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Journal of Chemical and Pharmaceutical Research
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J. Chem. Pharm. Res., 2011, 3(2):605-616
ISSN No: 0975-7384 CODEN(USA): JCPRC5

A kinetic method for the determination of diazepam based on ligand-exchange reaction
Snežana S. Mitić1, Aleksandra N. Pavlović1*, Snežana B.Tošić1, Emilija T. Pecev1, Milan N. Mitić1, Ružica J. Micić2
Faculty of Sciences and Mathematics, Department of Chemistry, University of Niš, Višegradska 33, P.O.Box 224, 18000 Niš, Serbia 2 Faculty of Sciences and Mathematics, University of Priština, Lole Ribara 29, 38220 Kosovska Mitrovica
1

______________________________________________________________________________ ABSTRACT
New kinetic-spectrophotometric method has been developed to determine diazepam (DZP) in pharmaceutical formulations and human control serum. The method is based on ligand-exchange reaction. The reaction was followed spectrophotometrically by measuring the rate of change of absorbance at 425 nm in ethanolic sodium hydroxide solution. The optimum operating conditions regarding concentration of reagents and temperature were established. The initial-rate method is adopted for constructing the calibration curve, which was found to be linear over the concentration range 0.28-3.70 µg mL-1. The optimized conditions yielded a theoretical detection limit of 0.14 µg mL-1 based on the 3Sb criterion. The interference effects of certain drugs, foreign ions and amino acids upon the reaction rate were studied in order to assess the selectivity of the method. The method described in this work was validated following the analytical performance parameters required by the USP-27/NF-22. Statistical Students t-test and F-test have been used and satisfactory results were obtained.

Keywords: diazepam; ligand-exchange reaction; kinetic spectrophotometry; validation; pharmaceutical preparations ______________________________________________________________________________ INTRODUCTION Diazepam (DZP), first marketed as Valium by Hoffmann-La Roche, is a widely prescribed benzodiazepemine in the world for the past 40 years. It is effective for the symptomatic relief of tension and states of anxiety as well as for the treatment and prevention of seizures. It may also be used in some surgical procedures to induce amnesia [1]. It is inexpensive, widely available in 605

21. there is not any kinetic-spectrophotometric method for the determination of DZP related in the literature. Res. Also. insoluble additives should be removed to prevent the columns from becoming blocked. The absorbance of the solution was measured at the wavelength of 425 nm. The procedure is easier to execute and requires less sample handling than methods currently described in the literature.02 ºC before the beginning of the reaction.0×10-3 mol L-1) of diazepam was prepared in absolute ethanol from pharmaceutical 99. Reagents Stock solution (1. the wide use of diazepam necessitates a rapid. Spectrophotometry is the technique of choice even today due to its inherent simplicity. a.02 º C) was used to control the reaction temperature at 22. Belgrade. The present work describes a kinetic method for the determination of DZP in commercial pharmaceutical preparations and human control serum. The various analytical methods such as spectrophotometry [2-5].. reliable and sensitive method for its quantitation. thin layer chromatography [14]. or by derivatization with phthaldehyde and fluorescamine.00 ± 0. 2011. Serbia. 3(2):605-616 ______________________________________________________________________________ developing countries and effective when given by the intravenous or rectal routes.00 ± 0. The acute toxity of diazepam is a result of its central nervous depressant effect. Also.Aleksandra N. Pharm. A model 1200 Agilent Technologies was used for HPLC analysis. complex. The analytical column was C18 (Zorbax. occasionally suffering from lack of selectivity and require procedures that may increase the amount of the hydrolyzed product during manipulation of the sample. as far we know.22]. photochemical degradation. IR spectrometry [23]. EXPERIMENTAL SECTION Apparatus The reaction rate was monitored spectrophotometrically. kindly provided by a pharmaceutical laboratory Galenika. polarography [18-20]. A Julabo MP-5A model thermostatic bath (operating in a temperature range 20. gas chromatography [15-17]. Fluorimetric methods are simple. The drug is notably more toxic by the intravenous route than by other modes of administration. For the GLC methods.26] have been reported for the determination of DZP. 606 .. All these methods are time consuming.6 mm). The readings were done on a Perkin-Elmer Lambda 15 UV/Vis spectrophotometer. but these methods have not been widely used in practice because the species that can be detected are limited. However. with this technique. fluorimetry [2. connected to a thermo-circulating bath. Diazepam solution was stored at 4ºC. 250x4.00 ± 0. Pavlović et al J. chemical derivatization is essential. It is frequently used in the laboratories of the developing countries to overcome a variety of analytical problems. 5µm.92 % certified products. HPLC [6-13]. This type of the reactions is quite recent and has thus been studied less. The solutions were thermostated at 22. The method is based on ligand-exchange reaction. Using methods such as HPLC. solid-phase microextraction (SPME) [24] and capillary zone electrophoresis (CZE) [25. accurate and precise.00-60. fluorescent species should be generated either by thermal heating in acidic solvent. Chem.02 ºC.d.

