Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

DEVELOPMENT AND VALIDATION OF SPECTROPHOTOMETRIC METHOD FOR

DETERMINATION OF CEFIXIME AND GLIMEPIRIDE BY TERNARY COMPLEX
FORMATION WITH EOSIN AND CU(II)
Ihab M. Almasri , Mohammad K. Al-Laham
Department of Chemistry and Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Gaza, Gaza Strip.
ARTICLE INFO
Article history
Received 25/11/2014
Available online
16/12/2014

Keywords
Spectrophotometry;
Ternary Complex;
Eosin; Cu(II);
Cefixime,
Glimepiride.

ABSTRACT
A simple, accurate and sensitive UV-Visible spectrophotometric method have been developed
and validated for the quantitative determination of cefixime and glimepiride in either pure
form or in their dosage forms. The method is based on the formation of a ternary complex
with copper(II) and eosin. The method does not involve solvent extraction. Appropriate
conditions were examined for the reaction to obtain maximum absorptivity and sensitivity.
The color of the produced complex is measured at 550 and 544 nm with apparent molar
absorptivities of 1.49 X 104 Lmol-1cm-1 and 1.657 X 104 Lmol-1cm-1 and Sandell's sensitivities
of 3.1 X 10-2 and 2.9 X 10-2 g/cm2 for cefixime and glimepiride, respectively. The method is
applicable over concentration range of 4-28 and 5-50 gmL-1 for cefixime and glimepiride,
respectively. The analytical performance of the developed method was fully validated and the
results obtained revealed high accuracy (recovery values, 100 1%) and precision (with
relative standard deviation <1.50%). Furthermore, the developed methods hold their accuracy
and precision well when applied to the determination of cefixime and glimepiride in their
dosage forms, therefore, they could be used for routine analysis of the two drugs in their
pharmaceutical dosage forms.

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Ihab M. Almasri et al. Development and Validation of Spectrophotometric Method for
Determination of Cefixime and Glimepiride by Ternary Complex Formation with Eosin and Cu(II. Indo American Journal of
Pharm Research.2014:4(12).

5670

Corresponding author
Ihab M. Almasri
Department of Chemistry and Pharmaceutical Chemistry
Faculty of Pharmacy, Alazhar University-Gaza
Palestine
i.masri@alazhar-gaza.edu
972599403757
97082823180.

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INTRODUCTION
Cefixime (CEF) chemically is (6R,7R)-7-[[(Z)-2-(2-Aminothiazol-4-yl)-2[(carboxymethoxy) imino]acetyl]amino]-3-ethenyl8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate (Figure 1). Cefixime is white to almost white, slightly
hygroscopic powder and is classified as a third generation oral -Lactam cephalosporin antibiotic. It has broad spectrum activity
against Gram positive and Gram negative bacteria. It acts by inhibiting the transpeptidase enzyme involved in the building of bacterial
cell walls [1]. It is used in lower respiratory tract Infections [2], acute urinary tract infections [3], acute otitis media [4], peptic ulcer
[5]. Various methods have been reported in the literature for the analysis of cefixime including: spectrophotometry [6-8],
spectrophotometry with chromogenic reagent [9], spectrofluorometry [10], High-performance liquid chromatography [1113], High
performance thin layer chromatography HPTLC [14,15], Voltammetry [16].
Glimepiride (GLI) chemically is 1-[[4-[2-(3-Ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido)-ethyl] phenyl]sulphonyl]-3trans-(4-methylcyclohexyl)urea (Figure1). Glimepiride is a third-generation sulfonylurea antidiabetic drug exerts its insulin-releasing
effect mainly by inhibiting ATP-sensitive potassium channels in the pancreatic -cell. It is given orally for the treatment of type 2
diabetes mellitus [17]. A literature survey revealed that few liquid chromatography [18,19] and derivative spectrophotometry [18-20]
methods has been developed for the determination of glimepiride in pharmaceutical formulations. Spectrophotometry [21-22] and
Liquid chromatography [23-25] methods have also been developed for the estimation of glimepiride in combination with other drugs
in pharmaceutical formulations. Liquid chromatography- mass spectrometry method has been developed for the quantification of
glimepiride in human plasma [26].
In Gaza Strip there is a serious problem regarding counterfeit drugs and pharmaceutical products smuggling, therefore, there
is increasing need to analyze the quality of the products in the market to ensure their efficacy and safety. Most methods that developed
for determination of cefixime and glimepiride are sophisticated, expensive and require modern instruments that are not available in
our laboratories, so there is a need for simple, accurate and available method for analysis.
The purpose of this study was to determine the two drugs in their bulk and pharmaceutical dosage forms without prior
extraction by the development of simple, rapid and selective spectrophotometric assays for quality control and routine analysis
purposes based on ternary complex formation between the studied drug-metal ion [Cu(II) (drug)n] as
cation and an organic dye (eosin) as anion.

