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Application of simultaneous Equation Method for the Determination of

Azithromycin and Cefixime Trihydrate in Tablet Formulation

Article in Research Journal of Pharmacy and Technology · January 2017

DOI: 10.5958/0974-360X.2017.00025.7

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 Research J. Pharm. and Tech. 10(1): January 2017

ISSN 0974-3618 (Print) www.rjptonline.org

0974-360X (Online)

RESEARCH ARTICLE

Application of simultaneous Equation Method for the Determination of

Azithromycin and Cefixime Trihydrate in Tablet Formulation
1 2 1 1
Jagdish V. Manwar , Bhagwat U. Nagargoje , Vishal C. Gurumukhi , Dipti G. Ratnaparkhi ,
1 1 1 3
Poonam P. Warade , Dipak D. Kumbhar *, R.L. Bakal , Rahul S. Manmode
1 2
KYDSCT College of Pharmacy, Sakegaon (Bhusawal) 425 201, Dist. Jalgaon, MS, India
Maharashtra Institute of Pharmacy, MIT Campus, Kothrud, Pune 411038, MS, India
3
Genzyme Corporation, 500 Soldiers Field Road, Allston 02134, MA, USA
*Corresponding Author E-mail: dipakk16@gmail.com

ABSTRACT:
A simple, accurate, and precise uv-spectrophotometric method has been developed for the simultaneous estimation
of azithromycin (AZI) and cefixime trihydrate (CEFI) in tablet formulation. The method was based on employing
simultaneous equation method for the analysis of both drugs. AZI and CEFI have shown absorbance maxima at 222
and 289 nm in methanol, respectively. The linearity was obeyed in the concentration range of 10-50µg/ml for both
2
drugs, with a significantly high correlation coefficient ( r = 0.999). The limits of detection for AZI and CEFI were
0.81 and 1.52 µg/ml, respectively, and the limits of quantitation for AZI and CEFI were 2.40 and 4.60 µg/ml,
respectively. The suitability of the developed method for quantitative determination of drugs was proved by
validation. The method was successfully used to analyze a tablet formulation.

KEYWORDS: Azithromycin, Cefixime trihydrate, Simultaneous equation method, Uv-spectrophotometry,

Validation.

1. INTRODUCTION:
There are number of analytical methods for the
determination of drugs from bulk and various
formulations like tablets, capsules, injections, etc. These
1-3 4-8
methods include Uv-spectrophotometry , HPLC ,
9-10 11-12
UPLC , Gas chromatography , etc. Azithromycin
(AZI) is a chemically
(2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-
trihydroxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-11-
{[3,4,6-trideoxy-3-(dimethylamino)-β-D-xylo-}oxy}-1-
oxa-6-azacyclopentadec-13-yl- 2,6-dideoxy-3-C-methyl-
13
3-O-methyl-α-L-ribo-hexopyranoside (Fig. 1). It is an
azalide category of drug, which is subclass of the
14-16 Fig. 1: Structure of azithromycine.
macrolide antibiotic. It is mainly used in upper
17
respiratory tract infections. Cefixime trihydrate (CEFI) is chemically (6R,7R)- 7-
{[(2Z)-2-(2-amino- 1,3-thiazol-4-yl)-2-methoxyimino-
acetyl]amino}-3-(methoxymethyl)-8-oxo-5-thia-1-
azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid (Fig. 2).
18
CEFI is a third-generation cephalosporin antibiotic. It is
Received on 02.12.2016 Modified on 19.12.2016 used in treatment of lower respiratory tract, urinary tract,
Accepted on 24.12.2016 © RJPT All right reserved 19-20
Research J. Pharm. and Tech. 2017; 10(1): 108-112.
and other soft tissues infections. AZI and CEFI
DOI: 10.5958/0974-360X.2017.00025.7
108
 Research J. Pharm. and Tech. 10(1): January 2017

are official in Indian Pharmacopoeia and United States Substituting for c y in eq. (1), and rearranging gives
13,21
Pharmacopoeia.
cx = A2ay1 – A1ay2 / ax2ay1 –
ax1ay2 and
c =Aa Aa /a a a a
y 1 x2 – 2 x1 x2 y1 – x1 y2