General Procedure In order to obtain good mechanical and thermal stability. The rate of the reaction at different concentrations of each of the reactants was obtained by measuring the slope of the linear part of the kinetic curves to the absorbancedA / dt . and then finally with deionised water. Res.45 µm membrane filter (Millipore) directly in a 50. slope = dA / dt . and the absorbance at 425 nm was measured spectrophotometrically every 30 s over a period of 5-6 min after mixing against the reagent blank prepared similarly.50 mL of this solution was transferred into a 25.6×10-3 mol L-1) was prepared by dissolving CuCl2×2H2O (Merck) in water. copper(II). distilled water. 4 rectal solutions (appropriate amount of diazepam 20 mg) were transferred and filled up to a volume with ethanol. A working solution (1. Ionic strength was kept constant at 0. Then. All the glassware used was washed with aqueous HCl (1:1) and then thoroughly rinsed with running. Pavlović et al J. 607 . was dissolved in 25. dc / dt = ε ⋅l rate = dc / dt ). equivalent to 2 mg of DZP. the instruments were run for 10 min before the first measurement. sodium hydroxide in the second.1 by adding the appropriate amount of NaCl solution (mol L-1) Analytical grade chemicals and deionised water (MicroMed high purity water system.0 mL volumetric flask and filled up to a volume with ethanol to obtain a solution whose expected DZP concentration was 40.Aleksandra N. 2011. The vessel was thermostated at 22.. The reaction solution was put into a cell.02 °C and the reaction was initiated by mixing. dA / dt = ε ⋅ l ⋅ dc / dt . 3(2):605-616 ______________________________________________________________________________ Sodium hydroxide solution (NaOH. Stock solution of copper(II) (1. In the reaction mixture vessel with four compartments. 1-nitroso-2-naphthol solution (1. Chem.0 mL volumetric flask. µg mL-1). The reaction was carried out in the following way.0 µg mL-1.0 µg mL-1. An accurately weighed portion of the resulting powder.00 ± 0.0×10-3 mol L-1) (Merck) was prepared by dissolving a known amount in 5 ml absolute ethanol and diluting it with water (total volume 50 mL). Then it was centrifuged at 3500 rpm for 5 minute and filtered through a 0. versus concentration of DZP ( c DZP . 2. Pharm. TKA Wasseraufbereitungssysteme GmbH) were used for the preparation of all solutions. 1. the solution of 1-nitroso-2-naphthol was placed in one compartment. diazepam in the third.0 mol L-1) was prepared from NaOH (Merck). time plot (from Beer`s law A = ε ⋅ l ⋅ c . Into a 50. The calibration graph was constructed by plotting the slope of the linear part of the kinetic curve. Procedure for tablets A total of ten tablets of each one of the different used pharmaceutical preparations containing DZP were weighed and finely powdered using a mortar and pestle.6×10-4 mol L-1) was obtained by appropriately diluting the stock copper solution with water.0 mL volumetric flask and added ethanol to obtain a solution whose expected DZP concentration was 40. electrolyte for the ionic strength and ethanol (total volume 10 mL) in the fourth compartment.0 mL of ethanol.