Materials and reagents

Pure samples
Pure dug standards were kindly donated by Pharmacare Pharmaceutical company (Ramallah, Palestine).

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MATERIALS AND METHODS

Instrumentation
All absorbance measurements were carried out using Shimadzu 1601 double beam scanning UV-Vis spectrophotometer, with
1 cm matched quartz cell. A digital pH meter (HM-60V; TOA, Japan) provided with a glass electrode (GST-5421C; TOA) was used
for checking the pH (range 4.08.0) of acetate buffer solutions.

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Figure 1: Structure of: (A) cefixime drug, (B) glimepiride drug.

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Pharmaceutical formulations
Commercially available pharmaceutical dosage forms were purchased from the local market.
- Rizacef (cefixime) 400 mg capsules manufactured by Pharmacare Pharmaceutical company (Ramallah, Palestine)
- Amiran (glimepiride) 3 mg tablets manufactured by Birzeit Pharmaceutical Company (BPC) (Ramallah, Palestine).
Reagents
All chemicals were of analytical grade with high purity.
1- Eosin B (Sigma, Austria), 0.1% w/v prepared by dissolving 100 mg of eosin in 100 mL double distilled water.
2- Copper (II) sulfate (Merck, Germany), 0.1% w/v prepared by dissolving 100 mg of copper (II) sulfate [CuSO 4 . 6H2O] in 100 mL
distilled water.
3- Acetate buffer solutions (100 mL) of pH 4, 5 and 5.6 were prepared by mixing different volumes of acetic acid 0.1 M and sodium
acetate solution 0.1 M and its pH was checked periodically.
4- Methylcellulose (MC) (Sigma-Aldrich, Germany), 0.3% w/v prepared by dissolving 300 mg of MC in 100 mL distilled water.
Preparation of standard solutions
Cefixime stock solution (400 g mL1) was prepared by dissolving 40 mg in a 100 mL methanol with good shaking for 5
minutes. Glimepiride stock solution (500 g mL-1) was prepared by dissolving 50 mg of the drug in a 100 mL DMF with good
shaking for 5 minutes. Working standard solutions containing 428 g mL-1 and 550 g mL-1 were prepared by suitable dilution of
the stock solutions with methanol and water for CEF and GLI, respectively.
General Procedures
For pure drugs
Accurately measured aliquots of the stock solutions of drugs were transferred into separate 10 mL volumetric flasks and then
1 mL copper (II) sulfate solution was added to each flask followed by 1 mL acetate buffer solution (pH 5.6 for CEF, 5.0 for GLI ) and
1 mL eosin solution. For GLI 1 mL MC solution was added. The contents of each flask were shaken well and each mixture was
diluted to 10 mL with the suitable solvent. The mixtures were left at room temperature for 20 minutes and then the absorbance of the
resulting solutions were measured at 550 and 544 nm, for CEF and GLI respectively, against reagent blank treated in a similar way.
The measured absorbances vs., the final concentrations were plotted to get the calibration curves and the regression equations were
calculated.
Assay of cefixime capsules
Twenty capsules were emptied and a known weight of the powder content equivalent to 40 mg of the drug was dissolved in
60 mL methanol with good shaking for 10 minutes and filtered. The filtrate solution was then transferred into 100 mL volumetric flask
and diluted to 100 mL with methanol. The assay determination for the content of CEF was performed as described under General
procedures for pure drugs (section 2.4.1). A triplicate measurements were done for each concentration and the percentage of recovery
was calculated from the calibration graph.
Assay of glimepiride tablets
The contents of twenty tablets were weighed and powdered and a known weight of the powder equivalent to 25 mg of
glimepiride was dissolved in 25 mL DMF with good shaking for 10 minutes and filtered. The filtrate solution was then diluted to 50
mL with DMF. The assay determination for the content of GLI was performed as described under General procedures (section 2.4.1).

