Cloud point extraction of Cefixime drug by direct (UV- Vis) spectrophotometer and indirect(Flame Atomic Absorption) technique

A new method for the determination of the Cefixime drug in some Pharmaceuticals using ultra violet-visible (UV- Vis) and indirect Flame Atomic Absorption Spectrophotometry (FAAS) by Cloud point Extraction by using Triton X-114 as surfactant, the method based to form chelating complex CEF – Pd (II) at 456nm, variables parameters were studied such as the concentration of metal ion, effect of pH, Triton X-114 amount, equilibration temperature and incubation time. The best pH for the formation of chelating complex was (9). The best temperature on cloud-point extraction was 55 0C at 20 min. then complex extracted with ethanol. The mole-ratio method has been used to determine the structure of chelate CEF – Pd (II) and found to be 1:1 L:M (Ligand : Metal). Beer’s Law was obeyed in the range 2.5-30 and 2.5-32.5μg/mL for UVVis and FAAS respectively. Limit of Detection and Limit of Quantitation LOD and LOQ values for these methods were (0.456592, 0.33374) μg/mL (1.521976, 1.112471) μg/mL respectively.the method was validated and successfully applied to drug formulations like Cefix capsules marketed in Iraq. The results of analysis have been validated statistically and by recovery studies and were found satisfactory


1.
Antibiotics are the chemotherapeutic agents that kill or inhibit the growth" of microorganisms. These chemical agents are used to treat disease by destroying pathogenic microorganisms or inhibiting their growth at concentration low enough to avoid" undesirable damage to the host. Antibiotics are drugs preparations which contain some chemical substances that are produced by microorganisms and by chemical synthesis. These substances at very low concentrations are known to totally destroy or partially inhibit microorganisms . Antibiotics have wide spread appli-cation in the treatment of bacterial disease " [1] Cefixime is Chemically, (CEF) (6R, 7R)-7-[2-(2-amino-4-thiazolyl) glyoxylamido]" -8-oxo-3-vinyl-5-1-azabicyclo [4.2.0] oct-2-ene-2carboxylicacid, 7-9z)-[o-carboxymethyl)-oxime] trihydrate. Molecular formula of Cefixime is C 16 H 15 N 5 O 7 S 2 , molecular weight (453.45 g/mol), It is third generation cephalosporin antibiotic. It is under the category of -Lactam Antibiotics, Cell Wall inhibitor. It acts by inhibiting an enzyme Transpeptidase, involved in the building of Bacterial Cell Walls. It is used in Lower Respiratory Tract Infections, Acute Urinary Tract Infections", Biliary Tract Infections, Sinusitis, Acute Otitis Media, Peptic ulcer [2] " It is used to treat or prevent infections that are proven or strongly suspected to be caused by bacteria. One of the major problems with this drug is its very poor solubility in biological fluids that results" into poor bioavailability after oral administration. It shows erratic dissolution problem in gastric and intestinal fluid due to its poor water solubility. Rate of absorption and/or extent of bioavailability for such insoluble drugs are controlled by rate of dissolution in gastrointestinal fluids " [ 3] The structures of drug are shown in Figure.  The cloud point procedure" (CPE) is based on the following phenomenon: an aqueous solution of some surfactant becomes turbid and separation to two isotropic phases if some condition such as temperature or pressure is changed or if an appropriate substance is added" to the solution [5]"which describes a liquid chromatographic method for its assay in bulk form. In order to assure the quantity"" of cefixime in dosage forms, several methods have been reported which include liquid chromatography-mass spectrometry " [6] ,high performance liquid chromatography [7][8][9][10] ,high performance thin layer chromatography " [11][12]""derivative spectrophotometry " [13] , voltammetry [14] , and capillary electrophoresis " [15] .

Interference Solutions of 1000 ppm
An amount of 1000 g mL -1 stock solution of interferences is prepared by dissolving 0.1g of the different organic compound such as [Lactose, Starch, Arabic Gum, Glucose and Talc] and inorganic compound such as [0.2579g, 0.2500g] of Ca 3 (PO 4 ) 2 and CaCO 3 respectively in distilled water and diluting them to the mark in 100 ml volumetric flask.

General procedure for CPE by UV-VIS Method
Aliquots 10 ml of a solution containing known amount of Cefixime drug was mixed with 1ml of 250 g mL -1 Pd +2 metal ion Then pH was adjusted by using 0.1M NaOH and 0.1 HCl then added 1mL of 10% (v/v) Triton x-114. The mixture was shaken for 1 min and left to stand in a thermo-stated bath at 50 o C, for 10 min. Separation "of the phases was achieved by centrifugation at 3000 rpm for 10 min, Test tube taken in ice bath to increased viscosity micelles layer for 1min. then become easily separated was dissolved by 1mL of ethanol, the measurements of absorbance of the complex were followed by UV-Vis spectrophotometer with used 1.0 cm quartz cell to get max for CFX -Pd (II) " complex against blank which was prepared in the same way but without drug" .