Its chelate copper complex is more stable than that formed with R(NO)OH. Fe3+ ions were masked by adding the appropriate amount of F. In all cases it was assumed that the actual content of the tablet corresponds to that reported by the manufacturing laboratories.5 mL of serum.6 mm Zorbax C18 (5µm) analytical column operating at room temperature. the residue was reconstituted with mobile phase and 10 µL was transferred into glass vial for automatic injection into the HPLC system. The complex agrees with the empiric formula [Cu (DZP ) 2 ]2+ . Cu[R( NO )O ]2 + 2Cl − + 2 DZP → [Cu (DZP )2 ]Cl 2 + 2 R( NO )OH The plot of the reaction as a function of the diazepam concentration is a straight line which can be used as a calibration graph.00 mL standard volumetric flask and filled up to the mark with the same solvent. The IR and electronic spectroscopy suggest an octahedral symmetry around metal ions (coordination via the nitrogen atoms 1. 30:70 (by vol).00 mL volumetric flask. 608 . Br ) . [Cu ( DZP) 2 ]X 2 ( X = Cl . Flow rate was 1 mL min-1. Serum sample preparation Human lyophilised control serum (Lyotrol N) was used. Comparative Method HPLC was used as a parallel method [9]. For HPLC determination. the appropriate amount of the stock solution of DZP (1. Res. The rate of the reaction was obtained by measuring the slope of the linear part of the kinetic curves to the absorbance-time plot (slope = dA/dt).0 mg mL-1) and 25 mL of ethanol was added and after brief vortex mixing it was centrifuged for 5 min at 3000 rpm to deposit the protein precipitate. Pharm. Serum sample was spiked at one concentration level listed in Table 5.. The reaction moves to the right and DZP was determined by monitoring the rate of appearance of 1-nitroso-2-naphthol in basic medium at 425 nm. Serum sample was contained 200. 2011. RESULTS AND DISCUSSION Mechanism of the Reaction Diazepam shows complexing ability with Cu(II).Aleksandra N. Diazepam was detected and quantified on a 250x4. 3(2):605-616 ______________________________________________________________________________ Aliquots of these solutions were transferred into vessels covering the concentration range listed in Table 4. To 0.ions (1×10-4 g mL1 ).4 of the diazepemine ring) [27]. For kinetic determination. The eluate was monitored at 254 nm. Chem.0 µg mL-1 of DZP. The mobile phase was a mix of acetonitrile-water. aliquots of DZP solution were transferred in a 10. Pavlović et al J. Aliquots of this solution were transferred into vessels covering the concentration range listed in Table 5. The separated supernatant was collected in a 50. Injection of the samples (20 µL) was performed using an autosampler. evaporated to dryness in a water bath. Cu 2+ + 2Cl − + 2 R ( NO )OH → Cu[R ( NO)O ]2 + 2Cl − Kinetic studies A tangent method was used for the processing of the kinetic data.

Cu(II) concentration of 3. 2.5 slope x 10 . Res. The reaction rate increased with increasing the concentration of Cu(II) from 0. mol L Figure 1.5×10-5 mol L-1 (Fig 2). 3.5×10-5 mol L-1 and becomes constant at higher concentrations than 2.5×10-5 mol L-1.6-2. c DZP = 1. It can be seen that the reaction rate increases linearly with increasing of the1-nitroso-2-naphthol concentration from 0. c NaCl = 0.1 × × mol L-1..2×10-5 mol L-1.02 °C The effect of the concentration of 1-nitroso-2-naphthol on the rate of reaction was studied in the range 0.3-1.5 1 2 3 4 2 5 -1 cNaOH x 10 . min 3 -1 2.0 1.0×10-5 mol L-1 and become constant at 1.4×10-5 mol L-1. 2011.0×10-5 mol L-1. Chem. cCu ( II ) = 1. the dependence of the rate of reactions on the concentration of each of the reactants was determined.0×10-5 mol L-1.0 0. Therefore. a concentration of 1.6-4. Initial concentrations: c R ( NO ) OH = 1. the conditions needed to be optimized. 3(2):605-616 ______________________________________________________________________________ Effect of variables In order to determine the lowest possible determinable concentration of diazepam. this was recommended for the determination process. The correlation between slope and the Cu(II) concentration is given on Fig. 609 . The dependence of the reaction rate on the alkalinity of the solution (Fig. The influence of the concentration of Cu(II) on the rate of reaction examined in the range of 0. Pavlović et al J.5 1.6 ×10-5 mol L-1.3-1. t = 22.0×10-5 mol L-1. Effect of sodium hydroxide concentration on the slope of the absorbance-time curve.2×10-5 mol L-1 was chosen as the optimum concentration. Pharm. Thus.Aleksandra N.5×10-2 mol L-1 and this concentration was selected for further work. 1) shows a maximum at the concentration of sodium hydroxide of 1.00 ± 0.

