Spectrophotometer Determination of Cefixime in pure form and pharmaceutical preparation by Using Cloud point Extraction

Two simple methods spectrophotometric were suggested for the determination of Cefixime (CFX) in pure form and pharmaceutical preparation. The first method is based without cloud point (CPE) on diazotization of the Cefixime drug by sodium nitrite at 5Co followed by coupling with ortho nitro phenol in basic medium to form orange colour. The product was stabilized and measured 400 nm. Beer’s law was obeyed in the concentration range of (10-160) μg∙mL -1 Sandell’s sensitivity was 0.0888μg∙cm -1 , the detection limit was 0.07896μg∙mL -1 , and the limit of Quantitation was 0.085389μg∙mL -1 .The second method was cloud point extraction (CPE) with using Trtion X-114 as surfactant. Beer’s law was obeyed in the concentration range of (10-160) μg∙mL -1. Sandell’s sensitivity was 0.1470μg∙cm -1 , the detection limit was 0.06680μg∙mL -1 , and the limit of quantitation was 0.07293μg∙mL -1. All variables including the reagent concentration, reaction time, colour stability period, and mole ratio were studied in order to optimize the reaction conditions. The composition of product (1:1). The methods were effectively useful to the determination of Cefixime in pharmaceutical dose form, and the attained results were in good agreement with the official result and other methods in literature .No interference was observed from the commonly encountered additives and excipients Key word: Cloud Point Extraction, Cefixime, Diazotization, Orthonitrophenol, Triton X-114. Introduction: Sulfa drugs were the first antibiotics used regularly and paved the way for the revolution of antibiotics in medicine. The first sulfonamide, named Prontosil (red dye), was discovered in 1932 by Gerhard Dumagk (1) .Antibiotics are the chemotherapeutic agents to inhibit the microorganisms growth.The chemical agents were used to treatment the disease by destroy pathogenic microorganisms orInhabitation their growth at concentration low enough to shun undesirable damage to the host.Antibiotics are drugs measures which have some chemical substance that are produced by microorganisms and by chemical synthesis. These substances at very low concentrations are well-known to totally raze or partly inhibit microorganisms. Antibiotics include broad spread application in the cure of bacterial disease (2). Department of Chemistry, College of Science for Woman, University of Baghdad, Baghdad, Iraq. * Corresponding author: assrasalam@yahoo.com * ORCID ID: https://orcid.org/0000-0002-6384-3967 Cefixime :.an antibiotic that belongs to the third generation of cephalosporin and is taken orally to treat bacterial infections, including pharyngitis, middle ear, sore throat and urinary tract infection. It is approved for medical use in 1989. Over-all characterizes of Cefixime are given in Table 1 (3). Table 1. General properties of Cefixime (CFX).

Uncharacteristic cloud point extraction needs the subsequent step:Taking (10mL) of volumetric flask having the optimal conditions for diazotization and coupling reaction of [CFX] gotten from first batch with [10%(v/v) surfactant ] then ending it to the blot by ethanol and the comfortable of volumetric flask transmission to centrifuge test tube . The mix is shuddered for 1min and left in thermos bath at 60 C o for 20 min, then detached by centrifuge at 4000rpm for 20 min. Test tube was set in ice bath to rise thickness micelles coat , at that point the informal detached. The outstanding micellar was softened by 1mL ethanol later that the absorbance is unhurried spectrophotometrically UV-VIS at maximum wavelength.

Sample
Preparation of Pharmaceutical Determination Cefixime: A process on medication Cifixime has been useful, the production company is [Pharma International Co.Amman. Jorden]. Five mL have been taken from drug in volumetric flask 100mL and the volume is completed by distilled water, so it is given (1000 µg mL -1 ) from CFX.

Result and Discussion:
First methods: Spectrophotometric determination of sulphadimidine sodium (SDMS) by oxidation coupling reactions. Optimization Parameters for Reaction.
All of the factors that affect the absorbance of formation of azo dye product are optimized to improve the sensitivity and detection limit for the determination of the drugs. All optimization work under wavelength at 400 nm is shown in Fig. 1.

