Method Development
and Validation for Simultaneous Estimation of Esomeprazole and itopride
Hydrochloride in Combined Pharmaceutical Dosage Form and bulk by RP- HPLC
Sowjanya Vadrevu
Block 1, Building 18, Al
Farwaniya, Kuwait.
*Corresponding Author E-mail: vvlsps999@gmail.com
ABSTRACT:
The
developed RP- HPLC method allows rapid and precise determinations of
simultaneous estimation of esmoprazole and itopride hydrochloride in combined
pharmaceutical dosage forms. The objective of the proposed method is to develop
simple and accurate methods for the determination of simultaneous estimation of
esmoprazole and itopride hydrochloride in combined pharmacetcal dosage forms
and it’s stability indicative studies. A series of mobile phases were tried,
among the various mobile phases, Separation of the drug was achieved on ACE C18
(150×4.6) mm, 5μ using mobile phases, (A) 6.5 pH di hydrogen ortho
phosphate buffer, (B) Acetonitrile and Water in the ratio of 35:65v/v. The flow
rate was 1.2 ml/min and the detection wavelength was 272 nm.The Linearity and
correlation coefficient of esomeprazole and itopride hydrochloride at
10-50µg/ml and 10-50µg/ml was found to be 0.999 and 0.999 respectively. The LOD
was found to be 0.57 mg/ml and 0.56mg/ml and LOQ was found to be 1.74mg/ml and
1.69 mg/ml for Esomeprazole and itopride hydrochloride respectively which
represents that sensitivity of the method is high. The method was known to be
accuratewith the assay method. The % assay was found to be 99.58 % and 99.98%.
The developed method was showed to a good accuracy and precision. The % RSD is
for Esomeprazole and itopride hydrochloride is 0.55 and 0.75. This method shows
that good reproducibility of the results. Furthermore this method was simple,
sensitive, and accurate. This method can be employed for routine quality
control analysis of tablets of esomeprazole and itopride hydrochloride in
combined pharmacetcal dosage forms in various educational institutions.
KEYWORDS: Esomeprazole,
itopride hydrochloride, Optimization, RP-HPLC.
INTRODUCTION:
Esomeprazole
(esomeprazole) is a proton pump inhibitor that decreases the amount of acid
produced in the stomach. esomeprazole is used to treat symptoms of gastroesophageal reflux disease (gerd) and
other conditions involving excessive stomach acid such as zollinger-ellison
syndrome. Itopride hydrochloride hasanticholinesterase
(AchE) activity as well as dopamine D2 receptor
antagonisticactivity. It is well established that M3 receptors exist on the
smooth muscle layerthroughout the gut and acetylcholine (ACh) released from
enteric nerve endingsstimulates the contraction of smooth muscle through M3
receptors. Several methods have been developed using various
chromatographic studies and the scope of the present work is to expand and
optimization of the chromatographic conditions, to develop RP-HPLC method. A series
of mobile phases were tried, among the various mobile Separation of the drug
was achieved on ACE C18 (150×4.6) mm, 5μ using mobile phases, (A) 6.5 pH
di hydrogen ortho phosphate buffer, (B) Acetonitrile and Water in the ratio of
35:65v/v.,since it gave a good resolution and peak shapes with perfect
optimization. These drugs are evaluated for linearity, precision, accuracy,
LOD, LOQ, Specificity, %assay, system suitability, robustness and ruggedness.
HIGH PERFOMENCE LIQUIID
CHROMATOGRAPHY1-4:
HPLC
is a type of liquid chromatography that employs a liquid mobile phase and a
very divided stationary phase. In order to obtain satisfactory flow rate liquid
must be pressurized to a few thousands of pounds per square inch.
The
rate of drugs between stationary and mobile phase is controlled by diffusion
process, if diffusion is minimized, a faster and effective separation can be
achieved. The technique of liquid chromatography is called because of its
improved performance in compared to classic column chromatography. Advanced in
column technology, high- pressure pumping system and sensitive detectors have
transformed liquid column chromatography into high speed, efficient, accurate
and highly resolved method of separation.
HPLC
is the method of choice in the field of analytical chemistry, since this method
is specific, robust, linear, precise and accurate and the limit of detection is
low.
Advantages of HPLC:
·
Speed (many analysis can be accomplished in 20min orless)
·
Grater sensitivity (various detectors can beemployed)
·
Improved resolution (wide variety of stationaryphases)
·
Reusable column (expensive columns but can be used for manyanalysis)
·
Ideal for the substances of lowviscosity
The
various components of a HPLC system are here with described.
