172 PLASTIC BUMETANIDE MEMBRANE SENSOR AND ITS APPLICATIONS IN PHARMACEUTICAL PREPARATIONS AND BIOLOGICAL FLUIDS Maha El-Tohamy, Magda El-Maamly and Abdalla Shalaby* تم تطوير إلكترود غشائي بالستيكي (مسرى كهربيي ديييي (ميا ميادل بيولي يلييا ك)ويرييي يرتكيي ن)يل ررييو رل تيررب ل ادل تلجستو وسفات بيوميتانيي ك ادل نشطة كهربيا ً وذلك لتقيير مادل بيوميتانيي بشك)ه رللقي و ي رل ستحضررت رلصيييننية وكانت رل ادل رل )ينة هي دري بيوتيا سييباكات وديي غطيت رنسيتجابة رل طيية مييى تركييي ل)ييور،و ي رلسورئا رلبيولودية وديي تيم8-5 نقيي وكيا مييى ررل رلهييييوديلي رل )يي هيو/ مي))يي ولت58.5 نييايي ب ييا مقييري10-3x1 – 10-6x1 ومسيوغات رلييور ك يا تيم تطبييا رإللكتيرود رل قتير، ورلق)ويييرت،رإلبالغ نا رلتييرالالت رللاتجية ميا رلكاتيونيات رلشيائ ة لتقيير مادل بيوميتانيي بشك)ها رللقي وتجانس رل حتوى ي رردررص وكانت رللتائج متور قة دير ً مع رللتائج رلتيي تيم رلحصيو NFXX رلرس ي وكتيب رلوصفات ررمريكيUSPXXV ن)يها باست يرم طريقة دستوي رردوية ررمريكي A novel plastic poly (vinyl chloride) membrane electrode based on bumetanide-tungstophosphate ion association as electroactive material for the determination of bumetanide in pure form, pharmaceutical formulations and biological fluids is developed, in which the plasticizer is di-butyl sebacate. The linear response covers the range of 1x10-6-1x10-3M drug concentration with a slope of 58.5 mV/decade. The practical pH range is 5-8. Interferences from common cations, alkaloids and drug excipients are reported. The proposed electrode has been successfully applied to determine bumetanide in pure form and the content uniformity for tablets. The results are correlated well with those obtained by the official USP 25, NF 20 method. Key words: Bumetanide, bumetanide plastic membrane, pharmaceutical analysis, potentiometric methods, pontent uniformity. Introduction Bumetanide [3- ( butyl amino) - 4- phenoxy - 5 sulfamoyl benzoic acid] is an important pharmaceutical compound classified as a strong diuretic agent (loop diuretic). It is indicated for the treatment of oedema associated with congestive heart failure, hepatic and renal diseases, including the nephritic syndrome (1). The content of bumetanide tablets is usually only 1.0 mg, therefore, the analytical methods used for the assay should be very sensitive. Several analytical methods have been reported for the determination of bumetanide in pure form, pharmaceutical formulations or in biological fluids including spectrophotometry (2,3), capillary zone electrophoresis (4), high Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig Egypt * To whom correspondence should be addressed. Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 performance liquid chromatography (5-9), gas chromatography-mass spectrometry (10-12), fluorimetry (13), liquid chromatography-mass spectrometry (14). Bumetanide as a diuretic agent sometimes misused by competitors for two main reasons, namely: To reduce weight quickly in sports where weight categories are involved. Also to reduce the concentration of abused drugs by diluting the urine. Health risks are involved in such misuse because of serious side effects which might occur. Therefore, due to the increasing importance of bumetanide and its abused in sports a fast method for routine analysis and quality control of commercial formulations is very desirable. The aim of this work was to develop a novel sensitive, fast and low cost for the routine quality control (assay, content uniformity) and dissolution study of bumetanide in pure form and in pharmaceutical formulations. PLASTIC BUMETANIDE MEMBRANE SENSOR Experimental Reagent and Materials: All chemicals used of analytical or pharmacopoeial grade. Doubly distilled water was used throughout the experiments. Bumetanide was provided by Memphis-Delagrange, (France), poly (vinyl chloride) (PVC) was from Aldrich (Germany), dibutyl sebacate was from Fluka (Buchs, Switzerland), tungstophosphoric acid and tetrahydrofuran (THF) were from Memphis-Delagrange (France), (Burinex® tablets, Edemex® tablets and ampoules) were purchased form local drug stores. Stock bumetanide solution (1x10-1M) was prepared daily by dissolving an appropriate amount of the drug in double distilled water. More dilute solutions were prepared by appropriate dilution. All solutions were stored at darkness in airtight containers. Apparatus: Jenway 3010 pH/mV meter (U.K.) with a bumetanide-PVC membrane electrode