Simultaneous Spectrophotometric Determination of Metronidazole and Diiodohydroxyquine

AAnnaallyyttiiccaaISllS N : 0974-7419 Volume 13 Issue 1 CCHHEEAnMM IndIIiSaSnT TJoRuRrnYaYl

Full Paper ACAIJ, 13(1) 2013 [23-32] Simultaneous spectrophotometric determination of metronidazole and diiodohydroxyquine

Hesham Salem1, Safa,a M.Riad2, Mamdouh Reda2, Kholoud Ahmed1* 1Pharmaceutical and Analytical Chemistry Department,Faculty of Pharmacy, October University for Modern Sciences and Arts, 6thof October city, (EGYPT) 2Analytical Chemistry Department, Faculty of Pharmacy-Cairo University, Kasr El-Aini Street, 11562 Cairo, (EGYPT) E-mail : [email protected]

ABSTRACT KEYWORDS

Four sensitive and precise spectrophotometric methods were developed Metronidazole; and validated for the simultaneous determination of metronidazole (MTR) Diiodohydroxyquinoline; and diiodohydroxyquinoline (DIQ) in their mixture and in their pharmaceu- Determination; tical formulations. Among the methods adopted were, second-derivative Spectrophotometry. (2D), third-derivative (3D), derivative ratio spectroscopy (1DD) and isosbestic point technique. The selectivity of the proposed methods was checked using laboratory prepared mixtures. The proposed methods were simple, not expensive and applicable that make them suitable for the analysis of MTR and DIQ in their mixture and in pharmaceutical formulations for routine unknown analysis in quality control labs.  2013 Trade Science Inc. - INDIA