Fig No -1: Diagram of HPLC
System
The
main objective of the proposed method is to develop simple and accurate methods
for the determination of esmoprazole and itopride hydrochloride in combined
pharmaceutical dosageforms.
MATERIALS AND METHODS:5-8
Instruments:
Waters
2695 HPLC, UV visible double beam spectrophotometer labindia UV-300, pH1500
Eutech electronics limited SE 60 US ultra sonicator, melting point apparatus.
Reagents
and chemicals used:
Potassium
Di hydrogen ortho phosphate , Acetonitrile, Milli –Q water, Methanol (99.9%)
HPLC grade obtained from fischer scientific labs Drugs Used: Esmoprazole
and itopride hydrochloride are obtained from krish care laboratories.
METHOD
DEVELOPMENT9-14:
Proper
selection of HPLC method development depends upon the nature of the sample, its
molecular weight and solubility. For successful method development various
Chromatographic parameters such as pH, mobile phase, its composition and
proportion, detection wavelength and other factors were exhaustivelystudied.
Selection of Chromatographic
method:
Preparation of the
Esomeprazole and Itopride hydrochloride Standard Solution: Standard Solution
Preparation:
Accurately
weigh and transfer 20 and 75mg of Esomeprazole and Itopride hydrochloride
working standard into a 10mL clean dry volumetric flask add Diluent and
sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stocksolution).
Further
pipette 1.0ml of Esomeprazole and Itopride hydrochloride of the above stock solution
into a 10ml volumetric flask and dilute up to the mark with diluent.
Further
pipette 1.5ml of Esomeprazole and Itopride hydrochloride of the above stock solution
into a 10ml volumetric flask and dilute up to the mark with diluent
Initialization of the instrument
The
HPLC instrument was switched on. The column was washed with HPLC water for 45
minutes. The column was then saturated with mobile phase for 45 minute. The
mobile phase was run to find the peaks. After 20 minutes the standard drug
solution was injected inHPLC.
Different chromatographic
conditions used and their Optimizations
The
different HPLC chromatographic conditions were used to find out the optimum
chromatographic condition for best elution of drug
TRAIL
AND ERROR METHOD:
Six
number of trials were conducted and the results are Based up on the results the
optimized method was determined.
OPTIMIZED METHOD FOR HPLC
CHROMATOGRAPHIC CONDITIONS:
Chromatogram
was shown in Fig.No. 2 and the parameters are shown in Table .1 and results in
Table.2
Table.1. Optimized
chromatographic conditions
|
Stationary phase (column) |
Inertsil -BDS C18(250 x 4.6 mm, 5 µ) |
|
Mobile Phase |
buffer: acn (35:65) |
|
Flow rate (ml/min) |
1.2 ml/min |
|
Run time (minutes) |
10 min |
|
Column temperature (°C) |
250c |
|
Volume of injection loop (l) |
20 µL |
|
Detection wavelength (nm) |
272 nm |
|
Drug RT (min) |
Eso is2.1 snd itopride hydrochloride is |
ANALYTICAL METHOD VALIDATION:
The
following parameters were considered for validating the developed method as per
ICH guidelines.
SYSTEM SUITABILITY:
A
Standard solution was prepared by using esomeprazole and itopride hydrochloride
working standards as per test method and was injected Five times into the HPLC
system.
The
system suitability parameters were evaluated from standard chromatograms by
calculating the % RSD from five replicate injections for esomeprazole and
itopride hydrochloride, retention times and peak areas.
Acceptance criteria:
1. The % RSD for the
retention times of principal peak from 5 replicate injections of each Standard
solution should be not more than 2.0%
2. The % RSD for the
peak area responses of principal peak from 5 replicate injections of each
standard Solution should be not more than 2.0%.
3. The number of the oreticalplates (N) for the
esomeprazoleandi to pride hydrochloride peaks is NLT3000.
4. The Tailing factor
(T) for the esomeprazole and itopridehydrochloride peaks is not more then 2.0
Observation:
The
%RSD for retention times and peak areas were found to be within the limit.
Refer Table: 3.
SPECIFICITY:
esomeprazole
and itopride hydrochloride standard and sample solutions were prepared as per
the test method are injected into chromatographic system.
Acceptance criteria:
Chromatograms
of standard and sample should be identical with near Retention time.
Observation:
The
chromatograms of standard and sample were same identical with same retention
time.
PRECISION:
Repeatability:
System
precision: esomeprazole and itopride hydrochloride Standard solution prepared
as per test method and injected five times.
