Amera Abbas Mohammad Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 94 Determination of Cefalexin by Direct (UV- Vis) Spectrophotometer and Indirect(Flame Atomic Absorption) Technique Amera A. Mohammad* ,1 *Department of pharmaceutical chemistry,College of Pharmacy ,University of Baghdad ,Baghdad ,Iraq Abstract A new method for the determination of the drug cefalexin in some Pharmaceuticals using (UV- Vis) and indirect Flame Atomic Absorption Spectrophotometer (FAAS) , Fe III should forms a chelating complex with cefalexin (CEX –Fe III) at pH (1-8) and the best pH for the formation of (CEX –Fe III) chelating complex was (2) .The complex extracted with Methanol and Dimethy-Sulphoxide .The mole-ratio method has been used to determine the structure of chelate (CEX - Fe III) and found to be 2:1 LM ( Ligand : Metal.) . Keywords : Cefalexin , chelating complex. الخالصة انًسئُت و انطسَقت -طسَقت حدَثت نخقدَس انسُفانكسٍُ فٍ بعض انعقازاث انصُدالَُت و ذنك باسخخداو حقُُخٍ األشعت فىق انبُفسجُت pHغُس انًباشسة ناليخصاص انرزٌ أنههبٍ .حُث وجد أٌ انحدَدَك َسخطُع أٌ َكىٌ يعقد كُهُخٍ يع انسُفانكسٍُ فٍ يدي يٍ قُى اسخخهص باسخخداو يزَج و انًعقد انُاحج ( = ٢pH ) حعطٍ اعهً يًخصُت كاَج عُد pH( و أٌ افضم قًُت نــ ٨ -١ح يا بٍُ )حخساو ( عهً انخىانٍ.١:٢( هٍ ) ( CEX-FeIII انًُثاَىل و ثُائٍ يثُم انسهفىكساَد و كاَج انُسبت انًىنُت نهخعقُد يٍ Introduction Cephalosporins are β –lactam antibiotics obtained originally From a cephalosporium mold.These antibiotics have the same mechanism of action as the Pencillins, but differ in antibacterial spectrum 1 . Cephalosporins discovered by Bortzu 2 in 1948. Cefalexin is the First generation of caphalosporins .The chemical names for cefalexin are :-7-(D – α -aminophenyl acetamido)des-acetoxy cephalosporic acid. 3 Or 7-(D-2-Amino-2-phenylacetamido)-3-methyl- 3-cephem-4- carboxylicacid. 4 The chemical formula is C16 H17 N3 O4 S . H2O . And the chemical structure for the drug : Cefalexin monohydrate Molecular weight: 365.4 gm Melting point: 326.8 0 C Cefalexin is a white to faint yellow powder slightly soluble in water, insoluble in ethanol , chloroform and ether . Anacona et. al 5 prepared complexes for β –lactam antibiotics with some metals, and they identify it by spectroscopic and physio- chemical methods ,also they studiedthe reaction of these antibiotics with some transitional elements like Mn II , Cu II ,Zn II, and confirmed the structures of products using I .R. and N.M.R. spectra. on the other hand, different solutions of cephalosporins and chloride salts of essential and trace elements prepared in a study using different temperatures ( 37˚Cand 60 ˚C) in order to accomplish the complex formation in molar ratio of the drug and metal salts 1:1 6 . Also Abdel Gaber et.al 7 studied the complexes of ions ZnII , CuII, Ca II with cephalosporins potentially they calculated the Molar – Ratio for (Liganed- Metal) at 25 ºC (1:1). 1 Corresponding author E-mail : Am-Ab-Mo @ yahoo . com Received : 16/11/2008 Accepted : 28 / 3/2009 Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 05 Lozano and Borras 8 studied titrametically the complexes of Cefalexin with metals ZnII ,Cd II, they analysis the result by Electrical methods using ( Least squares computer. program Super quad). They prepared complexes of cefalexin like Zn (CEX)2 , 3H2O and Cd (CEX)(OH) H2O , these complexes have been identified by I.R and N.M.R Spectra . In addition Abo El-Maali et-al 9 determined some cephalosporins antibiotic with Cd II or Zn II by using voltimeteric mothed which determined the presence of (liganed - Metal)1:1 like in complexes cefataxime , Cefuroxime and 2:1 like ceftazidime. Moreover, Iqbal and Ahmad 10 prepared complexes of cephalexin with copper II and ZnII these complexes have been characterized by microanalysis and by magnetic and spectroscopic analysis. The complexes, were found five-coordinate monohydrate, and ML 2 .On complexation with copper and zinc the antimicrobial activity of cephalexin improved Significantly. These results suggested that metallic elements should be seriously considered during drug design .Also, Alekseev et al. 11 studied the complex formation in solutions containing Nickel (Ni ++ ) cations,glycine anions (Gly - ),and β-lactam antibiotics, they found mixed-ligand complexes is formed of [ Ni Gly