Analytical method development and validation of spectrofluorimetric and spectrophotometric determination of some antimicrobial drugs in their pharmaceuticals

Accepted Manuscript Analytical method development and validation of spectrofluorimetric and spectrophotometric determination of some antimicrobial drugs in their pharmaceuticals

F. Ibrahim, M.E.K. Wahba, G. Magdy PII: DOI: Reference:

S1386-1425(17)30597-8 doi: 10.1016/j.saa.2017.07.033 SAA 15320

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

29 January 2017 13 July 2017 20 July 2017

Please cite this article as: F. Ibrahim, M.E.K. Wahba, G. Magdy , Analytical method development and validation of spectrofluorimetric and spectrophotometric determination of some antimicrobial drugs in their pharmaceuticals, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.07.033

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Analytical method development and validation of spectrofluorimetric and spectrophotometric determination of some antimicrobial drugs in

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their pharmaceuticals

Department of pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura

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F.Ibrahim1, M.E.K.Wahba1*, G.Magdy2

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University, 35516, Mansoura, Egypt. Department of pharmaceutical Chemistry, Faculty of Pharmacy,

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Delta University for Science and Technology, Gamasa, 35712, Egypt.

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*Corresponding author: M.E.K.Wahba E-mail: [email protected] Tel & Fax: +20/050/2247496

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Abstract In this study, three novel, sensitive, simple and validated spectrophotometric and spectrofluorimetric methods have been proposed for estimation of some important antimicrobial drugs. The first two methods have been proposed for estimation of two important third-generation cephalosporin antibiotics namely, cefixime and cefdinir. Both

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methods were based on condensation of the primary amino group of the studied drugs with

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acetyl acetone and formaldehyde in acidic medium. The resulting products were measured by spectrophotometric (Method I) and spectrofluorimetric (Method II) tools. Regarding

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method I, the absorbance was measured at 315 nm and 403 nm with linearity ranges of 5.0140.0 and 10.0-100.0 µg/mL for cefixime and cefdinir, respectively. Meanwhile in method

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II, the produced fluorophore was measured at λem 488 nm or 491 nm after excitation at λex 410 nm with linearity ranges of 0.20 -10.0 and 0.20 -36.0 µg/mL for cefixime and cefdinir,

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respectively. On the other hand, method III was devoted to estimate nifuroxazide spectrofluorimetrically depending on formation of highly fluorescent product upon

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reduction of the studied drug with Zinc powder in acidic medium. Measurement of the

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fluorescent product was carried out at λem 335 nm following excitation at λex 255 nm with linearity range of 0.05 to 1.6 µg/mL. The developed methods were subjected to detailed validation procedure, moreover they were used for the estimation of the concerned drugs in

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their pharmaceuticals. It was found that there is a good agreement between the obtained

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results and those obtained by the reported methods.

Keywords: Cefixime; cefdinir; Nifuroxazide; Hantzsch reagent; Spectrophotometry; spectrofluorimetry.

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1. Introduction Cefixime (CFX) and cefdinir (CFD) are oral third-generation cephalosporins. They are bactericidals with a broad spectrum of activity. They are stable to hydrolysis by many beta-lactamases and have higher activity than first- or second-generation cephalosporins against Gram-negative bacteria. However, cefdinir is reported to be much more active in

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vitro than cefixime against Staphylococcus aureus, but not meticillin-resistant strains, and

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it is less active against some Enterobacteriaceae [1].

Cefixime is designated chemically as (6R,7R)-7-[[2-(2-amino-1,3-thiazol-4-yl)-2-

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(carboxymethoxyimino)acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2ene-2-carboxylic acid [2] (Fig.1A).

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While cefdinir is designated chemically as (6R,7R)-7-[[(2Z)-2-(2-amino-1,3-thiazol-4-yl)2-hydroxyiminoacetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-

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carboxylic acid [2] (Fig.1B).

Nifuroxazide (NX) is an intestinal nitrofuran antibiotic with a broad spectrum which used

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designated

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infections[1].It

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for the treatment of colitis and diarrhoea. It is also used to manage skin and urinary tract chemically

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4-hydroxy-N-[(E)-(5-nitrofuran-2-

yl)methylideneamino]benzamide (Fig.1C) [2]. Literature survey revealed different methods for estimation of CFX, CFD and NX whether

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alone or in combination with other drugs. As for CFX, it has been determined by:

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spectrophotometry [3-6], spectrofluorimetry [7-9], HPLC [10-13], HPTLC [14, 15], LC/MS [16, 17], high-performance capillary electrophoresis [18, 19] and stripping voltammetry [20, 21].

While CFD has been estimated by : spectrophotometry [22-24], spectrofluorimetry [25, 26], HPLC [27-30], LC/MS [31, 32] and stripping voltammetry [33]. From literature, up till now no spectrophotometric or spectrofluorimetric methods have been reported for estimation of the CFX or CFD using Hantzsch condensation reaction which encouraged us to develop the first two methods.

