World Journal of Pharmaceutical Research

Wadher et al. World Journal of Pharmaceutical Research

SJIF Impact Factor 7.523

Volume 6, Issue 10, 1002-1014. Research Article ISSN 2277–7105

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING

ASSAY METHOD FOR SIMULTANEOUS ESTIMATION OF
AMOXICILLIN TRIHYDRATE AND CLOXACILLIN SODIUM IN
PHARMACEUTICAL DOSAGE FORM BY USING RP-HPLC

Wadher Shailesh J.*, Kalyankar Tukaram M., Shivpuje Shivraj S.,

Khandre Supriya S., Lamture Sima S. and Shaikh Isak

Department of Quality Assurance, School of Pharmacy, Swami Ramanand Teerth

Marathwada University, Vishnupuri, Nanded- 431606, (M.S.) India.

ABSTRACT
Article Received on
11 July 2017, A simple, accurate, precise, sensitive and stability indicating reverse
Revised on 01 August 2017, phase high performance liquid chromatography method has been
Accepted on 21 August 2017
DOI: 10.20959/wjpr201710-9410 developed for simultaneous determination of amoxicillin trihydrate and
cloxacillin sodium in bulk and in combined capsule dosage form.
Chromatographic separation was performed on C18 column, with
*Corresponding Author
Dr. Wadher Shailesh J.
mobile phase consist of water, acetonitrile, and methanol in the ratio of
Department of Quality 70:20:10 (v/v/v), at flow rate 1.4 ml/min. Quantification of both drugs
Assurance, School of was achieved at UV detector at 238.8 nm.The retention time of
Pharmacy, Swami amoxicillin trihydrate and cloxacillin sodium was found to be 8.240
Ramanand Teerth
and 14.036 minute respectively with run time 20 minutes. Amoxicillin
Marathwada University,
Vishnupuri, Nanded-
trihydrateand cloxacillin sodiumfollowed the linearity in concentration
431606, (M.S.) India. range 20-100 µg/ml for both with correlation coefficient (r2) values
0.998 for amoxicillin trihydrate and 0.998 for cloxacillin sodium. The
proposed method was validated according to ICH guidelines in terms of linearity, accuracy,
precision, LOD and LOQ. Percentage assay was found to be 98.92 % and 99.61 % for
amoxicillin trihydrate andcloxacillin sodium respectively. In precision % RSD was found to
be < 2% for both. The percentage recovery was found in range 99.18%-99.42%. The LOD
and LOQ values werewithin limit. The degradation studies carried under condition of acid,
base, neutral, oxidative, photolysis, thermal degradation and no attempt was made to identify
the degradation product.

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KEYWORDS: Amoxicillin trihydrate, cloxacillin sodium, RP-HPLC, validation, stability

indicating assay method.

INTRODUCTION
Amoxicillin trihydrate (AMO) is a broad spectrum semi-syntheticantibiotic. It is effective
against a wide range of infections caused by wide range of Gram-negative bacteria and
Gram-positive bacteria in human and animals. It acts by inhibiting synthesis of bacterial cell
wall. Chemically AMO is (2S,5R,6R)-6-{[2R)-2-Amino-2-(4-hydroxyphenyl)acetyl]amino}-
3,3-dimethyl-7-oxo-4-thia-1-azabicycloheptane-2-carboxylic acid.[1]

Cloxacillin sodium (CLO) is semi synthetic antibiotic used against staphylococci that
produce beta-lactamase. It is less active against pnG sensitive organisms. ChemicallyCLO
(2S,5R,6R)-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl] amino}-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.[2] Structure of AMO and CLO are
shown in figure 1.

According to literature survey AMO and CLOwas estimated by UV[3-11], RP-HPLC[12-21],

HPTLC[22], and stability indicating HPLC[23] in combination, but no stability indicating RP-
HPLC method has been reported for simultaneous estimation of AMO and CLO in
combination by using water:acetonitrile:methanol (70:20:10) as mobile phase.Present work
described the stability indicating HPLC method for simultaneous estimation of AMO and
CLO in bulk and dosage form.