Initial concentrations: c NaOH = 1.5 1.1 mol L× × 1 .5 -1 1.0 1.0×10-5 mol L-1.5 0.5 1. Pharm.0 0.0 0.0×10-5 mol L-1.2 5 1.5×10-2 mol L-1.0 1.5×10-2 mol L-1.0 1.0 5 2+ 3.5 2.Aleksandra N. 2011. c NaCl = 0.02 °C 610 . t = 22 ± × × × 0.0 2.2×10-5 mol L-1.0 0.5 0. Effect of 1-nitroso-2-naphthol concentration on the slope of the absorbance-time curve.5 -1 4. c DZP = 1. c NaCl = 0.02 °C 2. cCu ( II ) = 1. Initial concentrations: c NaOH = 1. mol L Figure 2.0 0. 3(2):605-616 ______________________________________________________________________________ slope x 10 . mol L Figure 3. min 3 -1 2.6 ×10-5 mol L-1. Res. t = 22 ± 0.9 1.3 0. c DZP = 1.5 1.. Chem. min 3 -1 2.5 3.5 slope x 10 .6 0.8 cR(NO)OH x 10 . Pavlović et al J. c R ( NO )OH = 1.0 cCu x 10 .1 mol L-1. Effect of copper(II) concentration on the slope of the absorbance-time curve.5 2.

70 µg mL-1 under the optimal reaction conditions ( c R ( NO ) OH =1. percent recoveries.Aleksandra N. 25. precision. Res. selectivity [28]. cCu ( II ) = 3. Beer’s law) and c DZP is the diazepam concentration expressed in µg mL-1. Chem. c NaCl = 0. × -2 -1 -5 -1 -5 -1 -5 -1 c NaOH = c R ( NO ) OH = 1.7×10 mol L . Pharm.5×10-2 mol L-1. It was found that the calibration graph obtained at 22ºC possessed good linearity and is recommended that the determination can be carried out at 22ºC. 3(2):605-616 ______________________________________________________________________________ The effect of temperature on the reaction rate was studied at 19.5×10 mol L . Effect of the temperature on the slope of the absorbance-time curve. c NaOH =1. where b and a are slope and intercept.37589 r = 0. C 0 33 Figure 4. 22. Pavlović et al J. t = 22.2×10-5 mol L-1.1 × × × mol L-1 For the validation process the following parameters were characterized: linearity and range.53509 ⋅ c DZP + 0. 7 slope x 10 . min -1 3 6 5 4 3 2 1 0 18 21 24 27 30 t. Initial concentrations: 1. 28 and 31ºC (Fig. limit of detection.2×10 mol L . 2011. respectively) for the calibration graph and correlation coefficient (r) [29] for the determination of DZP in the interval 0. cCu ( II ) = 3.00 ± 0. The least squares' equation (y = bx + a.9984 where slope is the slope of the linear part of the kinetic curve to the absorbance-time plot ( slope = dA / dt = ε ⋅ l ⋅ dc / dt . 4). The rate for different concentrations of DZP at each temperature was calculated and utilized for plotting the calibration curve. repeatability. c DZP = 0.02 °C) were calculated: tgα ⋅ 10 3 = 0. The absorbance-time curves obtained at these temperatures indicated the temperature dependence of the reaction rate.2×10-5 mol L-1.2×10 mol L . 611 .28 to 3..

