DEVELOPMENT AND VALIDATION OF HIGH PERFORMANCE
LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS
ESTIMATION OF CEFOPERAZONE SODIUM AND
SULBACTUM SODIUM IN COMBINED DOSAGE FORM
MADHURI A. HINGE*, AVNI I.MEHTA, RAJVI J. MAHIDA, ALISHA P. PATEL
*
Department of Quality Assurance, Rofel Shri G.M.Bilakhia College of Pharmacy, Vapi
email id: madhuri_shreyal@yahoo.co.in
ABSTRACT
A simple, rapid and sensitive isocratic reversed-phase (RP) high-performance liquid
chromatography (HPLC) method was developed and validated for the simultaneous
determination of cefoperazone sodium (CEF) and sulbactum
sodium (SUL) in various
pharmaceutical formulations. Compound separation was achieved in less than 10 min with a
Thermo Hypersil C18 (250 x 4.6 mm, 5 μm) and a mobile phase consisting of 0.05 M
Potassium Dihyrdrogen Phosphate (pH 4.5): Methanol (60:40%V/V). Analyses were
performed at a flow rate of 1 mL min−1 and at a detection wavelength of 240 nm. The method
was shown to be selective, linear, accurate, and precise in intra-day and inter-day analyses.
The robustness of the method was shown by slightly changing the flow rate, column oven
temperature, and proportion of acetonitrile in the mobile phase. The method was found,
however, to be very sensitive to the pH of the mobile phase buffer. The method was
successfully validated following the guidelines of the International Conference on
Harmonization (ICH).
Keywords
Cefoperazone sodium ; Sulbactum sodium; HPLC method; Force degradation study.
INTRODUCTION
Cefoperazone sodium (CEF)1-4, sodium;(6R,7R)-7-[[(2R)-2-[(4-ethyl-2,3-dioxopiperazine -1carbonyl) amino] -2-
(4-hydroxyphenyl) acetyl] amino] -3- [(1-methyltetrazol-5-yl)
sulfanylmethyl] -8 -oxo -5 -thia -1 -azabicyclo [4.2.0] oct -2 -ene -2 -carboxylate. It is a
Cephalosporin 3rd generation Anti-Bacterial Agents. Cefoperazone sodium is official in IP
20076 and assay method for this drug is Liquid chromatographic method.
Sulbactum
sodium
(SUL)6-8,
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid,3,3-
dimethyl-7-oxo-,4,4-dioxide,sodiumsalt,(2S-cis)-Sodium(2S,5R)-3,3-dimethyl-7-oxo-4-thia1-zabicyclo[3.2.0]heptane-2-carboxylate 4,4-dioxide.It is used as an anti-bacterial agent.
Sulbactum sodium is official in BP 20099 and USP 200710.Assay method for this drug is
Liquid chromatography in BP 2009 and USP 2007.
The assay of this antibacterial combination of CEF and SUL was the subject of several
analytical reports. The literature survey reveals that several methods are reported for
cefoperazone sodium alone and in combination with other drugs are spectrophotometric
methods11-12, HPTLC method14 and HPLC methods13-16 and for Sulbactum sodium various
Spectrometric methods17-22, HPTLC method23 and HPLC methods24-30 are reported alone and
in combination with other drugs. RPHPLC method are reported for determination of
Cefoperazone sodium and Sulbactum sodium in plasma31 and pharmaceutical dosage form3233
.
MATERIALS AND METHODS
Equipments used:
HPLC Model was Jasco 980 pump, Column was Thermo Hypersil C18 (250 x 4.6 mm, 5
μm),Injector: An Rheodyne – 7725 injector, Detector: UV Detector used for analytical work.
The analysis was carried by using Oracle 2 software. All the weighing was carried out on the
Electronic analytical balance (Mettler Toledo), Sonication of samples was carried out by
sonicator.
Chemicals and reagents:
Cefoperazone Sodium was gift sample by Zen Pharmaceuticals, Vapi . Sulbactum sodium
was supplied as gift sample by Zen Pharmaceuticals, Mumbai.The analytical
methanol was purchased from
grade
Fisher Scientific (India). Tablets (Cefotroy-SB) were
purchased from local pharmacy. The distilled water was used for analytical work and
rinsing of clean glasswares.