MATERIALS AND METHOD

Instrumentation and reagents
Chromatographic separation was performed on HPLC-system (model Shimadzu SCL- 10)
C18 column (250 mm x 4.6 mm, 5 µm) UV Detector, equipped with a solvent delivery pump,
sample injector and column thermostats. Lab solution (Version 1.25) software was applied
for data collecting and processing. Water, acetonitrile, and methanolused were of HPLC
grade. Pure drug sample of AMO and CLO was procured from Macleods Ltd., Daman.

Chromatographic condition
Mobile phase: Water: acetonitrile: methanol (70:20:10 v/v/v)
Column : C18column
Detector wavelength : 238.8 nm

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Wadher et al. World Journal of Pharmaceutical Research

Injection volume : 20 µl
Flow rate : 1.4 ml/min
Run time : 20 minutes

Selection of detection of Wavelength

Accurately weighed 10 mg AMO and CLO were transferred into 100 ml volumetric flask
separately, dissolved in mobile phase, sonicated and filtered through Whatman filter paper
No. 41 and volume was made up to the mark with mobile phase. Pipette out 2 ml of this stock
solution and diluted to 10 ml to get a concentration of 20 µg/ml of AMO and CLO each. The
λ max was determined on Shimadzu UV-visible spectrophotometer (Shimadzumodel UV-
1800) in the range 200-400 nm. Theoverlain spectra showed iso-absorptive point at 238.8 nm
was selected as wavelength. The overlain spectra of AMO and CLO are shown in figure 2.

Preparation of standard solution

Standard solution was prepared by transferring 50 mg of AMO and 50 mg of CLO in 50 ml
of volumetric flask separately and dissolved in mobile phase. It was sonicated for 20 min to
dissolve completely, and then volume was made up to the mark with mobile phase to get
concentration 1000 µg/ml for both. From the above stock solution pipette out 1 ml and
transferred to 10 ml volumetric flask and volume was made up with mobile phase and 20 µl
was injected into HPLC system to develop the chromatogram. Typical chromatogram of
AMO and CLO standard is shown in figure3.

System Suitability test

System suitability was performed by injecting standard solution and determines the various
parameters such as theoretical plates, tailing factor, resolution.Results are shown in table1.

Method Validation
Method was validated as per ICH guidelines.

Linearity
Linearity was performed at five different concentration (20, 40, 60, 80, 100 µg/ml) for both
AMO and CLO. Standard calibration curve was plotted between peak area against
concentration of drug shown in figure 4 and results of linearity are shown in table 2.

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Accuracy
The accuracy of the proposed method was performed by recovery studies at three different
levels (80%, 100%, and 120%) of concentration by adding a known amount of standard drug
to pre-analyzed sample. Each determination was repeated three times at each level and
injected into HPLC system.Results of accuracy are shown in table3.

Precision
Precision of an analytical method is usually expressed as the standard deviation or relative
standard deviation. The precision was determined at different parameter like repeatability,
intermediate precision (intra-day, inter-day). Repeatabilitywas determined by analyzing
AMO (100 µg/ml) CLO (100 µg/ml) three times. Intraday precision was determined by
analyzing same concentration of solution for three times within day and Interday precision
was determined daily for three days. Then % RSD was calculated and it was within limit (less
than 2%). Results of precision are shown in table 4.

Limit of detection (LOD) and Limit of quantification (LOQ)

LOD is the lowest concentration of analyte in sample that can be detected but not necessarily
quantified. LOQ is the lowest concentration of analyte in sample that can be quantitatively
determined with precision and accuracy. Results of LOD and LOQ are given in table 5.

LOD and LOQ was calculated by using following formulae

LOD = 3.3 σ/slope, LOQ = 10 σ/slope
(Where σ = the standard deviation of the response and S = slope of calibration curve).

Robustness
The robustness was performed by assaying test solutions after slight but deliberate change in
analytical conditions. The study was performed by changing the flow rate, temperature and
wavelength. Results of robustness study are shown in table 6.