Effect of Acid Type
In this study, using 1mL of (0.5M) from different acids [HCl, H 2 SO 4 , HNO 3 , H 3 PO 4 and CH 3 COOH] and added [1mL of CFX(100 μg•mL -1 ), 1 mL of each acid, 1mL of NaNO 2 , 1mL of H 3 NSO 3, 1mL ONP and 1mL of NaOH] in 10 mL of volumetric flask and complete the volume by distilled water to form diazonium salt(azo dye). Then the absorbance was measured at 400 nm, the resulting absorbance is shown in Table 2. It is clear from this study that the hydrochloric acid gave a higher absorbance, This acid is used in subsequent experiments, as shown in the Tabl 2.
Effect of Optimum Volume of 0.5M of acid.
The same addition is done with CFX[1mL CFX, with varying volumes of 0.5M HCl from (0.1-1) mL ,1 mL NaNO 2 , 1mL H 3 NSO 3 , 1mL ONP and 1mL of NaOH] in 10 mL volumetric flask and complete the volume by distilled water. Then, the absorbance and the optimum volume were measured for higher absorbance fixed for sequence experiment. The resultant absorbance is shown in Table 3. It is obvious that absorbance increased with increasing the acid volume, suddenly the absorbance decreased because the primary amine became inactive (4). The optimum volume for higher absorbance was fixed in subsequent experiments (for HCl with CFX) which affects the composition of diazonium salt( azo dye).

Base Type.
In this experiment different basics have been used [NaOH, KOH, K 2 CO 3 , Na 2 CO 3 , NH 4 OH, NaHCO 3 ] and that followed the addition [1mL CFX, 0.8mL HCl, 1mL NaNO 2 , 1mL H 3 NSO 3 , 1mL ONP and 1mL of each base(1M) ]in volumetric flask 10 mL and completed to the mark by distilled water .The absorbance was measured and the absorbance results are shown in Fig. 2 .

Figure 2. Data of absorbance for different bases with CFX
It is clear that Sodium hydroxide gave the higher absorbance, Therefore, this base was fixed in subsequent (5) show in Fig. 2.

Optimum Volume of 0.5M [NaOH].
The same addition was done for CFX in 10 mL volumetric flask and the volume was completed by distilled water. Then the absorbance and the optimum volume were measured for higher absorbance and fixed for sequence experiment. The absorbance results are shown in Table 4. It is evident that absorbance increased with increasing the volume of NaOH, but suddenly it decreased because the decomposition happened when increasing the volume of NaOH and formation of diazotate ions may form coupling. This shows agreement with previous studies (6). The optimum value of 1 mL for NaOH is with CFX . Optimum Volume of 1% Sodium Nitrite.
The same additions were [1mL for CFX,0.8 mL HCl, with varying volume of 1% NaNO 2 from (0.1-1) mL, 1mL H 3 NSO 3 , 1mL ONP and 1 mL NaOH] in volumetric flask 10 mL and complete d to the mark by distilled water. Then the higher absorbance of optimum volume was fixed for sequence experiment as shown in Table 5. It is clear in from Table 5 that the absorbance increased with increasing the volume of NaNO 2 , but the absorbance decreased because the increase of nitrate concentration causes the decomposition of diazonium salt (7). The optimum value of Sodium Nitrate 0.8 mL is with CFX.

Effect of Optimum Volume of 1% Sulphamic Acid.
The additions for experimental were [1mL for CFX,0.8 mL HCl, 0.8 mL1% Na 2 NO 2 with varying volume of 1% H 3 NSO 3 from (0.1-1) mL,1mL ONP and 1 mL NaOH] in a volumetric flask 10 mL and the volume was completed by distilled water. Then the higher absorbance of optimum volume was fixed for sequence experiment. Table 6 shows the data of the absorbance. It clear from this Table that the absorbance increased with increasing the volume of Sulphamic acid, but the signals decreased suddenly because this volume removed nitrite and released nitrogen gas (8).The optimum volume of Sulphamic acid was 0.2 mL.