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When comparing the developed methods for quantitation of CFX with the previously reported method [5] which depends on spectrophotometric determination by formation of a complex with Cu(II) at pH = 7.8 in acetate buffer, It was found that the developed spectrophotometric method has wider linearity range and the developed spectrofluorimetric method has higher sensitivity and wider linearity range.

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For Cefdinir, the developed methods are more simple, rapid and with wider linearity range

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than the reference method [25] which depends on spectrofluorimetric determination of CFD in 1M sodium hydroxide at 95°C for 1 hour.

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Also, NX has been estimated by various methods including spectrophotometry [34-36], spectroflourimetry [37, 38], HPLC [35, 39], polarography and stripping voltammetry [40,

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41].

In spite that two spectrofluorimetric methods [37, 38] have been reported to determine NX,

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we were encouraged to perform this study since it carries many advantages over the previously reported methods mentioned in literature, such as simpler procedure, wider

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linearity range and higher sensitivity. All these criteria of the proposed method make it a

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candidate for routine work in quality control laboratories. Reduction of nitro group to amino group as a tool to develop fluorescence of many pharmaceutical compounds was reported in many research articles [42, 43]. By analogy to these studies; fluorescence of NX could

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be obtained through a simple reduction procedure.

2. EXPERIMENTAL 2.1. Instruments

- A Shimadzu UV-Visible 1601 PC spectrophotometer (Kyoto, Japan) P/N 206-67001. One cm cell of quartz was used for measurements. - A Perkin-Elmer UK model LS 45 luminescence spectrometer, equipped with a 150 Watt Xenon arc lamp. Quartz cell 1 cm was used (for Method II).

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- Shimadzu RF-1501 Spectrofluorimeter with an emission slits set at 5 mm and Xenon arc lamp was utilized for excitation. One cm cell of quartz was used for measurments, Japan (for Method III).

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- A Jenway 3503 digital pH meter (Stone, Staffs, UK) was used for pH adjustment.

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2.2. Materials

- Cefixime (Batch # KCI03140046) sample was obtained from Sigma Pharmaceutical Co.,

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Cairo, Egypt.

- Cefdinir (Batch # CDRN150004) sample was supplied by Hikma Pharma Co., 6th of

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October City, Egypt.

- Nifuroxazide (Batch # 20090301) sample was supplied by EVA Pharmaceutical

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Company, Cairo, Egypt.

- Suprax® capsules contain 400 mg of cefixime/capsule, batch # 1520160, Kahira for

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Pharmaceutical and chemical industries, Cairo, Egypt.

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- Suprax® suspension contains 100 mg cefixime/5mL, batch # 1520157, Kahira for Pharmaceutical and chemical industries, Cairo, Egypt. - Omnicef® capsules contain 300 mg of cefdinir/capsule, batch # 150414, Hikma Pharma

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Co., 6th of October City, Egypt. - Omnicef® suspension contains 125 mg cefdinir/5mL, batch # 150417 , Hikma Pharma

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Co., 6th of October City, Egypt. - Antinal® capsules, batch # 153011, each capsule contain 200 mg NX , Amoun Pharmaceutical Company, Cairo, Egypt - Antinal® suspension, batch # 152130, contain 220 mg NX/5mL, Amoun Pharmaceutical Company, Cairo, Egypt. All dosage forms were obtained from municipal pharmacy.

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2.3. Chemicals and Reagents All chemicals and reagents were of analytical grade, solvents were of spectroscopic grade for spectrophotometric method (Method I) and high performance liquid chromatography grade for spectrofluorimetric methods (Method II and III) and the water utilized in the study was bidistilled.

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- Acetate buffer solutions (0.2M ) were obtained by adjusting the pH of sodium acetate

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trihydrate, El Nasr Co. (ADWIC, Egypt) (0.2M ) with acetic acid, El Nasr Co. (ADWIC, Egypt) (0.2M ) [44].

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- Acetyl acetone with purity 99%, batch # 523711745, Merck-Schuchardt, Honenbrunn, Germany.

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- Formaldehyde (34%), El Nasr Co. (ADWIC, Egypt). It was prepared as 20% v/v by mixing 5 mL formaldehyde (34%) with distilled water to 25 ml.

Methanol was obtained from TEDIA company (USA).

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-

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- Zn powder was obtained from BDH Chemicals (Poole, UK).

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Preparation of acetyl acetone/acetate buffer reagent for method I and II: Acetyl acetone/acetate buffer reagent was freshly prepared as 8.4% v/v solution in a 25.0 mL volumetric flask, where 2.1 mL acetyl acetone was mixed with appropriate volume of

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acetate buffer of optimum pH and diluted to volume with distilled water.

