ASIAN JOURNAL OF CHEMISTRY

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1 Asian Journal of Chemistry; Vol. 9, o. 1 (17), ASIA JOURAL OF CHEMISTRY htts://doi.org/1.1433/ajchem Sensitive and Validated Voltammetric Methods for Determination of Zileuton in Serum, Urine and Pharmaceutical Dosage Forms at Activated Glassy Carbon Electrode ABIL A. ALHEMIARY 1,,* and MOUSTFA A. RIZK 1,3 1 Deartment of Chemistry, College of Science and Arts - Sharurah, ajran University, Sharurah, Saudi Arabia Deartment of Chemistry, College Science, Ibb University, Ibb, Yemen 3 Deartment of Chemistry, College of Science, Suez Canal University, Ismailia, Egyt *Corresonding author: alhemyri1@yahoo.com Received: 7 Aril 17; Acceted: 11 Setember 17; Published online: 3 October 17; AJC-1864 A simle, recise and sensitive voltammetric methods determination of zileuton were resented at activated glassy carbon electrode in Britton-Robinson buffer of H 8 by differential ulse voltammetry and square wave voltammetry methods. A study of the variation of the eak current with solution variables such as H, ionic strength, concentration of drug, ossible interference and instrumental variables such as scan rate, ulse amlitude, accumulation otential, has resulted in the otimization of the reduction signal for analytical uroses. Linear calibration lots were obtained over the concentration ranges of. 1-6 to and to mol/l, resectively, using differential ulse voltammetry and square wave voltammetry methods. The correlation coefficients were.9977 and.9947 for differential ulse voltammetry and square wave voltammetry methods, resectively. The linear resonse was obtained in Britton-Robinson buffer in the range of and mol/l for siked serum and urine samles, resectively. The limit of detection (LOD) and limit of quantification (LOQ) were and mol/l for differential ulse voltammetry method and and mol/l for square wave voltammetry method. The RSD for five measurements were.489 and.885 using 5 mv/s scan rate. The methods were alied for the determination of zileuton drug in dilute serum, urine samles and harmaceutical dosage form with satisfactory results and comared with the official reference method. Comlete validation of the roosed method was also done. Keywords: Zileuton, Differential ulse voltammetry, Square wave voltammetry, Cyclic voltammetry, Activated glassy carbon electrode. ITRODUCTIO Zileuton [R,S-(±)--(1-(benzo[b]thien--yl) ethyl)-hydroxyurea] (Fig. 1) and is an anti-asthma drug that differs chemically and harmacologically from other anti-asthmatic agents [1,]. Zileuton is indicated for the rohylaxis and chronic treatment of asthma in adults and children 1 years of age and older [3]. It blocks leukotriene synthesis by inhibiting 5-lioxygenase, an enzyme of the eicosanoid synthesis athway [1]. Current data indicates that asthma is a chronic inflammatory disorder of the airways involving the roduction and activity of several endogenous inflammatory mediators, including leukotrienes. Sulfido-etide leukotrienes (LTC4, LTD4, LTE4, also known as the slow-releasing substances of anahylaxis) and LTB4 [4,5]. A chemoattractant for neutrohils and eosinohils are derived from the initial unstable roduct of arachidonic acid metabolism, leukotriene A4 (LTA4) and can be measured in a number of biological fluids including broncho alveolar lavage fluid (BALF) from asthmatic atients [6]. In humans, retreatment with zileuton attenuated broncho- S HO CH 3 Fig. 1. Structure of zileuton O H constriction caused by cold air challenge in atients with asthma [7]. Zileuton is a very slightly soluble comound without any ionizable functional grou [8]. Recently, the molecule has been reorted in the literature as a new efficient and safe antiacne drug[9]. The methods available for analysis of zileuton in harmaceutical dosage forms and biological fluids are those utilizing the rinciles of UV and VIS-sectrohotometry [1,11], HPLC [1-14], HPTLC [15], RP-HPLC [16,17], LC/MSMS [18,19] and olarograhy []. The advantages of electrochemical techniques in drug analysis are their simlicity, low cost and relatively short analysis time comared with other routine analytical techniques. In