Criteria for obtaining maximum precision, based upon

absorbance ratios, have been suggested that place limit on
the relative concentrations of the components of the
mixture. The criteria are that the ratios,

A2/A1/ ax2/ ax1

and
Fig. 2: Structure of cefixime trihydrate.
ay2/ay1/ A2/A1

Combination of AZI sodium and CEFI is available in the For the precise determination of Y and X respectively,
these ratios should lie outside the range of 0.1-20. These
market which is used to treat or prevent bacterial
22-23 criteria are satisfied only when the λmax of the two
infections especially throat infection . Literature
components are reasonably dissimilar. An additional
survey revealed only two Uv-spectrophotometric methods
criterion is that the two components must not interact
were reported for simultaneous estimation of AZI and
24-25 chemically, thereby negating the initial assumption that
CEFI in bulk drug and Pharmaceutical dosage forms. the total absorbance is the sum of individual absorbance.
Nevertheless, there was no report of simultaneous
equation method for the simultaneous determination of 3.MATERIALS AND METHODS:
AZI and CEFI in combined dosage form. Thus, the prime
3.1. Instrument:
objective of present work was to develop new uv-
A Shimadzu UV-Visible spectrophotometer model 1600
spectrophotometric method for the simultaneous
with matched pair of quartz cell (1.0 cm path) was used
determination of AZI and CEFI in combined dosage
forms. After examining the overlain spectra of both the for absorption measurements.
drugs, simultaneous equation method was applied for the
simultaneous determination both the drugs in tablet dosage 3.2. Chemical and reagents:
forms. Pure drug sample of AZI and CEFI were obtained as a gift
sample from Ajanta pharmaceutical Ltd, Mumbai.
2.THEORY: Methanol AR grade of Rankem Ltd., New Delhi was used.
Simultaneous equation method method is used where a Drug product samples (tablets) were obtained from local
sample contain two absorbing drugs (X and Y) each of market. Tablet use for analysis was HYFEN-AZ
manufactured by Hetro labs (Hyderabad, India) containing
this absorbs at the λmax of each other i.e. λ1 and λ2, it may AZI 250 mg and CEFI 200 mg.
be possible to determine both the drugs by the technique
of simultaneous equation method provided that certain
3.3. Selection of common solvent:
criteria apply. The information required is (i) absorptivity
of X at λ1 and λ2 and ax1 and ax2 respectively, (ii) Methanol was selected as common solvent for studying
spectral characteristics of the selected drugs.
absorptivity of Y at λ1 and ay1 and ay2 respectively, (iii)
absorbance of the diluted sample at λ1and λ2, A1 and A2
3.4. Preparation of standard stock solutions:
respectively. Let Cx and Cy be the concentration of X and
Y respectively in the diluted sample. Two equations are Stock standard solutions of AZI and CEFI were separately
constructed based upon the fact that at λ1 and λ2 the prepared by dissolving 1000 mg in 1000 ml volumetric
absorbance of the mixture is the flask containing 30 mL methanol, shaken for 30min and
sum of the individual absorbance of X and Y. the volume was made up to the mark with methanol (1000
µg/ml of each drug).
At λ1 A1 = ax1 bcx + ay1 bcy ………(1)
At λ2 A2 = ax2 bcx + ay2 bcy ……….(2) 3.5. Selection of analytical wavelength:
Appropriate volume, 10 ml of AZI and 10 ml CEFI
Rearrange eq. (2) cy standard stock solution was transferred to two separate
= A2 - ax2cx / ay2 100 ml volumetric flasks and the volume was adjusted to
mark with methanol to get concentration 10µ g/ml and
10µ g/ml, respectively. The solutions were scanned
separately in the UV-region i.e. 200-400nm.
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 Research J. Pharm. and Tech. 10(1): January 2017