–MS/ INTRODUCTION plasma, saliva and gingival crevicular fluid by LC MS[9]. Simultaneous multi residue determination of met- Metronidazole (MTR), is 2-(2-methyl-5-nitro-1H- ronidazole and spiramycin in fish muscle was done us- imidazol-1-yl) ethanol (Figure 1)[1]. It is used as anti- ing HPLC with UV detection[10]. Microsized graphite bacterial and antiamaebiasis. Diiodohydroxyquinoline sensors for potentiometric determination of metronida- (DIQ), 5, 7 -diiodoquinolin-8-ol (Figure 2). It is widely zole and spiramycin was done[11]. DIQ was determined known by the trade name Diodoquin, is a quinoline in pharmaceutical formulations using HPLC[12]. MTR derivative which can be used in the treatment of amoe- and DIQ were determined by bivariate spectrophoto- biasis[2]. metric method[13]. The literature survey reveals several analytical meth- In modern analytical laboratory, there is always a ods for quantitative estimation of MTR in body fluids need for simple, rapid and accurate methods for simul- and in pharmaceutical formulations these methods in- taneous determination of drug combinations that could clude ultaviolet spectrophotometry[3-5], high performance be used for routine analysis. The present work aimed liquid chromatography (HPLC)[6,7] and voltammetry[8]. to develop simple instrumental methods for simultaneous Quantitation of metronidazole and spiramycin in human determination of MTR and DIQ in combination. 24 Spectrophotometric determination of m. etronidazole and diiodohydroxyquine ACAIJ, 13(1) 2013 Full Paper DIQ (1 mg mL-1) in methanol were prepared for the proposed spectrophotometric methods. All solutions were freshly prepared on the day of analysis. Procedures Direct spectrophotometric method Spectral characteristics of MTR and DIQ Two aliquots (1 mL) of each MTR and DIQ were M.W. 171.15 gm separately transferred into two 100 mL volumetric flasks. Each flask was completed to volume with metha- Figure 1: Chemical structure of metronidazole (MTR) ìg mL nol to obtain final concentration of 10 -1 of MTR ìg mL and 10 -1 of DIQ. The spectrum of each solution was scanned and recorded separately. Linearity Portions of MTR standard solution and of DIQ standard solution each was separately transferred to a series of 10 mL volumetric flasks. Each flask was com- M.W. 396.951 gm pleted to the volume with methanol to reach the con- ìg mL ìg mL Figure 2 : Chemical structure of Diiodohydroxyquine (DIQ) centration range of 2-24 -1and 1-12 -1, respectively. The absorbance was measured at 311 nm EXPERIMENTAL and 254 nm. Calibration graphs were constructed by plotting the absorbance versus concentrations. The re- Instruments gression equations were then computed for MTR and DIQ at the specified wavelengths. A double beam UV-visible spectrophotometer 2 (Shimadzu, Japan) model UV-1601PC, with 1 cm Second-derivative ( D) method quartz cells, connected to an IBM-compatible com- Linearity puter was used. The software was UV-PC personal Standard serial concentrations in the range of 2-24 ìg mL ìg mL spectroscopy software version 2.32. The spectral band -1 of MTR and of 1-12 -1 of DIQ were width was 2 nm with wavelength-scanning speed of prepared as described under section 2.4.1.2. The am- -1 2800 nm min . plitudes of the second-derivative peaks of MTR were Materials and reagents measured at 311 nm and the amplitudes of the second- derivative peaks of DIQ were measured at, 255.3 nm, Reference metronidazole hydrochloride powder Ä= 8 nm and 238.5 nm with scaling factor =100. (MTR) and reference diiodohydroxyquine (DIQ) were Calibration graphs were constructed by plotting the kindly donated by Al Qahira Pharmaceuticals Co. The µg mg”1 peak amplitudes versus concentrations. The regression potency was certified to contain 1002 for MTR µg mg”1 equations were then computed for MTR and DIQ at and 1009 for DIQ. Pharmaceutical dosage form the specified wavelengths. (Paramibe compound, 500 mg tablets were kindly sup- plied by Chemical Industries Development (CID) and Third -derivative (3D) method were claimed to contain 250 mg of MTR and 250 mg Linearity of DIQ per each tablet. Methanol was spectroscopic Standard serial concentrations in the range of 1-12 ìg mL grade. -1 of DIQ were prepared as described under Standard solutions section 2.4.1.2. The amplitudes of the third -derivative Stock standard solutions of MTR (1 mg mL-1) and peaks of DIQ were measured at 260 nm and 267.6 nm Anallyttiicall CHEAMn IIndSianT JoRurnYal ACAIJ, 13(1) 2013 Kholoud Ahmed et al. 25 Full Paper Ä= with 8 nm and scaling factor =1000. tions were