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Optimization of experimental conditions

In order to prove the formation of a ternary complex between copper(II) (A), eosin (B) and the drugs (cefixime or
glimepiride) (C), a series of absorption spectra have been obtained for each component, separately, and compared with their mixtures
under the experimental conditions previously described. The spectra revealed that aqueous solution of eosin, (B) absorbs in the visible
region at max 515 nm, while the other two components (A and C) have no absorbance maximum in the visible region. The drugcopper mixture has the same maximum absorbance as that of drug and copper, separately and the mixture of copper and eosin has the
same maximum absorbance as that of eosin. Furthermore, complexes formed have absorption maximum at 544 nm and 550 nm for

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RESULTS AND DISCUSSION

The main purpose of this work was to develop a simple spectrophotometric method for the determination of cefixime and
glimepiride in their pharmaceutical formulations without prior extraction through ternary complex formation. Ternary complexes of
the general formula (LNMXSY) was used in spectrophotometric analysis of many drugs [27-29]. In the ternary complexes formed in
this study, the drug and eosin are the ligands and copper(II) is the metal. The mechanism of ternary complex formation includes
coordinate bond formation between copper ion and the drug molecule through atoms carrying lone pairs of electrons, and
subsequently, a ternary complex is formed by interaction with eosin. The major advantage of ternary complex formation in
spectrophotometric analysis is that it often has higher values of molar absorbtivities and therfore improves the sensitivity of the
method. Furthermore, it shifts the absorption peak towards higher wavelengths and thus could improve the selectivity as well. In the
present study, appropriate conditions were selected for the color reaction between eosin, Cu(II) and the drug in order to reach
maximum sensitivity.

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glimepiride and cefixime (Figure 2), respectively. According to these results, the reaction would be a ternary complex system
formation, ABC, having different spectral characteristics from that of AB or AC binary mixtures.

1.0

0.8
0.6
A

0.4
0.2
0.0

400

450

500

550

600

nm

2.0
1.8
1.6

1.4
1.2
A

1.0
0.8
0.6
0.4
0.2
0.0
400

450

500

550

600

nm
B

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To optimize the assay variables, the effects of surfactant, pH, reaction time, temperature and the concentrations of eosin,
copper (II) on the absorbance of the ternary complex formed, with respect to maximum sensitivity, adherence to Beers law and
stability, were studied through controled experiments. The effect of the type of surfactant and its concentration on the absorbance of
the complex was studied using various dispersing agents, e.g., an ionic surfactant sodium lauryl sulphate (SLS) and the non ionic
surfactant methylcellulose (MC). The effect of aqueous surfactant solutions were compared with methanol alone as a solvent.
Regarding CEF, the use of surfactants and methanol prevented precipitate formation and therefore excluded prior extraction steps.
However, the use of methanol alone as a solvent improved the complex stability and produced the highest sensitivity when compared
to SLS and MC. On the other hand, for GLI, the addition of MC (1 mL of 0.3% w/v) was found to improve the complex stability and
to produce the highest sensitivity when compared to SLS and methanol. The effect of pH on the absorbance of the ternary complex
was also investigated. The absorbance of the drugCu(II)eosin complex solution was investigated over the pH ranges 47. The
optimum absorbance was achieved at pH 5.4 and 5.0 for CEF and GLI, respectively. The effect of the concentration of the reagents
(eosin and Cu (II) sulfate) on the absorbance of the ternary complex was studied. Maximum color intensity was produced when the

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Figure 2: Absorption spectrum of ternary complex of: A) Cefixime (8-24g mL-1), B) Glimepiride (5-50 g mL-1).