Ampicillin], [ Ni Gly Amoxicillin] ,and [Ni Gly Cephalexin]. Alekseev 12 , in another study, found that the complex formation of Neodymium III (Nd 3+ ) ions with Ampicillin, Amoxicillin and Cephalexin anions (L) in aqueous solution at 20˚C and ionic strength of 0.1 (KNO3) by pH titration was in form of NdL and Nd(OH)L complexes using weak alkaline solution. A new study discussed the spectrophotometric determination of Cephalexin as intact cephalexin or its degradation product, cephalexin was determined in the range (1×10 - 5 -18×10 -5 M) and the limits of detection were 0.3×10 -5 M. These results were compared with reversed- phase HPLC determination, the UV- Vis spectrophotometric method was improved to be selective and reproducible 13 . Instruments , Materials and Method A - Instruments Shimadzu ( AA- 670 ) Flame Atomic Absorption Spectrophotometer . 1. U.V–Visible Spectrophotometer(CARY 100)wave length 200 -1100 nm . 2. PH meter type 60 A . B - Materials All the chemical stock solution were prepared from analytical grade BDH,SDI ,India and Germany . C- Method Stock solution of Fe III A solution of 1000 ppm of Fe +3 was prepared by dissolving 0.2896 gm of Fecl 3 in small amount of Water and complete the volume to 100 ml by using volumetric flask ,. Then 10 ml of the stock solution was diluted to 100 ml with distilled water to Prepare 100 PPMsolution . stock solution of cefalexin (CEX) A solution of 1000 ppm of CEX was prepared by dissolving 0.05 gm of (CEX) in water and complete the volume to 50 ml by using volumetric flask .Then a stock solution of 100 ppm was prepared as above . Spectral study Cefalexin spectrum 10 ml of stock solution (CEX) was transferred to a volumetric flask 5 ml and diluted with distilled water then the Absorbance was measured at 200-600 nm wave length using water as blank . Ferric spectrum 1ml of stock Ferric solution was transferred to a volumetric flask 5ml and diluted with distilled water, then the Absorbance was measured at 200-1100 nm using water as blank. Formation complex with Fe III (2-5) ml from stock solution of (CEX) 1000 PPM was transferred to a volumetric flasks 5ml and 1ml of Fe III 1000 PPM was added .The chelating complexes can be extracted by using 2ml mixture of Methanol + Dimethyl sulphoxide (0.5 :1.5) .The formation of complex was stutied at room temperature The reaction was heated at different temperature with different pH . Results and discussion Drug spectra : measuring (CEX) spectra as in figure (1) we see one Absorption peak at λ g water as blank . Metal spectra : figure (2) for Fe III spectra show Absorption peak at λ water as blank.Chelating complex spectra : CEX- FeIII.Figure(3) for chelating complex show two new peaks,the first at λ 0) nm and the second at (358) nm which indicate the Formation of complex between drug and FeIII using organic solvent as blank. Table (1) show different compounds and the wave length at λmax for each drug and FeIII. Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 05 Fig(1) The UV absorption spectrum for cefalexin Figure(2): The UV absorption spectrum for Fe III solution Figure (3) :The UV absorption spectrum for the complex CEX-FeIII Table(1) Color and λ max for the drug FeIII and complex Optimum condition for complex formation The experimental work showed that the reaction was not proceed at room – temperature so heating was necessary, media must be acidic, for these reasons we study the effect of pH, reaction time , extraction time, concentration of ion Fe III , temperature effect ,suitable solvent for extraction and number of extraction . pH Effect: Figure (4) show the Absorbance of (CEX –Fe III ) using different pH (1-8) the optimum pH for the complixation was (pH:2). Figure(4) : Effect of pH on the absorbanc of CEX-FeIII by using UV spectrum Reaction time It was found that higher Absorbance of extracted complex was happened After 25 minutes, after that the Absorbance decrease due to dissociation part of complex With increase in heating time . Figure (5) , show that 25 minutes is the best time for reaction togive maximum Absorption for ( CEX-Fe III) at 80ºC and pH 2. Color Max(nm) λ Compound White 264 (CEX) Orange 208 Fe III Yellow 341+358 Complex 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 A