calculated. Calibration graphs were constructed by plotting the Assay of pharmaceutical formulations (Paramibe peak amplitudes versus concentrations. The regression compound, 500mg tablets) equations were then computed DIQ at the specified Twenty tablets were weighed from the dosage form wavelengths. and the average weight was calculated, tablets were 1 First-derivative of ratio spectra ( DD) method crushed to furnish a homogenous powder and certain Linearity amount of powdered tablets were dissolved in metha- Standard serial concentrations in the range of 2-24 nol for 15 minutes and filtered. The solutions were di- ìg mL ìg mL -1 for MTR and 1-12 -1 for DIQ were pre- luted to the same concentration of the appropriate work- µg mL pared as under section 2.4.1.2. and accurately 5 - ing solutions then the procedures were followed as de- 1 of DIQ standard solution to be used as a divisor for scribed under each method. µg mL MTR and 12 -1of MTR to be used as a divisor for DIQ. The spectra of the prepared standard solutions were RESULTS AND DISCUSSION scanned (200-400 nm) and stored into the PC. The stored spectra of MTR were divided (the am- Direct spectrophotometric method ìg plitude of each wavelength) by the spectrum of 5 The UV spectra of MTR and DIQ allow direct de- mL-1 of DIQ. The first-derivative of the ratio spectra termination of MTR at 311 nm with interference from Ä=4 nm (1DD) with and scaling factor of 10 was ob- DIQ which can be determined directly at 254 nm (Fig- tained. The amplitude of the first-derivative peaks of ure 3). A linear relationship was obtained in the range ìg mL MTR were measured at 286nm and 328.5 nm. of 2-24 -1 for MTR. The corresponding regres- The stored spectra of DIQ were divided (the am- sion equation was computed and found to be: A = 0.05 ìg plitude of each wavelength) by the spectrum of 12 C - 0.01 (r=0.9997), at 311 nm Where, A is the absor- mL-1 of MTR. The first-derivative of the ratio spectra bance of MTR at 311 nm, C is the concentration of 1 Ä=4 nm and scaling factor of 10 was ob- ìg mL ( DD) with MTR ( -1) and r is the correlation coefficient. A ìg tained. The amplitude of the first-derivative peaks of linear relationship was obtained in the range of 1-12 DIQ were measured at 250 nm and 260.7 nm mL-1 for DIQ. The corresponding regression equation Calibration graphs were constructed relating the was computed and found to be: A = 0.2013 C - 0.042 peak amplitudes of (1DD) to the corresponding con- (r=0.9998), at 254 nm Where, A is the absorbance of ìg mL centrations. The regression equations were then com- DIQ at 254 nm, C is the concentration of DIQ ( - puted for MTR and DIQ at the two specified wave- 1) and r is the correlation coefficient. lengths. Second-derivative (2D) method Isosbestic point The second-derivative (2D) ultraviolet spectropho- In this method DIQ is determined by derivative ra- ’t interfere tometry was applied for the determination of MTR and tio technique at 260.7 nm where MTR don DIQ, either in raw material or in pharmaceutical formu- while total is measured at the isosbestic point at 280 lations. nm then concentration of MTR is determined by sub- The absorption spectra of MTR and DIQ showed traction. overlapping, interference and error probability affect Analysis of laboratory prepared mixtures the use of direct spectrophotometry and first-deriva- Laboratory prepared mixtures containing different tive method (1D)for determination MTR and DIQ in ratios of MTR and DIQ were analyzed using the sug- presence of each other. When the second derivative gested methods, aliquots of MTR and DIQ were mixed spectra were examined (Figure 4), it was found that to prepare different mixtures and the procedures were MTR could be determined at 311nm where DIQ has followed as mentioned under each method, the con- no contribution (zero crossing) at 311nm the clear zero centrations from the corresponding regression equa- crossing of DIQ allowed accurate determination of MTR Anallyttiicall CHEAMn InIdSianT JoRurnYal 26 Spectrophotometric determination of m. etronidazole and diiodohydroxyquine ACAIJ, 13(1) 2013 Full Paper in presence of any level of DIQ. A linear relationship Where 2D is the peak amplitude of the second-deriva- ìg mL was obtained in the range of 2-24 -1 for MTR. tive curve at the corresponding wavelengths, C is the ìg mL The corresponding regression equations were computed concentration of MTR ( -1) and r is the correla- and found to be: tion coefficient. 2D = 0.012C +0.0007 (r=0.9999), at 311 nm The mean percentage recovery was found to be