08 2.5)×10-5 mol L-1.4)×10-5 mol L-1.Aleksandra N.0-1.14 µg mL-1.39 -0.14 The following kinetic equation for the reaction was deduced on the basis of the graphic correlations obtained.3 × S 0 / b where S 0 is the residual standard deviation of the calibration line. Table 2 Accuracy and precision of the determination of DZP Taken µg mL-1 Founda) -1 x ± SD . Pavlović et al J.constant proportional to the rate constant of the reaction The equation is valid for the following concentrations: R(NO)OH (1.01 4. b)relative standard deviation. r Variance ( S 0 × 10 ) / (µg mL ) Detection limit / µg mL-1 3 2 -1 2 0.2x10-10 (µgmL-1)2.70 µg mL-1. NaOH (0.14 1. Chem. the reaction rate was studied at 19.99 1.37589 ± 0. The limits of detection (LOD) [30] was evaluated using the following equation: LOD = 3. Table 1 Quantitative parameters of the analysis Parameters Calibration range / µg mL-1 Regression equation (Slope ± SD)×103 (Intercept ± SD)×103 Correlation coefficient. DZP 0. c)accuracy of the method The effect of temperature on reaction rate is well known and important in understanding the various activation parameters of the reaction products.9984 6.37589 3 0.2×10-4 0. Cu(II) (2. rate = k ⋅ c NaOH ⋅ c DZP k. The low value of variance indicated negligible scattering of the experimental data points around the line of regression. The precision and accuracy of the above system were studied by performing the experiment 5 times for different concentrations of diazepam..30 ± 0. 2011.93 7.5)×10-2 mol L-1. 28 and 31ºC at c NaOH = 1.70 3. Res.06 3. µg mL 0.67 ± 0. 25. 22.97 ± 0.5×10- RSDb) (%) ( x − µ ) / µ ⋅ 100 c 612 . b is the slope of the calibration line (analytical sensitivity) and found to be 0.Quantitative parameters of the analysis are given in Table 1.00828 0.53509 ± 0.28 – 3. 3(2):605-616 ______________________________________________________________________________ The variance ( S 02 ) of the calibration line was evaluated to be 6.53509 ⋅ c DZP + 0.4-1. Pharm. In order to evaluate the apparent activation parameters.28-3.81 a) Mean and standard deviation of five determinations and 95 % confidence.019 r = 0.28 0.01 3.23 -1.5-4. The results of accuracy and precision of the recommended procedure are given in Table 2.70 n = 7 tgα ⋅ 10 = 0.

Table 3 Tolerance ratio for foreign species in the determination of 1. citric acid.303R) and found to be 99. Zn2+ Met. Trp. Lys).14 ± 0. Chem. Ala. Arg. Mg2+. 613 . According to Hammond [31] and Smith [32].2×10-5 mol L-1. sorbitol. some of the amino acids (Phe.99 µg mL-1 of diazepam Ia (%) Tolerance level (µg mL-1 interferent/ µg mL-1 DZP) 102 2 Foreign species citric acid fructose. cCu ( II ) = 3. lactose. Pharm. Ser. B6. Gly) interfere with the method. Met. Mg2+. Fmannitol. Gly. the interference of those species accompanying DZP in pharmaceuticals was studied. B6 and B12. 2011. Activation energy (Ea) can be calculated from the slope (Ea/2. Asp. Arg. Res. the usual ingredients of powdery drugs (fructose. C2O42Ca2+. Tyr. sorbitol. 3(2):605-616 ______________________________________________________________________________ mol L-1. Trp Phe. c DZP = 0. B1. Arrhenius curve was constructed by plotting log k versus 1/T and found to be linear with coefficient of correlation. lactose).2×10-5 mol L-1. stearic acid. because the amounts tolerated are much higher than those usually present in pharamceuticals. B12. K+.anions. glucose.Aleksandra N.. stearic acid. indicated that the reaction is feasible. r = 0. The tolerance limits (expressed as w/w ratio).and C2O42. Ions Ca2+. Interference studies To assess the selectivity of the method. No interference was found when including up to 100-fold mannitol. Amino acids (His.08 kJ mol-1. The ability to predict changes in the rate of the reaction is based on a knowladge of the value of the activation energy. Asp.52 kJ mol-1). Li+. most reactions which have observable rates at ordinary temperatures have activation energies of 15-30 kcal mol-1 (62.14 kJ mol-1 is in accordance with this postulate.A value of 99. for the species studied on the determination of 1. will not interfere with the method. c R ( NO ) OH = 1. More severe intrference was observed for Fe3+ ion. Lys His.10 <5 5 – 10 <5 5 -10 interference 10 1 1 interference 1 Interference coefficient.ions As can be seen. Ser Fe3+ b) a) 10 – 15 5 . I = (cºDZP – cDZP) / cºDZP cºDZP and cDZP are measured concentrations of DZP without and with the interfering species b) Masking with F.76-125.9997. Tyr. It also should be noted that a higher tolerance level exists to the presence of vitamins B1. glucose. F. Zn2+ interfere when present in approximately 10-fold excesses. Pavlović et al J.7×10-5 mol L-1.99 µg mL-1 of DZP were given in Table 3. Ala.