3.6. Linearity and Range: Selection of Concentration
In order to select the final concentration of each drug, a
separate calibration graph for AZI and CEFI was plotted
as absorbance versus concentration by measuring the
absorbance of each solution at 289 and 222 nm and
regression equations were established. For this purpose,
a series of dilutions (10, 20, 30, 40 and 50 μg/ml) were
made separately for each drug using standard stock
solution. From calibration graph, final concentration for
AZI and CEFI were 50μg/ml and 40μg/ml, respectively.
The limit of detection and limit of quantitation were
measured on the basis of signal-to-noise ratios.
filtered and was further diluted to get a final
concentration of about 50µg/ml of AZI and 40µg/ml of
CEFI. The response of sample solutions was measured at
222 nm and 289 nm against blank. The content of AZI
and CEFI in tablet dosage form was calculated using two
framed simultaneous equations and derivative method.
4. VALIDATION OF METHOD:
The method was validated according to the ICH
26
guidelines. Parameters studied for validation were
accuracy, precision, linearity, LOD, LOQ and specificity
for the analyte.

3.7. Determination of Absorptivities: 4.1. Accuracy:

(A (1%, 1 cm)). The absorbance (a) of selected To study the accuracy, reproducibility and precision of
concentration for both drugs was taken at selected the proposed method, recovery study was carried out by
wavelength. These absorbance values were then divided addition of standard drug solutions to preanalysed
by concentration in gm/l to get absorptivities A (1%, 1 sample. Recovery studies of proposed method was
cm). carried out on the basis of standard addition method at
different levels (i.e. 80, 100, 120%). Tablet solution
3.8. Methodology and Simultaneous Equation: containing 50 mcg/ml of AZI and 40 mcg/ml of CEFI
For each drug, the absorptivity value A (1%, 1 cm) was considered at 100% and from that three different levels
determined at both the wavelengths for each drug. A set i.e. 80%, 100%, 120% was prepared by using standard
of two simultaneous equations was formed as: stock solution of each drug. Then by taking the
c =Aa Aa /a a a ay absorbance calculate the amount added and find out
x 2 y1 – 1 y2 x2 y1 – x1 2 percentage recovery.
and
c =Aa Aa /a a a a
y 1 y2 – 2 y1 x2 y1 – x1 y2 4.2. Limit of Detection (LOD) and Limit of
Quantitation (LOQ):
where, The LOD and LOQ values were determined by diluting
A1 and A2 are absorbances of mixture at 289 nm and 222 known concentrations of drug until the average
nm; responseswere approximately 3 or 10 times the standard
a
x1 and ay1 are absoptivities of AZI and CEFI deviationof the responses of the blank for six replicate
respectively at 289 nm; determina-tions. The signal/noise ratios 3:1 and 10:1
a
x2 and ay2 are absoptivities of AZI and CEFI were taken asthe LOD and LOQ, respectively.
respectively at 222 nm;
4.3. Precision:
3.9. Calculation of Concentration: precision of method was checked by measuring the
The values cx and cy were calculated by putting the absorbance of standard solution for AZI (50 µg/ml) and
values of A1 and A2 to solve the simultaneous equations CEFI (40 µg/ml) repeatedly (n = 6).
1 and 2.
5. RESULTS AND DISCUSSION:
3.10. Assay of Tablet Formulation: Simultaneous equation method was applied for the
Twenty tablets of marketed combination were weighed determination of AZI and CEFI in tablet formulation.
accurately and crushed to fine powder. A quantity of Based on the solubility of both the drugs, methanol was
tablet powder equivalent to 50 mg of AZI was accuratly selected as a common solvent in the present study. From
weigheed and transferred to 100 ml volumetric flask. To the overlain spectra (Fig. 3), two wavelengths 222 nm
this flask, about 20-30 ml of methanol was added. The (λmax of AZI) and 289nm (λmax of CEFI) were selected
flask was shaken for 30 min and then sonicated for 30 for the formation of simultaneous equation.
min. After complete dissolution, final volume was made
to 100 ml with the same solvent. The solution was

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 Research J. Pharm. and Tech. 10(1): January 2017

