WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Mrudul et al.
World Journal of Pharmacy and Pharmaceutical Sciences
Volume 3, Issue 2, 1340-1348.
Research Article
ISSN 2278 – 4357
A NEW SPECTROPHOTOMETRIC METHOD FOR
DETERMINATION OF AMOXICILLIN USING BROMOCRESOL
GREEN
Mrudul R. Keskar*, Ravin M. Jugade
Department of Chemistry, R. T. M. Nagpur University, University Campus, Amravati Road,
Nagpur, 440 033, Maharashtra, India.
Article Received on
29 November 2013,
Revised on 17 December
2013,
Accepted on 15 January
2014
ABSTRACT
Simple and rapid spectrophotometric method has been developed for
the determination of amoxicillin in bulk and pharmaceutical
formulations. The method is based on the formation of blue ion-pair
complex
between
amoxicillin
and
bromocresol
green
in
*Correspondence for
dimethylsulphoxide medium. The studies have been carried out with
Author:
Spectronic 20D+ (Thermospectronic) visible spectrophotometer. The
Mrudul R. Keskar
Department of Chemistry, R. T.
M. Nagpur University,
absorption maxima of the complex was found to be 630 nm. The
composition of complex was determined by Job’s method was found to
University Campus, Amravati
be 2:1 (Drug: Reagent). Different variables affecting the reaction were
Road, Nagpur, 440 033,
studied and parameters such as time, reagent concentration etc were
Maharashtra, India.
optimised. The formed complex was quantified spectrophotometrically
at absorption maxima. Concentration of bromocresol green was found
to be optimum at 9.6µM. Under the optimum conditions calibration graph was found to be
linear over the range of 1-13 µg/ml with detection limit 0.06µg/ml. Thermodynamic studies
were carried out including stability constant (log K=4.97) and Gibb’s free energy change (∆G
= –2.8167 x 104 kJ/mol). Validation parameters such as Sandell’s sensitivilty, correlation
coefficient, coefficient of determination, standard deviation and relative standard deviation
were determined. Recovery studies were carried out. The method was successfully applied
for the determination of amoxicillin in pharmaceutical formulations such as tablets, capsules
and oral suspension. The proposed method is simple, direct, sensitive and do not require any
extraction process. Thus, this method could be readily applicable for the quality control and
routine analysis.
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Keywords: Amoxicillin, bromocresol green, ion-pair complex, spectrophotometry.
INTRODUCTION
Amoxicillin, semi-synthetic drug belongs to a class of antibiotics called as Penicillin. It is
shown to be effective against a wide range of infections caused by wide range of Grampositive and Gram-negative bacteria in both human and animals. Structure of amoxicillin is
as shown in Fig. 1(a). In the past decade, it is used to treat infections of the middle ear (otitis
media), tonsilis (tonsillitis and tonsillopharyngitis), throat, larynx (laryngitis), pharynx
(pharyngitis), brosnchi (bronchitis), lungs (pneumonia), urinary tract, (UTI), skin etc[1-3].
Several methods have been reported in the literature for the analysis of amoxicillin such as
HPLC[4],
potentiometry[5],
spectrofluorometry[6],
flow-injection[7],
Thin
layer
chromatography[8], voltammetry[9] etc.
Bromocresol green (BCG) as shown in Fig. 1(b) is a sulphonphthalein dye commonly used as
indicator and spectrophotometric reagent.
(a)
(b)
Fig. 1 Structure of (a) amoxicillin, (b) bromocresol green.
Spectrophotometric method is based on the formation of coloured (charge transfer or ionpair) complex between drug and reagent which can be estimated by visible
spectrophotometry.
Charge transfer complex is also called as electron donor-acceptor
complex in which a fraction of electric charge is transferred between the molecular entities.
In ion-pair complex formation, ions of opposite electric charge are produced in the solution
due to the electron transfer. Reported spectrophotometric methods of amoxicillin include
complex
formation
[11]
thiocyanate
with
chloranilic
acid[4],
, Folin-Clocaltue phenol reagent
[12]
hematoxyline[10],
[13]
, methylene blue
molybdenum
and
, iodine and wool fast
blue[14].