rapid.30 98. Belgrade.09 97.48 ± 0.o.06 ± 0. the proposed kinetic-spectrophotometric method for the determination of DZP in pharmaceutical samples and human control serum reported in this work is simple.39 and 2.05 2.56 3. They were treated as described in the Experimental section.72 3. Belgrade.36 ± 0. Statistical analysis of the results (Tables 4 and 5) showed that calculated F.129 3. ν2=4) and t-value (ν=8) at 95 % confidence level are 6. inexpensive.83 ± 0. Also.o.Aleksandra N.d.81 97.13 DZP found by the proposed methoda) x ± SD .04 1.62 0.306. Pavlović et al J. bioMérieux® sa.51 ± 0. good recovery was observed in the case of serum sample (Table 5).03 1.35 1.06 2. the results obtained by this method are in accordance with the HPLC method. Table 4 Determination of diazepam by the kinetic and HPLC method Pharmaceutical Preparation Diazepam Bensedin®d) Diazepame) c) Taken µg mL-1 1. indicating that the constituents of the human control serum do not interfere (Fe3+ ions were masked with F. The results of the proposed method were statistically compared with those of the HPLC method using a point hypothesis test [33.02 2.05 2.06 4.89 1. pharmaceutical industries and research institutions.21 98.42 2.39 ± 0.values at 95 % confidence levels are less than the theoretical ones. µg mL-1 Fvalueb) tvalueb) 1.07 1. Serbia) containing diazepam 2 mg and excip.85 HPLCa) RSDa) % Recoverya) % x ± SD x ± SD .and t. Pharm. 2011. France) by standard addition method Proposed method µg mL-1 Founda) Added 0.306.80 ± 0. is very suitable for developing countries. 614 . 3(2):605-616 ______________________________________________________________________________ Applicability of the Proposed Method The proposed method was applied to the determination of diazepam in three pharmaceutical formulations and human control serum using the direct calibration curve. e) Tablets (from Panfarma d. Also. respectively c) Rectal solution (from Pharmacy Belgrade.. Serbia) containing diazepam 2 mg and excip Table 5 Determination of diazepam in human control serum ("Lytorol N". Therefore.and the protein precipitate was deposited) in any way with the detection of diazepam. ν2=4) and t-value (ν=8) at 95 % confidence level are 6.34].730 2. Serbia) containg 5 mg of diazepam in 2. Res. As can be seen in Table 4.01 0.968 a) Data are based on the average obtained from five determinations b) Theoretical F-value ( ν1=4.03 3.5 mL of solution d) Tablets (from Galenika a. µg mL-1 Fvalueb) tvalueb) 0. thus being very appropriate for routine quality control analyses of active drug in the laboratories of hospitals. Statistical comparison of the results with the HPLC method shows good agreement and indicates no significant difference in accuracy and precision. the proposed method could be used for the determination of diazepam in serum samples.. confirming no significant differences between the performance of the proposed and the HPLC method.81 1.757 a) Data are based on the average obtained from five determinations b) Theoretical F-value ( ν1=4.39 and 2.65 0.09 ± 0. µg mL-1 RSDa) % Recoverya) % HPLCa) x ± SD . Chem. respectively CONCLUSION In conclusion..