Chromatographic Condition
C18 column, 250mm × 4.6 mm, 5μm was used for the chromatographic separation at a
detection wavelength of 240 nm. Mobile phase of composition 0.05 M KH2PO4 Buffer :
Methanol (60:40 v/v, pH- 4.5 adjusted with orthophosphoric acid) was selected for elution
and same mixture was used in the preparation of standard and sample solutions. Flow rate
was adjusted to 1.0 ml/min and the injection volume was 20.0 μl.
Preparation of mobile phase
A degassed mixture of Methanol and water in the ratio 80:20 (v/v) was prepared and pH was
adjusted to 3.5 with orthophosphoric acid. The mixture was then filtered through 0.45 μ
membrane filters and it was degassed.
Preparation of standard solution of binary mixtures of CEF and SUL
Accurately weighed 100 mg of CEF and 100 mg of SUL were transferred to 100 ml
volumetric flask and diluted up to mark with mobile phase to give concentration of 1000
μg/ml of CEF and 1000 μg/ml of SUL.
Preparation of working solution of binary mixtures of CEF and SUL
10 ml of standard stock solution was taken in 100 ml volumetric flask and diluted up to the
mark with mobile phase to get concentration of 100 μg/ml of CEF and 100 μg/ml of SUL.
Preparation of Sample Solution
Twenty tablets of Cefotroy-SB were weighed and crushed. Tablet powder equivalent to 10
mg of CEF and 50 mg of SUL was weighed accurately and transferred to a 100 ml volumetric
flask. From this 60 ml was transferred to 100 ml volumetric flask and volume was made up to
mark with mobile phase. Solution was then filtered through 0.45μ membrane filter. The
diluted solution was analyzed under optimized chromatographic conditions. The areas of
resulting peak were measured at 240 nm.
Optimization of RP-HPLC method
The HPLC method was optimized with an aim to develop a simultaneous estimation
procedure for the assay of Cefoperazone Sodium and Sulbactum sodium. For the method
optimization,
different mobile phases
were tried, but acceptable
retention
times,
theoretical plates and good resolution were observed with 80:20 (v/v) was prepared and
pH was adjusted to 3.5 with orthophosphoric acid.using Thermo Hypersil C18 (250 x 4.6
mm, 5 μm)
Validation of RP-HPLC Method35
System Suitability Study
Diluent was used as blank. Standard and sample containing 60 μg/ml CEF and 60 μg/ml
SUL were prepared and was injected in system as per stated condition. The mean values of
system suitability parameters are shown in following table 1
Linearity and Range
For the determination of linearity, appropriate aliquots were pipetted out from working stock
solution to a series of 10ml volumetric flasks and volume was made up with the solvent to
obtain concentration ranging from 20-100 g/ml of CEF and 20-100 g/ml of SUL. Each
solution was injected in triplicate. Calibration curves were plotted with observed peak areas
against concentration followed by the determination of regression equations and calculation
of the correlation coefficients. The calibration curves for CEF and SUL were shown in figure
2 and figure 3 and their corresponding linearity parameters were given in table 2.
Precision
The repeatability of the method was verified by calculating the %RSD of six
replicate injections of (60 g/ml of CEF and 60 g/ml of SUL) on the same day
and
for intermediate precision % RSD was calculated from repeated studies on
different days. The results were given in table 3.
Accuracy
To ensure the reliability and accuracy of the method recovery studies were carried out by
standard addition method. A known quantity of pure drug was added to pre-analysed
sample and contents were reanalysed by the proposed method and the percent recovery was
reported. The results were given in table 4.
Specificity
Specificity of a method was determined by testing standard substances against potential
interferences. The method was found to be specific when the test solution was injected and no
interferences were found because of the presence of excipients.
Limit of Detection (LOD) and Limit of Quantitation (LOQ).
The LOD and LOQ were calculated from the slope(s) of the calibration plot and the standard
deviation (SD) of the peak areas using the formulae LOD = 3.3 σ/s and LOQ = 10 σ/s. The
results were given in table 2.
