Stability indicating analytical method development and validation for estimation of Ceftazidime and Avibactam in bulk and pharmaceutical dosage form using RP-HPLC

Syeda S F et al / Int. J. of Farmacia, 2016; Vol-(2) 1: 70-78
70
International Journal of Farmacia
Journal Home page: www.ijfjournal.com
Stability indicating analytical method development and validation for
estimation of Ceftazidime and Avibactam in bulk and pharmaceutical dosage
form using RP-HPLC
Syeda Saniya Fatima1*
, R. Vani2
Deccan School of Pharmacy, Aghapura, Dar-Us-Salam, Hyderabad, Telangana
Corresponding Author: Syeda Saniya Fatima
Email: ainas_tremendous.queen@yahoo.co.in
ABSTRACT
A new simple, rapid, specific, accurate and precise stability indicatind RP-HPLC method has been developed for the
estimation of Ceftazidime and Avibactam in bulk & pharmaceutical dosage form. From UV spectrophotometric
method selected wavelength for estimation of drugs were 231 nm as isobestic point and 256 nm, 210 nm as λ max of
CAZ and AVY respectively by using methanol as a solvent. RP-HPLC method was developed by using Hypersil
ODS (150×4.6mm) 5µ. The samples were analyzed by using mixed phosphate buffer (PH
adjusted to 4 using
orthophosphoric acid): Acetonitrile (60:40 % v/v) as the mobile phase at the flow rate of 1.0 ml/min and detection
wavelength is 231 nm. Both the drugs were eluted within 5 minutes and gave sharp peaks with high theoretical plate
count and low tailing factor. The retention time for Ceftazidime and Avibactam was found to be 2.523 and 4.410
min respectively. Calibration curve was linear with correlation coefiicient of 0.996 and 0.999 over a concentration
range of 240-560 µg/ml and 60-120 µg/ml for Ceftazidime and Avibactam respectively. The percent recovery was
100.10 and 99.75 for Ceftazidime and Avibactam respectively indicating accuracy and reliability of the method.
Forced degradation studies were carried out and drug peaks were well resolved without any significant degradation
products when subjected to stress conditions. So the developed stability indicating method could be utilized for
routine analysis of Ceftazidime and Avibactam in bulk and pharmaceutical dosage form.
Keywords: Ceftazidime, Avibactam, Stability indicating, UV, RP-HPLC.
INTRODUCTION
Avibactam (AVIB) is chemically named as
(1R,2S,5R)-2-Carbamoyl-5-methyl-7- oxo-1,6-
diazabicyclo [3.2.1]octan-6-yl hydrogen sulphate.
Avibactam is a non β lactam β-mlactamase inhibitor
antibiotic used for treating complicated urinary tract
and complicated intraabdominal infections caused by
antibiotic resistant-pathogens, includind those caused
by multi-drug resistant gram negative bacterial
pathogens.[4,5] Ceftazidime (CEF) is chemically
named as (7R,Z)-7-(2-(2-aminothizol-4yl)-2- (2-
carboxypropan-2-yloxyimino) acetamido)-8-oxo-3-
(pyridinium-1-ylmethyl)-5-thia-1aza bicycle[4.2.0]oct-
2-ene-2-carboxylate. [1-3]
Ceftazidime is a semi synthetic broad spectrum
beta lactam antibiotic for parentral administration.
Ceftazidime is bactericidal in action exerting its effect
by inhibition of enzymes responsible for cell-wall
synthesis, primarily penicillin binding protein 3
(PBP3). It is a third generation cephalosporin. As a
class cephalosporin’s have activity against gram-
positive and gram-negative bacteria. The balance of
activity tips toward gram positive organisms for earlier
generations; later generations of cephalosporin’s have