Effect of Optimum Volume of (100 µg mL -1 ) Reagent.
The same additions are [1mL for CFX, 0.8 mL HCl, 0.8 mL 1% NaNO 2 ,0.2mL H 3 NSO 3 , with varying volume of (100 µg mL -1 )ONP from (0.1-1) mL and 1 mL NaOH] in 10 mL of volumetric flask and the volume was completed by distilled water. Then the higher absorbance of optimum volume at maximum wavelength is fixed for sequence experiment as shown in Table 7. The absorbance increased with the reagent volume increase until 0.6(0.5) mL which had a decrease in absorbance. Therefore 0.5 ML was chosen as optimum volume of the regent for drug coupling.

Effect of Reaction Time on Stability Colour Product.
The optimum volumes of parameters were completed [1mL for CFX, 0.8 mL HCl, 0.8 mL 1% NaNO2,0.2 mL H3NSO3, 0.5 mL ONP and 1 mL NaOH]. The time on stability colour of product is one of the most important factors to Cloud Point Extraction and diazotization. So, this factor through time (0-60) min needs to be studied. Then, absorbance was measured and the higher absorbance at maximum wavelength was fixed as show in Table 8.

Effect of Order Addition.
When completing the volume of parameter, the sequence of addition with optimum volume needs to be studied but in a different order . The effect of order addition is shown in Table 9. The optimum order addition is [ D+H+N+S+R+B]

, B=Base(NaOH), R=Reagent(O-Nitro Phenol).
At the maximum wavelength the absorbance is measured, and the higher absorbance was fixed for the best order addition as displayed in Table 9.

Effect of Solvents.
All additions of diazotization and coupling reaction were done for CFX with optimum condition. This was followed by diluting with polar solvent [water, ethanol, methanol,1-propanol ,acetonitrile & acetone ] in volumetric flask 10 mL. At maximum wavelength, the absorbance was measured and recorded for the best solvent. The effect of absorbance is shown in Table 10. The data shows that ethanol is the best solvent as shown in Table 10 .

Effect Temperature in the Formation of Colour Product and Stabilization.
The conclusion of different temperatures on colour product have been studied from (5-60)C 0 .
And the rest of addition are optimal settings then dilution done with distilled water except for the CFX dilution which is by ethanol in volumetric flask 10 mL. Then absorbance was measured at the maximum wavelength Table 11. The best temperature (15C 0 & 20 C 0 ) was the greatest absorbency for CFX , on the other hand when the temperature rises the absorbency , it starts dissociation of product and can be noticed from strength of color. The results are in arrangement with literatures (9), and this temperature is stable in later experiment. (10).

Stoichiometric Determination of Product. Continuous Variation Method
The conformation of the azo dyes product ratio is supported by using the slope analysis method. In this method,the absorbance is planned against

Mole Ratio Method.
Mole Ratio Method is useful to study the coloured of product (11) .In this method the volume of drug is constant in 1 mL with

Absorbance [R]/[R]+[D]
Open concentration (6x10 -4 M) for CFX and concertation of ONP is [6X10 -4 ] M in volumetric flask 10 mL, the rest of addition was optimum conditions and the volume was completed by ethanol. That followed the absorbance which is measured at the maximum λmax=400 nm for CFX. The stoichiometric ratio between drug with reagent 1:1 results are displayed in Fig.4

Calibration Curve for CFX-ONP:
Aliquots of 10 mL solution is prepared ,having increasing concentration of CFX [varying volume of CFX (0.1-1.6 mL) with concentration (10-160 µg mL -1 ), 0.8 mL HCl, 0.8 mL 1% NaNO 2 ,0.2mL H 3 NSO 3 , 0,5 mL ONP and 1 mL NaOH]. The volume was complete by ethanol, then the absorbance was measured at maximum wavelength against a blank solution able under alike condition without drug. Linear calibration graph was plotted by scheming absorbance against concentration of CFX in figure 5 the Concentration (10-160) µg mL -1 obeys the Bear law as shown in Fig. 5 .

Effect of Interference.
The effect of interference ordinary was present in [CFX] to identify the method fussiness under learning by addition 1mL (1000 ppm) from interference with 1mL (1000 ppm) from CFX 1mL (1000μg•mL -1 ). It necessity lies in the size of interference which is lesser for a sample to limit the dilution of sample and use using the maximum concentration probable in the sample (12). The results are shown in Table 12. The results in this tables that there were displayed +no interference to present with drug in pharmaceuticals.