Method
precision: esomeprazole and itopride hydrochloride Prepared six sample
preparations individually using single as per test method and injected each
solution.
Acceptance
criteria:
The
% relative standard deviation of individual esomeprazole and itopride
hydrochloride , from the six units should be not more than 2.0%.
The
individual assays of esomeprazole and itopride hydrochloride should be not less
than 98% and not more than 102.0%.
Observation:
Test
results are showing that the test method is precise.as shown in figure 5
Intermediate precision
(analyst to analyst variability):
To
evaluate the intermediate precision (as known as ruggedness) of the method,
precision was performed on different day by using different make column of same
dimensions. The standard solution was injected for six times and measured the
area for all six injections in HPLC. A study was conducted by two analysts as
per test method the %RSD for area of six replicate injections was found to be
within the specified limits
Acceptance criteria:
The
individual assays of esomeprazole and itopride hydrochloride should be not less
than 98% and not more than 102% and %RSD of assays should be not more then 2.0%
by bothanalysts.
Observation:
Individual
% assays and % RSD of Assay are within limit and passes the intermediate
precision, shown in fig.6.
ACCURACY (RECOVERY):
Drug
Assay was performed in triplicate as per test method with equivalent amount of
and esomeprazole and itopride hydrochloride into each volumetric flask for each
spike level to get the concentration of Esomeprazole and itopride hydrochloride
equivalent to 50%, 100%, and 150% of the labeled amount as per the test method.
The average % recovery of esomeprazole and itopride hydrochloride
werecalculated.
Acceptance criteria:
The
mean % recovery of the Esomeprazole and itopride hydrochloride at each spike
level should be not less than 98.0% and not more than 102.0% for both the drugs
separately.
Amount found
%
Recovery = ––––––––––––––– × 100
Amount added
Observation:
The
recovery results indicating that the test method has an acceptable level of
accuracy.
LINEARITY OF TEST METHOD:
A
Series of solutions are prepared using Esomeprazole and itopride hydrochloride
working standards at concentration levels from 20µg/mL to 80µg/mL of target
concentration .Measure the peak area response of solution at Level 1 and Level
6 six times and Level 2 to Level 5 two times.
Linearity:
Linearity
solutions were prepared such that 0.2mL, 0.3 mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL,
0.8mL from the stock solutions esomeprazole and itopride hydrochloride were
taken in to 7 different volumetric flasks and diluted to 10mL with diluents to
get 20µg/mL, 30µg/mL, 40 µg/mL, 50µg/mL.
Acceptance criteria:
Correlation
Coefficient should be not less than 0.9990.
%
of y- Intercept should be ±2.0.
%
of RSD for level 1 and Level 6 should be not more than 2.0%.
Observation:
The
linear fit of the system was illustrated graphically. The results are shown,
Figure; 3 and 4
RUGGEDNESS OF TEST METHOD:
a) System to systemvariability:
System
to system variability study was conducted on different HPLC systems, under
similar conditions at different times. Esomeprazole and itopride hydrochloride
solutions Six samples were prepared and each was analyzed as per test method.
Comparison of both the results obtained on two different HPLC systems, shows
that the assay test method are rugged for System to system variability.
Acceptance criteria:
The
% relative standard deviation of Esomeprazole and itopride hydrochloride from
the six sample preparations should be not more than 2.0%
The
% assay of Esomeprazole and itopride hydrochloride should be between 98.0%-
102.0%.
Observation:
The
% RSD was found within the limit, shown in figure no.7.
b)
Column
to columnvariability:
Column
to column variability study was conducted by using different columns.
Esomeprazole and itopride hydrochloride solutions Six samples were prepared and
each was analyzed as per test method
Acceptance criteria:
The
% RSD of Esomeprazole and itopride hydrochloride tablets should be not more
than 2.0%.The % assay of Esomeprazole and itopride hydrochloride should be
between 98.0% and 102.0% for individual drugs.
Observation:
The
results obtained by comparing with both two types were within limit.
ROBUSTNESS:
a) Effect of variation of flowrate:
A
study was conducted to determine the effect of variation in flow rate.
Esomeprazole and itopride hydrochloride Standard solution prepared as per the
test method was injected into the HPLC system using flow rates, 1.0 mL/min and
1.2 mL/min. The system suitability parameters were evaluated and found to be
within the limits for 1.0 mL/min and 1.2 mL/min flow. Esomeprazole and itopride
hydrochloride and was resolved from all other peaks and the retention times were
comparable with those obtained for mobile phase having flow rates 1.0mL/min.