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amount of the reagents mentioned in the construction of the calibration graphs have been used. Higher concentration of reagents did
not affect the colour intensity.
In order to examine the effect of temperature and reaction time on the absorbance of the ternary complex, the above
mentioned procedure was carried out at different temperatures using thermostatic water bath. Maximum and constant absorbance was
obtained at room temperature after 20 min from the addition of the reaction contents. The color formed under the above mentioned
optimum conditions was stable for at least 2 hours.
Stoichiometry of the Reaction
The nature of the complex (drug-Cu-eosin) was determined using Jobs method of continuous variation. The result of
applying this method can be summarized for both drugs as follows: the [drug:copper(II)] ratio in presence of excess eosin was 1:1, the
[drug:eosin] ratio in presence of excess Cu(II) sulfate was 1:1, and the [eosin:copper(II)] ratio in presence of excess drug was 1:1.
Hence the composition of the ternary complex formed may be expressed as [drug-Cu-eosin] (1:1:1) for both CEF and GLI.
Validation of the proposed method
Linearity
Under the above experimental conditions, the calibration curve was constructed by plotting the absorbance of the studied
drugs versus the drug concentration within the specified range. A linear correlation was found between concentration and absorbance
in the range given in Table 1. The correlation coefficients, intercepts and slopes for the calibration data for the tested drugs were
calculated using least squares method (Table 1).
Sensitivity
The limit of detection (LOD) and the limit of quantitation (LOQ) for the proposed method were calculated using the
following equations:
LOD = 3.3 SD/S
LOQ = 10 SD/S
SD is calculated as the standard deviation of the residuals around the regression line, S is the slope of calibration curve. LOQs and
LODs for the studied drugs are listed in Table 1. Sandell's sensitivity was also calculated and listed in table 1.
Table 1: Assay parameters and spectral data for spectrophotometric determination of cefixime and glimepiride ternary
complex formation with Cu(II)-eosin.
Parameters
Cefixime
Glimepiride
max nm
550
544
Beers law limits, g mL-1
4 28
5 50
Molar absorptivity, L mol-1 cm-1
1.491 X 104
1.657 X 104
2
Sandell's sensitivity, g/cm
0.030
0.029
Limit of detection, g mL-1 (LOD)
0.900
1.70
Limit of quantitation, g mL-1 (LOQ) 2.70
5.10
Regression equation*
Y = 0.0361 + 0.03X Y = 0.0093 + 0.0338X
Intercept (a)
0.036
0.0093
Slope (b)
0.030
0.0338
Correlation coefficient (r)
0.9997
0.9998
Y= a + bX, where Y is the absorbance, a intercept, b slope and X concentration in gmL-1.

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Precision and accuracy

The precision and accuracy of the proposed method were tested by means of replicate measurements of the tested drug
within Beers law limits. The precision of the analytical procedure is usually expressed as the standard deviation of a series of
measurements. Intraday and interday precision were assessed using three concentration and three replicates of each concentration. The
calculated relative standard deviation values were found to be small (< 2%) indicating good repeatability and reliability of the
proposed methods. The results and their statistical analysis were summarized in Table 2. Accuracy of the proposed method was further
confirmed by performing recovery studies at three levels by standard addition method. The determination with each concentration was
repeated three times and average percent recovery of the added standard was calculated and results are tabulated in Table 3. The
results obtained in tables 2 and 3 showed excellent mean recovery percent values, close to 100 %, and low standard deviation values
(S.D. < 1.0) which indicate high accuracy of the proposed analytical methods.