µg mL ————) showing maximum absorbance at 254 nm and MTR 8µg mL ——) showing maximum Figure 3 : DIQ 8 -1 ( -1 ( µg mL absorbance at 311 nm and mixture containing 4 -1(...... ) of each showing isosbestic point 100.347 at 311 nm. contribution (zero crossing), the clear zero contribution When the second derivative spectra for DIQ were of MTR allowed accurate determination of DIQ in pres- examined (Figure 4), it was found that DIQ could be ence of any level of MTR. A linear relationship was ìg mL determined at 255.3nm and 238.5nm, where MTR has obtained in the range of 1-12 -1 for DIQ. The no contribution, the clear zero contribution of MTR al- corresponding regression equations were computed and lowed accurate determination of DIQ in presence of found to be: any level of MTR. A linear relationship was obtained in 3D = 0.8405 C - 0.2326 (r=0.9998), at 260 nm ìg mL-1 the range of 1-12 for DIQ. The corresponding 3D = 0.1932 C + 0.1467 (r=0.9998), at 267.6 nm regression equations were computed and found to be: Where 3D is the peak amplitude of the third-derivative 2 D = 0.0973 C - 0.0229 (r=1), at 238.5 nm curve at the corresponding wavelengths, C is the con- 2 ìg mL D = 0.3023C -0.0911 (r=0.9998), at 255.3 nm centration of DIQ ( -1) and r is the correlation Where 2D is the peak amplitude of the second-deriva- coefficient. tive curve at the corresponding wavelengths, C is the The mean percentage recoveries were found to be ìg mL-1 concentration of DIQ ( ) and r is the correlation 99.978 at 260nm and 100.041 at 267.6 nm. coefficient. The mean percentage recoveries were found to be Derivative ratio spectrophotometric method 100.053 at 238.5 nm, 99.929 at 255.3 nm. Derivative ratio spectrophotometric method was Third-derivative (3D) method used to determine MTR in presence of DIQ. The zero- order of the derivative ratio spectra of MTR and the The third derivative (3D) ultraviolet spectrophotom- first-order of the derivative ratio spectra were presented etry was applied for the determination of DIQ, either in raw material or in pharmaceutical formulations. in Figure 6 & Figure 7, respectively. The concentration When the third derivative spectra for DIQ were of the divisor was studied. It was found that upon di- ìg mL-1 examined (Figure 5), it was found that DIQ could be viding by 5 of DIQ product led to the best determined at 260nm, 267.6nm, where MTR has no results in terms of sensitivity, repeatability and signal to Anallyttiicall CHEAMn IIndSianT JoRurnYal ACAIJ, 13(1) 2013 Kholoud Ahmed et al. 27 Full Paper