in conjunction with double Junction Ag/AgCl electrode (Orion 9002) (Taiwan, R.O.C.) containing 10 % w/v potassium nitrate in outer compartment. An Orion 91-02 glass-calomel combination electrode, (Taiwan, R.O.C.) was used for pH adjustment. All potentiometric measurements were carried out at 25±1ºC with constant magnetic stirring. Recommended procedures: Preparation of bumetanide ion-pair: The ion-pair was prepared by mixing 50 ml aliquots of 1x10-2 M bumetanide and tungstophosphoric acid. The resulting white precipitate was filtered through G4 sintered glass crucible and washed thoroughly with deionized water then dried at room temperature for 24 hours. Membrane composition: The membrane was prepared by dissolving 190 mg of powdered PVC, 0.35 ml of the plasticized (dibutyl sebacate) and 10 mg of the ion-pair in 5 ml tetrahydrofuran (THF). The solution was poured into a petri dish (3 cm in diameter), covered with a filter paper and the solvent was allowed to evaporate slowly at room temperature. Electrode Construction: A punched circular membrane was attached to a poly–ethylene tube (8 mm in diameter) in an electroSaudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 173 de configuration by means of PVC-THF solution. A mixture containing equal volumes of 1x10-3 M bumetanide and potassium chloride was used as internal reference solution in which the Ag/AgCl reference electrode was dipped. The constructed electrode was pre-conditioned after preparation by soaking for at least 24 hours in 1x10-3 M drug solution and stored in the same solution. The electrochemical system is represented as follow: Ag/AgCl/inner solution/membrane/test solution/KCl salt bridge//SCE. Electrode Calibration: 10 ml aliquots of 1x10-1-1x10-6 M standard bumetanide solution were transferred into 50 ml beaker and the sensor in conjunction with Ag/AgCl reference electrode were immersed in the solution. The measured potential was plotted against the logarithm of drug concentration. The electrode was washed with deionized water and dried with tissue paper between measurements. Effect of pH: The effect of pH on the potential of the electrode was measured using two pH/mV meters. The combined glass calomel electrode was connected to one instrument and the PVC-bumetanide membrane with the double junction Ag/AgCl reference electrode was connected to the second instrument. Thirty ml aliquots of 1x10-6 M, 1x10-5 M, 1x10-4 M, 1x10 -3 M, drug solutions were transferred to a 100 ml beaker where the three electrodes were immersed, the potential readings corresponding to different pH values were recorded. The pH was gradually increased or decreased by the addition of small aliquots of dilute solutions of 0.1 N sodium hydroxide or 0.1N hydrochloric acid respectively and the pH-mV was measured and plotted. 6. Selectivity of the electrode: Selectivity coefficients were determined by the separate solution method (15) in which the following equation was applied. Pot. 1/z log K bumet.Jz+=(E2–E1)/S + log [Bumet.]– log [Jz+]. Where E1 is the electrode potential in 1x10-3 M bumetanide solution. E2 is the potential of the electrode in 1x10-3 M solution of the interferent ion Jz+ and S is the slope of the calibration plot. The selectivity of the electrode towards sugars, amino acids, certain cations and alkaloids was studied. In some cases, when the selectivity coefficients were 174 not very high, both matched potential and mixed solution methods (16) were used. The tested interferents are listed in Table 2. Standard addition method: This technique is usually employed when the sample matrix has an unknown but probably significant effect on the cell potential. First, the cell potential in the sample is recorded and then a volume of a solution containing a known concentration of the primary ion is added. This is commonly a small volume of concentrated primary ion solution to minimize dilution effects. The cell potential in this altered sample is also recorded. The change in response is related only to the change in primary ion, and from the Nernst equation the difference in electrode potential can be calculated. Therefore, an electrode was immersed into a sample of 50 ml with unknown concentration (ca. 10-4M) and the equilibrium potential of E1 was recorded. Then 0.1 ml of 10-1 M of bumetanide standard was added into the testing solution and the equilibrium