Microsoft Word - 221-234 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |221   Use of Acidic Hydrolysis and Diazo Coupling Reaction for Spectrophotometric Determination of Furosemide in Urine and Pharmaceutical Formulation Hawraa Ali Sumayha muhammed sumayha.mu@gmail.com Dept. of Chemistry/ College of Education for Pure Science (Ibn Al-Haitham / University of Baghdad Abstract Simple and rapid spectrophotometric determination of furosemide (FUR) has been investigated .The method is based on acid hydrolysis of FUR to free primary aromatic amine and diazotization followed by coupling with 3, 5 di methyl phenol (3, 5-DMPH) at basic medium. The absorbance was measured at 434 nm, the method was optimized for best condition, and beers’ law is obeyed over the range of 0.4-50 µg.mL-1 with molar absorptivity and sandal’s sensitivity 1.3899 x104 L moL-1 .cm-1 and 0.0238x104 µg.cm-2 respectively. Analysis of solution containing nineteen different concentrations of FUR gave a correlation coefficient of (0.9999) and limit of detection, limit of quantitation were 0.127, 0.464µg.mL-1 respectively. The reaction stoichiometry was evaluated by Job’s and mole ratio method was found to be 1:1(diazotized FUR: 3, 5-DMPH) .The method was applied in synthetic urine and pharmaceutical formulation. The recovery of FUR in spiked urine was satisfactory resulting in the values of (99±3.32) %, the results of the suggested method was compared with available official literature method. Keywords: Furosemide, 3,5-Dimethyl Phenol Spectrophotometry, Diazotization. IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |222   1. Introduction Furosemide or Frusemide (4-chloro-N-furfuyl -5-sulfamayl- anthranilic acid is formally a sulfonamide, an antibacterial agent. However, the intense and fast dieresis produced by this drug, has extended its application as a powerful acidic diuretic for diverse treatment in human and veterinary medicine. Furosemide is often classified as a loop diuretic due to its predominate action in the nephron [1]. Fur acts inhibiting of sodium on Henle, s 100g and inhibiting the co-transportation of sodium, potassium and chloride, and causes excretion of calcium, magnesium and bicarbonate ion. Intense and fast dieresis may also mask the ingestion of other doping agents by reducing their concentration in urine [2]. Fur have a large number of analytical technique to determinate it in pharmaceutical and biological samples a number of spectrophotometric method have also been reported for furosemide [3-8]. Also electrochemical detection and capillary electrophoresis have been used to quantify fur [9-11]. HPLC is generally the method of choice for diuretics quantitation, due to the required time and cost of the analysis [12-13]. The aim of this study was to develop and validate a new spectrophotometric method for quantitative determination after acidic hydrolysis with strong acid media to yield salumine that can be azotization and coupled with 3.5 dimethyl phenol to form a new orange complex have a maximum absorption at 434nm. Scheme (1): Chemical structure of furosemide 2. Experimental Apparatus 1-All spectrophotometric measurements were made on Shimadzu U.V- visible double beam spectrophotometer equipped with 1 cm quartz cells (Kyoto, japan) the uv-vis spectra of standard and solution were recorded in 1cm quartz cell at wave length of 434nm. 2-Sartorius BL210 electronic balance was used for weighing the samples. 3-pH measurements were taken with Jan way pH-meter 3310. IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |223   Material and reagents: All chemicals used were of analytical grade or chemically pure, distilled water was used for all dilutions of reagents and samples Expect 3, 5- DMPH which was dissolved in methanol. Standard Grade of furosemide was kindly provided by State Company for Drug Industries and Medical Appliances (S.D.I), Samara-Iraq. Standard hydrolyzed furosemide (100 µg.mL-1) Standard FUR was prepared by dissolving 0.25 g of pure drug in methanol and diluting to the mark in 250ml volumetric flask with distilled water to prepare (1000 µg.mL-1). Transfer 150mL of prepared solution with 25mL HCl (M 11.8 to volumetric flask of 250mL and diluting to the mark with distilled water to prepare (600µg.mL-1) of FUR. The obtained solution was reflexed 3hr at 70C° until it would be clear and yield a light yellowish solution pointed to complete the acidic hydrolysis, leave it to cool in ice bath then transfer 16.6mL to 100mL volumetric flask then diluted to the mark with distilled water to prepare (100µg.mL-1) of hydrolyzed FUR . Reagent solutions 1-Sodium nitrate solution (1%) was prepared by dissolving 1g of NaNO2 (Merck) in distilled water and dilute to the mark in 100mL volumetric flask. 