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Both buffer pH and volume were selected so as to reach maximum absorbance or relative fluorescence intensity values of CFX and CFD. Based on this concept, acetate buffer of pH ranges (2.5-5.5) were tried, and it was found that pH 5 (CFX) and 5.5 (CFD) for both methods gave maximum results. Besides, buffer volumes were also investigated for the same reason. We found that 15 mL of buffer resulted in optimum result for both methods, so it was used for preparing the reagent.

2.4. Standard Solution

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Stock solutions of CFX, CFD and NX containing 200.0µg/mL were obtained by dissolving 20.0 mg of the pure drug in 100 mL of methanol. With this solvent, further dilution was performed to get the required concentration range for method I,II and III.

2.5. General procedures

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2.5.1 Construction of calibration graphs for method I and II

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Accurate volumes of CFX and CFD standard solutions were measured and transferred into a series of 10.0 mL volumetric flasks. Appropriate volume of 8.4% v/v

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acetyl acetone was added followed by appropriate volume of 20% v/v formaldehyde. The solutions were heated in a boiling water bath for a specified time, then the solutions are

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cooled and completed to the mark with distilled water. The optimum conditions for method I and II are listed in Table 1.

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For method I, The absorbance of each solution was determined at 315 nm and 403 nm for CFX and CFD respectively against a reagent blank. The absorbance values were plotted

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against drug concentrations (µg/mL) to obtain the calibration plot. Then, the corresponding

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regression equation was derived.

For method II, The fluorescence intensities were detected at λem 488 nm and 491 nm following excitation at λex 410 nm for CFX and CFD respectively. Fluorescence intensities

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(FI) were plotted against the final drug concentrations in µg/mL to obtain the calibration

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plot. Then, the corresponding regression was derived.

2.5.2 Construction of calibration graphs for method III 10.0 mL of the stock solution of NX were transferred into a 100 mL conical flask. 2.5 mL of conc.HCl and 0.4 gm of Zn powder were added. This mixture was left to stand for half an hour with occasional shaking, then it was filtered into a 100.0 mL volumetric flask and completed to the mark with methanol. Accurate volumes of reduced nifuroxazide standard solutions were measured and transferred into a group of 10.0 mL volumetric flasks, to get concentration range of 0.05 -

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1.6 µg/mL. The solutions were completed with methanol to the mark. The fluorescence intensities were detected at 335 nm following excitation at 255 nm. The fluorescence Intensities (FI) were plotted against the reduced NX concentrations in µg/mL to obtain the

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calibration graph. Then, the corresponding regression was derived.

2.5.3. Procedure for Capsules

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The content of 10 capsules were weighed and thoroughly mixed. A weighed

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quantity of the powder equivalent to 20.0 mg either of the three studied drugs was transferred into a 100.0 mL volumetric flask and the volume was completed to the mark

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with methanol. The flask contents were sonicated for 30 minutes, filtered and different volumes of the filtrate were quantitatively transferred into 10.0 mL volumetric flask. Then

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the procedure utilized to construct calibration graphs applying any of the three methods was followed. From the derived regression equation, the capsules nominal contents were

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estimated.

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2.5.4. Procedure for Suspension

An accurate volume of the freshly reconstituted oral suspension equivalent to 20.0 mg

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of any of the three drugs was extracted with about 25 mL of methanol, sonicated for about 45 minutes then filtered into a 100.0 mL volumetric flask. The solution was then

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made up to volume with methanol. This extract was further diluted with methanol. For all methods, the procedure was completed as mentioned for preparing the calibration plots. From the corresponding regression equation, the nominal contents of the suspension were determined.

3. Results It was possible to carry out two simple methods which enable us to assay CFX and CFD in their dosage forms using simple procedures and relatively economic and available

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reagents. Both CFX and CFD react with Hantzsch reagent to form a product with a high molar absorptivity (method I) (Fig. 2). Besides, the same products exhibit fluorescence (method II) as demonstrated from (Fig. 3). Such reactions depend mainly on condensation of the primary amino group of CFX or CFD with acetyl acetone and formaldehyde in acidic medium resulting in the condensation products as illustrated in scheme 1.

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Nifuroxazide, being a drug carrying a nitro group, doesn’t exhibit fluorescence as could be

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deduced through scanning of its fluorescence spectrum (Fig. 4).The basic idea of the third developed method is the reduction of the NX nitro group into the corresponding amino

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group using Zn/HCl. The reduced Nifuroxazide has intense fluorescence (Fig. 5) due to the formation of stable resonance product of NX as suggested by a previous report [42] and

Optimization of experimental conditions for method I and II

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3.2.

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presented in scheme 2.

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were studied and optimized.