71
more gram-negative coverage. Ceftazidime is one of
the few in this class with activity against
pseudomonas. It is not active against methicillin-
resistant Staphylococcus aureus. [4-6] The review of
literature revealed that several analytical methods have
been reported for ceftazidime in spectrophotometry,
HPLC, HPTLC, LC/MS individually and in the
combination with other drugs. [6-12] To date there
have been no published reports about the stability
indicating method for estimation of avibactam and
ceftazidime by HPLC in bulk and in pharmaceutical
dosage forms. This present study report for the first
time stability indicating simultaneous estimation of
avibactam and ceftazidime by RP-HPLC in bulk drug
and in pharmaceutical dosage form.
Fig 1: Structure of Ceftazidime Fig 2: Structure of Avibactam
MATERIALS AND METHODOLOGY
Chemicals
Avibactam and ceftazidime were obtained as gift
sample from Chandra Pharma Research Laboratory in
Hyderabad and marketed formulation was purchased
from local market.
Instrument and chromatographic condition
All solvent used in this work are HPLC & AR
grade. Instrument and chromatographic condition RP-
HPLC Agilent 1220 infinity series separation model
equipped with UV Detector was employed in this
method. The EZchrom software was used for LC peak
integration along data acquisition and data processing.
The column used for separation of analyte is Hypersil
ODS (150×4.6mm) 5µ. Mobile phase consisting of
phosphate buffer: Acetonitrile in the ratio of 60:40 %
v/v at flow rate 1 ml/min. it was filter through 0.45 μm
nylon filter and sonicated for 5 min in ultrasonic bath.
Sample was analyzed at 231 nm at an injection volume
of 20μl.
Preparation of phosphate buffer
1.625 gm of Potassium Di Hydrogen ortho
phosphate and 0.3 gms of Di Potassium Hydrogen
ortho phosphate was weighed and dissolve in 100 ml
of water and volume was made up to 550 ml with
water. Adjust the PH
using ortho phosphoric acid. The
buffer was filtered through 0.45µ filters to remove all
fine particles and gases.
Preparation of Solutions
Preparation of Mixed Standard Solution:
(400µg/ml & 100µg/ml)
Weigh accurately 400 mg of Ceftazidime & 100
mg of Avibactam and dissolve in 100 ml of diluents.
From the above stock solution 400 µg/ml of
Ceftazidime and 100 µg/ml of Avibactam is prepared
by diluting 1 ml to 10 ml with mobile phase. This
solution (400 µg/ml of Ceftazidime & 100 µg/ml of
Avibactam) is used as working standard concentration
for estimation of IV infusion.
Preparation of Sample Solution
5 bottles were weighed & calculate the average
weight of each bottle then the weight equivalent to 1
bottle was transferred into a 500 ml of volumetric
flask. Add sufficient quantity of diluents & sonicated
for 25 minutes further the volume is made up with
diluents and filtered. From the filtered solution 1 ml
was pipette out into a 10 ml with diluents.

72
Table 1: Summary of Chromatographic conditions
S. No Parameter Description
1. Column ODS Hypersil C-18 (150×4.6mm) 5µ
2 Mobile Phase & PH
Phosphate Buffer : Acetonitrile PH
4, 60:40 v/v
3 Flow rate 1 ml/min
4 Column & sample temperature Room temperature (20-25o
C)
5 Detection Wavelength 231 nm
6 Detector UV
7 Injection Volume 20 µl
8 Retention time Ceftazidime – 2.523 Min
Avibactam - 4.410 Min
9. Run time 10 mins
Fig 3: Typical chromatogram of Avibactam and Ceftazidime
Method Validation
The validation of method was carried out as per
ICH guideline. The parameters assessed were
specificity, linearity, precision, accuracy, stability,
LOD and LOQ. Specificity is the ability of the
analytical method to measure the analyte response in
the presence of interferences including degradation
product and related substances.
Accuracy
The accuracy was determined by calculating %
recoveries of avibactam and ceftazidime. It was