2-Sodium hydroxide solution (1.5M) was prepared by dissolving 1.5g of NaOH (Merck) in distilled water and dilute to the mark in 25mL volumetric flask. 3- Buffer solution (pH11) was prepared by mixing 100mL of 0.025 M borax with 41mL of 0.1 M HCl 4-Sodium carbonate solution (1.5M) was prepared by dissolving 3.9g of salt in distilled water and dilute to the mark in 25mL volumetric flask. Pharmaceutical and synthetic urine solutions A_ synthetic urine sample was spiked in order to achieve concentration close to (4.0X10-4) M. A complete description of the components of the synthetic urine is given in Table (1) B_ Solutions for tablets were weighted out grinded and mixed well. A portion of the result powder (containing 0.25 g FUR) was used for the preparation of the solution as described before. Calibration Curve procedure. Calibration curve was prepared by taking appropriate aliquots (0.1_4) mL of working standard solution of hydrolyzed FUR in 5mL volumetric flask to give (0.4_50) µg.mL-1. Samples for each test were prepared by adding 0.5 mL of freshly 1% NaNO2, then 0.5 mL of 3,5_DMPH added to the resulting solutions followed addition of 0.7 mL buffer solution. The solutions were making up to the mark with distilled water and mixed well, the absorbance of the light orange dye was measured at 434 nm against the reagent blank. Standard addition procedure A_ In synthetic urine The method was used for assay FUR present in synthetic urines samples. To a series of seven (5mL) calibrated flasks , transfer 0.5 ml of synthetic urine sample containing 4.25 mL of 600.µg.mL-1 hydrolyzed FUR followed by addition (0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |224   mL)of standard FUR solution of 100 µg.ml-1. The result mixtures were then treated as described in calibration curve procedure and measured the absorbance at 434nm. B_ In pharmaceutical samples To a series of seven 5mL volumetric flask, transfer 0.5mL of 100µg.ml-1 tablet solution followed by addition (0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6mL)of standard FUR of 100 µg.ml-1 .The result mixture were then treated as described in calibration procedure and measure the absorbance at 434 nm. 3. Result and discussion Absorption spectra The primary test of the present method involved diazotization of hydrolyzed FUR (The pH of FUR 5) with sodium nitrate then reacted with 3, 5-DMP in basic medium to form colored azo dyes product. The absorbance and ⋋ max of azo dye was measured against the reagent blank. Figure (1) shows the maximum absorption obtained at wavelength 434nm. Optimization of reaction conditions The optimum conditions for color development were established by varying the parameters one at time keeping the others fixed and observing the effect produced on the absorbance of the colored product. Preliminary investigation showed that the light orange product resulted upon treating FUR solution with 3, 5_DMPH after diazotization in alkaline medium. Study of the variables affecting the color development of reaction product, namely : the amount of reagent for the range ( 0.1 _ 2 )mL , the amount of NaNO2 for the range ( 0.1 _ 2 ) mL, the pH of the coupling reaction by using different volumes (0.1_ 2) mL of borax buffer, the effect of diazotization reaction time was studied at different times (0_30 min) , and the order of mixing reagent solutions, were performed on 10 µg.mL1 of FUR solution. 