2 68 Alhemiary et al. Asian J. Chem. the resent study, electrochemical behaviour and oxidationreduction mechanism of zileuton at an activated glassy carbon electrode was investigated using cyclic voltammetry (CV), differential ulse voltammetry (DPV) and square wave voltammetry (SWV). Determination of zileuton in siked urine, siked serum samles and in harmaceutical dosage forms was also investigated using DPV and SWV. EXPERIMETAL The electrochemical analyzer comuterized system with 797 VA comuterized software (1.) from Metrohm, Swizerland was used. A three-electrode system was used, including a glassy carbon electrode as the working electrode. A digital H/mV meter (Jeanway 351) with a glass combination electrode was used for the rearation of the buffer solution. An Oxford adjustable microiette (Huawei, Ireland) was used to measure microliter volumes of the drug standard solutions. The sigma 4-5 L centrifuge, Sigma (USA) was used to centrifuge the urine and serum samles, which were then suitable for voltammetric analysis. The chemicals and reagents used were of analytical reagent grade and some of them were used as such without any further urification. Distilled water was used throughout all exeriments. Zileuton (urity 99.8 %) were sulied by Simaco Addwaeih, Al-Qassim, Saudi Arabia. Stock solution of mol/l zileuton was freshly reared by dissolving a calculated weighed of the active ingredient drugs in deionized water and stored at 4 C in PVC containers. More diluted solutions were freshly reared daily by accurate dilution..4 mol/l Britton-Robinson buffer between H 4-1 were used as the suorting electrolytes. All solutions were reared from AnalaR grade reagents (Sigma-Aldrich) in doubly distilled water. The calibration curves for DPV and SWV analysis were constructed by lotting the eak current against zileuton concentration. Otimizations: To obtain the otimum H, an aroriate amount of zileuton working standard solution mol/l was laced in the electrolytic cell, which contained 5 ml of Britton-Robinson buffer solution and the cyclic voltammogram was recorded. The exeriment was reeated by using buffer solutions of different H values 4-1 and the otimum H was obtained. The effect of different tyes buffer such as acetate, hoshate and Britton- Robinson buffer on the eak current (I ) of zileuton was studied. The working electrode was immersed in buffer solution of the otimum H containing an aroriate amount of the drug stock solution mol/l, the cyclic voltammogram was recorded. For analytical alication, the following arameters being emloyed: DPV-ulse amlitude 5 mv, ulse width 5 ms, scan rate 5 mv/s, SWV-ulse amlitude 5 mv, frequency 15Hz, otential ste 4 mv, electrochemical analyzer does the background subtraction automatically. For cyclic voltammetry, the initial and final otential values were variable, deending on the H value and the cut-off the electrolyte. Scan rate measurements in the range of 1- mv/s were carried out. Pre-treatment of glassy carbon electrode: The electrode was retreated by cycling a square-wave otential with a frequency of 35 Hz between the otential limits of ± 6 V followed by the alication of triangular otential swee between ± 6 V (frequency 35 Hz) in.1 mol/l otassium nitrate solution. Finally, the electrode was subjected to an electrochemical retreatment by alying a otential of +1.5 V for 5 min and then -1. V for s in.1 mol/l otassium nitrate solution. These stes were reeated until the voltammetric resonse of the electrode became reroducible. At the end of the rocedure, the electrode surface was so stable that for 4 measurements, the electrochemical retreatment alone was sufficient before each scan [1]. General rocedure: Voltammetric analyses were erformed in 5 ml of Britton-Robinson buffer. The solution was continuously stirred at 1 rm when accumulation otential (usually oen circuit conditions) was alied for a certain time to the working electrode. At the end of