potential of E2 was obtained. From the change of Δ E (E2-E1) one can determine the concentration of the testing sample (17). Analytical applications: 1. Determination of bumetanide in dosage forms: 1.1 Tablets The content of ten tablets were shaken with 100 ml distilled water to obtain different concentrations in the range of 1x10-3–1x10-6M. The prepared solutions were adjusted to pH 5 using 0.1 N dilute hydrochloric acid. The PVC bumetanide membrane electrode was immersed in the solution. The electrode system was allowed to equilibrate with stirring and the e.m.f. recorded and compared with the calibration graph. The standard addition (spiking technique) was also applied by recording the electrode potential after the addition of 0.1 ml of standard 1x10-1M bumetanide solution to the above drug test solutions. 1.2.Ampoules The prepared ampoules solution was diluted with deionised water to give serial dilutions ranging from 1x10-3-1x10-6M. These solutions were transferred into 50 ml beaker, adjusted to pH 5 and the procedure was completed as described under tablets. Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 EL-TOHAMY ET AL 1.3. Content uniformity assay of bumetanide tablets: Ten individual tablets of 1.0 mg per tablet were placed in separate 100 ml beaker and dissolved in 90-100 ml of distilled water. The electrode was directly immersed into 10 ml of each sample for five times and should be washed with deionized water to reach steady potential between the individual measurements. The mean potential was used to evaluate the content uniformity from the calibration graph. Fig. 4, showed content uniformity of bumetanide in tablets. 1.4. Dissolution test Place 900 ml of water into the dissolution vessel and equilibrate to 37 + 0.5, place one tablet in the dissolution vessel and immediately operate the apparatus at 2:50 rpm after the stated time, withdrown 20 ml of the dissolution medium. For the potentiometric determination, after an appropriate time interval (0.5-3 min), the potential values were recorded, and the amount of the bumetanide was calculated from the calibration graph. In order to investigate all the important physical processes during the dissolution period, the released profile was numerically simulated by a typical equation (18). Fig. 5, showed the dissolution profile of bumetanide 1.0 mg tablets. 2. Determination of bumetanide in biological fluids: 2.1. Preparation of urine samples: Place 10 ml urine in 100 ml volumetric flask. Add distilled water and adjust the pH to 5 using 0.1N dilute hydrochloric acid. Known amount from stock solution of bumetanide 1.0x10-1M was added to form serial dilutions from 1x10-6 M to 1x10-3 M. The potential response for the concentrations was measured using bumetanide-tungstophosphate-PVC membrane electrode, with constant magnetic stirring. The calibration graph was plotted. The unknown concentrations of the drug were calculated from the calibration graph using standard addition method. 2.2. Preparation of serum samples: Place 5 ml human serum in 25 ml volumetric flask complete to volume using normal saline 0.9N sodium chloride. The pH was adjusted to 5 using 0.1N dilute hydrochloric acid. The samples were found free from bumetanide, so they were prepared by spiking with known amounts of analyte, using PLASTIC BUMETANIDE MEMBRANE SENSOR 175 1x10-1 M stock solution of bumetanide . The calibration graph was plotted, then determination of different samples was performed from adequate aliquot by standard addition method. COOH I O II H2N-S II O Results and discussion Table 1: Critical response characteristic of bumetanide-tungstophosphate-PVC membrane electrode. Parameter Value decade)a Slope (mV per Intercept E (mV)b Correlation coefficient, r. Linear range (mole-1) Lower limit of detection (mole-1) Working pH range. Response time for 1x10-4M bumetanide/sec. Life time /day. 58.5±0.2 499.3 0.9999 1x10-3-1x10-6 4x10-7 5-8 30 45 a Standard deviation of average slope values for multiple calibration (n=6). b Standard deviation of values recorded over a period of 45 days (n=6). Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 NH(CH 2 )3 -CH3 w12 PO40 Fig. 1. Bumetanide-tungstophosphate ion pair complex. 