0.5 ml of 3, 5_DMPH solution was selected as an optimum amount Figure (2), while 0.5mL was the optimum volume of NaNO2 Figure (3), of buffer solutions different volumes are tested to control the pH of coupling reaction medium. The study showed using 0.7 mL of borax gave the best absorbance of the colored product Figure ( 4) while the study of reaction time showed the reaction spontaneously occur at zero time .The optimum order which gave the highest absorbance was ; FUR+ reagent + buffer. The suggested mechanism for the hydrolysis and diazotization with coupling reaction is shown in scheme (2): IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |225       Final absorption spectra The absorption of the spectrum colored produced under optimum reaction condition, shows, maximum absorption at 434 nm against reagent blank Figure (5) Calibration curve and analytical data Employing the optimum conditions, the measured absorbance at 434 nm versus different standard concentrations of FUR were plotted to construct calibration curve. The linearity of the obtained plot was in the concentration range (0.4 _ 50) µg.mL-1 as shown in Figure (6). Evaluating the liner regression Evaluating the liner regression of the suggested method is done by plotting the standardized concentration residuals vs the predicated concentration of the tested standard solutions. The residuals for FUR in all points appear to be randomly distributed around zero as show in Figure (7) Stoichiometry of the product Jobs and mole ratio methods have been used to determine the stoichiometry of the colored product. FUR and 3, 5_DMPH solution of 1.38x10-3M were prepared and mixed in various molar ratio in 5mL volumetric flask. According to the suggested procedure, the absorbance was measured at434nm. The graph of the results obtained as in Figure (8) & (9) gave maximum mole ratio Xmax= 0.5, X= 1 in Jobs and mole ratio method respectively and showed that1:1 FUR: 3, 5_DMPH. Scheme (2): The hydrolysis and diazotization with coupling reaction of FUR with 3, 5-DMPH IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |226   Precision and accuracy The precision and accuracy of the proposed method was tested by analyzing five replicate of FUR in three different levels (within Beers' law range). The result list in Table (3) indicates an acceptable accuracy and precision to suggested work. Interferences study The presence of different quantity (2, 6 and 12ml) of 1000µg.mL-1 for Excipients can be tolerates in the analysis 5µg.mL-1 FUR since it would cause relative error percent less than 5% as it shown in Table (4). Application in pharmaceutical forms In order to demonstrate the applicability of the proposed method in determination of FUR, the method was applied to different manufacturing source containing FUR: 1_ Furosemide (Tablet 40 mg) Ajanta (India) 2_ Lazine (Furosemide injection 20mg) (Syria) To apply the suggested method on pharmaceutical formulation, an accurately weighed 1.212g from tablet (40mg) and 12.5mL-1 (20mg) from ampule which contains 0.25g of pure furosemide to prepare the solutions as described before. Five replicate of three concentration levels (2, 6 and 12µg.mL-1) were taken and calculated the recovery and relative standard deviation. The result indicated the suitability of suggested work for routine analysis of FUR asset shown in Table (5). Standard additions method The proposed method was applied to the determination of FUR in synthetic urine as described before. 4.25mL of 600µg.mL-1 hydrolyzed FUR was transferred to the prepared urine sample and completed the volume to the mark in 25mL volumetric flask with distilled water. Seven solutions were prepared in seven-5 mL volumetric flasks, by the addition of 0.5mL of synthetic urine solution and different volumes (0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL) of 100 µg.mL-1 standard FUR drug solution and the same procedure of calibration curve were to give a good recovery in drug sample which indicate no interferences from the matrix affect the determination of FUR. Figure (10) and table (11) show the standard addition plots and Table (6) shows the recovery. Comparison with the reported methods Table (7), shows the between the proposed method and other literature spectrophotometric methods throughout some measured analytical parameter. IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |227   Table (1): The Components of the synthetic urine [22]. Table (2): Summary of optical characteristics, validation parameters and statistical data for the proposed method. Value Parameter 434 (nm)max λ Light orange Color 0.4-50 )1-Linear range (μg.mL y =0.042x+0.0112 Regression equation 0.042 Slop 0.0112 Intercept 4x10 1.3889 )1-.cm1-Molar absorptivity (L.mol 0.9999 Correlation coefficient 0.127 )1-Detection Limit (μg.mL 0.464 )1-(μg.mLQuantitative limit 40.0238x10 )2-sensitivity (μg.cm Shandell’s Table (3): Evaluation of the accuracy and precision of the propose method FUR Conc. ( µg.mL-1) Relative Error % R.S.D.