accumulation eriod, the stirring was stoed and after 5. s rest eriod was allowed for the solution to become quiescent. The used drug was determined by using DPV method. Aliquots of the drug solution of mol/l were introduced into the electrolytic cell and the rocedure was reeated. The voltammograms were recorded. The eak current was evaluated as the difference between each voltammogram and the background electrolyte voltammogram. All measurements were carried out at room temerature. Analysis of harmaceutical dosage form: Tablets containing 6 mg of zileuton er tablet were urchased from local Saudi Arabia market. Ten tablets of zileuton were crushed into a fine owdered in a mortar. A suitable amount of this owder was accurately weighed and then dissolved in 5 ml methanol. It was sonicated for 5 min. The content was allowed to settle after stirring magnetically for 5 min. The samle solution was filtered through a Whatman no. 4 filter aer. Aroriate solutions were reared by taking suitable aliquots of the clear suernatant liquid and diluting them with bidistilled water in order to obtain a final solution of mol/l zileuton. Each solution was transferred to a voltammetric cell and the voltammograms were subsequently recorded following the otimized conditions. The amount of zileuton er tablet was calculated using linear regression equation obtained from the calibration curve of ure zileuton. Analysis of siked biological samles: The urine and serum samles, obtained from healthy volunteers was centrifuged (4 rm) for 3 min at room temerature and searated. Serum samles were stored frozen until assay. After gentle thawing, an aliquot volume of samles were fortified with drug samle dissolved in deionized water to achieve final concentration of mol/l and treated with.4 ml acetonitrile as serum denaturing and reciitating agent. Acetonitrile is known to remove serum roteins more effectively. The volume was comleted to 3 ml with the same serum samle. The tube was vortexed for 1 min and then centrifuged for 1 min at 4 rm to get rid of serum rotein residues and suernatant was taken carefully. Aroriate volumes of the clean solution were analyzed in the voltammetric cell containing Britton- Robinson buffer, H 8.. Quantification were erformed by means of calibration curve methods [,3]. RESULTS AD DISCUSSIO Cyclic voltammetry: Anodic cyclic voltammogram for the oxidation-reduction of zileuton in Britton-Robinson buffer at activated glassy carbon electrode is shown in (Fig. ). It is

3 Vol. 9, o. 1 (17) Voltammetric Determination of Zileuton in Serum, Urine and Pharmaceutical Dosage Forms at Activated GCE 69 clear that the voltammogram exhibits one anodic and cathodic eaks at 943 mv and 4 mv, resectively, which shows a quasi- reversible behaviour, with eak-to-eak searation ( E = E c-e a ) of -53 mv. The results suggest the redox coule which shows reversible behaviour with equal number of rotons and electrons articiated in the working buffer. The zileuton may undergoes rotonation with bond cleavage and one electrone loss in the first oxidation ste. Then the oxidized roduct was reduced via gaining electron and derotonation of the oxidized roduct. The roosed mechanism of oxidationreduction reaction is shown in Scheme-I (b) -1 (a) Fig.. Cyclic voltammogram of mol/l zileuton in Britton-Robinson buffer at H 8.; scan rate = 5 mv/s Effect of H: The H of the electrolyte medium is one of the variables that commonly and strongly influenced the shae of the voltammogram and therefore it was imortant to investigate the effect of the H on the electrochemical behaviour of the drug. The effect of H on the reduction of the drugs under investigation at activated glassy carbon electrode was studied over the H range 4-1 at same concentration mol/l of zileuton solution in various electrolytes, such as acetate, hoshate and Briton-Robinson buffers of different ionic strength in the range of.4-. mol/l. It was found that the maximum eak size was obtained at H 8 in Britton-Robinson buffer, which allows wider range of determination than acetate