furosemide is acidic due to the presence of a sulfonamide group and a carboxylic acid. It dissolves easily in 0.1 M NaOH and other diluted basic solutions. This solvent is a great option because it guarantees full drug dissolution and is transparent in the UV region of interest[22]. The solvent for furosemide analysis in many official pharmacopoeial procedures, such as the British Pharmacopoeia, is 0.1 M NaOH. Methanol: Methanol dissolves furosemide. Because of its low UV cutoff wavelength (around 205 nm), which prevents it from absorbing light in the area where furosemide has its main absorption peaks, methanol is a frequently used solvent for UV spectroscopy. It works well as a substitute for aqueous basic solutions[23]. Ethanol: Similar to methanol, ethanol can also dissolve furosemide and has a UV cutoff of about 205 nm, making it a viable option[23]. The most strongly advised solvent for method development is 0.1 M sodium hydroxide. Complete sample solubilisation is ensured by its easy dissolution of furosemide by producing the sodium salt, which is essential for precise and repeatable results. It is also easily accessible and reasonably priced. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 477 Selection of wavelength The wavelength of maximum absorbance (lambda max) should be the analytical wavelength since it minimises divergence from the Beer-Lambert equation and offers the highest sensitivity. Because pH affects the chromophores in the molecule, the solvent utilised determines the lambda max of furosemide. Furosemide usually exhibits two or three absorption maxima in its UV spectrum. There are noticeable peaks in the furosemide spectrum in an alkaline media, such as 0.1 M NaOH. For furosemide in 0.1 M NaOH, the primary absorption maximum (lambda max) is usually detected at about 271 nm[32]. There are further absorption maxima at between 228 and 333 nm[32]. Before developing the procedure, first experimentally find the \lambda_{max} by making a diluted solution of furosemide in 0.1 M NaOH and scanning it across the UV spectrum (from 200 nm to 400 nm) to verify the peak absorbance. After confirmation, this wavelength would be utilised for all further measurements, such as sample analysis and calibration curve construction. 1.4. Method validation One of the most important steps in developing an analytical method is method validation. It offers verified proof that the created approach is appropriate for the goal for which it was designed. Validation would be carried out in accordance with the International Council for Harmonization's (ICH) Q2(R1) guideline for the UV spectroscopic examination of furosemide. The following are the main parameters and steps for verifying the UV spectroscopic approach for furosemide using the wavelength (271 nm) and solvent (0.1 M NaOH) that were previously suggested. • Specificity: The capacity of the procedure to precisely quantify the analyte (furosemide) in the presence of additional elements like excipients, contaminants, or degradation products is known as specificity[12]. Acceptance Criteria: There should be no interference (i.e., no discernible absorbance) at 271 nm in the placebo spectrum. The absorbance should be attributed to furosemide if the sample's spectrum matches the standard's. • Range and Linearity: Over a given range, linearity shows that the absorbance, or response, of the technique is exactly proportional to the analyte concentration. The range is the distance between the highest and lowest concentrations for which the method's linearity, accuracy, and precision are demonstrated[12]. Acceptance Criteria: o The plot should be linear. o The correlation coefficient (R²) should be ≥ 0.999. o The y-intercept should be close to zero. • Accuracy: The degree to which test findings closely resemble the actual value is known as accuracy. Recovery studies usually determine it[12]. Acceptance Criteria: At each concentration level, the mean recovery percentage should fall between 98.0% and 102.0%. • Precision: The degree of spread between a set of measurements is measured by precision. Two stages of assessment are used: Repeatability precision and Intermediate precision. Acceptance Criteria: The relative standard deviation (RSD) should be not more than 2.0%. • Limits of Quantitation (LOQ) and Detection (LOD) : Although they are typically not necessary for the assay of a medication in a final product, LOD and LOQ are significant for the identification of contaminants or degradation products. They are able to comprehend the method's limitations, though. Acceptance Criteria: It must be shown that a solution at this concentration can be examined with sufficient precision and accuracy in order for the LOQ value to be validated. • Robustness: Robustness gauges a method's ability to withstand minor, intentional changes in its parameters, giving an indicator of how reliable it is under typical operating conditions[12]. Acceptance Criteria: The outcomes of the various conditions shouldn't be appreciably different from those of the standard procedure. The results' RSD should stay within reasonable bounds, usually ≤ 2.0%. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 478 1.5. Application of UV Spectroscopy in Furosemide Analysis: UV-based qualitative analysis One effective technique for verifying the authenticity of a material such as furosemide is visible spectroscopy. The idea is that each UV-absorbing chemical has a distinct absorption spectrum that functions as a "molecular fingerprint." We can verify the identity of an unknown sample by comparing its spectrum to that of a recognised reference standard. Recognition and Verification : Two requirements must be fulfilled for the sample to be positively identified as furosemide Position of Maxima: The Test Solution's UV absorption spectra needs to show absorption maxima at wavelengths that match those of the Reference Solution[11]. The spectra of furosemide in 0.1 M NaOH will exhibit distinctive peaks at roughly 228 and 271 nm (the major and most noticeable peak). 333 nanometres Spectral Concordance: The Test Solution's whole spectrum needs to be superimposable, or identical to, the Reference Solution's spectrum. This demonstrates that the same absorbing organisms are present in both solutions. The test sample is identified as furosemide if its spectrum closely resembles that of the reference standard in terms of the position and overall form of the absorption peaks. For routine quality control of a pure furosemide drug substance or a simple formulation where interfering substances are not expected, UV spectroscopy is an excellent choice due to its speed, simplicity, and low cost[12]. 1.6. Quality control and quality Assurance application In the pharmaceutical sector, analytical techniques provide the foundation of both quality assurance (QA) and quality control (QC). They offer the unbiased information required to guarantee that each batch of a medication, such as furosemide, is safe, efficient, and satisfies all necessary quality standards. Assay (Potency Assessment) : The purpose of the assay is to precisely measure the amount of furosemide, the active pharmaceutical ingredient (API), in a specific dosage form (such as tablets or injections). Uniformity of Content : This test guarantees that the active ingredient is present in a constant and uniform level in each dose unit. This is essential to guaranteeing that each tablet or injection gives patients the right dosage. Testing for Dissolution : A crucial quality control metric is dissolution testing, which gauges how quickly and how completely a medication dissolves in a given medium from its dosage form. The drug's in vivo bioavailability can be accurately predicted by this in vitro assay. 5. Result For the quantitative analysis of furosemide, UV-Vis spectroscopy is a well-established, straightforward, quick, and economical technique. Numerous studies' findings consistently demonstrate that techniques, most frequently using 0.1 M NaOH as a solvent with a lambda max about 271 nm, are effectively validated in accordance with ICH Q2(R1) criteria. With negligible interference from common pill excipients, these techniques consistently show exceptional precision (%RSD < 2%), high accuracy (usually 98-102% recovery), and excellent linearity (R^2 > 0.999). This review demonstrates that HPLC is still required for the analysis of furosemide in combination medication products, even if UV spectroscopy is very appropriate and dependable for the routine quality control, assay, and dissolution testing of furosemide in bulk drug and simple pharmaceutical formulations. 6. Conclusion This review demonstrates that UV-Vis spectroscopy is a reliable, valid, and strong analytical technique for furosemide measurement. The approach is a highly ideal tool for routine applications due to its simplicity, speed, and costeffectiveness, which have been consistently validated in accordance with ICH principles. For the assay and in vitro dissolution testing of furosemide in bulk medication (API) and its single-component pharmaceutical forms, including tablets, this approach is advised. However, more advanced separative methods like UV-Vis are required to guarantee specificity and accuracy for the analysis of more complicated formulations containing furosemide in conjunction with other UV-absorbing medications. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(01), 472-480 479 It concluded, UV spectroscopy for furosemide analysis is evolving from a straightforward, single-wavelength measurement to a method that is information-rich and data-driven. The emphasis will be on using the full spectrum data to ensure quality in real-time, analyse complex mixtures effectively, and enable rapid analysis anywhere from the production line to the point of care rather than just verifying the quantity. 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