400 300 Electrode potential/mV Nature and response characteristics of the electrode bumetanide reacts with tungstophosphoric acid to form a stable bumetanide-tugstophosphate ion-pair complex ( Fig. 1), which is water insoluble but readily soluble in an organic solvent such as tetrahydrofuran. The complex was prepared and tested as active material with di-butyl sebacate as a solvent mediator in a poly (vinyl chloride) membrane response for bumetanide. The critical response characteristics of bumetanide-PVC membrane electrode were determined and results are summarized in Table 1. The electrode exhibits a Nernstain response over the concentration range from 1x10-3–1x10-6M bumetanide with a cationic slope of 58.5±0.2 mV/decade change in concentration (Fig. 2). The choice of membrane solvent to achieve the required selectivity is based on its electric permittivity and its immiscibility with aqueous phase, high viscosity, low solubility of the matrix in the membrane and ability to dissolve ion-pair complex. The response time of the electrode was tested for 1x10-3–1x10-6M bumetanide solutions. The sequence of measurements was from low to high concentrations. This electrode exhibits a fast dynamic response of about 30-40 seconds. The electrode used for a period of 45 days without significant change in the electrode parameters. I O I + 200 100 0 1 2 3 4 5 6 7 - log conc. of bumetanide Fig. 2: Typical calibration graph of bumetanidetungstophosphate-PVC membrane electrode. Table 2: Selectivity coefficient for some sugars, amino acids and common cations with bumetanidetungstophosphate-PVC membrane electrode. Pot Interfe- -log KBumet rent SSMa MSMb + Na 2.54 2.85 K+ 2.39 2.42 NH+4 2.72 2.96 Mg2+ 2.58 3.01 Ca2+ 2.97 3.12 Sr2+ 3.89 — Co2+ 4.12 — Cu2+ 3.97 — Pb2+ 4.58 — Mn2+ 4.01 — Ni2+ 2.60 2.98 Ba2+ 2.65 2.87 Al3+ 3.86 — a Separate solution method. Pot Interferent -log KBumet SSMa MSMb Glucose 2.67 2.94 Lactose 2.73 2.92 Maltose 3.91 — Dextrose 3.98 — Quinidine 2.43 2.65 Atropine sulphate 2.55 2.74 Sulfathiazole 2.53 2.76 Urea 2.68 3.01 Tryptophan 2.61 2.95 Starch 3.67 — Glycine 4.17 — b Mixed solution method. 176 EL-TOHAMY ET AL Table 3: Determination of bumetanide in pure form using bumetanide-tungstophosphate-PVC membrane electrode in comparison with reference method (18). 100.5 Statistical Reference Direct Potentiometry Parameters method Calibration Standard (18) graph addition method Mean % recovery 99.74 99.62 99.67 N 6 7 6 Variance 0.220 0.144 0.121 S.D. 0.469 0.379 0.348 S.E. 0.191 0.143 0.142 R.S.D. 0.470 0.380 0.349 (0.503)(2.201)* (0.294)(2.228)* (1.53)(4.39)* (1.82)(5.05)* "t" F % of bumetanide content 100 99.5 99 98.5 98 97.5 97 10 9 8 7 6 5 4 3 2 1 Numbers of Bumetanide 1.0 mg Tablets Fig. 4 . Content Uniformity of Bumetanide in Tablets. * Theoretical values of ''t'' and F at P=0.05 200 180 160 140 NUMBER 120 4.00 3.00 100 2.00 80 1.00 3 4 5 6 7 8 9 PH Fig. 3: The effect of pH on electrode potential/mV of bumetanide-tungstophosphate-PVC membrane electrode. 1.0x10-3(1), 1.0x10-4(2), 1.0x10-5(3),1.0x10-6M(4). Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 1. Quantification, accuracy and precision: Direct potentiometric determination of bumetanide using the bumetanide- tungstophosphate-PVC membrane electrode was performed and calculated from the calibration curve. The statistical data of the analytical results obtained by the proposed method (direct and standard addition techniques) for the drug in pure form are listed in Table 3. Furthermore, the results obtained were encouraging so the proposed method was applied for the determination of bumetanide in some of its pharmaceutical preparations. In both cases, the results obtained were compared with the reference method (Dissolve about 1.00 g of bumetanide accurately weighed in 150 ml of methanol. In 250 ml conical flask, add phenol red and titrate with 0.1 N NaOH. Perform a blank determination and make any necessary correction (18). The results were listed in Table 4. PLASTIC BUMETANIDE MEMBRANE SENSOR 177 Table 4: Comparative analytical results of the proposed and official method for the tested drug in some pharmaceutical preparations. Sample and Source Statistical parameters Mean % recovery N Variance S.D. S.E. R.S.D. t F Mean % recovery N Variance S.D. S.E. R.S.D. t F Mean % recovery N Variance S.D. S.E. R.S.D. t F Burinex® tablets 1mg/tablet (Leo/Minapharm) Edemex® tablets 1mg/tablet (Memphis-Delagrange) Edemex® ampoules 0.5mg/ml (Memphis-Delagrange) Direct potentiometry Official method (18) Calibration Standard