and hoshate buffers. Hence, it was used as a favourable buffer throughout the study. Fig. 3 shows the effect of H on the current eak in a Briton-Robinson buffer of H range from 4 to 1 for. 1-6 mol/l zileuton. The effect of solution H on eak otentials of zileuton at GCE was also investigated. Cyclic voltammograms at different H values of 4-1 were shown in Fig. 3., which show that an increase in H of the solution caused shift in the oxidative eak otential to the negative direction, indicating that the electrode rocess is influenced by rotonation reactions. A linear correlation between the eak otential and solution H was obtained as shown in Fig. 4 with a linear equation and correlation of: (c) (d) (e) Fig. 3. Cyclic voltammogram obtained for. 1-6 mol/l zileuton in Britton-Robinson buffer solution at (a) H 4., (b) H 6., (c) H 8, (d) H 1 and (c) H 1 with otential; scan rate: 5 mv/s E a (mv/s) = H K r =.9911 The sloe was found to be.114 mv/h, which suggested that the number of roton taking art in the electrode reaction is similar to the number of electrons. Hence, the oxidation of zileuton involves one electron and one roton. E (V) y = -.114x R = H Fig. 4. A linear correlation between the eak otential and H Effect of scan rates: The relationshi between measured eak current and scan rate was studied over range 1- mv/s. It was found to be directly roortional the eak current and the eak otential of zileuton were evaluated as shown in Fig. 5. Scan rate studies were carried out to assess whether the rocess at the glassy carbon electrode was under diffusion or adsortion controlled rocess as shown in Fig. 6. The anodic and cathodic eak current of zileuton increases linearly with the square root of scan rate, in the otential range from 1 to mv, with eegression r = A lot of logarithm of eak current versus logarithm of scan rate gave a straight line with a sloe very close to the theoretical value of.5, which is exressed for an ideal reaction of diffusion controlled electrode rocess (Fig. 7) [4]. Also the anodic and cathodic eak otentials of the electrode were shifted toward ositive and negative otential, (b) (a) HO O HO OH + HO O H +H+ e H + H +e H S CH 3 S CH 3 Scheme-I: Proosed mechanism of the oxidation-reduction reaction of zileuton drug S CH 3

4 63 Alhemiary et al. Asian J. Chem (6) (1) log i y =.518x R = Fig Cyclic voltammograms of. 1-6 mol/l zileuton in Britton- Robinson buffer at H 8 at scan rates of: (1) 1, () 3, (3) 5, (4) 8, (5) 1, (6) mv/s A: Anodic eak otentials Scan rate (V s ) Scan rate (V s ) y = 3.6x R = Fig. 6. y = 95.95x R =.996 B: Cathodic eak otentials The anodic (a) and cathodic (b) eak current of zileuton with the square root of scan rate resectively. This may be attributed to the accumulation of the oxidation or reduction roducts on the electrode surface. Effect of accumulation oerators: The accumulation of zileuton at GCE surface deends on oerational factors, which were additional investigations to ensure sensitive detections of this drug [5]. So, the effect of accumulation time on the efficiency of the collection of mol/l zileuton drug on the working electrode surface was evaluated by rising the accumulation time over the range of -4 s. The resulting eak current-otimum accumulation time is exhibited in 3 s (Fig. 8) and as can be seen from this lot, a steady enhancement in the eak current was observed over the range -4 s and after that the eak intensity nearly decreased robably due to the satu- log i log scan rate (V s ) log scan rate (V s ) y =.5857x R = Fig. 7. Relation between log i (µa) and log scan rate (V/s) for oxidation of zileuton drug at activated glassy carbon electrode Fig Accumulation time (s) Effect of accumulation time (t acc) of 1-6 mol/l zileuton eak current at Britton-Robinson buffer, H = 8, E =.1 V and scan rate 5 mv/s ration of the GCE surface. Hence, 3 s accumulation time was selected for all the future exeriments. In a different study, the accumulation otential effect on the eak current of this drug was recorded by variation of accumulation otential from.15 V to -.6 V. The maximum anodic eak current value was obtained with an accumulation otential of.1 V, which was selected as the otimum value in future exerimental studies. Analytical alications: While the sensitivity enhancement associated with the interfacial accumulation is significant, the main advantage of the method is its inherent selectivity towards the surface-bound analyte. For this urose the working electrode with the extracted drug was transferred from the comlex