The proposed method is based on formation of ion-pair complex of amoxicillin with
bromocresol green. Bromocresol green has been used for the first time with significantly low
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World Journal of Pharmacy and Pharmaceutical Sciences
detection limit, high sensitivity and wider dynamic range for the determination of
amoxicillin. An important feature of the method is that no extraction is required and it is
feasible at room temperature.
MATERIALS AND METHODS
Reagents and Chemicals
All chemicals and reagents used were of analytical grade. Amoxicillin was kindly provided
by ZIM laboratories. Bromocresol green, dimethyl sulphoxide (DMSO) were obtained from
LOBA Chemie. 0.01M solutions of amoxicillin and bromocresol green were prepared in
dimethyl sulphoxide as stock solutions. The solutions were further diluted as per requirement.
Apparatus
Spectrophotometric studies were carried out with Spectronic 20D+ (Thermo-Spectronic)
visible spectrophotometer. A Mettler balance (Ner-Parma instrument Corp. L.C. =0.01mg)
was used for weighing purpose.
Procedure for Calibration Curve
Suitable aliquots of amoxicillin in dimethyl sulphoxide were transferred into series of 10ml
of volumetric flasks. Bromocresol green solution was added to make final concentrations of
9.6µM making up the volume with dimethylsulphoxide. The absorbance of blue coloured
solution was measured at 630nm against the appropriate reagent blank.
Procedure for dosage forms (tablet, capsules and oral suspension)
Five tablets or capsules were weighed and average weight of one tablet or one capsule was
determined. They were powdered and about 0.05g was exactly weighed and shaken with
30ml dimethylsulphoxide for 30 minutes. It was filtered with Watmann filter paper no. 40
and made up 50 ml with dimethylsulphoxide. The same procedure was applied for oral
suspension of drug using 1ml suspension. Suitable aliquots were analysed using general
procedure describe above. The antibiotic content of tablet, capsule and oral suspension were
calculated from calibration curve and also by standard addition method.
RESULTS AND DISUSSION
Absorption Spectra
Amoxicillin form blue coloured ion-pair reaction product with bromocresol green having
absorbance maxima 630 nm as shown in Fig. 2. Under the experimental conditions, the
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reagent blank showed negligible absorbance at 630 nm. Different experimental parameters
affecting the formation and stability of complex were studied to determine the optimum
conditions.
Fig. 2. Absorption spectra of amoxicillin (3700µg/ml), bromophenol blue (8µM) and
their complex.
Effect of Solvent
Various solvents like methanol, ethanol, acetone, dichloromethane, dichloroethane,
dimethylsulphoxide, chloroform and acetonitrile were studied for solubility, complex
formation, to achieve maximum sensitivity and product stability. Dimethylsulphoxide
(DMSO) was found to be most suitable solvent for amoxicillin and bromocresol green. Hence
it was used as a solvent.
Stoichiometric Relationship
Composition of ion-pair complex was established by applying Job’s method of continuous
variation as shown in Fig. 3 using equimolar solutions of the drug and the reagent. The results
indicated that the complex was formed in the ratio of 2:1 (D: R), the absorbance of the
complex was used to calculate stability constant and Gibbs free energy change.
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Fig 3. Continuous variation plots for the ion-pair complex of amoxicillin with
bromocresol green in DMSO solvent at 630 nm (a) 1x10-4M, (b) 2x10-4M, (c) 3x10-4 M.
Effect of Time
Mixture of drug and reagent was prepared; the optimum reaction time was determined by
recording the absorbance of the formed complex at different time intervals. The variation has
been shown in Fig. 4. It was found that the ion-pair complex was formed instantaneously at
room temperature and stable for near about 40 minutes.
Fig. 4 Effect of time on the absorbance of amoxicillin (3700µg/ml) and bromocresol
green (8µM) in DMSO solvent at 630 nm.