µg mL µg mL –––– Figure 4 : Second derivative of DIQ 12 -1 (------) and of MTR 12 -1 ( ) where MTR could be determined at 311 nm when DIQ has no contribution, DIQ could be determined at 255 .3 nm, 238.5 nm, where MTR has no contribution

Magnification of MTR Peak at 311nm

µg mL-1 (-----) and of MTR 12 µg mL-1(–––––) where DIQ could be determined at 260 Figure 5 : Third derivative of DIQ12 nm and 267.6 nm when MTR has no contribution Anallyttiicall CHEAMn InIdSianT JoRurnYal 28 Spectrophotometric determination of m. etronidazole and diiodohydroxyquine ACAIJ, 13(1) 2013 Full Paper noise ratio. Linear calibration graphs were obtained for The mean percentage recoveries were found to be ìg mL MTR in concentration range of 2-24 -1 by re- 99.878 at 286 nm and 99.974 at 328.5 nm cording the peak amplitude at 286nm and 328.5nm Derivative ratio spectrophotometric method was ìg mL using 5 -1 of DIQ as a divisor. The regression used to determine DIQ in presence of MTR. The zero- equations were computed and found to be: order of the derivative ratio spectra of DIQ and the first- (1DD) = 0.3036 C - 0.0944 (r=0.9998), at 286 nm order of the derivative ratio spectra were presented in (1DD) = 0.1769 C+ 0.0173 (r=0.9999), at 328.5 nm Figure 8 & Figure 9, respectively. The concentration of Where 1DD is the peak amplitude of the first-deriva- the divisor was studied, it was found that upon dividing 1 ìg mL tive curve for (MTR/DIQ), C is the concentration of by 12 -1 of MTR product led to the best results in ìg mL MTR ( -1) and r is the correlation coefficient. The terms of sensitivity, repeatability and signal to noise ratio. precision of the proposed method was checked by the Linear calibration graphs were obtained for DIQ in con- ìg mL analysis of different concentrations of authentic samples centration range of 1-12 -1 by recording the peak