5 Vol. 9, o. 1 (17) Voltammetric Determination of Zileuton in Serum, Urine and Pharmaceutical Dosage Forms at Activated GCE 631 samle to an electrolytic blank solution between the reconcentration and measurement stes. Differential ulse voltammetry: To develo a quantitative voltammetric evaluation methodology for determining the drug was established on the linear correlation between the eak current and concentration. For analytical uroses we selected the DPV and SWV mode. Differential ulse exeriments were erformed on the GCE in Britton-Robinson buffer solution at H = 8 with exerimental conditions were: scan rate 5 mv s -1 ; ulse amlitude 5 mv; ulse width of 5 ms and ulse eriod 4 ms. The otential was scanned anodically from an initial to a final otential of 4-1 mv resulting voltammograms shown in Fig. 9, which show that while the eak otential remained almost constant at.89v, the DVP data for the determination of the drug under investigation (Fig. 1) shows a linear relation between the eak current (I ) and zileuton concentration (C) and was found in the following range: mol/l mv resulting voltammograms are shown in Fig. 11. It shows that while the eak otential remained almost constant at.866v, the SWV data for the determination of the drug under investigation (Fig. 1) shows linear relations between the eak current (I ) and zileuton concentration (C) and was found in the following range: to mol/l Fig. 11. Square-wave voltammograms with increasing concentration ( mol/l) of zileuton in H 8 buffer solution on GCE electrode y = x +.45 R = Fig. 9. Differential-ulse voltammograms with increasing concentration of zileuton ( mol/l) in H 8 buffer solution on GCE electrode y =.918x R = Concentration (mol L ) Fig. 1. Calibration grahs of zileuton drug by using DPV method Square wave voltammetry: The SWV exeriments were erformed at GCE in Britton-Robinson buffer solution at H = 8 with exerimental conditions were: scan rate 5 mv s -1 ; ulse amlitude 5 mv; 4 mv otential ste and otential range of 5 to 1 mv and frequencies 5 Hz. The otential was scanned anodically from an initial to a final otential of Concentration (mol L ) Fig. 1. Calibration grahs of zileuton drug by using SWV method Validation of analytical rocedure: The linearity of calibration curve was obtained for both DPV and SWV techniques and the loss of linearity was robably due to the adsortion of zileuton on the electrode surface. The characteristics of these grahs are given in Table-1. The recision of the method was investigated by reeatedly (n = 5) measuring eak otential and eak current of zileuton within a day and over three consecutive days for both techniques. LOD and LOQ were calculated as (3.3 s/m) and (1 s/m), resectively where s is standard deviation of resonse (five runs) and m is the sloe of the calibration curve. LOD and LOQ values confirmed the sensitivity of the roosed methods, These results demonstrated good recision and accuracy [3,6]. Determination of zileuton in harmaceutical: Both roosed methods (DPV and SWV) were alied to the direct determination of zileuton in tablet using the related calibration curve of the straight lines without samle rearation and after