Robustness
Robustness of the method was verified by altering the chromatographic conditions like
mobile phase composition, flow rate, detection wave length, etc. and the % RSD should be
reported.
Small changes in the operational conditions were allowed and the extent to which the method
was robust was determined. A deviation of ±2nm in the detection wave length and
±0.2ml/min
in
the
flow
rate,
were
tried individually. A solution of 100% test
concentration with the specified changes in the operational conditions was injected to the
instrument in triplicate. %RSD was reported in Table 5.
Assay of Marketed Formulations
10.0 l of sample solution of concentration of 60 g/ml CEF and 60 g/ml of SUL was
injected into chromatographic system and the peak responses were measured and shown in
the figure 5. The solution was injected three times in to the column. The amount of drug
present and percentage purity was calculated by comparing the peak areas of the standards
with that of test samples.
Force Degradation Study
Force degradation Study was performed as Acid Hydrolysis, Alkali Hydrolysis, Oxidative
Hydrolysis, Thermal Hydrolysis and Sunlight degradation.
RESULTS AND DISCUSSION
After a number of trials with mobile phases of different composition, 0.05 M potassium
dihydrogen phosphate : methanol 60:40 v/v pH adjusted to 4.5 with orthophosphoric acid
was selected as mobile phase because of better resolution and symmetric peaks. CEF and
SUL were found to show appreciable absorbance at 240.0 nm when determined
spectrophotometrically and hence it was selected as the detection wavelength. An
optimized chromatogram showing the separation of CEF and SUL at different RTs was
shown in figure 5.
System suitability was carried out by injecting 5 replicate injections of 100% test
concentration, number of theoretical plates, HETP and resolution were satisfactory. The
chromatograms confirm the presence of CEF and SUL at 4.08 min and 6.40 min
respectively without any interference. The parameters were given in table 1.
Concentration range of 20-100 µg/ml for CEF and 20-100 µg/ml for SUL were found to be
linear with correlation coefficients 0.9989 and 0.9966 for CEF and SUL respectively. The
results were given in table 2.
The proposed method was found to be precise and reproducible with % RSD of 0.597 1.959 and 0.506 -1.993 for CEF and SUL respectively. %RSD was reported in table 3.
Accuracy of the method was verified by performing recovery studies by standard addition
method. The percent recovery of the standard added to the pre-analysed sample was
calculated and it was found to be 98.00 – 101.75 %w/w and 98.94 -100.04 % w/w for CEF
and SUL respectively. This indicates that the method was accurate. Values obtained were
given in Table 4.
The limits of detection for CEF and SUL were found to be 1.8337 µg/ml and 0.0549 µg/ml
respectively and the limits of quantitation were 5.5569 µg/ml and 0.1665 µg/ml
respectively. Values were represented in Table 2.
The method was found to be specific for the combination of interest after verifying the
chromatograms showing no interference of the excipients present. Hence, the method was
well suitable for the estimation of the commercial formulations of the selected combination.
Values obtained were given in Table 6.
The method was found to be robust after changing the conditions like, flow rate (±0.2 ml),
mobile phase composition and pH (± 2). %RSD was calculated for each variation and
reported. Values obtained were given in Table 5.
CONCLUSION
A new RP-HPLC method has been developed for simultaneous estimation of CEF
and SUL in marketed formulation. The method gave good resolution for both the drugs
with a short analysis run time within 6 min. The developed method was validated. It was
found to be novel, simple accurate precise, sensitive and cost effective. Hence
the
proposed RP-HPLC method is suitable for routine assay of CEF and SUL in
pharmaceutical dosage form in quality control laboratories. The stress testing study for CEF
and SUL indicates that CEF is significantly degrade under acidic and oxidative condition
SUL was significantly degradable under acidic and alkaline degradation. All degradants
forms were separated and did not interfere with estimation of CEF and SUL.
Acknowledgment
The authors are thankful to the Zen Pharmaceuticals, Vapi for providing the gift sample of
CEF and Zen Pharmaceuticals, Mumbai for providing gift sample of SUL. The authors are
also thankful to the Principle, ROFEL Shri G. M. Bilakhia College of Pharmacy, VAPI,
India, for providing the required facilities to carry out this research work.