* % Taken Found* 2 2.021 1.050 0.043 6 5.891 -1.816 0.016 12 11.931 -0.575 0.021 *Average of five replicate Compound Weight ( g) NaCl 0.05 Na2SO4 0.05 KH2PO4 0.05 Kcl 0.05 CaCl2 0.05 NH4(SO4)2 0.05 Urea 0.62 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |228   Table (4): The relative error value for 10µg.mL-1of FUR in The presence of different excipients Relative error (%) Taken 1000 Excipients 0.2783 2 Acacia 0.3671 6 0.4559 12 0.1214 2 Glucose 0.3801 6 0.7345 12 0.3889 2 Starch 0.6100 6 0.9842 12 0.1851 2 Vanillin 0.4141 6 0.8148 12 0.1960 2 Maltose 0.3944 6 0.5784 12 0.0971 2 Sucrose 0.1784 6 0.3012 12 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |229   Table (5): Recovery data obtained by application the present method in drug formulations Table (6): Recovery data obtained by standard additions method for FUR in rigin tablet and synthetic urine Pharmaceutical preparation Conc.Taken µg/ml Conc.found µg/ml Recovery Sample FUR +0.5ml tablet 10 10.01 100.3 Sample FUR with synthetic urine 12 11.82 98.5 *Average of three replicate Pharmaceutical preparation Labeled amount mg Conc.taken µg/mL Conc.found* (µg) Recovery% S.D Movineer, Tablet ,actavis England 40 6.000 5.902 98.366 0.130 12.000 12.000 100.000 0.044 Movineer,Tab,ajanta/India 40 6.000 5.734 95.566 0.033 12.000 11.947 99.558 0.175 Lazine Ampule Syria 20 1.000 0.968 96.800 0.049 2.000 1.993 99.650 0.086 IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |230   Table (7): Analytical parameter for the determination of Fur by the proposed method comparing to other methods FUR against reagent blank, (B) blank 1-A) 10µg.mL ( (1): Absorption spectra of Figure solution Technique used D.L Liner range Rec.% Ref. Electrochemical M )7-3.8x10 ( )M7-5.0x10-2-(1.0x10 - 14 Electrochemical ----) 8-7.0x10 ( ----) 6-7.0x10 –7-1.0x10( - 15 HPLC - 1-µg.mL)200-20( 99.9 16 HPLC 1-ng.mL) 5.2 ( 1-ng.mL ) 25.00-5.20( - 17 Fluorescence 1-µg.mL )0.007( 1-g.mLµ )140.00 –0.03( 97.8-100.8 18 Spectrophotometric - ------ )21.20x10-3-8X10( 99.14 19 Spectrophotometric (0.50)------ (20-160)------- - 20 Spectrophotometric 0.23) ---- ( (5-30)------ 99.69-100.83 21 Present method 0.12 )----- ( 0.4-50) ------( 99 - IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |231   Figure (4): Effect of the volume of Buffer solution on color development of colored compound under optimum 1-(A) 10µg.mL ,Figure (5): Absorption spectra of condition, (B) lank solution against solvent 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 1 2 3 A b s V buffer solution(mL) 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 A b s V. of NaNO2 (mL) 0 0.1 0.2 0.3 0.4 0 1 2 3 A b s V.of Reagent (mL) Figure (2): Effect of the volume of 3, 5- dimethyl phenol on colour Figure (3): Effect of the sodium nitrate volume on colour development AB  IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |232       Figure (8): Job's curve of 1.38×10-3M FUR and 3, 5-DMPH  Figure (9): Mole ratio of 1.38×10-3M for each FUR and 3, 5-DMPH     Figure (10): Plot of standard additions method for determination of Fur in tablet   ‐0.04 ‐0.02 0 0.02 0.04 0 0.5 1 1.5 2 2.5R e si d u a l fitted value 0 0.1 0.2 0.3 0.4 0 0.5 1 A B S V L FUR. /VML FUR. +VM L REAG.  y = 0.042x + 0.0113 R² = 0.9999 0 1 2 3 0 20 40 60 A b s µg/ml 0 0.1 0.2 0.3 0.4 0 0.5 1 1.5 2 A b s VmL Reg./VmL Fur. Figure (6): Calibration curve for the determination of FUR under optimum conditions Figure (7): The residual error of liner regression model  y = 0.8314x + 0.4074 R² = 0.9992 0 0.2 0.4 0.6 0.8 1 ‐0.6 ‐0.4 ‐0.2 0 0.2 0.4 0.6 0.8 A b s v.(ml) IHSCICONF 2017 Special Issue Ibn Al-Haitham Journal for Pure and Applied science https://doi.org/ 10.30526/2017.IHSCICONF.1795 For more information about the Conference please visit the websites: http://www.ihsciconf.org/conf/   www.ihsciconf.org   Chemistry |233   Figure (11): Plot of standard additions method for determination of Fur. in urine   References [1] Maria Jose Ruiz –Angle and Alain Berthod, Analytical Techniques for Furosemide Determination, Separation & Purification Reviews, 35, (2006)39-58. [2] M. Espinosa Bosch; A.J. Ruiz Sánchez; F. Sánchez Rojas; C. Bosch Ojeda Analytical Determination for Furosemide: The Last Researches Review Article Pharmaceutical Sciences, IJPBS,(2013). [3] Jasmin shah, M. 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