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Various experimental conditions influencing the condensation reaction in both methods

3.2.1. Effect of acetyl acetone volume

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The effect of volume of 8.4% v/v acetyl acetone was studied from 0.1 to 2 mL. Regarding method I, 0.2 mL and 1.5 mL for CFX and CFD respectively were found to give

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optimum results. While for method II, 0.2 mL and 0.5 mL for the two drugs respectively were found to be optimum for maximum values of the formed product (Fig. 6).

3.2.2. Effect of formaldehyde volume The effect of volume of 20% v/v formaldehyde was also investigated from 0.5 to 3 mL. Regarding method I, 2 mL and 1 mL for CFX and CFD respectively were chosen, while for method II, 1.5 mL for both drugs were found to be optimum for maximum absorbance or RFI values of the formed product (Fig. 7).

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3.2.3. Effect of Diluting solvents Diluting solvents influence on the fluorescence intensity or absorbance of the reaction products was studied including methanol, distilled water, butanol, dimethylformamide and acetonitrile. It was found that distilled water is the best diluting solvent giving the

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maximum absorbance or fluorescence intensity, which adds another advantage to the

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proposed methods.

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3.2.4. Effect of temperature and time The reaction is kinetically catalyzed as no color was developed at room temperature. The

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optimum heating time for the reaction was determined by investigating the maximum absorbance or fluorescence intensity at different temperatures from room temperature to

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100°C. Maximum fluorescence intensity or absorbance was obtained at 100°C (Fig. 8). The time of heating was also observed in the range 10-60 minutes. The absorbance or

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fluorescence intensity increased until 45 minutes and then remained constant (Fig. 9). So,

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heating the reaction mixture at 100°C for 45 minutes was the best condition to obtain optimal absorbance or fluorescence intensity for method I and II for both drugs.

Optimization of experimental conditions for method III

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3.3.

The conditions used to perform the reduction process include: the amount of Zn powder,

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volume of conc.HCl and the reduction time. These parameters were studied and optimized to choose the most suitable reduction system giving the maximum fluorescence intensity.

3.3.1. Amount of zinc powder: The influence of the amount of Zn powder (0.1-0.6 gm) was studied. It was observed that increasing the amount of Zn powder result in an increase in RFI till 0.4 gm then no more increase was obtained (Fig. 10A), therefore 0.4 gm was used through the study.

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3.3.2. Hydrochloric acid volume: The influence of conc.HCl volume (1- 4 mL) on the RFI was observed. As the volume of conc.HCl increase, the RFI increase up to 2.5 mL after this value no more increase was obtained. As a consequence, 2.5 mL conc.HCl is the optimum value for complete reduction

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of NX (Fig. 10B).

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3.3.3. Reduction time

The influence of time on the fluorescent product development was observed.

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Complete reduction was developed after 30 minutes as observed from maximum values of

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RFI. The produced fluorophore remained stable for at least one hour (Fig. 11).

Discussion

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Both CFX and CFD have low molar absorptivity values (ξ) as could be concluded from Figure 2. Method Ι succeeded to increase ξ through the formation of a condensation

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product between the primary amino group of both drugs and Hantzsch reagent. The

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formed products exhibit both bathochromic and hyperchromic shift (Fig. 2). Hyperchromic shift could be explained by the marked increase in the conjugation system which in turn leads to enhancement of absorbance readings of both drugs.

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While bathochromic shift could result due to the formation of more energetic products,

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since the reaction takes place in acidic medium provided by acetate buffer, yielding positive charges held by the secondary and teriary amino groups of the formed products, hence rendering them more energetic and as expected their need for energy to be excited for higher energy levels will remarkably decrease, and as a consequence a positive shift in the wavelength results. The same reaction products were found to exhibit fluorescence, which may have also resulted from the extended conjugated system and expected to have a rigid structure, as it is well known that fused ring structures containing heterocycles usually fluoresce , and rigidity decreases nonradiative relaxation to a degree where relaxation by fluorescence has time to take place.

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The study carried out for both method Ι, and ΙΙ, regarding the volume of the reagent (acetyl acetone and formaldehyde), the reaction temperature and time, all reflect the conditions needed for the completeness of the reaction, and consequently the highest values for either the absorbance or RFI readings. The absorbance spectra appear as single broad peaks, which may be attributed to the fact that in polar solvents, the

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frequent collisions and interaction between CFX or CFD and water cause an energetic

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modification in the vibrational levels in an irregular form. Similarly speaking, the factors discussed for method ΙΙΙ, influence the complete reduction of the nitro group

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in NX - which is well known to cause quenching- to amino group yielding a fluorophore.

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5. Methods validation

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The developed procedures were validated according to ICHQ2 (R1) recommendations

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5.1. Linearity and range

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[45], including the following validation parameters.