73
carried out by adding known amount of each analyte
corresponding to three conc. Levels (80, 100, and 120)
of the label claims to the excipients. At each level, six
determinations were performed and the accuracy
results were expressed as percent analyte recovered by
proposed method.
Precision
Method precision
Precision of an analytical method is usually
expressed as the standard deviation. Method precision
was demonstrated by preparing six samples as per test
method representing single batch and were
chromatographed. The precision of the method was
evaluated by computing the %RSD of the results. The
individual values and the low % RSD observed on the
values are within acceptance criteria and indicates that
method is precise.
Linearity
The purpose of the test for linearity is to
demonstrate that the entire analytical system
(including detector and data acquisition) exhibits
linear responses and directly proportional over the
relevant conc. Range for the target conc. of the
analyte. The linear regression data for the calibration
plot is the indicative of a good linear relationship
between peak and concentration over wide range. The
correlation coefficient was indicative of high
significance.
Robustness
Robustness of method was investigated under a
variety of conditions including changes of
composition of buffer in the mobile phase, flow rate
and temperature. This deliberate change in the method
has no effect on the peak tailing, peak area and
theoretical plates finally the method was found to be
robust.
Ruggedness
The ruggedness of the method was studied by the
determining the analyst to analyst variation by
performing the Assay by two different analysts. %
RSD Assay values between two analysts not greater
than 2.0%, hence the method was rugged.
Limit of Detection and Limit of Quantitation
The LOD can be defined as the smallest level of
analyte that gives a measurable responses and LOQ
was determined as the lowest amount of the analyte
that was reproducibly quantified. These two
parameters were calculated using formula based on
standard deviation of the response and slope. LOD and
LOQ were calculated by equation, LOD=3.3 x ϭ/s and
LOQ= 10 x ϭ/s, where s = standard deviation, S =
slope of calibration curve.
Assay of avibactam and ceftazidime in
pharmaceutical dosage form
Assay of marketed product was carried out by
using the developed method. Sample solutions were
prepared and injected into RP –HPLC system. The
sample solution was scanned at 231 nm. The
percentage drug estimated was found to be 100.23%
and 101.21% respectively as ceftazidie avibactam. The
chromatogram showed two single peaks of ceftazidime
and avibactam was observed with retention time 2.523
and 4.410 min respectively.
Forced degradation studies
Stress studies are performed according to ICH
guidelines under following conditions.
Acid degradation
To 5 ml of sample solution add 1 ml of 0.1 N HCL
and sonicate place it aside for 3 hrs, then neutralize the
solution with 1ml of base and then transfer above
solution into 10 ml volumetric flask dilute with mobile
phase and record the chromatogram.
Alkaline degradation
To 5 ml of sample solution add 1 ml of 0.1 N
NaOH place it aside for 3 hrs, then neutralize the
solution with 1ml of acid and then transfer above
solution into 10 ml volumetric flask dilute with mobile
phase and record the chromatogram.
Peroxide degradation
To 5 ml of sample solution add 1 ml of 3% H202
and sonicate place it aside for 3 hrs, then transfer
above solution into 10 ml volumetric flask dilute with
mobile phase and record the chromatogram.
Photolytic degradation
Expose about 100 mg of sample in UV light
chamber at 231 nm for 3 hrs. weigh accurately this