µg mL Figure 6 : Divisionof MTR (2-24) -1 using 5ug mL-1 of DIQ as divisor

µg mL µg mL Figure 7 : First derivative of ratio spectra MTR (2-24) -1 using 5 -1 DIQ as divisor Anallyttiicall CHEAMn IIndSianT JoRurnYal ACAIJ, 13(1) 2013 Kholoud Ahmed et al. 29 Full Paper ìg mL amplitude at 250nm and 260.7 using 12 -1 of MTR The mean percentage recoveries were found to as a divisor. The regression equations were computed be 99.949 at 250 nm and 99.998 at 260.7 nm. and found to be: Isosbestic point –3.0204 (r=0.9999), at 250 nm (1DD) = 12.9746 C In this method DIQ is determined by derivative ra- 1 –8.3705 (r=0.9999), at 260.7 nm ( DD)= 18.3730 C tio technique at 260.7 nm where MTR don,t interfere Where 1DDis the peak amplitude of the first-de- while total is measured at the isosbestic point at 280 rivative curve for (DIQ/MTR), C is the concentra- nm then concentration of MTR is determined by sub- ìg mL tion of DIQ ( -1) and r is the correlation coef- traction. To ensure the validity of the chosen isosbestic ficient. The precision of the proposed method was point Figure 3 shows the isosbestic point of MTR, DIQ checked by the analysis of different concentrations and their mixture in which each sample either single or of authentic samples. in mixture contains 8 ug/mL.

µg mL Figure 8 : Division of DIQ (1-12) -1 using 12 ug mL-1 of MTR as divisor

µg mL µg mL Figure 9 : First derivative of ratio spectra DIQ (1-12) -1 using 12 -1 MTR as divisor Anallyttiicall CHEAMn InIdSianT JoRurnYal 30 Spectrophotometric determination of m. etronidazole and diiodohydroxyquine ACAIJ, 13(1) 2013 Full Paper Statistical analysis tory prepared mixtures with good precision as shown The suggested methods were successfully applied in TABLE 3 & 4. The proposed methods were also for the determination of MTR and DIQ in their labora- used for estimating the concentration of both drugs in TABLE 1a : Assay parameters and validation sheet for de- TABLE 1b : Assay parameters and validation sheet for termination of metronidazole (MTR) and diiodohydroxy- determination of metronidazole (MTR) and quine (DIQ) by second derivative and Isosbestic point tech- diiodohydroxyquine (DIQ) by third derivative and first niques derivative of ratio spectra techniques

Isosbestic Parameter 2D-method Parameter 3D-method 1DD-order method point method DIQ at MTR DIQ at MTR at DIQ at MTR at Range 260 nm 267.6 nm 286 nm 328.5 nm 250 nm 260.7 nm Range 311 nm 238.5 nm 255. 3 nm 280 nm 1-12 1-12 2-24 2-24 1-12 1-12 2-24 1-12 1-12 1-12 ìg -1 ìg -1 ìg -1 ìg -1 ìg -1 ìg -1 ìg mL-1 ìg mL-1 ìg mL-1 ìg mL-1 mL mL mL ml mL mL

Slope 0.841 0.193 0.304 0.158 12.975 18.373 Slope 0.012 0.097 0.302 0.02 Intercept -0.233 0.147 -0.094 0.017 -3.020 -8.371 Intercept 0 -0.022 -0.091 -0.007 Mean 99.978 100.041 99.878 99.974 99.949 99.998 Mean 100.347 100.053 99.929 100.433 ±S.D. 0.995 0.701 0.821 0.433 0.586 0.641 ±S.D. 0.847 0.463 0.939 0.603 Variance 0.990 0.491 0.674 0.187 0.343 0.411 Variance 0.717 0.214 0.882 0.364 Coefficient of 0.995 0.701 0.822 0.433 0.586 0.641 Coefficient of variation 0.844 0.463 0.939 0.600 variation Correlation 0.9998 0.9998 0.9998 0.9999 0.9999 0.9999 Correlation coefficient (r) 0.9999 1 0.9998 0.9998 coefficient (r) R.S.D. (%) 0.844 0.463 0.939 0.600 R.S.D. (%) 0.995 0.701 0.822 0.433 0.586 0.641 TABLE 2a : Statistical comparison for the results obtained by the proposed methods and the official method for analysis of MTR and official method for analysis of DIQ 2D-method Isosbestic point official method official method Parameters MTR DIQ MTR MTR DIQ 311 nm 238.5 nm 255. 3 nm 280 nm Mean 100.347 100.053 99.929 100.433 100.07 100.18 ±S.D 0.845 0.463 0.939 0.603 0.507 0.641 Variance 0.714 0.214 0.882 0.364 0.257 0.411 N 12 12 12 8 6 6 F-test 2.78 (4.74)a 1.92 (4.74)a 2.15 (4.74)a 1.42 (4.88)a Student,s t-test 0.866 (2.120)a 0.432 (2.120)a 0.667 (2.120)a 1.22 (2.179)a a The values in the parenthesis are corresponding theoretical t- and F-values at P = 0.05[16] TABLE 2b : Statistical comparison for the results obtained by the proposed methods and theofficial method for analysis of MTR and official method for analysis of DIQ