The calibration plots obtained by plotting the absorbance (method I) or RFI (methods II and III) against the final concentrations (µg/mL) were found to be linear over the

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concentration ranges presented in Table 2. The validity of the developed methods were

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proven by statistical regression line [46] and it was found that percentage relative standard deviation (%RSD) was small while correlation coefficient values (r) were high (Table 2) indicating the good linearity of calibration plots. 5.2. LOQ and LOD LOD and LOQ were determined in accordance to ICH Q2 (R1) requirements [45] and their values are presented in Table 2.

5.3. Precision and accuracy

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The proposed methods accuracy could be demonstrated by comparing the obtained results with those obtained by comparison methods [5, 25, 37] showing good agreement between them (Table 3). On the other hand, precision was tested by using three different concentrations of the tested drugs whether during one day (intra-day precision), or on three different times (inter-day precision). Remarkable precision is noted from low RSD values

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shown in Table 4.

5.4. Robustness

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It was established by consistency of absorbance (method I) or RFI (method II and III) with deliberately small changes in different experimental conditions. Regarding method I

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and II, these changes involve: volume of acetyl acetone (optimum volume ± 0.1 mL) and volume of formaldehyde (optimum volume ± 0.1 mL).While for method III, these changes

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involve: the amount of zinc powder (0.4 ± 0.1 gm), hydrochloric acid volume ( 2.5 ± 0.2 mL) and reduction time (30 ± 2 minutes). These small changes which can occur during

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experimental process didn’t influence the RFI or absorbance of the studied drugs,

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demonstrating the robustness of these methods.

5.5. Specificity

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It was demonstrated by investigation of any interference from the common capsule and

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suspension excipients. No interfering peaks were observed during analysis. 6. Pharmaceutical applications The developed methods were used to estimate the three drugs in their dosage forms. The statistical comparison of the obtained results with those of reference methods [5, 25, 37] revealed that no significant difference was found between them regarding t and F values as presented in Table 5.

7. Conclusion

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Three

simple,

sensitive,

precise

and

inexpensive

spectrofluorimetric

and

spectrophotometric methods were developed for estimation of CFX, CFD and NX in their pharmaceuticals without interference from common excipients and high recovery of the studied drugs was obtained in different formulations. Furthermore, the developed methods have an economical advantage and do not need complexed requirements needed in

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chromatographic methods. In addition to the reproducibility as well as the simplicity and

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convenience. So they can be applied in quality control laboratories for analysis of the

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studied drugs.

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chromatographic determination of cefdinir in bulk powder and dosage form using monolithic stationary

[29] P. Ravisankar, G.D. Rao, M.K. Chaitanya, Development and validation of RP-HPLC method for the

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determination of cefdinir in bulk and capsule dosage form, Indian J. Res. Pharm. Biotech., 1 (2013) 255263.

[30] G.M. Shahed, M.A. Ullah, A.A.A. Al, M.U. Ahmed, M.S. Islam, Z. Nahar, A. Hasnat, A Simple RP−

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HPLC Method for the Determination of Cefdinir in Human Serum: Validation and Application in a Pharmacokinetic Study with Healthy Bangladeshi Male Volunteers, Dhaka Univ. J. Pharmaceut. Sci., 10

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(2012) 109-116.

[31] Z.-j. Chen, J. Zhang, J.-c. Yu, G.-y. Cao, X.-j. Wu, Y.-g. Shi, Selective method for the determination

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of cefdinir in human plasma using liquid chromatography electrospray ionization tandam mass spectrometry, J. Chromatogr. B., 834 (2006) 163-169. [32] H.E. Jin, I.B. Kim, C.K. Kim, H.J. Maeng, Determination of cefdinir levels in rat plasma and urine by

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high‐performance liquid chromatography–tandem mass spectrometry: application to pharmacokinetics after oral and intravenous administration of cefdinir, Biomed. Chromatogr., 27 (2013) 1423-1430.

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[33] S.-Y. Dong, Z.-Q. Yu, X.-F. Han, T.-L. Huang, J.-B. Zheng, Voltammetric behavior of degradation product and determination of cefdinir, Chem. Res. Chinese Unversities, 25 (2009) 807-811. [34] F.H. Metwally, Simultaneous determination of nifuroxazide and drotaverine hydrochloride in pharmaceutical preparations by bivariate and multivariate spectral analysis, Spectrochim. Acta Mol. Biomol. Spectrosc., 69 (2008) 343-349. [35] F.H. Metwally, M. Abdelkawy, I.A. Naguib, Determination of nifuroxazide and drotaverine hydrochloride in pharmaceutical preparations by three independent analytical methods, J. AOAC Int., 89 (2006) 78-87. [36] M.A. Hegazy, Stability Indicating Spectrophotometric and Chemometric Methods for Determination of Nifuroxazide in Presence of Its Alkaline Degradation Products, Pharm. Anal. Acta, (2011) 2:127.

