Visible Quantitative Methods for the Estimation of Furosemide in Pure form and Pharmaceutical Formulations

Aims: Design of technical methods for the determination of Furosemide in its pure and pharmaceutical dosage form using spectral methods. Study Design: planned and executed to estimate Furosemide by using Visible spectrophotometric in pure and pharmaceutical dosage form. Place and Duration of Study: Laboratory of Analytical Research, chemistry department, college of Science, University of Mosul ,Mosul-Iraq, during the period of April 2021 to August 2021. Methodology: Furosemide, the commercially known drug Lazix, which is important in the treatment of heart diseases and high blood pressure. This study was carried out using JASCO V – 630, double-beam computerized UV-Visible spectrophotometer, with 1 cm matched cell, and HANA pH meter was used for reported pH readings. Results: The reaction between Furosemide and bromo-phenol blue, xylenol orange, and chromazorol S. The decreasing in the intensity of the resulted colored complex was measured using bromo-phenol blue, xylenol orange, While the increasing of the color intensity was measured in the method (C). These three methods were based on charge transfer reaction. The limits of Beer's law for method (A) 0.4-32μg. mL -1 , method (B) 1-32 and method (C) were 0.8-32 depending on the level of concentration, while the values of the molar absorption coefficient 1.4×10 4 , 2.1×10 4 and 1.57×10 4 l.mol -1 .cm -1 for the first, second and third method respectively. Sandel's significance Original Research Article Saleem et al.; JPRI, 33(47B): 200-209, 2021; Article no.JPRI.76295 201 also was calculated for these three methods, 0.0157 μg.cm -2 for the first method, 0.0236 μg.cm -2 for the second method, while the third method was 0.0210 μg.cm -2 . The method has been successfully applied for the determination of furosemide in its pure form and in some of its pharmaceutical preparations Conclusion: The proposed methods were validated in terms of linearity, range, Accuracy, precision, Specificity, Robustness. The proposed methods were successfully applied to the estimation of Furosemide in pharmaceutical dosage form, method (B) was experimentally considered as a best method depending on the best values of molar absorptivity, stability of the resulted complex, and the linearity of the method (B) .


INTRODUCTION
Furosemide (FSD) is known commercially as Lasix, and chemically as 4-chloro-en-furfuryl-5sulfamoylantranilic acid or 5-(aminesulfonyl)-4chloro-2[(2-furanylmethyl) amino] benzoic acid. FSD is used to treat high blood pressure as well as a diuretic where it is used to treat edema associated with heart failure, cirrhosis, and kidney disease [1][2][3][4][5]. FSD is an important drug for human health, as it is used to treat the most important organs of the body, starting with the heart, kidneys and liver. Therefore, researchers have dealt with this drug in many studies, which have dealt with its solubility, uses and methods of estimation it, mostly of these method used various techniques, and among of these techniques, a spectrophotometric and chromatographic methods have been used for determination of FSD depending on: charge transfer complex method [6], mass spectroscopy [7], infra-red spectroscopy [8], Also, first order derivative spectroscopy method and absorbance ratio (Q-Absorbance) method were used [9], other spectrophotometric method was using principal component regression [10], Validated RP-HPLC Method was used for estimation Furosemide in tablet [11], Some researchers depending on diazotized method to assay FSD [12], liquid chromatography were reported for estimation FSD in plasma and urine samples [13,14], as well as polarographic method [15]. Other method was based on using schiff's bases to estimate FSD spectrophotometrically [16]. Also, liquidliquid extraction and high-performance liquid chromatography were used for estimation FSD [17,18]. Spectrophotometric methods adopted different reactions for the determination of FSD in pharmaceutical dosage forms [19][20][21][22], finally, reverse-phase high-performance liquid chromatography [23], and flow injection with HPLC [24], as well as HPLC method were used in the estimation of FSD [25][26][27].

Apparatus
The final spectrum of FSD was measured and drawn using JASCO V -630 double-beam computerized UV-Visible spectrophotometer, for all spectrophotometric measurements, 1 cm matched cell was used, and HANA pH meter used for reported pH readings.

Analytical Chemicals
All chemicals used were of the purest analytical grade.

Preparation of Furosemide from Tablets
Three different brands of pharmaceutical preparations were used for furosemide, where 10 tablets (each tablet contains 40 mg) were ground into a very fine powder, then weighed precisely about 0.01 g of the powder, this quantity was then dissolved in methanol, filtered and completed the volume of filtration with methanol mixed with warm distilled water at a ratio of 1:1 in a 100 ml volumetric flask.

DISCUSS THE EXPERIMENTAL RESULTS
100 μg of FSD in a final volume of 25 mL was used to study the experimental optimal conditions

Study of Optimum Conditions
In this research paper, the optimal conditions suitable for the formation of the colored complex of furosemide were studied and selected.