Acceptance
criteria:
The
Tailing Factor of Esomeprazole and itopride hydrochloride standards should be
not more then 2.0 for Variation inFlow.
Observation:
The
tailing factor for Esomeprazole and itopride hydrochloride was found to be
within the limits, as shown in figure.no.8
b) Effect of variation
oftemperature:
A
study was conducted to determine the effect of variation in temperature.
Esomeprazole and itopride hydrochloride Standard solution prepared as per the
test method was injected into the HPLC system at 20şC temperature. The system suitability
parameters were evaluated and found to be within the limits for a temperature
change of 20şc. Similarly sample solution was chromatographed at 25şC
temperature. Esomeprazole and itopride hydrochloride were resolved from all
other peaks and the retention times were comparable with those
Acceptance
criteria:
The
Tailing Factor of Esomeprazole and itopride hydrochloride standard and sample
solutions should be not more than not more then 2.0 for Variation in
temperature.
Observation:
The
tailing factor for esomeprazole and itopride hydrochloride, x is found to be
within the limits,
LIMIT OF DETECTION AND
QUANTITATION (LOD and LOQ):
From
the linearity data calculate the limit of detection and quantitation, using the
following formula.
LOD:
It
is the lowest amount of the analyte in a sample that can be detected but not
necessarily be quantitated as an exact concentration or amount
LOD=
3.3 σ
S
LOQ:
It
is the lowest amount of an analyte that can be measured quantitatively in a
sample with acceptable accuracy and precision. The LOQ is a parameters for
tests measuring impurities in the drugproduct.
σ
= standard deviation of theresponse
S
= slope of the calibration curve of the analyte.
LOQ
= 10 σ
S
σ
= standard deviation of the response
S
= slope of the calibration curve of the analyte.
DEGRADATION STUDIES:
The
International Conference on Harmonization (ICH) guideline entitled stability
testing of new drug substances and products requires that stress testing be
carried out to elucidate the inherent stability characteristics of the active
substance. The aim of this work was to perform the stress degradation studies
on the Esomeprazole and Itopride hydrochloride using the proposed method. the
results of degradation are shown intable.4
Preparation of stock:
Accurately
weigh and transfer 15mg and 50mg of Esomeprazole and Itopride hydrochloride
working standard into a 10ml clean dry volumetric flask add Diluent and
sonicate to dissolve it completely and make volume up to the mark with the same
solvent. (Stocksolution)
Further
pipette 1ml of Esomeprazole and Itopride hydrochloride of the above stock
solution into a 10ml volumetric flask and dilute up to the mark with diluent.
Hydrolytic degradation under
acidic condition:
Pipette
1.5ml of above solution into a 10ml volumetric flask and 3ml of 0.1N HCl was
added. Then, the volumetric flask was kept at 60şC for 6 hours and then
neutralized with 0.1 N NaOH and make up to 10ml with diluent. Filter the
solution with 0.22 microns syringe filters and place in vials.
Hydrolytic degradation under
alkaline condition:
Pipette 1.5ml of above
solution into a 10ml volumetric flask into a 10ml volumetric flask and add 3ml
of 0.1N NaOH was added in 10ml of volumetric flask. Then, the volumetric flask
was kept at 60şC for 6 hours and then neutralized with 0.1N HCl
and
make up to 10ml with diluent. Filter the solution with 0.22 microns syringe
filters and place in vials.
Thermal induced degradation:
Esomeprazole
and Itopride hydrochloride sample was taken in petridish and kept in Hot air
oven at 1100C for 24 hours. Then the sample was taken and diluted
with diluents and injected into HPLC and analysed.
Oxidative degradation:
Pipette
1.5ml above stock solution 2 into a 10ml volumetric flask solution into a 10ml
volumetric flask 1 ml of 3% w/v of hydrogen peroxide added in 10ml of
volumetric flask and the volume was made up to the mark with diluent. The
volumetric flask was then kept at room temperature for 15 min. Filter the
solution with 0.45 microns syringe filters and place in vials.