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[37] T.S. Belal, A simple and sensitive spectrofluorimetric method for analysis of some nitrofuran drugs in pharmaceutical preparations, J. Fluoresc., 18 (2008) 771-780. [38] A.A. El-Zaher, M.A. Mahrouse, A validated spectrofluorimetric method for the determination of nifuroxazide through coumarin formation using experimental design, Chem. Cent. J., 7 (2013) doi:10.1186/1752-153X-7-90. [39] H.M. Maher, T.S. Belal, HPLC-DAD stability indicating determination of the fixed-dose combination

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of nifuroxazide and drotaverine hydrochloride in capsules, J. Liq. Chromatogr. R. T., 35 (2012) 2001-2020. [40] A. Radi, S. El-Laban, I. Kenawy, Determination of Nifuroxazide in Capsules by Differential Pulse

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Polarography, Anal. Sci., 14 (1998) 607-608.

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[41] J. Mozo, J. Carbajo, J. Sturm, L. Núñez‐Vergara, P. Salgado, J. Squella, Determination of Nifuroxazide by Flow Injection Linear Adsorptive Stripping Voltammetry on a Screen‐Printed Carbon

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Nanofiber Modified Electrode, Electroanal., 24 (2012) 676-682.

[42] F. Belal, S. Julkhuf, N.Y. Khalil, A.A. Al-Majed, Spectrofluorometric determination of nimodipine in dosage forms and human urine, Pharmazie, 3 (2003) 874-876.

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[43] A. Smith, R. Manavalan, K. Kannan, N. Rajendiran, Improved liquid chromatographic method for the determination of flutamide in pharmaceutical formulation, (2009).

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[44] H.T.S. Britton, Hydrogen Ions, Revised and Enlarged, 4th ed., Chapman & Hall, London, 1955.

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[45] ICH Harmonized Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology, Q2 (R1), Current Step 4 Version, Parent Guidelines on Methodology Dated November 6 1996, Incorporated

in

November

[http://www.fda.gov/downloads/Regulator%20yInformation/Guidances/UCM128049.pdf]website

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(Accessed April 8, 2016).

[46] J.N. Miller, J.C. Miller, Statistics and Chemometrics for Analytical Chemistry, Prentice Hall/Pearson,

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Harlow, England, 2010.

9. List of Figures: Figure 1A: Structural formula of Cefixime.

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Figure 1B: Structural formula of Cefdinir.

Figure 1C: Structural formula of Nifuroxazide.

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product of CFX (40.0 µg/mL) with Hantzsch reagent.

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Figure 2A: Absorption spectra of: (a) CFX (40.0 µg/mL) in methanol, (b) Condensation

Figure 2B: Absorption spectra of: (a) CFD (60.0 µg/mL) in methanol, (b) Condensation

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product of CFD (60.0 µg/mL) with Hantzsch reagent.

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Figure 3A: Fluorescence spectra of condensation product of CFX (6.0 µg/mL) with Hantzsch reagent :

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(a, a`) are excitation and emission spectra of reagent blank (b, b`) are excitation and emission spectra of the condensation product.

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Figure 3B: Fluorescence spectra of condensation product of CFD (10.0 µg/mL) with

(a, a`) are excitation and emission spectra of reagent blank

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(b, b`) are excitation and emission spectra of the condensation product.

Figure 4: Fluorescence spectra of: (a) NX (1.0 µg/mL) without reduction , (b) reagent blank.

Figure 5: Fluorescence spectra of: (a, a`) are excitation and emission spectra of reagent blank (b, b`) are excitation and emission spectra of reduced NX (1.0 µg/mL).

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Figure 6: Effect of 8.4% v/v acetyl acetone volume on the absorbance or fluorescence intensity of CFX and CFD (40.0 µg/mL for method I and 10.0 µg/mL for method II ).

Figure 7: Effect of 20% v/v formaldehyde volume on the absorbance or fluorescence

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intensity of CFX and CFD (40.0 µg/mL for method I and 10.0 µg/mL for method II ).

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Figure 8: Effect of heating temperature on the absorbance or fluorescence intensity of

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CFX and CFD (40.0 µg/mL for method I and 10.0 µg/mL for method II ).

Figure 9: Effect of heating time on the absorbance or fluorescence intensity of CFX and

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CFD (40.0 µg/mL for method I and 10.0 µg/mL for method II ).

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Figure 10A: Effect of the amount of Zn powder on RFI of reduced NX (1.0 µg/mL).

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Figure 10B: Effect of the volume of conc.Hcl on RFI of reduced NX (1.0 µg/mL).

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Figure 11: Effect of the reduction time on RFI of reduced NX (1.0 µg/mL).