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Table 1: Mean values of system suitability study (n=5)
CEF
± % RSD
4.080
1.34
3378
Parameters
Retention time
Tailing factor
Theoretical plates
Resolution
SUL
± % RSD
6.04
1.43
7205
7.06
Table 2: Results for Linearity (n=3)
Parameters
CEF
SUL
Slope
Y intercept
132654.0050
198077.70
382481.5800
1450734.40
Correlation
coefficient
(r2)
0.9989
0.9966
Regression
equation
Y=132654.0050x+198077.70
Y=1450734.40+382481.5800
20-100 g/ml
20-100 g/ml
1.8337 g/ml
5.5569g/ml
0.0549g/ml
0.1665g/ml
Linearity
range
LOD
LOQ
Table 3: Results of precision (n=6)
Drug
Intraday
Interday
Precision(%RSD)
Precision(%RSD)
CEF
0.597 - 0.977
1.829 - 1.959
SUL
0.506 - 0.737
1.758 - 1.993
n=No. of determinants
Table 4: Results for Accuracy (n=3)
CEF
SUL
Recovery
level
Amount
Added
(µg/ml)
Amount
Found
(µg/ml)
%
Recovery
Amount
Added
(µg/ml)
%
Recovery
Std
Test
0%
0
40
40.70
101.75
0
40
39.98
99.95
80%
32
72
32.76
98.00
32
72
31.66
98.94
100%
40
80
39.62
98.00
40
80
40.81
102.04
120%
48
88
47.46
98.87
48
88
48.07
100.14
Mean
recovery
Std
Amount
Found
(µg/ml)
Test
98.00 – 101.75 %w/w
98.94 -100.04 %w/w
n= No. of determinants
Table 5: Results for Robustness
%RSD
Parameters (n=3)
CEF
SUL
Mobile Phase +2
1.7508
0.1262
Mobile Phase -2
0.3713
1.1001
Flow rate +2
0.3033
1.1825
Flow rate -2
0.4579
0.3874
pH +2
1.1503
0.3549
pH -2
0.3670
0.3874
n= No. of determinants
Table 6 : Analysis of marketed formulation (n=3)
MARKETED
Actual Conc.
Mean conc. obtained ± S.D.
% Conc. Of
FORMULATION
(μg/ml)
(μg/ml)
Label claim
CEF
SUL
CEF
SUL
CEF
CEFOTROY*SB
60
60
60.45 ± 0.227 60.44 ± 0.293
100.75
100.73
ZOSTUM
60
60
59.49 ± 0.066 59.67 ± 0. 717
99.16
99.45
Table 7. Force Degradation Study of CEF and SUL in formulation
Sr.No.
Stress Type
CEF
SUL
%Degradation
Rt(min)
Rt(min)
CEF
SUL
1
Acid
4.35
5.95
24.74
14.12
2
Alkali
4.12
6.41
16.26
22.42
3
Oxidation
4.16
6.47
28.93
9.66
4
Thermal
4.10
6.41
4.11
3.16
5
Sunlight
4.10
6.41
1.35
1.66
SUL
Fig 1 Structure of CEFOPERAZONE SODIUM
Fig 2 Structure of SULBACTAM SODIUM
Fig. 3 Calibration curve for Cefoperazone Sodium
Fig. 4 Calibration curve for Sulbactum Sodium
Fig 5: Optimized Chromatogram of CEP and SUL(0.05 M KH2PO4 Buffer : Methanol
(60:40 v/v, pH- 4.5 adjusted with orthophosphoric acid)
Figure 6: A typical chromatogram for assay of marketed formulation containing 60
g/ml of CEF and 60 g/ml SUL.
A) Acid Hydrolysis
Fig 7. Chromatogram of SAMPLE under Acid Hydrolysis
B) Alkali Hydrolysis
Fig 8. Chromatogram of SAMPLE under Alkali Hydrolysis
C) Oxidative Degradation
Fig 9. Chromatogram of SAMPLE under Oxidative degradation
D) Thermal Degradation
Fig 10. Chromatogram of SAMPLE under Thermal Degradation
E) Sunlight Degradation
Fig 11 Chromatogram of SAMPLE under Sunlight