Addition graphs method 99.69 99.42 99.76 6 5 6 0.113 0.232 0.089 0.336 0.482 0.298 0.137 0.216 0.122 0.337 0.485 0.299 (0.382)(2.228)* (1.371)(2.262)* (1.27)(5.05)* (2.61)(5.19)* 99.68 99.86 99.62 6 5 6 0.149 0.428 0.358 0.386 0.654 0.598 0.158 0.292 0.244 0.387 0.655 0.600 (0.206)(2.228)* (0.631)(2.262)* (2.40)(5.05)* (1.20)(5.19)* 99.91 99.71 99.72 6 5 5 0.250 0.438 0.548 0.500 0.662 0.740 0.204 0.296 0.331 0.500 0.664 0.742 (0.489)(2.262)* (0.023)(2.306)* (2.19)(5.19)* (1.25)(6.39)* * All values of ''t'' and F are in P=0.05. Table 5: Determination of bumetanide in pure form in human urine ''spiking technique'' using bumetanide-tungstophosphate-PVC membrane electrode. Calibration graphs method Taken (M) 1.0 x 10-6 5.0 x 10-6 1.0 x 10-5 5.0 x 10-5 1.0 x 10-4 5.0 x 10-4 1.0 x 10-3 Mean ± S.D ( P= 0.05) N Variance S.D. S.E. R.S.D. Standard addition method Recovery* Found [-log conc.] (M) % 5.99 99.83 5.29 99.79 4.97 99.40 4.31 100.21 3.99 99.75 3.26 98.76 3.01 100.33 99.72 ± 0.525 7 0.276 0.525 0.198 0.526 *Average of three experiments. Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 Added ( M) 9.0 x 10-6 1.0 x 10-5 5.0 x 10-5 9.0 x 10-5 1.0 x 10-4 5.0 x 10-4 9.0 x 10-4 Found [-log conc.] (M) 5.02 4.99 4.27 4.01 3.97 3.31 3.03 99.53 ± 0.394 7 0.155 0.394 0.149 0.396 Recovery* % 99.49 99.80 99.28 99.12 99.25 100.27 99.48 Mean % of bumetanide 178 EL-TOHAMY ET AL Table 6: Determination of bumetanide in pure form in human serum ''spiking technique'' using bumetanidetungstophosphate-PVC membrane electrode. Calibration graphs method Taken (M) Found [-log conc.] (M) 1.0 x 10-6 5.0 x 10-6 1.0 x 10-5 5.0 x 10-5 1.0 x 10-4 5.0 x 10-4 1.0 x 10-3 Mean ± S.D ( P= 0.05) N Variance S.D. S.E. R.S.D. Standard addition method Recovery* % Added ( M) 9.0 x 10-6 1.0 x 10-5 5.0 x 10-5 9.0 x 10-5 1.0 x 10-4 5.0 x 10-4 9.0 x 10-4 6.01 100.17 5.29 99.79 4.99 99.80 4.28 99.51 3.97 99.25 3.29 99.67 2.98 99.33 99.65 ± 0.315 7 0.099 0.315 0.119 0.316 Found [-log conc.] (M) 5.03 4.97 4.29 4.01 3.99 3.32 3.04 99.73 ± 0.449 Recovery* % 99.69 99.40 99.74 99.12 99.75 100.58 99.81 7 0.202 0.449 0.170 0.450 *Average of three experiments. and in this range the electrode can be safely used for bumetanide determination. 120 100 80 60 40 20 0 -20 0 10 20 30 40 Time/min Fig. 5. Dissolution profile of bumetanide in tablets. All values are the average of six determinations. 2. Effect of pH: The effect of pH of the bumetanide solutions (10-6 ,10-5, 10-4, 10-3, M bumetanide) on the electrode potential was investigated. The solutions were acidified by the addition of very small volumes of HCl then the pH value was increased gradually using NaOH (0.1 or 1.0 M) for each pH value, the potential was recorded and thus the potential–pH curves for four bumetanide concentrations were constructed (Fig.3). As is obvious, within the pH range 5-8, the electrode potential is practically independent of pH, Saudi Pharmaceutical Journal, Vol. 14, Nos. 3-4, July-October 2006 3. Selectivity of the electrode: The selectivity of the ion-pair associates based membrane electrodes depends on the selectivity of the ion-exchange process at the membrane-test solution interface and the mobilities of the respective ions within the membrane. The selectivity coefficients obtained by the separate solution method (15). Table 2, showed that the proposed bumetanidetungstophsphate-PVC membrane is highly selective toward bumetanide ion. The inorganic cations did not interfere due to the differences in their mobilities and permeabilities as compared with bumetanide cation. In the case of sugars and amino acids, the high selectivity is mainly attributed to the difference in polarity and lipophilic character of their molecules relative to bumetanide. The matched potential and mixed solution methods( rather time consuming due to the preparation of many solutions and performance of many steps) were only used in cases when – log KpotBumet. was less than 3.5. It was demonstrated by these methods that the electrode shows high selectivity. . 4. The electrode response in pharmaceuticals and biological fluids: The use of bumetanide drug in various fields, from clinical to abuse in sports have necessitated an PLASTIC BUMETANIDE MEMBRANE SENSOR 179 accurate and rapid, quantitative analysis in various matrices (dosage forms and biological fluids). 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