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Scheme 1: Reaction mechanism proposal for condensation of cefixime and cefdinir with acetyl acetone and formaldehyde

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Scheme 2: Reaction mechanism proposal between NX and Zn/Hcl

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Table (1): Assay parameters for determination of CFX and CFD by Method I and II.

Spectrophotometry (Method I)

Spectrofluorimetry (Method II)

CFD (Method IB )

CFX (Method IIA)

CFD (Method IIB)

Volume of Acetyl acetone (8.4% v/v)

0.2 mL

1.5 mL

0.2 mL

0.5 mL

Volume of formaldehyde (20% v/v)

2 mL

1 mL

Diluting Solvent

Distilled water

Distilled water

Heating Temperature

100°C

100°C

Heating Time

45 minutes

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45 minutes

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CFX ( Method IA )

1.5 mL

1.5 mL

Distilled water

Distilled water

100°C

100°C

45 minutes

45 minutes

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Table (2): Analytical performance data for the proposed Methods.

Spectrophotometry

Spectrofluorimetry CFD

CFX

CFD

NX

(Method IA)

(Method IB)

(Method IIA)

(Method IIB)

Concentration range (µg/mL)

5.0-140.0

10.0-100.0

0.20 -10.0

0.20 -36.0

(Method III) 0.05-1.6

Limit of detection LOD (µg/mL)

0.6202

1.2734

0.0168

0.0902

0.0046

Limit of Quantitation LOQ (µg/mL)

1.8795

3.8587

0.0510

0.2734

0.0139

Regression equation*Y=a+bX

Y= 0.0086X + 0.037

Y= 0.0064X + 0.0354

Y= 94.015X + 63.155

Y= 23.415X + 68.552

Y= 518.12X + 9.4413

Correlation coefficient Sy/x ,S.D. of the residuals

0.9999

0.9999

0.9999

0.9999

0.9999

0.0035

0.0032

1.3107

1.6188

1.99

0.0025

0.4796

0.6402

0.722

0.0001

0.1295

0.0418

1.195

0.740

0.485

0.665

0.713

0.265

0.301

0.161

0.221

0.225

85.77

63.82

3889.22

2523.41

0.0001

%RSD

0.796

A%(dl.gm-1.cm1)

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%Error(%RSD/√ n)

Molar absorpitivity (l.mol.-1cm-1)

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* Y: absorbance; a: intercept; X: Concentration (µg/mL); b: slope.

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Table (3): Application of the proposed methods to the determination of CFX, CFD and NX in pure form. Spectrophotometry

Parameter

CFX (Method IA)

Spectrofluorimetry

CFD (Method IB)

CFD (Method IIB)

NX (Method III)

T P

I R

for CFX [5]

C S U

for CFD [25]

for NX [37]

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL)

% founda

Conc. Taken (µg/mL

% founda

5.0 10.0 20.0

100.93 101.28 100.58

10.0 20.0 40.0

99.38 98.43 100.04

0.20 0.40 0.80

99.15 99.30 99.38

0.20 0.80 2.0

99.05 99.55 99.19

0.05 0.1 0.2

100.60 99.50 100.90

5.0 10.0 20.0

98.97 101.21 99.45

0.20 0.80 2.0

99.15 100.98 99.62

0.2 0.4 0.6

100.70 99.95 99.35

40.0 60.0 80.0 100.0 120.0

99.71 99.42 99.27 99.19 100.10

60.0 80.0 100.0

99.90 100.51 99.16

1.60 2.0 4.0 6.0 8.0

99.55 99.80 100.21 100.31 100.36

6.0 12.0 18.0 24.0 32.0

40.0

100.01

6.0

100.03

0.8

100.34

140.0

99.92

10.0

99.65

36.0

Mean± SD

100.06 + 0.80

99.57 +0.74

t

0.16(2.306)*

0.39(2.571)*

F

1.47(8.85)*

1.11(9.01)*

a

CFX (Method IIA)

Comparison Method

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C A

D E

N A

M

100.68 100.88 99.52 99.55 99.89

0.4 0.6 0.8 1 1.2

100.88 99.25 100.25 99.12 100.05

100.30

1.4

100.51

1.6

99.90

99.76+ 0.48

99.79+ 0.66

100.07+ 0.71

0.99(2.306)*

0.99(2.306)*

0.99(2.262)*

4.08(8.85)*

1.39(8.85)*

1.49(8.81)*

Each result is the average of three separate determinations

*Values between brackets are the tabulated t and F values, at p = 0.05 [46]

99.91+0.97

99.95+ 0.78

100.09 + 0.58

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Table (4): Precision data of the proposed methods for the determination of CFX, CFD and NX in pure form

Conc. Taken

Mean |%found| + SD

Inter-day precision %RSD

%Error

(µg/ml)

IA

Conc. Taken

Mean |%found| + SD

%RSD

%Error

( µg/ml)