6 63 Alhemiary et al. Asian J. Chem. TABLE-1 CHARACTERISTICS OF ZILEUTO CALIBRATIO PLOTS USIG PROPOSED VOLTAMMETRIC METHODS Parameters Differential ulse Square wave voltammetry voltammetry Concentration range (mol/l) Sloe (µa. mol/l) Intercet (µa) Correlation coefficient (r) SD RSD (%) LOD (mol/l) LOQ (mol/l) an adequate dilution (Table-). The roosed analysis rocedure was successfully alied for the assay of zileuton in its harmaceutical dosage form. As far as, there is no official method in any harmacooeias related to harmaceutical rearations of zileuton. For this reason, HPLC method [15] was used for comarison and for the reliability of the develoed rocedures. The results obtained for the formulation are listed in Table- and comared with HPLC. The recovery studies were carried out by adding the known amount of ure drug to earlier analyzed harmaceutical formulations of zileuton. The recovery of the drug was calculated by comaring the concentration obtained from the siked mixtures with those of ure drug [3]. Table- shows a good result, which demonstrates the selectivity of roosed method for the determination of zileuton in commercial tablet forms. Alication on siked biological samles: The otimized rocedure has been successfully alied for the determination of zileuton in rotein-free siked human serum and urine samles. Acetonitrile is tried as a serum and urine reciitating agent. o extraction stes other than the centrifugal rotein searation are required rior to the assay of drug. Calibration equation arameters and validation data are shown in Table-3. The obtained recovery results of siked human serum samles are given in Table-3. The recovery results of zileuton in serum and urine samles are calculated from the related linear regression equations. DPV and SWV curves of zileuton in serum and urine samles are examined and tyically comared with that obtained from row material. It could be seen that no oxidation of zileuton drug occurs and no extra eaks are detected in tested biological material in the otential range; whereas the analytical eak aeared. Conclusion In conclusion, the resent work demonstrates that highly sensitive electrochemical measurement of zileuton drug is feasible utilizing its extraction on to GCE. The roosed methods showed advantages, such as a short eriod of the real time of drug analysis and no retreatment or time consuming in extraction stes were required rior to analysis. Moreover, because of its very limits of detection and quantification, the roosed methods alied in harmaceutical dosage forms. It also has been successfully alied to the determination of drug in serum and urine. ACKOWLEDGEMETS Deanshi of Sicentific Research of ajran University has suorted this work (Project o.: U/MID/15/1). REFERECES 1. R.B. Bell, P.R. Young, D. Albert, C. Lanni, J.B. Summers, D.W. Brooks, P. Rubin and G.W. Carter, Int. J. Immunoharmacol., 14, 55 (199); htts://doi.org/1.116/19-561(9)918-k.. S.E. Wenzel and A.K. Kamada, Ann. Pharmacother., 3, 858 (1996). 3. E. Israel, R. Dermarkarian, M. Rosenberg, R. Serling, G. Taylor, P. Rubin and J.M. Drazen,. Engl. J. Med., 33, 174 (199); htts://doi.org/1.156/ejm E. Gounaris, M.J. Heiferman, J.R. Heiferman, M. Shrivastav, D. Vitello,.R. Blatner, L.M. Knab, J.P. Phillis, E.C. Cheon, P.J. Grio, K. Khazaie, H.G. Munshi and D.J. Bentrem, PLoS One, 6, 1 (15); htts://doi.org/1.1371/journal.one Morina, G. Bocari, A. Iljazi, K. Hyseini and G. Halac, Acta Inform. Med., 4, 16 (16); htts://doi.org/1.5455/aim W. Berger, M.T. De Chandt and C.B. Cairns, Int. J. Clin. Pract., 61, 663 (7); htts://doi.org/1.1111/j x. 7. P. Lu, M.L. Schrag, D.E. Slaughter, C.E. Raab, M. Shou and A.D. Rodrigues, Drug Metab. Disos., 31, 135 (3); htts://doi.org/1.114/dmd TABLE- EVALUATIO OF THE ACCURACY AD PRECISIO OF THE PROPOSED AD OFFICIAL METHODS FOR THE DETERMIATIO OF ZILEUTO I ITS PHARMACEUTICAL FORMS AT GCE Method Drug Taken Proosed method Official method (µg/ml) ± % RSD, n = 5 ± % RSD, n = 5 F-test T-test Differential ulse voltammetry 6 mg/tablet.5 1. ± ± Square wave voltammetry 6 mg/tablet ± ± T-value =.77, Tabulated F-value = 6.39 at 95 % confidence limit. TABLE-3 AALYTICAL RESULTS FOR ZILEUTO DRUG RECOVERIES FROM URIE AD SERUM SAMPLES Technique Differential ulse voltammetry Square wave voltammetry Serum Urine Serum Urine Concentration range (mol/l) Sloe (µa. mol/l) Intercet (µa) Correlation coefficient (r) Recovery (%) RSD (%)

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