2. Optimization of CPE Methodology
"A group of experiments has been conducted to study the effect of several variables that affect the extraction efficiency of the CPE and maximize the sensitivity of the detection system for drug under study using a classical optimization. The variables such as the concentration of metal ion , best of pH ,best of buffer ,best of volume buffer, Triton X-114 amount, equilibration temperature and incubation time".

2.1. Effect of metal ions concentration
The effect of palladium ion concentrations was studied by measuring the absorbance values according to the geranial procedure containing 1mL of 250 g mL -1 Cefixim, 1mL of 10% (v/v) Triton x-114 of and varying volumes of the250 g/mL Pd (II) ranged from (0.2-1.8) mL .The optimum volume of the metal ions that gave maximum absorbance was 1.2 mL as shown in Figure 3 .this volume was used throughout this study. The

Effect of pH
The pH plays a unique role on metal-ligand formation and subsequent extraction, and is proved to be a main parameter for CPE [16]. Set of similar experiments in the pH range of 2-14 were accomplished according to the described procedure The maximum sensitivity and the best separation for the complexes drug-Pd (II) was obtained at pH 9 .The results are shown in Figure  4 and table1.  6 It was shown that the sensitivity increase at pH 9. At higher pH, the drug is deprotonated and it behaves like a hydrophilic molecule and easily gets solubilized in the micelles while at lower pH, the drug is protonated and its ionic characteristics increase and led to decrease in its solubilization in the hydrophobic micelles.

Effect of buffer solutions
The effect of buffer solutions tris (hydroxymethyl) aminomethane and Sodium tetra borate (borax) with acidic function 9 was studied on the intensity of complex formation CEF+Pd(II) The best intensity values of buffer Sodium tetra borate (borax) as shown in table 2.

2.4.Effect of Volumes buffer solutions
Figure (5) shows the value of absorbance intensity for the CEF-Pd +2 complex against the value of volumes of Sodium tetraborate (borax) buffer solutions. It is evident that absorbance increase with increase the volume of buffer up 1.2 mL, but suddenly decrease the absorbance because the decomposition happen when increase basicity. 1.2 mL was selected for the subsequent  at 50 0 C for 10 min incubation time then it centrifuged at 3000 rpm for 10min , separated the surfactant-rich phase and dissolved in 1ml ethanol then measured by UV-Vis at max = 456nm for CEF -Pd(II) complex the results shown in Table 3 . Table 3. Data of absorbance of type of surfactant It was observed that Triton X-114 which has maximum absorbance at 456 nm .It is clear from the results that the nonionic surfactant Triton X-114 is of high absorbance and the surface increases of the efficiency of the cloud point extraction.

Effect of Triton X-114 Amount
Most studies confirm that the amount of an nonionic surfactant type Triton X-114 as an extracting medium plays an important role for maximizing the extraction efficiency by minimizing the phase volume ratio (Vs/Va) and therefore improving the pre-concentration factor of the CPE procedure, Therefore, the amount of Triton X-114 was investigated by varying the volume of 10% Triton X-114 between (0.2-1.6 mL) for CEF -Pd(II) complex. The results are presented in Figure 7. It was noticed that the absorbance values of CEF drug continued to increase dramatically and reached maximum at 1.2mL of 10% Triton X-114. These values were selected as optimal amount and used in the proposed methods for the detection of CEF. Plotting the absorbance values of the cloud point versus the volume of Triton X-114 is shown in Figure 7 Figure 7: Effect of the Triton X-114 amount on absorbance of CEF-Pd (II ) complex

Effect of the Equilibration Temperature and Time
In order to optimize the method, it was necessary to examine the effect of the temperature on cloud-point extraction. Temperature that enhances higher range of (30 -70)°C and (5 -35)min, respectively, while keeping all other parameters constant. Excellent absorbance was found at temperature 55 0 C as shown in figure 8 and table 4, therefor choose 55 0 C, higher than 55 0 C is probably due to the decomposition of the complex.