Effect of Reagent Concentration
The optimum reagent concentration was determined by adding various volumes of
bromocresol green to the drug. The colour intensity was found to be increase with addition of
bromocresol green up to 9.6 µM and decreased thereafter. Fig. 5 indicates the effect of
concentration of bromocresol green on absorbance. The absorbance was found to be
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World Journal of Pharmacy and Pharmaceutical Sciences
maximum at bromocresol green concentration of 9.6 µM therefore; this concentration was
selected for the preparation of calibration graph.
Fig. 5 Effect of bromocresol green concentration on ion-pair complex of amoxicillin (a)
15 µg/m, (b) 7 µg/ml in DMSO solvent at 630 nm.
Calibration curve for amoxicillin was constructed by plotting absorbance versus
concentration in µg/ml. Under optimum conditions, various analytical parameters were
obtained and presented in table 1. The value of correlation coefficient indicates good linearity
of the present method. High value of molar absorptivity and lower value of Sandell’s
sensitivity reflect good and high sensitivity of the method.
Table 1. Collective data for the Qualitative and Statistical parameters using proposed
method.
Drug
Amoxicillin
Solvent
Dimethyl sulphoxide
λmax(nm)
630
Molar ratio(D:R)
2:1
Linear range (µg/ml)
1-13
Slope
0.0339
Intercept
-0.0066
Linear regression equation
A=0.0339C -0.0066
2
Sandell’s sensitivity (µg/cm )
0.0294
Correlation coefficient (r)
0.9994
Coefficient of determination(r2)
0.9988
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a
World Journal of Pharmacy and Pharmaceutical Sciences
Stability constant (log K)
4.97
Molar absorptivity (L mol-1 cm-1)
1.2553 x104
LOD a(µg/ml)
0.06
LOQ b(µg/ml)
0.20
%RSD c (at 10 µg/ml)
2.24
ΔGo d(kJ mol-1)
-2.8167 x104
Limit of detection,
b
Limit of Quantification, c Relative standard deviation,
d
Free energy
change, A = Absorbance, C= Concentration of drug (µg/ml).
Recovery studies were carried out for amoxicillin using calibration curve at three different
concentrations. These results are presented in table 2.
Table 2. Recovery studies of Amoxicillin.
a
S.N
Sample
Method
1.
Amoxicillin BCG
Taken
(µg/ml)
2.5
7.5
12.5
Found
(µg/ml)a
2.5
7.5
12.6
Recovery
(%)
100.00
100.00
100.80
Mean±SD
100.26±0.46
Average of three determinations.
APPLICATION
The proposed method has been successfully applied to the determination of amoxicillin in
pharmaceutical formulation such as tablet, capsules and oral suspension. Calibration curve as
well as standard addition method was adopted for quantitative analysis. The results are
presented in table 3.
Table 3. Analysis of pharmaceutical formulations
S.
N.
a
Pharmaceutical
Preparationsa
Labelled
Amount
(mg)
Found Amount (mg)
1
2
Rexel capsules
Moxkid tablets
250
250
Calibration curve
method b
246.33±1.50
251.10±2.13
3
Moxkid tablets
125
124.03±0.63
Standard addition method
126.73±1.01
b
241.66±4.92
253.73±3.96
4 Novamax Liquid
125
123.66±1.25
120.86±1.61
b
Ranbaxy laboratories products , (Average±SD) of three observations
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The developed method was found to be versatile and have many advantages over the
previously reported methods. The proposed method is more sensitive compared to the
established method as shown by the molar absorptivity[4]. The detection limit is lower than
the reported methods [4, 13]. The method is utilized a single step reaction with no extraction
process and so simpler compared to reported methods [11, 13].
CONCLUSION
This method requires only dye and solvents which are comparatively cheaper and readily
available. The method is simple as it does not involve adjustment of critical conditions like
temperature, pH or tedious sample preparation. This method has many advantages over other
analytical methods due to its simplicity, sensitivity, rapidity, low cost instrumentation,
accuracy. Due to these advantages this method can be used for quality control and routine
analysis.
ACKNOWLEDGEMENT
The authors are thankful to UGC, New Delhi for financial assistant under Start-up-Grant.
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