74
power equivalent to 40 mg of Ceftazidime and 10 mg
of Avibactam into a 100 ml volumetric flask and make
up the volume and sonicate for 30 minutes with
intermittent shaking and volume is made up to the
mark with mobile phase and record the chromatogram.
Thermal degradation
Expose about 100 mg of sample in to dry heat at
800
C for 3 hrs. weigh accurately this power equivalent
to 40 mg of Ceftazidime and 10 mg of Avibactam into
a 100 ml volumetric flask and make up the volume and
sonicate for 30 minutes with intermittent shaking and
volume is made up to the mark with mobile phase and
record the chromatogram.
Records the peak area of stressed samples then
compare it with peak area of unstressed sample to
determine the % degradation.
Response of unstressed sample – response of stressed sample
% degradation = ------------------------------------------------------------------------------ X 100
Response of unstressed sample
RESULTS & DISCUSSION
Ceftazidime/avibactam (AVY-CAZ) is a recent
combination in the market used in the treatment of
complicated intra-abdominal infections (cIAI),
complicated urinary tract infections (cUTI), acute
pyelonephritis (AP). Literature survey reveals that
various methods for the estimation of Ceftazidime
individually and in combination with other drug is
reported, but no method has been reported for the
estimation of the Ceftazidime and Avibactam in
combine dosage form by RP-HPLC method. So here
an attempt has been made to develop simple, accurate,
sensitive, rapid and economic method for estimation of
Ceftazidime and Avibactam from combine dosage
form using HPLC. Specific objectives includes
The conditions in HPLC were optimized in order
to obtain the drugs separation of eluted compounds.
Initially various composition of mobile phase & PH
range were tried in order to have a good separation of
the titled ingredients. The composition of Mobile
phase & PH selections were based on peak parameters
like height, tailing, capacity factor, symmetric factor,
theoretical plates, run plates, run time & resolution.
The system with phosphate buffer: acetonitrile of
70:40 was found to be robust with PH 4. The optimum
wavelength for detection was 231 nm at which both
drugs have good response. CAZ was eluted at 2.523
min and AVI was eluted at 4.410 min.
System suitability test were carried out on a freshly
prepared stock solution % RSD of peak area of five
replicated injections of standards were taken & was
found to be less than 2%. Specificity of the method
developed was performed & it was found that there is
no interference of excipients with the analyte which
indicate that the method is specific for the analysis of
the analytes in their dosage form. Assay was
determined for both Standard & sample solution & the
% assay was found to be 100.23% & 101.21% and
respectively.
A calibration curve was found to be linear in the
concentration range of 240-560 µg/ml and 60-140
µg/ml for CAZ & AVI. Accuracy studies of the
method developed wad determined by spiking the
known amount of analyte to sample solution and the
percentage mean recovery was found to be 100.10%
and 99.75% for CAZ & AVI respectively. Precision of
the method & system were found to be within limits.
Limit of detection was found to be 11.2 and 2.5 µg/ml
for CAZ & AVI respectively and Limit of
Quantification was found is to be 33.8 and 7.69 µg/ml
for CAZ & AVI respectively. Robustness of the
method was determined by varying flow rate and
wavelength & Ruggedness of the method was
determined by carrying out the determination by two
different analyst.
Samples containing Ceftazidime and Avibactam
were subjected to various stress conditions and it was
found that overall net degradation was within the
limits without any significant degradation products.
A RP-HPLC method developed for Ceftazidime
and Avibactam shows that the results obtained for RP-
HPLC are promising with better resolution in set
chromatographic conditions. The developed methods
were statistically validated which suggested that the
methods were within the acceptable limits hence these
methods can be used for the routine determination of
Ceftazidime and Avibactam in bulk drug and
pharmaceutical formulation.

75
Fig 4: Linearity curve of Ceftazidime Fig 5: Linearity curve of Avibactam
Table: 2 Results of Linearity
CEFTAZIDIME AVIBACTAM
S.No. Conc.(µg/ml ) Area Conc.(µg/ml ) Area
1 240 1344.606 60 108.783
2 320 1849.853 80 159.306
3 400 2338.421 100 204.849
4 480 2563.186 120 222.682
5 560 3106.591 140 243.873
Table: 3 Precision method of proposed RP- HPLC method
S.No. Rt Area Rt Area
1 2.510 2192.417 4.397 267.545
2 2.523 2322.573 4.410 211.442
3 2.523 2321.138 4.413 202.102
4 2.523 2333.196 4.413 200.853
5 2.507 2350.119 4.397 202.888
6 2.497 2341.355 4.390 198.551
Avg 2.513833 2310.133 4.403333 213.8968
Stdev 0.010926 6.248003 0.009893 2.001001
%RSD 0.433746 0.270460 0.224216 0.935498
Table: 4 Recovery data for Ceftazidime
Recovery
level
Amount
taken
(mcg/ml)
Accuracy of Ceftazidime
Area Average
area
Amount
recoverd
%Recovery Average %
Recovery
80% 80+320=400 2608.241 2607.639 10.8 111.3
2609.160
2605.517
100% 80+400=480 2194.643 2244.257 13.25 95.85
y = 1.538x + 33.44
R² = 0.999
0
50
100
150
200
250
300
0 50 100 150

