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CHAPTER-4 METHOD DEVELOPMENT AND VALIDATION
METHOD DEVELOPMENT AND VALIDATION
List of Instruments:
● HPLC – Waters Model No.2690/5 Compact System consisting of panel with Inertsil C18
ODS.
● Electronic balance (SARTORIOUS)
● Sonicator (CLEAN FAST)
List of Chemicals:
● HPLC Grade Methanol
● HPLC Grade buffer (KH2PO4)
● HPLC Grade Acetonitrile
● HPLC Grade Water
Standards:
Cefepime and Enmetazobactam working standards.
HPLC METHOD DEVELOPMENT:
Based on a review of the literature, the goal of the experiment is to refine the assay procedure for
estimating Cefepime and Enmetazobactam. The trails that were discussed here therefore describe
how optimization was carried out.
Preparation of Stock solution:
10 mg of Cefepime and 10 mg Enmetazobactam was weighed accurately and transferred to a
10ml volumetric flask, dissolved in 10 ml of mobile phase. The mixture was then sonicated for
20 minutes to obtain 1000 ppm.
Preparation of Working Standard Solution:
From the above stock solution take 1ml and dilute to 10ml with mobile phase.
Method Development: It was done by changing various mobile phase ratios etc.
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CHAPTER-4 METHOD DEVELOPMENT AND VALIDATION
Trail 1
Table 4.1: Chromatographic conditions for Trail 1
Parameters Method
Flow rate 1.0ml/min
Column Inertsil-C18, ODS system
Detector wavelenght 262nm
Column Temperature Ambient
Injection Volume 20μl
Run time 12min
Retention time Cefepime for 3.658 min and
Enmetazobactam for 6.460
Mobile phase ratio Water:Degassed
Acetonitrile 50:50
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CHAPTER-4 METHOD DEVELOPMENT AND VALIDATION
Fig.4.1: Chromatogram for Trail 1
S.No. Name of the Drug Retentiontime(min)
1 Cefepime 3.658
2 Enmetazobactam 6.460
Inference: Peak forms are undesirable.
Trial :2
Mobile Phase: Water and methanol were degassed in a 75:25 v/v ratio.
Table 4.2: Chromatographic conditions for Trail 2
Parameters Method
Flow rate 1.0ml/min
Column Inertsil-C18, ODS system
Detector Wave length 262 nm
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CHAPTER-4 METHOD DEVELOPMENT AND VALIDATION
Column temperature Ambient
Injection volume 20μl
Run time 12 min
Retention time Cefepime for 5.262 min and
Enmetazobactam for 7.269 min.
Mobile phase ratio Water:Degassed
Acetonitrile 50:50
S.No Name of the Drug Retention time (min)
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1 Cefepime 5.262
2 Enmetazobactam 7.269
Fig 4.2: Chromatogram for Trial
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CHAPTER-4 METHOD DEVELOPMENT AND VALIDATION
Inference: Shapes of peaks aren't healthy
Trial : 3
Mobile Phase: Acetonitrile and methanol were degassed at a 50:50 v/v ratio.
Table 4.3: Chromatographic conditions for Trail 3
Parameters Method
Flow rate 1.0ml/min
Column Inertsil-C18, ODS system
Detector wavelength 262 nm
Column temperature Ambient
Injection volume 20μl
Run time 12 min
Retention period Cefepime for 3.348 min and
Enmetazobactam for 3.941
min.
Mobile phase Water: Degassed
Acetonitrile 50:50
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Fig 4.3: Chromatogram for Trial 3
S.No Name of the Drug Retention time (min)
1 Cefepime 3.348
2 Enmetazobactam 3.941
Inference:An extra peak was observed with tailing for both the drug peaks. So, next trail was
performed.
Table 4.4: Chromatographic conditions for Optimized method
Parameters Method
Inertsil–ODS system C18(250 x 4.6 mm,
Column 5 µ)
Mobile Phase Methanol and Acetonitrile (80:20)
Flow rate (ml/min) 1.0 ml/min
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Duration of operation (minutes)
14 min
Temperature in the column (°C)
Ambient
Volume of injection loop (μl) 20
Wavelength of detection (nm)
262nm
6.120 min for Cefepime and 9.336 for
Drug RT (min)
Enmetazobactam.
Fig. 4.4: Optimized Chromatogram
S. No. Name of the Drug Retention time(min)
1 Cefepime 6.120
2 Enmetazobactam 9.336
Inference: Cefepime and Enmetazobactam were eluted at 6.120 min and 9.336 min
respectively with good resolution. Plate count and tailing factor was very satisfactory, so this
method was optimized and validated.
Preparation of solutions:
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Preparation of Mobile Phase: Methanol and Acetonitrile were degassed in 80:20 v/v ratios.
Preparation of stock solution:
10 mg of Cefepime and 10 mg Enmetazobactam was weighed accurately and
transferred to a 10ml volumetric flask, dissolved in 10 ml of mobile phase. The
mixture was then sonicated for 20 minutes to obtain 1000 ppm.