Selected the Optimum Medium of the Reaction
In order to choose the most appropriate type of acids and in the optimum quantity among sulfuric, acetic and hydrochloric acids, by studying the effect of adding different quantities to each type of these acids to determine the acidity function most appropriate for the three methods for estimating furosemide, a (0.1-3.0) of these acids was chosen with a concentration of 0.1 M as shown in Table 2.
The practically obtained results and illustrated in Table 1, that the addition of any type at any amount of all acids did not have a beneficial effect, therefore, this study was excluded from the subsequent experiments. Depending on this fact, the pH value of method A, B, and C in the absence of any quantity of acids or bases were 3.58, 4.51 and 4.77 respectively, so that, these pH value have adopted for the subsequent experiment.

Effect of Dye Quantity
The experimental results which were depending on the values of the correlation coefficient and absorbance values were considered the best factor to choose the optimum amount of dyes in the three proposed methods. 3 mL, 2 mL of 0.01 M and 2 mL of 0.001 M, have been selected as an optimum amount of these three dyes with correlation coefficient equals to (0.99863, 0.98972 and 0.9992) for method A, B, and C respectively.

Effect of Various Kinds of Surfactants
In many cases, the addition of any type of surfactant of different types may not lead to a shift in the wavelength or an improvement in the intensity of absorption, as happened in this study.
It was noted from the practical results that adding all types of surfactants (sodium dodecyl sulfate as a surfactant) Anionic, cetyltrimethylammonium bromide, cetylpyridinium chloride as cationic surfactants and non-ionic Triton X-100) to the staining regimen had no obvious effect either in increasing the absorption intensity or leading to the wavelength shift to higher values. Therefore, this study was not adopted in subsequent experiments.

Studying Order of Addition
The interaction components of the three methods do not exceed the drug and the dye, so there are no more than two sequences to study that lead to the same result, adding the dye to the drug or vice versa did not have a clear effect, so the addition of the dye to the furosemide drug was adopted in this study.

Studying Stability Period
The time required for the formation of the colored potion between FSD and the three dyes was studied, as it was found from the practical results that the color formed instantaneously and remained stable for more than 72 hours with a high stability of the three methods under optimal conditions, meaning that the colored compound was developed immediately and remains at a maximum and consistently and very stable more than 72 hours. Fig. 1 show a part of stability for this study for these three methods.

Beer's Law, Molar Absorption and Sensitivity
The standard curve of the proposed spectroscopic methods has been studied by adding different amounts ranging between (10-800), (25-800) and (20-800) for each method A, B and C respectively, then adding dyes and dilution to the mark with distilled water and after shaking the bottles. The absorbance was measured at the specified wavelength at 591, 583, and 525 nm, complied with Beer's law over the f and ppm of FSD While the molar absorbance was 1.

Absorption Spectra
Depending on the optimum conditions, the absorption spectrum of FSD was studied, as shown in Figs. 5, 6 and 7 which were indicate that the sample solution shows maximum absorption at 591, 583 and 525 nm for the three methods, respectively.

Accuracy and Precision
In order to verify the selectivity and efficiency of the proposed methods for FSD estimation, 100µg of FSD were determined using ten measurements for each method as shown in Fig.  8 which is illustrated that these three methods were almost reliable.

Mole Ratio
The determination of the interaction ratio between furosemide drug and the three dyes was studied by preparing equal concentrations for both drug and dyes, then taking volumes ranging from (0-5) mL of FSD, corresponding to (5-0) mL of each of the three dyes. Fig. 11 shows the result of using Job's method to study the reaction ratio in each method, so that, reaction ratio of FSD to bromophenol blue is 1:2, and 1:1 between FSD to xylenol orange dye, while the ratio of FSD to chromazurol S is 1:2 as shown in the Fig.  9.

Effect of Foreign Materials
This study was conducted by adding a number of potential substances present and use in pharmaceutical preparations, with concentrations of up to 1000 µg/mL. The results listed in Table 3 showed that the studied excipients do not seriously interfere in the determination of FSD in pharmaceutical preparations using the three proposed methods.

Application of the Method
The three proposed methods have been satisfactorily applied for the estimation of FSD in pharmaceuticals and the results are shown in Table 4.
It is noted from the results listed in Table 5 that the calculated value of the t-test measured at 95% confidence level and for five degrees of freedom (N1 + N2-2 = 5) did not exceed the theoretical values for that when compared with the theoretical values established in the references [32]. Table 6 shows some of the analytical variables measured for the current methods and their comparison with the spectroscopic methods proven in the references for the estimation of FSD.

CONCLUSION
Three spectroscopic methods are proposed based on the reaction of charge transfer and shortening of the dye using bromophenol blue in method 1, xylenol orange in method 2, and chromazorol S in rapid, sensitive and inexpensive methods that do not require expensive devices or any temperature control or any extraction process. Good recovery values for FSD are achieved upon successful application of the proposed methods for determining FSD in some of its pharmaceutical preparations.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.