RESULTS AND DISCUSSION:
DEVELOPMENT OF METHOD FOR THE
ESTIMATION OF ESOMEPRAZOLE AND ITOPRIDE HYDROCHLORIDE7-9:
Several
trails were conducted during the development of a method for the simultaneous
estimation of Esomeprazole and itopride hydrochloride in bulk form. The best
peak was shown in below chromatograms.based on these trials an optimized method
was developed and the chromatogram was shownbelow:
Fig 2: A typical chromatogram
for optimized method
Table 2. Optimized method
|
S. NO |
Name of the peak |
Retention time(min) |
|
1 |
2.149 |
|
|
2 |
itopride hydrochloride |
3.135 |
Observation:
This
is showing good peak resolution for two samples. For this reason it was taken
as a standard method procedure for the subsequent experiment
Table no 3: System suitability
parameters of Esomeprazole and itopride hydrochloride
|
Parameters |
Escitalopram |
Etizolam |
|
Area |
187069.4 |
577513.6 |
|
Retention time |
2.14 |
3.13 |
|
Theoretical plates |
3682 |
2859 |
|
Tailingfactor |
0 .93 |
1.2 |
Fig no: 3 and 4 Linear
calibration curve of Esomeprazole and itopride hydrochloride
Accuracy:
The
mean recoveries were found to be 99.73% for Esomeprazole and 102.0% for
itopride hydrochloride. The limit for
mean % recovery is 95-105% and as both the values are within the limit, hence
it can be said that the proposed method wasaccurate.
PRECISION:
Results of system precision
for ESOMEPRAZOLE (mg) AND ITOPRIDE HYDROCHLORIDE
Method precision
(Repeatability):
Fig 5: A typic al Chromatograms
for precision
Fig 6: A typic al Chromatograms
for Intermediate precision
Acceptance Criteria:
The
% RSD for the area of five standard injections results should not be more than
2%.the results are found to be with in limits.
Assay of Esomeprazole and
itopride hydrochloride In Dosage Form:
Assay
was performed and the results are with in limits and was found to be 99.59 for
esomeprazole and 99.98 for itopride hydrochloride.
RUGGEDNESS:
a) System to System
variability:
Fig .7. A typical Chromatograms
for system to system variability
Robustness:
a) Effect of variation of flow
rate for (1mL/min flow)
Fig 8: Atypical chromatograms
for (1.2 mL/min flow)
Acceptance criteria:
The
% RSD peak area of all peaks for the four replicates injections should be not
more then 2%.
Observation:
The
% RSD peak area was found to be within limit . So the method was robust.
LIMIT OF DETECTION AND LIMIT
OF QUANTITATION (LOD and LOQ): ESOMEPRAZOLE:
From
the linearity plot the LOD and LOQ are calculated:
LOD
= 3.3 σ
S
3.3×1774.524
= ------------------=0.94
6204
LOQ
= 10 σ
S
10×1774.524
= -----------------=2.86
6204
Itopride hydrochloride:
LOD = 3.3 σ
S
3.3×1840.015
= -------------------–=1.19
5097
LOQ
= 10 σ
S
10×1840.015
= ---------------––––=3.60
5097
The
LOD was found to be 0.94 mg/ml and 1.19mg/ml and LOQ was found to be 2.86 mg/ml
and 3.60mg/ml for Escitalopram and Etizolam respectively which represents that
sensitivity of the method is high.
Degradation:
RESULTS:
Table: 4 Table showing results
of amount of drug degradation:
|
|
||||
|
Area |
% Degraded |
|||
|
Standard |
92911 |
|
394495 |
|
|
Acid |
87708 |
5.6 |
380688 |
3.5 |
|
Base |
90867 |
2.2 |
362146 |
8.2 |
|
Peroxide |
82784 |
10.9 |
376743 |
4.5 |
|
Thermal |
82970 |
10.7 |
357412 |
9.4 |
Table 5. Summary of validation
parameters by HPLC method
|
PARAMETERS |
RESULTS |
|
|
ESOMEPRAZOLE |
Itopride hydrochloride |
|
|
Method precision |
%RSD = 0.641801 |
%RSD = 0.154822 |
|
Intermediate precision |
%RSD = 0.951074 |
%RSD =0.318806 |
|
Linearity |
10-50 µg/ml correlation coefficient =0.999 |
10-50 µg/ml correlation coefficient =0.999 |
|
Accuracy |
Recovery values =99.71 100% -102% |
Recovery values =100.28 97 %-101% |
|
LOD |
0.57 |
0.56 |
|
LOQ |
1.74 |
1.69 |
|
robustness |
0.55 |
0.75 |
CONCLUSION:
Hence,
the chromatographic method developed for Esomeprazole and itopride
hydrochloride is said to be rapid, simple, specific, sensitive, precise,
accurate and reliable that can be effectively applied for routine analysis in
research institutions, quality control department in industries, approved
testing laboratories, bio-pharmaceutics and bio-equivalence studies and in
clinical pharma cokineticstudies.
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Received on 15.02.2020 Modified on 08.03.2020
Accepted on 28.03.2020
©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(2):91-98.
DOI: 10.5958/2231-5675.2020.00016.2