Preparation of working solution:
From the above stock solution take 1ml and dilute to 10ml with mobile phase.
VALIDATION PARAMETERS
SYSTEM SUITABILITY:
Working standard solutions of Cefepime and Enmetazobactam were prepared and
were injected five times into the HPLC system. The system suitability parameters
were evaluated from standard chromatograms. %RSD for retention time and peaks
areas, number of theoretical plates, tailing factor for Cefepime and Enmetazobactam
were calculated.
Acceptance criteria:
The % RSD for the retention times for the Standard solution should be not more than 2.0 %
The % RSD for the peak area responses for the standard Solution should be not more than
2.0%.
The number of theoretical plates (N) should not be less than 3000.
The Tailing factor (T) should not be more than 2.0
SPECIFICITY:
Solutions of standard and sample prepared as per the test method were injected
into chromatographic system.
LINEARITY:
A series of solutions were prepared using working standards at various concentrations. The
peak area responses of solution at Level 1 to Level 6 were measured.
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Preparation of Solutions:
The linearity of the analytical procedure is the ability (within a given range) to obtain test
results which are directly proportional to the concentration of analyte in the sample.Linearity of
the detector response for Cefepime and Enmetazobactamwas established by analysing serial
dilutions of a stock solution of working standard. Seven concentrations such as
0,20,30,40,50,60,70 and 80µg/ml for Cefepime and 0,20,30,40,50,60,70 and 80µg/ml for
Enmetazobactam were prepared and analysed. The linearity graph was plotted using
concentration on x-axis and peak area on y-axis.
Preparation of Stock solution:
10.0 mg of Cefepime and 10.0 mg of Enmetazobactam API standards were accurately weighed
and transferred into two different 10 ml volumetric flasks, dissolved individually in methanol
and sonicated for 20 minutes to get 1000µg/ml of Cefepime and Enmetazobactam respectively.
From Cefepime and Enmetazobactam stock solutions different concentrations that range from
20µg/ml to 80µg/ml were prepared for both the drugs. These solutions were then sonicated and
filtered through a 0.45.
Acceptance criteria:
● Correlation Coefficient should be not less than 0.9990.
● % y- Intercept should be ±2.0.
● % RSD for level 1 and Level 6 should be not more than 2.0%.
ACCURACY (RECOVERY):
A study of Accuracy was conducted. Drug Assay was performed in triplicate as per
testmethod with equivalent amount of Cefepime and Enmetazobactaminto each
volumetricflask for each spike level to get the concentration of Cefepime and
Enmetazobactamequivalent to 50%, 100%, and 150% of the labelled amount as per
the test method. The average % recovery of Cefepime and Enmetazobactam was
calculated.
Acceptance criteria:
The mean % recovery of theCefepime and Enmetazobactam at each spike level
should be not less than 98.0% and not more than 102.0% for both the drugs separately.
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OBSERVATION:
Amount found
%Recovery = ---------------------- × 100
Amount added
PRECISION:
REPEATABILITY:
Precision is measured by the multiple samples obtain from homogenous sample under prescribe
condition. It is a closeness degree of samples. As per ICH, precision is performed as
repeatability, intermediate precision and reproducibility. The analytical procedures of precision
are commonly expressed as variance, standard deviations or coefficient of variation. At least
five duplicates are to be in process with including percent relative standard deviation.
Acceptance criteria:
The % relative standard deviation of individualCefepime and Enmetazobactam, should not be
more than 2.0%.
The individual assays ofCefepime and Enmetazobactam should be not less than 98% and not
more than 102.0%.
INTERMEDIATE PRECISION (analyst to analyst variability):
A study was conducted by two analysts as per test method.
Acceptance criteria:
The individual assays of Cefepime and Enmetazobactamshould be not less than
98% and not more than 102% and %RSD of assays should be not more than 2.0% by both
analysts.
LIMIT OF DETECTION AND QUANTITATION (LOD and LOQ):
From the linearity data the limit of detection and quantitation was calculated using the
formula.
3.3 σ
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LOD = --------------
S
σ = standard deviation of the response
S = slope of the calibration curve of the analyte.
ROBUSTNESS:
Effect of variation of flow rate:
A study was conducted to determine the effect of variation in flow rate.
Standard solution prepared as per the test method was injected into the HPLC system using
flow rates 0.8ml/min, 1.0ml/min and 1.2ml/min.
Acceptance criteria:
The % RSD ofCefepime and Enmetazobactam standards should be not more than
2.0 for Variation in flow rate.
RUGGEDNESS:
System to system variability:System to system variability study was conducted on
different HPLC systems, under similar conditions at different times. Six samples were prepared
and each was analysed as per test method.
Acceptance criteria:
The % relative standard deviationCefepime and Enmetazobactam from the six
sample preparations should be not more than 2.0% .
The % assay of Cefepime and Enmetazobactam should be between 98 – 102%
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CHAPTER- RESULTS AND DISCUSSIONS
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CHAPTER–7 REFERENCE
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