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0.40

0.23

99.97+0.59

0.59

0.34

140.0

100.28+0.83

0.83

0.48

10.0

100.1+0.70

0.70

0.40

5.0

99.94+0.25

0.25

0.14

5.0

60.0

99.88+0.40

0.40

0.23

60.0

140.0

99.76+0.46

0.46

0.26

10.0

100.03+0.58

0.58

0.33

40.0

99.97+0.40

0.40

0.23

40.0

100.57+0.61

0.61

0.35

100.0

100.1+0.53

0.53

0.31

100.0

100.40+0.67

0.66

0.38

0.20

99.98+0.73

0.73

0.42

0.20

99.83+0.63

0.64

0.37

4.0

99.71+0.62

0.62

0.36

4.0

99.57+0.57

0.57

0.33

10.0

99.85+0.35

0.35

0.20

10.0

100.02+0.41

0.41

0.24

0.20

99.93+0.40

0.40

0.23

0.20

99.99+0.66

0.66

0.38

12.0

99.96+0.67

0.67

0.39

12.0

100.08+0.79

0.79

0.46

36.0

100.30+0.47

0.47

0.27

36.0

100.51+0.67

0.67

0.39

0.40

100.25 ± 0.69

0.57

0.33

0.40

99.98 ± 0.78

0.78

0.45

99.85 ± 0.53

0.53

0.30

0.60

100.38 ± 0.98

0.98

0.57

100.06 ± 0.23

0.23

0.14

0.80

100.28 ± 0.80

0.80

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Table (5): Application of the proposed and comparison methods to the determination of CFX,CFD and NX in different dosage forms Pharmaceutical Preparation

Method II Conc. Taken (µg/ml)

% founda

% founda

100.30

0.80

99.55

99.79

40.0

99.03

1.60

98.29

98.85

80.0 100.0 120.0

99.14 98.72 99.68

4.0 6.0 8.0

100.72 100.72 99.49

100.63 100.99 99.33

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Mean+ SD

99.75+1.02

0.07(2.776)* 2.06 (6.39)* 98.66 100.22 99.00 99.38 99.48

99.92+0.89

099(2.776)* 1.31 (6.39)* 100.98 99.97 100.88 98.38 100.68

98.02 99.38 100.63 100.52 99.00

99.35+0.59

100.18+1.08

99.51+1.09

t

0.05 (2.776)*

0.99(2.776)*

F

3.41 (6.39)*

1.02 (6.39)*

98.61

2.0 4.0

100.63 101.68

101.08

40.0

99.31

6.0

99.86

99.56

60.0 80.0 100.0

100.15 99.88 99.17

8.0 12.0 16.0 20.0

100.58 98.31 99.82 100.57

99.71 100.51 100.02

99.42+0.61

100.21+1.04

100.18+0.62

0.15(2.776)*

0.99 (2.447)*

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Mean+ SD

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Comparison Method [25]

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Mean + SD

Omnicef® capsules (300 mg CFD/capsule)

0.80 1.60 4.0 6.0 8.0

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F Suprax® suspension (100 mg CFX/5mL)

Comparison Method [5]

Conc. Taken (µg/ml) 20.0

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Method I

F

1.03(6.39)*

2.81 (6.16)* 100.28 100.86

100.20

20.0

100.65

1.60 2.0

40.0

99.03

4.0

99.62

100.61

60.0 80.0 100.0

98.48 99.84 99.35

8.0 12.0 16.0 20.0

100.99 98.80 100.09 100.22

99.39 98.78 100.45

99.47+0.82

100.12+0.75

99.89+0.78

t

0.11(2.776)*

0.99(2.447)*

F

1.11(6.39)*

1.08(6.16)*

Omnicef® suspension (125 mg CFD/5mL)

Mean+ SD

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Comparison Method [37]

Method III

NX/capsule)

0.2 0.6

100.65 100.30

0.2 0.4

99.05 100.90

1 1.4 1.6

99.22 100.39 99.96

0.6 0.8

99.75 99.98

100.10+0.55

t

0.99(2.776)*

F

1.91(9.12)*

suspension (220 mg NX/5mL)

0.2 0.6 1 1.4 1.6

F

99.92+0.76

101.20 99.78 99.10 100.49 100.14+0.91

0.99(2.776)* 1.39(9.12)*

Each result is the average of three separate determinations

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0.2 0.4 0.6 0.8

99.99+0.77

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Mean+ SD

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Conc. Taken (µg/ml)

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Conc. Taken (µg/ml)

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Graphical Abstract

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Highlights

 Cefixime & cefdinir are determined by reaction with Hantzsch reagent  Nifuroxazide was estimated upon reduction with Zn2+/HCl  The developed methods were fully validated

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 The drugs were determined in their pharmaceuticals using the proposed methods

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