The Stability Constant of Coloured Product.
Dependent on two conducts, mole ratio and continuous variations methods revealed formerly, the composite product is[D: R] [drug: reagent] in the result is 1:1 as in the following equation.

K= [DR] / [D][R]
K= 1α/ α 2 c The stability constant K (13). It is clear the stability constant is high, so the dye formed is very stable display in Table 13.  It is clear that the stability constant is high, so the dye formed is very stable . Table 14 displays the accuracy and precision of CFX correspondingly. It is obvious that the results from this method have good accuracy and precision (14).

Applications of the Proposed Method on Pharmaceuticals.
The suggested method has been applied on pharmaceutical for CFX. Similar method was applied on Syrup Cefixime, the manufacture company is [Pharma International Co. Amman.
Jorden] that contains (200mg) in 100 mL and the sample is prepared in accordance with the preparation of pharmaceutical. The results are good and of great dependability in the analysis of samples in the pharmaceutical preparation. The results are shown in Table 15 for CFX.  Table 16. It is clear from the results above that surfactant Triton X-114 increases the absorbance and efficiently of cloud point extraction (15).

Effect of Triton X-114 Volume.
Sum of 10 mL solution is primed in 10 mL volumetric flask and custom changing volumes of 10% (v/v) Triton X-114 (0.2-2) mL , then the volume was completed by ethanol , and heated at 60 C 0 for 20 min to practice cloud point then centrifugation at 4000 rpm for 20 min. The surfactantopulent phase was softened by 1mL ethanol then the absorbance was measured at maximum wavelength at λmax = 400 nm. These results are displayed in Table 17. It can be noticed from the result above that the absorbance rises with the optimum volume of Triton X-114 but unexpectedly drops at higher amount. In conclusion, the volume of surfactant effected on the effectiveness of extraction and the enrichment factor (16). This can be explained due to the effect of surfactant best volume of Triton X-114 (1.4 mL) for CFX individually stable in following experimentations to complete high extraction efficiency.

Effect of Equilibrium Temperature:
Chains of solution are set in volumetric flask 10 mL and the volume was completed by ethanol, the temperature was varied from (35-65)C 0 and the incubation time ranged from (5-35)min for all drug. At the maximum wavelength the absorbance was measured and recorded. These results are displayed in Table 18. It is shown that the maximum absorption pointer of target drug is completed at (50) ºC for the azo dye product because the great number of micelles designed in cloud point layer important to the total transfer of the azo dye product into surfactant-rich phase that makes the most of the sensitivity (17) .

Effect of the Incubation Time:
Chains of solution are set in 10 mL volumetric flask and the volume was completed by ethanol, the temperature was 50 0 C for CFX and the incubation time from (5-35) min for all drug. At the maximum wavelength 400 nm, the absorbance was measured and the recorded result is shown in Table 19. CPE needs enough time to make balance between aqueous phase and surfactant-rich phase by more accumulation the micelles. This time signifies the amount of high temperature accumulated in the solution that lets Micelles drop water molecules in order to give small size hydrophobic with high viscosity easily entrap the product in it. It is perfect that the best incubation time is (20)min is carefully chosen to provide high extraction efficiency and no increases detected for longer time (18) .

Preparation of Calibration Curve in CPE.
Chains of solution are [changing volume of CFX (0.1-1.6mL) with concentration (100-160) g mL -1 ), 0.8 mL HCl, 0.8 mL 1% NaNO 2 ,0.2mL H 3 NSO 3 , 0,5 mL ONP, 1 mL NaOH and 1.4 mL 10%(v/v) Triton X-114] in 10 mL volumetric flask and full the volume by ethanol , at the best temperature and incubation time are heated in water bath to configuration cloud point and separated by centrifuge at 4000 rpm for 20 min as shown in Fig.6 .

Figure 6. (CFX+CPE) Calibration Curve
Accuracy and Precision: it can be noticed from the results below that the technique has good accuracy and precision as a significance of recovery rate which is 100.8182 % for CFX Table 20.  Table 21 for CFX

Conclusion:
Cloud point extraction demeans calm, safe and useful pre-concentration technique to determine Cefixime by UV/VIS. The planned method is a effective, selective and gives good RSD and low limit of detection.