2002. 282. 31(6). B Liawruangrath. MT Faucci. D Marot. Chem. [22] J Dolejsova. J Short. 2005. 13571360.. 43(4). G Mahuzier. 26. J.. MH Livertoux. Z Zhou. New York. Anal. E Civit. 36(4). BN Barsoum. Antiepileptic Drug. FA Aly.. 2011. [12] S Diallo. L Liu. Y Kato. Y Qiu. 2001. [23] J Moros. [6] K Chiba.. J. GO Kokwaro. 1993. J. Biomed. 447-458. MFM Tavares. EL Izake. Anal. The authors are grateful for the financial support provided by this Ministry. S Garrigues. 2007. B. 1700-1706. [8] A El-Mahjoub. C Staub. H Zhao. 2007. 110(1-3). E Santamarina. S Unger. CRJC Newton. 2004. 127-130. 132(7). Pharm. 20. CE Efstathiou. Acta. ERM Kedor-Hackmann. 47(1). Biomed. Biomed. Anal. 2006. Chromatogr. F Belal. The Analyst. ML Simoes Goncalves. 3(2):605-616 ______________________________________________________________________________ Acknowledgement This research was supported by grant number 142015 from the Serbian Ministry of Science and Environmental Protection. FM Musteata. 2001. [5] S Liawruangrath. Res. Egypt. [21] RS Guerrero. [20] W Guo. Chromatogr. Anal. J. J. 2004. J. J. [10] SN Muchohi. 493-499. Biomed.. J Pawliszyn. J. Anal. 2001. A Rose. T Hirano. 2001. J. Anal. [15] L Wang. 423-431. 2006. Pharm. Raven Press. 2007. 55-60. 77-84. J. Anal. 2007. 721-732. 771-780.. 1988. Biomed. JK Penry. V Famila. 1999. H Bagheri. Sci. SM Abd Allah. N Contel. H Horii. [25] S Furlanetto. Biomed. S Paterson. 1 st Edition. 1982. Pharm. J. Biomed. 850(1-2). G Massolini. 668(1).1185-1189. 1993. B Sebille. Anal. Chromatogr. [14] M Bakavoli. 2003. Biomed. Bioanal. Biomed. 509-518. [18] N Thuaud. 99-105.Aleksandra N. B. [7] MJ Koenigbauer. CG Benito.. [26] MSA Pardo. Chem. 357-362.. The Analyst. S Pinzauti. M Kaykhaii. 1277-1282. 34(5). [16] M Pujadas. R De la Torre. H Lin. Microchim. P Solich. 2004.. B. Part A. Chromatogr. 761(2). 672-678. X Zhang. J Makchit. [4] SM Khalile. J BessiereJ. RP Schmidt. S Pichini.. 2000. Pharm. 255-259. M Steppe. Pharm. F Senhadj-Raoul. 1074-1081. Anal. 1983. S Ren. A. 25(3-4). Chem. Pharm. E Bugni. 23(2-3). JM Calatayud. [2] A El-Brashy. REFERENCES [1] HR Mattson. 1-16. Anal. B. MC Dessalles. Anal. MIRM Santoro. C Ortiz. 594-601. Pharm. Pharm. Pharm. Pharm. 22(1). S Orlandini. Acta. K Na-Bangchang. 55(4). 871-876. B Kamanikom. 37(2). Pharm. T Ishizaki. Biomed. 374(6). 1137-1144. J. K Perez. [13] W Hanpitakpong. MA Curtis. 2004. S Gasdeblay. ChK Polydorou. A. 427. MA Zayed. DM Woodbury. 321-330. M De la Guardia. Chromatogr. 273-279. E La Porta. J. 277-285. J. S Grossman. [19] MM Correia dos Santos. Talanta. 126(10).. J. MA Koupparis. J. Pavlović et al J. V Banmairuroi. Biomed. [11] Z Liu. J. A Choemung. Spectrochim. [3] AA Salem. J Song. 497.. Anal. 44(2). [24] A Es-haghi. 615 .. Chromatogr. [17] R Cordero. T Chiba. J. Anal. Pharm. 1136(1). 1995.. [9] C Ferreyra.. 2001. 11(11-12). 60(4). BR Ogutu.

Res. Chem. 67(24). 616 .. Inorg. 24(5-6). 37(4). Nucl. DM West. 1991. Pharm. 1996. Chem. YV Heyden.Aleksandra N. 263-266. G Tosi. Chem. Inc. Analyst. Saunders College Publishing.. DL Massart. 7th Edition. FJ Holler. 2001. Pavlović et al J.. [31] GS Hammond. 4491-4499. Anal. 77(2). [33] C Hartmann. Phys.. [30] J Ermer. J Smeyers-Verbeke. 1979. J. Pharm. 2008.. P Vankeerberghen. 41(2). 1995. Rockville. Fundamentals of Analytical Chemistry.. [32] IWM Smith. 334-338. 755-767. MD. [34] DA Skoog. 116(1). Philadelphia. Chem. 11. [29] JN Miller. Chem. Anal. Authority of the United States Pharmacopoeia Convention. J. 2011. Rev.. Soc. Biomed. 1955. J. [28] The United States Pharmacopoeia USP-27/NF-22. 3(2):605-616 ______________________________________________________________________________ [27] C Preti C. 812-826. 2004. 3-14. W Penninckx.