Analysis of Marketed formulation

Twenty capsules weighed, average weight was determined, crushed to a fine powder and
mixed thoroughly. Accurately weighed powder equivalent to 50 mg of AMO and 50 mg of
CLO was transferred into 50 ml of volumetric flask and dissolved in mobile phase. This was
sonicated for 20 minutes, and then volume was made up to mark with mobile phase. Further
dilution was done with mobile phase to get final concentration of 100µg/ml of AMO and

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100µg/ml of CLO. The standard and sample solution was injected into HPLC system
todevelop the chromatogram. Typical chromatogram of AMO and CLO is shown in figure 5.
The content of AMO and CLO was calculated by using following formula.
Amount of drug (mg) = At/As x Ds/Dt x Ws/Wt x A ………….…..(i)
% Estimation = At/As x Ws/Wt x Avg.wt (A)/Lable claim x 100 ….….(ii)

Where,
At = Area count for sample solution
As = Area count for standard solution
Ds = Dilution factor for standard
Dt = Dilution factor for sample
Ws = Weight of standard (mg)
Wt = Weight of sample (mg)
A = Average weight of capsule
Result are shown in table 7.

Forced Degradation Study

Stress degradation studies were performed to check the stability of the AMO and CLO on
different conditions. The stress conditions for degradation study involved acid, base, neutral,
oxidative, thermal, and photolytic degradation.

Acid degradation
Accurately weighed 20 mg of AMO and CLO was transferred to 100 ml volumetric flask, to
it 20 ml mobile phase and 10 ml 0.1 N HCl was added. This flask was heated on water bath at
0 C for 4.30 hours.Solution was cooled and neutralized with 0.1 NaOH and volume was
made up to mark with mobile phase, finally this solution was diluted with mobile phase to get
concentration 20 µg/ml of AMO and 20 µg/ml of CLO. A 20 µl solution was injected into
HPLC system and analyzed under chromatographic condition.

Base Degradation
Accurately weighed 20 mg of AMO and CLO was transferred to 100 ml volumetric flask, to
it 20 ml mobile phase and10ml 0.1 N NaOH was added. This flask was heated on water bath
at 0 C for 4.30 hours. Solution was cooled and neutralized with 0.1 N HCland volume was
made up to mark with mobile phase. Finally thissolution was diluted with mobile phase to get

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concentration 20 µg/ml of AMO and 20 µg/ml of CLO.A 20 µl solution was injected into
HPLC system and analyzed under chromatographic condition.

Oxidative degradation
Accurately weighed 20 mg of AMO and CLO was transferred to 100 ml volumetric flask, to
it 20 ml mobile phase and 10 ml 3% H2O2 was added. This flask was refluxed for 4 hours.
Solution was cooled and volume was made up to mark withmobile phase, finally this solution
was diluted with mobile phase to get concentration20 µg/ml of AMO and 20 µg/ml of CLO.
A20 µl solution was injected into HPLC system and analyzed under chromatographic
condition.

Photolytic degradation
Pure drugs were exposed to UV radiations for 12 hours. The sample after exposure to light
were accurately weighed 20 mg of AMO and CLO was transferred to 100 ml volumetric
flasks diluted with mobile phase to get concentration 20 µg/ml of AMO and 20 µg/ml of
CLO.A 20 µl solution was injected into HPLC system and analyzed under chromatographic
condition.

Thermal degradation
Thermal degradation was carried out by exposing pure drugs to dry heat at 0 for 2 hours.
The samples after exposure to heat were accurately weighed 20 mg of AMO and CLO,
transferred to 100 ml volumetric flasks diluted with mobile phase to get AMO 20 µg/ml and
CLO 20 µg/ml. A20 µl solution was injected into HPLC system andanalyzed under
chromatographic condition.

Neutral hydrolysis
Accurately weighed 20 mg of AMO and CLO was transferred to 100 ml volumetric flask; to
it 50 ml water was added. This flask was refluxed for 4 hours at 0 , Solutions was cooled
and volume was made up to mark with mobile phase. Finally this solution was diluted with
mobile phase to get 20 µg /ml AMO and 20 µg/ml of CLO.A 20 µl solution was injected into
HPLC system and analyzed under chromatographic condition.

Chromatogram obtained under various degradation conditions are shown in figure 6. Results
of forced degradation studies are shown in table 8.

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RESULT AND DISCUSSION

Several mobile phase composition were tried to resolve the peak of AMO and CLO. The
mobile phase water:acetonitrile:methanol (70:20:10) was found ideal to resolve the peak of
AMO and CLO. The proposed method was found to be linear in concentration range of 20-
100 µg/ml for both. Retention time of AMO and CLO were 8.240 and 14.036 min
respectively. Percentage assay was found to be 98.92 and 99.61 for AMO and CLO.
Systemsuitability parameters were evaluated, which were within acceptance criteria. The
accuracy of the method was confirmed by recovery studies at three different level 80%,
100%, 120%, In precision % RSD was found to be < 2% for AMO and CLO. The forced
degradation showed AMO and CLO undergo degradation in acidic, basic, oxidative,
photolytic, thermal, neutral, condition and percentage degradation was found to be 7.48, 8.77,
7.1, 18.39, 4.23, 8.22, for AMO and 9.34, 11.05, 8.83, 17.43, 8.27, 12, for CLO.