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Specificity
The specificity of the developed method was investigated by observing any interference encountered from common
excipients of the pharmaceutical formulations such as starch, lactose, talc, and magnesium stearate and they were found not to
interfere in the analysis.

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Table2: Evaluation of the precision of the analytical procedure of cefixime and glimepiride.
Cefixime
4
16
g mL-1 g mL-1

Parameter

Glimepiride
24
gmL-1

10 gmL-1

25 gmL-1

50 gmL-1

24.74
24.73
24.81
24.76
99.04
0.0436
0.176
0.96%
24.80
24.82
25.09
24.90
99.6
0.162
0.651
0.4%

49.51
49.57
49.62
49.57
99.14
0.0551
0.111
0.86%
49.60
49.53
49.85
49.66
99.3
0.168
0.338
0.68%

Intraday
1
4.057
16.08
24.297 10.06
2
4.068
15.835
24.152 10.13
3
4.022
15.807
24.177 10.05
Mean recovery
4.049
15.907
24.209 10.08
Mean % recovery 101.22
99.42
100.87 100.8
S.D.
0.024
0.15
0.0775 0.0436
R.S.D. (%)
0.59
0.94
0.320
0.433
Relative error
1.22%
0.58%
0.87%
0.8%
Interday 1
4.050
15.907
24.210 10.08
2
4.065
15.914
24.220 10.065
3
4.024
16.070
23.921 10.146
Mean recovery
4.046
15.96
24.117 10.097
Mean % recovery 101.15
99.75
100.5
100.97
S.D.
0.021
0.092
0.170
0.043
R.S.D. (%)
0.51
0.58
0.705
0.43
Relative error
1.15%
0.25%
0.488
0.97%
S.D. = Standard Deviation, R.S.D. = Relative Standard Deviation.

Table 3: Results of recovery study for studied drugs.

Drug

Cefixime

Glimepiride

Base level
(g mL-1)
4
4
4
5
5
5

Amount spiked
(g mL-1)
4.00
12.0
20.0
5.00
20.0
40.0

Amount recovered*
(g mL-1)
4.036
12.078
19.92
5.047
20.068
39.88

% Recovery
SD
100.90.273
100.660.302
99.60.64
100.940.61
100.340.27
99.70.37

* Mean value of three determinations.

Application to pharmaceutical dosage forms
The proposed method has been successfully applied to the determination of the studied drugs in commercial dosage forms:
capsules and tablets for CEF and GLI, respectively. The estimated drug content with low values of standard deviation established the
precision of the proposed method. The results obtained are shown in Table 4. The recovery percentage obtained by the proposed
method was satisfactory when compared with the method developed by Frag et al, [30] for cefixime determination and Khan et al,
[31] for GLI analysis.
Table 4: Results of application of spectrophotometric method to the determination of cefixime and glimepiride in
pharmaceutical dosage forms.
Reference Methods
% Recovery

99.71%

99.7

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Proposed Method
Label claim % Recovery
(g mL-1)
SD
4
98.31.41
Cefixime
12
98.90.89
(Capsules 400 mg)
24
98.60.93
5
98.21.96
Glimepiride
15
98.331.3
(Tablets 3mg)
30
98.60.46
*Mean value of three determinations

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Pharmaceutical formulation

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CONCLUSION
The present study described the development and validation of a simple assay for the spectrophotometric determination of
CEF and GLI. The data obtained reveal that the proposed methods are accurate, time saving, do not require prior extraction procedure
nor heating and have the advantages of simplicity, sensitivity and reproducibility. The applicability of the proposed methodology for
the quality control of CEF and GLI was confirmed as indicated by the acceptable recovery values, therefore, can be used for routine
analysis of the two drugs in their pharmaceutical dosage forms.

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