76
2211.816
100.10
2326.313
120% 80+480=560 3109.681 3109.702 22.5 93.16
3112.744
3106.682
Table: 5 Recovery data for Avibactam
Recovery
level
Amount
taken
(mcg/ml)
Accuracy of Avibactam
Area Average
area
Amount
recoverd
%Recovery Average %
Recovery
80% 20+80=100 236.388 237.6637 9,56 92.65
99.75
234.158
242.445
100% 20+100=120 224.953 254.704 15.03 99.34
269.243
269.916
120% 20+120=140 280.051 275.7967 21.07 107.35
280.286
267.053
Table: 6 Robustness data
Parameter
Retention time(min) Tailing factor Retention time(min) Tailing factor
Flow Rate
0.8 ml/min
1.2 ml/min
3.130
2.090
1.258
1.036
5.443
3.663
1.167
0.943
Wavelength
229nm
233nm
2.513
2.517
1.222
1.179
4.380
4.380
1.088
1.125
Table: 7 Result of Ruggedness
Ceftazidime % Assay Avibactam % Assay
Analyst 01 99.3 Analyst 01 96.28
Analyst 02 99.30 Analyst 02 95.97
% RSD 1.82% % RSD 1.67%
Table: 8 Result of LOD and LOQ
DRUG LOD(μg/ml) LOQ(μg/ml)
Ceftazidime 11.2 33.8
Avibactam 2.5 7.69

77
Table: 9 Results of Forced Degradation studies
STRESS CONDITION PEAK AREA % DEGRADATION
CAZ AVI CAZ AVI
Unstressed sample 2192.417 267.545 0 0
Acid 3981.699 391.641 0.81 0.46
Alkaline 3934.512 396.009 0.79 0.48
Oxidation 3943.709 391.379 0.79 0.46
Photolytic 3951.770 397.622 0.80 0.49
Thermal 3971.642 385.227 0.81 0.43
CONCLUSION
Ceftazidime and Avibactam are essential for
treatment of complicated intra-abdominal infections
(CIAI), complicated urinary tract infections (cUTI),
acute pyelonephritis (AP) so it is therefore necessary
to know the quality of these drugs which is possible
through the use of simple, sensitive and cost effective
analytical methods so that the compounds can be
rapidly, routinely and consistently assessed. Literature
survey reveals that there are methods to estimate the
ceftazidime individually and with other combinations,
but not a single method is reported for estimation of
Ceftazidime and Avibactam in bulk and
multicomponent formulation. So, an attempt was made
to develop RP-HPLC method. A RP-HPLC method
proposed as a suitable method for the simultaneous
estimation of Ceftazidime and Avibactam. The
chromatographic conditions included mixed phosphate
buffer (PH
4): Acetonitrile in the ratio of 60:40 % v/v
as mobile phase. The optimum wavelength for
detection was 231 nm where as CAZ was eluted at
2.523 Min and AVY was eluted at 4.410 min. The
calibration curve of Ceftazidime and Avibactam is
linear in the range of 240-560 µg/ml and 60-140 µg/ml
respectively.
Accuracy study showed the percentage mean
recovery of Ceftazidime and Avibactam is 100.10%
and 99.75% respectively. The amount of Ceftazidime
and Avibactam present in the taken formulation was
found to be 100.23% and 101.21% respectively.
Forced degradation studies were carried under various
stress conditions. It was found to be stable and the net
degradation was within the limits without any
significant degradation products. From the above
experimental results and parameters it was concluded
that, this newly developed methods for the estimation
of Ceftazidime and Avibactam was found to be
simple, rapid, economic, precise, accurate and
reproducible. Forced degradation studies carried out
are helpful to determine stability of this drug in
combination. The analytical technique showed reliable
method hence it can be effectively applied for routine
analysis in research institutions & quality control
department in industries.
REFERENCES
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[2]. Marianna Z, Anna J, Agnieszka S and Wojciech M stability of ceftazidime pentahydrate in medicinal
preparations biotm and ceftium. Acta poloniae pharmaceutical and drug research, 62(1), 2005, 11-15.

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[3]. Cinzia A, Monica B, Roberto V, Pierangelo B, Rta P Determination of Ceftazidime concentration in Mueller
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[7]. International conference on harmonization, (ICH) “Q2A: Text on Validation of analytical procedure,” Federal
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Stability indicating analytical method development and validation for estimation of Ceftazidime and Avibactam in bulk and pharmaceutical dosage form using RP-HPLC

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