3 official official D-method 1DD-method method method Parameters DIQ MTR DIQ MTR DIQ 260 nm 267.6 nm 286 nm 328.5 nm 250 nm 260.7 nm Mean 99.978 100.041 99.878 99.974 99.949 99.998 100.07 100.18 ±S.D 0.995 0.701 0.821 0.433 0.586 0.641 0.507 0.641 Variance 0.990 0.491 0.674 0.187 0.343 0.411 0.257 0.411 N 12 12 12 12 12 12 6 6 F-test 2.41 (4.74)a 1.19 (4.74)a 2.62 (4.74)a 1.37 (4.74)a 1.198 (4.74)a 1 (4.74)a Student,s t-test 0.519 (2.120)a 0.420 (2.120)a 0.609 (2.120)a 0.395 (2.120)a 0.741 (2.120)a 0.568 (2.120)a a The values in the parenthesis are corresponding theoretical t- and F-values at P = 0.05[16] Anallyttiicall CHEAMn IIndSianT JoRurnYal ACAIJ, 13(1) 2013 Kholoud Ahmed et al. 31 Full Paper TABLE 3 : Determination of metronidazole and diiodohydroxyquine in laboratory prepared mixtures by the proposed methods 2D-method Method MTR DIQ 311 nm 238.5 nm 255.3 nm ± SD) ±0.471 ±0.358 ±0.311 (Mean 100.107 99.920 100.337 3D-method 1DD-order method DIQ MTR DIQ Method 260 267.6 286 328.5 250 260.7 nm nm nm ± SD) ±0.789 ±1.005 ±0.570 ±0.258 ±0.487 ±0.425 ( Mean 100.929 100.197 100.307 100.243 99.856 99.84 TABLE 4 : Determination of metronidazole (MTR) in lab prepared mixtures by proposed isosbestic point method Total MTR µg mL µg mL µg mL µg mL Mixture ratio DIQ: MTR Taken ( -1) Found ( -1) Taken ( -1) Found ( -1) R% 1:7 8 8.05 7 7.05 100.71 2:6 8 8.05 6 6.05 100.83 1:12 13 12.95 12 11.95 99.58 4:4 8 8.05 4 4.03 100.75 1:10 11 10.95 10 9.95 99.50 3:5 8 8.05 5 5.06 101.20 5:3 8 8.05 3 3.01 100.33 1:9 10 10.05 9 9.05 100.56 Mean 100.433 SD 0.603 TABLE 5 : Determination of MTR and DIQ in Paramibe compound tablet by proposed methods 2D-method Method MTR DIQ 311 nm 238.5 nm 255.3 nm ± SD) ± 0.388 ±0.275 ±0.179 (Mean 100.098 99.973 99.878 3D-method 1DD-order method DIQ MTR DIQ Method 260 267.6 286 328.5 250 260.7 nm nm nm ± (Mean ±0.448 ±0.563 ±0.394 ±0.415 ±0.450 ±0.510 100.535 100.190 99.753 99.66 99.535 100.253 SD) their pharmaceutical formulations. The results are shown CONCLUSION in TABLE 5. Assay parameters and a validation sheet for determination of the studied drugs are shown in Unlike the mostly recommended HPLC procedure, TABLE 1. Statistical comparison for the results ob- the proposed spectrophotometric methods are simple tained by the proposed methods and the reference ones and not expensive. The reagents used in the proposed for the studied drugs are shown in TABLE 2. The cal- methods are cheap and available. The procedures ap- culated t- and F-values were found to be less than the plied in each method do not involve any critical reac- tabulated ones confirming good accuracy and excellent tions or tedious sample preparations. This aspect of precision. spectrophotometric analysis is of major interest in ana- Anallyttiicall CHEAMn InIdSianT JoRurnYal 32 Spectrophotometric determination of m. etronidazole and diiodohydroxyquine ACAIJ, 13(1) 2013 Full Paper lytical pharmacy since it offers distinct possibility of as- [8] P.N.Bartlett, E.Ghoneim, G.El-Hefnawy, I.El- saying the studied drugs in their mixtures and in their phar- Hallag; Voltammetry and determination of metron- maceutical formulation without interference from the ex- idazole at a carbon fiber microdisk electrode, cipients. The suggested methods are found to be accu- Talanta, 66(4), 869-874 (2005). rate and selective with no significant difference of the pre- [9] C.Sagana, A.Salvadora, D.Dubreuila, P.Pierre, D.Pouletb, Duffautb, I.Brumptc; Quantitation of cision compared with the reference methods of analysis. metronidazole and spiramycin in human plasma, –MS/ MS, The proposed methods could be applied successfully, saliva and gingival crevicular fluid by LC for routine analysis of MTR and DIQ singly, in their mix- J. of Pharmaceutical and Biomedical Analysis, tures or in their pharmaceutical formulations 38(2), 298-306 (2005). [10] G.Guetens, H.Prenen, G.De Boeck, A.Van öffski, M.Highley, E.A.De Bruijn; REFERENCES Oosterom, P.Sch Simultaneous multi residue determination of met- [1] British pharmacopoeia; 1&2, 3952 (2009). ronidazole and spiramycin in fish muscle using high [2] Y.Laurent Galichet (Ed); Clarke,s Analysis of Drugs performance liquid chromatography with UV de- and Poisons, 3rd Edition, Pharmaceutical Press, tection, J. of Chromatography B Analytical Tech- (2005). nologies in the Biomedical and Life Sciences, [3] U.R.Atta, A.S.Rehmana, A.R.Ijazand; Spectropho- 809(2), 175-181 (2007). tometric determination of metronidazole in phar- [11] F.I.Khattab, N.K.Ramadan, M.A.Hegazy, maceutical pure and dosage forms using p-Benzo- N.S.Ghoniem; Microsized Graphite Sensors for Po- quinone, J.of the Iranian Chemical Society, 2, 197- tentiometric Determination of Metronidazole and 202 (2005). Spiramycin, Portugaliae Electrochimica Acta., [4] K.Siddappa, M.Mallikarjun, P.T.Reddy, M.Tambe; 29(2), 79-90 (2011). Spectrophotometric determination of metronidazole [12] Z.S.Gomaaa; Determination of Diiodohydroxyquin ’s base system using vanillin and through Schiff in Dosage Forms by High Performance Liquid PDAB reagents in pharmaceutical preparations, Chromatography, J. of Liquid Chromatography, ética Química, São Paulo, ISSN 0100-4670, Ecl 33, 10(6), 1175-1185 (1987). 4 (2008). [13] P.L.Lopez-de-Alba, K.Wrobel, L.Lopez-Martinez [5] O.A.Adegoke, O.E.Umoh; A new approach to the et al; Application of the bivariate spectrophotomet- spectrophotometric determination of metronidazole ric method for the determination of metronidazole, and tinidazole using p-dimethyl aminobenzaldehyde, furazolidone and diiodohydroxyquinoline in pharma- –419 Acta Pharm., 59, 407 (2009). ceutical formulations, J.Pharm.Biomed.Anal.Oct., [6] K.B.Mustapha, M.T.Odunola, M.Garba, 16(2), 349-355 (1997). O.Obodozie; Rapid cost-effective liquid [14] M.R.Spiegel, L.J.Stephns; Schaum Outline of chromatograghic method for the determination of Theory and Problems of Statistics, Schaum Out- metronidazole in biological fluids, African J. of Bio- line Series, New York, (1999). technology, 5(13), 1188-1191 (2006). [7] S.Coxa, C.Matthew, A.&J.Yarbrough; J.of Liquid Chromatography & Related Technologies, 33, 1 (2009).

Anallyttiicall CHEAMn IIndSianT JoRurnYal