200-250nm with peak maximum at the 222nm. For CEFI

the peak distribution of CEFI was range 200-400nm with
peak maximum at the 289nm. For calculation using
simultaneous equation, we have to find wavelength where
one component absorbs strongly while another absorbs
weakly. Because the two drugs were well separated
spectrally, one cannot use the maximum of one in the
analysis as the molar absorptivity of the other component
would be zero. Following the recommended procedure,
calibration curves were made at selected wavelength for
the standard of AZI and CEFI. The linearity and range was
studied by analyzing the standard solution of AZI and
CEFI separately in triplicate. The calibration curves for
AZI and CEFI are shown in Fig. 4 and Fig. 5,
Fig. 3: The overlain Spectra of AZI and CEFI in methanol.
respectively. The standard deviation of the slope value
was less than 2 (see Table 1).
Both the drugs had broad spectral features in the range of
wavelength from 200-400nm. Their spectral peaks were
well separated. The peak distribution of AZI was range
2.5
0.4 y = 0.041x + 0.023
y = 0.005x + 0.101 R² = 0.999
R² = 0.999 2
Absorban
Absorbanc

0.3
ce

1.5
e

0.2
1

0.1
0.5

0 0
0 10 20 30 40 50 60
0 10 20 30 40 50 60
Concentration of AZI (µg/ml)
Concentration of CEFI (µg/ml)
Fig. 4: Calibration curve for standard solution of AZI.
Fig. 5: Calibration curve for standard solution of CEFI.

The LOD for AZI and CEFI was found to be 0.81 µg/ml more number of chromophoric groups present on CEFI.
and 1.52 µg/ml, respectively. The LOQ for AZI and CEFI These groups –NH2, -NH-, and =N-O-.
was found to be 2.40 µg/ml and 4.60 µg/ml, respectively
(Table 1). Table 2: Estimated absorptivities for AZI and CEFI
Table 1: Results of linearity study (n=6) Components Absorptivity
Parameters Results at 222nm at 289 nm
AZI CEFI AZI 0.57 0.92
Linearity range (µg/ml) 10-50 10-50 CEFI 39.25 44.5
2
Correlation coeff. (r ) 0.999 0.999
Slope 0.005 0.041
LOD (µg/ml) 0.81 1.52 In the assay of tablet formulation, the % relative standard
LOQ (µg/ml) 2.40 4.60 deviation by proposed method in tablet for AZI and CEFI
were 0.0165 and 0.0165. The recovery for AZI and CEFI
Molar absorptivities of AZI and CEFI were obtained from were found to be 99.90% to 99.97% w/w, respectively.
the calibration curves made at selected wavelength. The method was validated to ensure accuracy and
Absorptivity values calculated for each drug at both the reproducibility. The recovery studies and statistical data
wavelengths are shown in Table 2. The AZI shows higher for the method were found to be satisfactory and therefore
absorptivity than CEFI. This is because of higher the method can be used for routine analysis. The results
sensitivity of CEFI as compare to AZI that is because of are shown in Table 3.

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 Research J. Pharm. and Tech. 10(1): January 2017

Table 3: Results of assay of tablet formulation and recovery study (n = 6)*

Level of Recovery Amt. present (mg/tab) Amt. of std. drug added Total amt. recovered Percent Recovery
AZI CEFI AZI CEFI AZI CEFI AZI CEFI
80% 250 200 200 160 449.50 359.87 99.88 99.96
100% 250 200 250 200 499.56 499.91 99.91 99.98
120% 250 200 300 240 549.60 439.85 99.92 99.96
Avg. 99.90 99.97
Std. Dev. 0.0164 0.0114
%RSD 0.0165 0.124
*Amount in milligram (mg)

5. CONCLUSION: form. Pharmaceutical Methods, 2012, 3(2), 57–61.

The results confirm the method is accurate and free from 11.
any negative or positive interfering of other compounds
present in formulation. Thus it concludes that proposed
simultaneous equation method is suitable for the
determination of AZI and CEFI from bulk drug and tablet 12.
formulation.
6. CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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