b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 Wave length 0 1 2 3 4 5 A b s A b s Wave length Wave length Fig. 2 The UV Absorption spectrum for Fe III solution Fig. 3 The UV absorption spectrum for the complex CEX-FeIII Fig. 1 The UV absorption sepctrum for cefalexin 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 A b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 Wave length 0 1 2 3 4 5 A b s A b s Wave length Wave length Fig. 2 The UV Absorption spectrum for Fe III solution Fig. 3 The UV absorption spectrum for the complex CEX-FeIII Fig. 1 The UV absorption sepctrum for cefalexin 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 A b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 Wave length 0 1 2 3 4 5 A b s A b s Wave length Wave length Fig. 2 The UV Absorption spectrum for Fe III solution Fig. 3 The UV absorption spectrum for the complex CEX-FeIII Fig. 1 The UV absorption sepctrum for cefalexin 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 A b s. PH 0 1.3 1.8 2.3 2.8 3.3 Time A b s. 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 A b s. 40 50 60 70 80 90 100 110 Temperature 3.5 Fig. 4 Effect of PH on the absorbanc of CEX-FeIII by using UV spectrum Effect of reaction time on formation of complex CEX-FeIII at PH 2 by using UV spectrum by using UV spectrum Fig. 6 Complex absorption value at defferent temp. Fig. 5 Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 05 Figure(5): Effect of reaction time on formation of complex CEX-FeIII at pH 2 by using UV Spectrum Temperature effect: the reaction of drug with metal was proceed slowly it may exceed hours to increase the reaction velocity we proceed the reaction at different temperature (50-100 ºC ), it was found that 80 ºC is the optimum temperature which gave the higher absorption as in figure (6) . Figure(6) Complex absorption value at different temp. by using UV spectrum Effect of Fe III concentration: The best concentration for Fe III ion is 80 µg /ml, it gave the maximum Absorbance figure (7) show that effect of concentration PPM on absorbance . Figure(7): The effect of Fe III Concentration on complex formation by using UV spectrum Phase Ratio: it was found 5ml of aqueous layer and 2ml of organic layer is enough to get higher Absorbance for complex as in figure (8), after that it was decrease as we increase the volume of organic layer. Figure(8): Effect of phase ratio on absorbance of complex CEX-Fe III at pH =2 by using UV spectrum Suitable solvent : Different organic solvent were used like Benzene, Chloroform ,Acetone Ethanol , Cyclohexanone , Carbon tetrachloride , Methanol , Dimethyl sulphoxide , and Diethyl ether for extraction of complex (CEX-FeIII) , it was found the mixture of Methanol and Dimethyl sulphoxide were the best solvents . Extraction time : The optimum time for shaking during the extraction of the complex was (4 min.) which give maximum Absorption . figure (9) showed 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 A b s . PH 0 1.3 1.8 2.3 2.8 3.3 Time A b s . 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 A b s . 40 50 60 70 80 90 100 110 Temperature 3.5 Fig. 4 Effect of PH on the absorbanc of CEX-FeIII by using UV spectrum Effect of reaction time on formation of complex CEX-FeIII at PH 2 by using UV spectrum by using UV spectrum Fig. 6 Complex absorption value at defferent temp. Fig. 5 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 A b s. PH 0 1.3 1.8 2.3 2.8 3.3 Time A b s. 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 A b s. 40 50 60 70 80 90 100 110 Temperature 3.5 Fig. 4 Effect of PH on the absorbanc of CEX-FeIII by using UV spectrum Effect of reaction time on formation of complex CEX-FeIII at PH 2 by using UV spectrum by using UV spectrum Fig. 6 Complex absorption value at defferent temp. Fig. 5 0 0.3 0.6 0.9 1.2 1.5 1.8 0 20 40 60 80 100 120 140 160 Conc. PPM A b s . 1.5 2 2.5 3 3.5 0 1 2 3 4 5 6 7 VL(ml ) A b s . Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 05 the maximum Absorption in the extraction was 4 minutes of shaking . Figure(9) : Effect of shaking time on the extraction process by using UV spectrum Number of extraction: extraction process for once is enough to extract the complex ,because the second extraction for the remaining concentration give very small Absorbance less than 0.08 ,so one extraction is enough. This illustrate in table (2) Table(2): Effect of number of extraction on absorbance of complex A0 Blank 0.005 A2 (Ex.no.2) 0.085 A1 (Ex.no.1) 2.84 PH 2 Fe III Mg/ml 60 CEX Mg/ml 800 Mole Ratio of complex: determination the mole ratio for complixation it means percent of moles of drug to the moles of metal , different volumes of CEX between (0.5-3.5)ml with constant volume of metal with all optimum conditions of complixation , it was found (1:2) (M:L) . Figure 10 show the relationship between absorbance . and VL / VM .The reaction of FeIII with drug occur in this equation :- FeIII +2CEX FeIII - 2CEX The suggested chemical structure for the reaction of FeIII with CEX. : Fig(10) mole ratio of complex CEX-FeIII by using UV Spectrum Calibration curve of the spectrum : For determination of direct calibration curve for (CEX-FeIII),a different concentration for complex were used as in figure (11) and the relationship between Absorbance and concentration PPM was draw, it was found that the maximum concentration which obey Beer- Lumbert law 200µg / ml . Fig(11) : Calibration curve for CEX-Fe III by using UV spectrum 2.4 2.7 3 3.3 3.6 0 1 2 3 4 5 6 7 Ti me (mi n ) A b s . Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 09 Determination of the drug concentration in pharmaceutical preparation : 6 capsules were mixed with each other then one capsule was weighted , and the Absorbance was measured of its (CEX-FeIII) using optimum condition then from the calibration curve determine the concentration , we carried the procedure for CEX . (SDI ,Ajanta, Germany) .Table (3)show the result of work .From these result , we suggested that the CEX . concentration in Germany capsules is less than the real concentration which is 500 mg while Ajanta and SD Iis O.K. Table(3):The concentration of Cefalexin capsule in 500mg cefalexin of different trade marks Determination of drug CEX – FeIII by using Flame Atomic Absorption Spectrophotometer: To be sure about the result obtained by U.V , we used another technical method, Flame Atomic Absorption Spectrophotometer (FAAS) , by indirect measurement the absorbance of Fe III in the complex to detect the cefalexin conc. as in figure (12) . The complex CEX-FeIII was prepared by using optimum condition of pH , temperature , proper solvent etc . (the same conditions mentioned previously in U.V spectro - photometer) except changing the conc.of ferric ion and phase ratio, it was found the best conc. of FeIII to give maximum absorbance 8 µg /ml , and 5ml of aqueous layer and 3ml of organic layer is enough to get higher absorbance for complex as in figure (13) and (14) .Also we measured the concentration of cefalexin in these pharmaceutical preparations using calibration curve of indirect (FAAS), we got the same result which obtained by U.V method Fig(12) : Calibration curve for (CEX) by indirect FAAS by using UV spectrum Fig(13): Effect of FeIII on complex by using FAAS Fig(14): Effect of phase ratio on absorption of complex by using FAAS Germany Ajanta SDI Capsules 1.29 1.3 1.4 Abs. 487 495 502 Conc.PPM 500 500 500 Standard 0.00 0.02 0.04 0.06 0.08 0.1 0.12 2 4 6 8 10 12 14 A b s. Conc. PPM Fig. 13 Effect of FeIII on complex by using FAAS 2 4 6 8 0.05 0.1 0.15 0.2 0.25 0.3 0 A b s. VL (ml) Fig. 14 Effect of phase ratio on Absorption of complex by using FAAS 0.00 0.02 0.04 0.06 0.08 0.1 0.12 2 4 6 8 10 12 14 A b s. Conc. PPM Fig. 13 Effect of FeIII on complex by using FAAS 2 4 6 8 0.05 0.1 0.15 0.2 0.25 0.3 0 A b s. VL (ml) Fig. 14 Effect of phase ratio on Absorption of complex by using FAAS Iraqi J Pharm Sci , Vol.18 (1) ,2009 Chelating complex of cefalexin with Fe III metal 00 References 1. Andres Goth , M.D.,(Cephalosporins), Medical Pharmacology , 1981 , Tenth Edition, p.641. 2. B.G.Katzung,(The origin of cephalosporin),Basic and clinical pharmacology ; Medical books, MeGraw- Hill ; Newyork 2001 , 8 th ed; p . 762-766. 3. Merck and Co ; Whitehouse station. N.J.USA; The Merck Index, 2006 , Fourteen edition, p.1974. 4. USP 27 – NF 22, Pharmacopeia, 2004, p.393. 5. J.R.Anacona and I.Rodriguez , (Synthesis and antibacterial activity of cephalexin metal complexes ),J.Coord . Chen ., 2004 ,57 (15), p. 1263-1269. 6. 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