Simultaneous Spectrophotometric Determination of Metronidazole and Diiodohydroxyquine
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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 analy- sis 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 stan- dard solution each was separately transferred to a se- ries 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, re- spectively. 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.