OH COONa
O
CH3
O N
, H2O

H2N CH3
NH O S
N
O Cl
N H H
OH H
S
N
CH3
H3C CH3 O
O

(a) Amoxicillin trihydrate (b) Cloxacilln sodium

Fig. 1: Chemical structure of AMO and CLO.

Fig 2: Overlay spectra of AMO and CLO.

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Fig 3: Chromatogram of standard solution.

Fig 4: Standard Calibration curve.

Fig 5: Chromatogram of AMO and CLO in marketed formulation.

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a. Acid hydrolysis b. Base hydrolysis

C.Oxidative degradation D.Photolytic degradation

E.Thermal Degradation F.Neutral Degradation

Fig 6: Forced degradation.

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Table 1: Result of system suitability test

Parameters Amoxicillin trihydrate Cloxacillin sodium
Retention time (min) 8.240 14.036
Peak area 303855 540700
Theoretical plates/meter 2216 3149
Tailing factor 1.5 1.25
Asymmetry 1.4 1.5
Resolution 6.1

Table No 2: Observation for standard curve

Amoxicillin Cloxacillin
Sr. No. Peak area Peak area
conc. (µg /ml) Conc.(µg/ml)
1 20 61771 20 112084
2 40 126548 40 226168
3 60 179185 60 329952
4 80 248074 80 448336
5 100 303855 100 540700

Table 3: Result of Accuracy.

Recovery % Recovery*
level Amount added(mg) Amount Recovered (mg) % RSD
(%) AMO CLO AMO CLO AMO CLO
80 40 40 39.67 39.8 0.141 0.411
100 50 50 49.79 49.71 0.333 0.536
120 60 50 59.83 59.65 0.430 0.165
*Average of three determination.

Table 4: Result of precision

(% RSD)
Parameters
AMO CLO
Repeatability 0.425 0.210
Precision
Intraday 0.433 0.827
(% RSD)*
Inter-day 0.425 0.210
*Average of three determination.

Table 5: Result of LOD and LOQ.

Drug LOD LOQ
Amoxicillin 0.103 0.310
Cloxacillin 0.082 0.247

Table 6: Result of Ruggedness

Retention time
Sr.No Parameter Condition (min)
AMO CLO
1.2 min/ml 9334 13.887
1 Flow rate
1.4 min/ml 8.240 14.036
236 nm 8.242 14.036
2 Wavelength
238.8 nm 8.240 14.036

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Table 7: Analysis of Marketed Formulation

Injection Label claim (mg) Assay (%)*
Amoxicillin trihydrate 250 mg 98.92
Cloxacillin sodium 200 mg 99.61
*Average of three determination.

Table 8: Result ofForcedDegradation study.

Sr. % Degradation % Assay
Condition
No. AMOX CLOX AMOX CLOX
01. Acid hydrolysis 7.48 9.34 92.52 90.66
02. Base hydrolysis 8.77 11.05 91.23 88.95
03. Oxidative degradation 7.71 8.33 92.29 91.67
04. Photo degradation 18.39 17.43 81.61 82.57
05. Thermal degradation 4.23 8.27 95.77 91.73
06. Neutral degradation 8.22 12 91.78 88

CONCLUSION
The developed HPLC method is found to be simple, specific, accurate and stability
indicating, hence it can be used for routine quality control analysis as well as stability studies
for the estimation of amoxicillin trihydrate and cloxacillin sodium in bulk and in combined
capsule dosage form. The degradation of AMO and CLO was determined by subjecting them
in various stress condition and no attempt was made to identify the degradation product.

ACKNOWLEDGEMENT
The authors are grateful to UGC, New Delhi for financial assistance to MRP and also
thankful RUSA Center for Herbo Medicinal Studies, S. R. T. M. University, Nanded, for
providing necessary research facilities to carry out the research work.

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