8. Order of Additions
The effect of order for additions of the metal on the absorbance of each analyte by the general CPE was tested. Figure 10 and Table 5 show that the best order of addition is the number 1 for target analytes due to giving a highest absorption signal among the others. It is noted that the best addition is the first order of complex CEF-Pd (II ) because if it's another order gets lost in the intensity of color and this order fixed in subsequent experiment

9. Effect of organic solvents
Different organic solvents are examined to evaluate their effects on the intensity of the resulting complex and the data are shown in Table 6 and figurer 11. It has been shown that water is the optimum solvent, economically, sensitivity method, cheap price, to provide and nontoxic. This solvent is fixed in subsequent experiment

.Effect of Interference
The "effect of some foreign organic compounds and Inorganic compounds which tabulated in table 7 were studied by adding 1mL of (100ppm) to the CEF-Pd (II) complex. The intensity of was developed follow the recommended procedure described earlier". It was observed from the table 6 and figure12 were not interfering with the determination at levels found in complex form.

.Selected Optimum Conditions
After the study of the effect of different optimum conditions on the absorbance intensity of the colored product, The optimum conditions for the proposed procedure were summarized in Table  7 and were used in all subsequent experiments.

Preparation of Calibration Curve of CPE
Amount of 10ml solution is prepared containing increasing concentration of drug Cefixime by taking (2.5-30 ) g mL -1 ,1.2mL Pd , 1.2mL buffer pH 9 and 1.2mL 10%(v/v)Triton X-114 then it is completed to the mark by distilled water, are mixed ,heated at optimum temperature in the thermostat water bath at optimum incubation time , to form cloud point then aqueous phase is separated by centrifugation at 3000 rpm for 20min ,1ml ethanol is added to the surfactant-rich phase to dissolve it then is measured by UV-Vis at max = 456 nm for palladium , triplicate manner The absorbance measurements are illustrated in table 9 .The calibration curve was .Plotting the mean absorbance values of the cloud point versus the concentration (ppm) of (CEFpalladium) as shown in Figure 13 .

Applications of the Cloud Point Extraction on Pharmaceuticals.
CPE has been applied on pharmaceutical Cefixime, the manufacture company [Novartis ] that contains (533.9mg) from Cefixime .The results are good and of high reliability in the analysis of samples in the pharmaceutical preparation. The results are summarized in the table 11 .

Determination of drug CEF-Pd(II )using Flame Atomic Absorption Spectrophotometer
To be sure about the result obtained by UV-VIS, we used another technical method, Flame Atomic Absorption Spectrophotometer (FAAS) , by indirect measurement the absorbance of Pd(II) in the complex to detect the cefixime conc. as in figure 15 . The complex CEF-Pd(II) was prepared by using optimum condition of pH , temperature , proper solvent etc . (The same conditions mentioned previously in U.V spectrophotometer) except changing the conc. of palladium ion, it was found the best conc. of Pd(II) to give maximum absorbance 25 μg /mL of organic layer is enough to get higher absorbance for complex as in figure 16 .Also we measured the concentration of cefixime in these pharmaceutical preparations using calibration curve of indirect (FAAS), we got the results close which obtained by UV method Figure 15 show the effect of Palladium ion volume upon the absorbance values of the extracted complexes using ( 250 g/mL) of drug solution . The optimum volume of the metal ions that gave maximum absorbance was 1mL of Pd( II )for the complex , the absorbance is measured and the absorbance results are shown in table 12 .

Preparation of Calibration Curve for CEF
In order to test the linearity of the method and under the optimized conditions established by CPE procedure, Calibration graphs were established by plotting absorbance versus concentration of Cefixime . of The calibration curve was .Plotting the mean absorbance values of the cloud point versus the concentration ( g mL -1 ) of (CEF-palladium) as shown in Figure 16

Comparison between the two methods of the proposed method
A simple comparison were make between the methods UV-Vis and AAS for determination of Complex and the calculation statistically parameter were illustrated in Table 14. The first and second methods characterized by simplicity , highly economic accurate and apply the green chemistry requirement two method characterized by high selectivity economic and not used any harmful chemicals .From the opinion of scientist analyst ,The first method prefer it because the absence of any interferences which may appear in the UV-Vis region and the best statistically calculation parameter. The proposed method was compared successfully with other literature methods and demonstrates which is the development of an excellent spectrophotometric method for the determination of cefixime drug , rapid, precise, high selectivity, and sensitive than other spectroscopic methods in the literature for the complex product of cefixime as shown in table 15. This method was successfully applied on pharmaceutical samples.

Conclusion
The proposed method is simple, sensitive and free from drastic experimental conditions such as heating. It is also accurate, precise enough to be successfully adopted as an alternative to the existing spectrophotometric methods and evaluation of cefixime in an metal Using CPE and in pharmaceutical Preparation samples determination Pd (II ) in some Pharmaceuticals ,the method gives a very low limit of detection and green chemistry