INTRODUCTION:
Omeprazole is a proton pump inhibitor that suppresses gastric
acid secretion by specific inhibition of the H+/K+ -ATPase in the gastric parietal cell. Domperidone
acts as a gastrointestinal emptying (delayed) adjunct and peristaltic
stimulant. 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 phases as methanol and 0.1%
ammonium acetate an ideal mobile phase, since it gave a good resolution and
peak shapes with perfect optimization.
These drugs are evaluated for linearity, precision,
accuracy, LOD, LOQ, Specificity, % Assay, Drug degradation etc
High
performance liquid chromatography:
High Performance Liquid Chromatography1-5
(HPLC) is the fastest growing analytical technique for the analysis of drugs.
Chromatographic separation in HPLC is the result of specific interaction
between sample molecules with both the stationary and liquid mobile phases.
HPLC has been rapidly developed with the introduction of new pumping methods,
more reliable columns and wide range of detectors. HPLC is also being automated
which involve automated sampling, separation, detection, recording, calculation
and printing of results. HPLC offers a wide choice of chromatographic
separation methodologies from normal to reverse phase and whole range of mobile
phases using isocratic or gradient elution techniques. Various detectors
available for HPLC are electrochemical detectors, refractive index detectors,
fluorescence detectors, radiochemical detectors, mass-sensitive detectors and
Ultra-violet (UV) detectors6-10. To develop a new HPLC method for
any drug, knowledge of its molecular weight, polarity, ionic character, pKa values, wavelength of absorption, purity of
compound and the solubility should be known. Method development involves
considerable effort and time. The most commonly applied method is reversed
phase and reverse coupled with ion-pairing. These two techniques probably
account for more than 85% of the applications for a typical pharmaceutical
compound. The typical pharmaceutical compounds are considered to be an active
pharmaceutical ingredient of molecular weight of less than 1,000 Daltons.
Depending on the number of active compounds to be resolved or separated, the
more complex is the separation, the more gradient elution will be advantageous
over isocratic mode. Optimization11-15 can be started only after
reasonable chromatogram has been obtained.
Fig: Schematic Diagram of HPLC Instrument
The
main objective was to develop a
sensitive, simple, rapid, reliable and accurate analytical method for the
simultaneous estimation of Omeprazole and Domperidone16
in formulations and validation of developed method by using RP-HPLC technique.
MATERIALS AND METHODS:
Instruments:
Shimadzu Separation Module LC-10AT
HPLC with UV detector, Waters C18 250×4.6mm. 5µm column, Mettle toledo
Electronic balance (sensitivity 0.1mg), Frontline ultra sonicator,
Sansel pH meter, Vaccum
filter pump (PCI analytics) were used for the measurements.
Software used:
LC
Solutions version 1.23
Reagents used:
HPLC
Grade Methanol was used as the solvent as well as the Mobile phase. It was
procured from Rankem, India. Ortho phosphoric acid
was purchased from Rankem, India. Pure water for the
analysis was prepared by using Millipore Milliq
purification system.
Chemicals used:
Methanol and ammonium acetate (HPLC Grade).
Method development:
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 exhaustively studied.
Selection of Chromatographic method:
Proper
selection of method depends up on the nature of the sample, molecular weight,
and solubility. The drug selected for the present study was Non polar. Non
polar compounds can be separated by either normal phase or reverse phase
chromatography Reverse phase chromatography was selected for initial
separations from the knowledge of properties of the compounds.
Selection of Diluent:
The
nature of the drug reveals certain information about the drug such as
solubility, pKa. Based on the solubility of the drug,
the diluents are selected. The solvent in which the drug has maximum solubility
is selected as the diluents. Omeprazole and Domperidone are soluble in Methanol. So methanol is used as
Diluent.
Selection of detection Wavelength:
Standard
solutions of Omeprazole and Domperidone
were injected separately as well as in combination into HPLC system then
scanned over entire the UV range (190-400nm).The spectra of Omeprazole
and Domperidone was recorded for determination of λmax. The λmax Of Omeprazole was detected at 280nm and the λmax of Domperidone was
detected at 288nm. Detection was carried out at 280nm.
Selection of column:
In
reverse phase chromatography non polar stationary phase is used for separation.
C8, C18 are the commonly used columns in reverse phase chromatography. Here C18
column of dimensions 250 × 4.6mm and particle size 5μm is used for the separation.
Selection of Mobile phase:
A
Number of trails were made to find out the mobile phase for eluting the drug.
The mobile phase containing Methanol: Ammonium acetate (70:30).
Mode of separation:
Several
trails were conducted by changing flow rate, injection volume and other
parameters till satisfactory separation were achieved, the resulting
chromatograms were recorded and the chromatographic parameters such as column
efficiency, theoretical plates were calculated. Finally the condition that
gives best result was selected for estimation.
Preparation of Standard Stock Solution:
Accurately
weigh and transfer 10mg of Omeprazole and 10mg of Domperidone working standard into two separate 10ml clean
dry volumetric flasks containing methanol. The solution was sonicated
for about 10mins and then made upto volume with
methanol.
Preparation of sample stock solution:
10
Tablets were taken, powdered and weighed. Average weight was taken. From this
amount of powder equivalent to 10mg Omeprazole of and
10mg Domperidone of was taken into 10ml volumetric
flask containing mobile phase. The solution was sonicated
for 10 mins and made up to volume with mobile phase.
Trail and error method:
Six numbers of trials were conducted and the
results are Based up on the results the optimized method was determined.
Optimized method:
Preparation of Buffers:
Prepared 0.1% Ammonium acetate by dissolving in 1000ml
of water.
Preparation of Mobile Phase:
Mobile Phase A:
Filtered
and degassed Methanol is used as mobile phase-A.
Mobile Phase B:
HPLC
Grade Ammonium acetate is used as Mobile Phase-B.
Optimized method for hplc
chromatographic conditions:
Chromatograms
are shown in fig.no.1 and 2
Instrument:
HPLC Shimadzu Separation Module LC- 10AT Liquid
chromatography.
Column:
Waters
C18 Column 250×4.6mm. 5µm
Column Temperature: Ambient
Auto sampler
Temperature: Ambient
Flow rate: 1ml/min
Wave length: 280nm
Run time: 4.8 mins
Injection volume: 20ul/min
Mobile phase: Methanol: 0.1% Ammonium acetate (70:30).
Analytical method validation:
The
following parameters were considered for validating the developed method as per
ICH guidelines.
System suitability parameters:
Mixed
working standard solutions were injected and chromatograms were recorded. The
system suitability studies were carried out as specified in USP. These
parameters include Column efficiency, Resolution, Capacity factor, Theoretical plates
and tailing factor. system suitability parameters was
shown in table no.1.
Acceptance criteria :
The
% RSD for the retention times and peak area responses of principal peak from 6
replicate injections of each standard solution should be not more than 2.0%.The
number of theoretical plates (N) for the drug peak is not less than 2500.The
Tailing factor (T) for the drug peak is not more than 2.0.
Linearity:
Preparation of standard stock solution:
Accurately
weigh and transfer 50mg of Omeprazole working
standard into a 10ml clean dry volumetric flask containing mobile phase and
80mg of Domperidone working standard into a 10ml
clean dry volumetric flask containing mobile phase. The solutions were sonicated for about 10mins and then made upto volume with mobile phase.
Preparation of Intermediate dilution:
2ml
of Omeprazole solution was taken from the prepared
standard stock solution in a 10ml volumetric flask and made upto
volume with mobile phase. 1.30ml of Domperidone
solution was taken from the prepared standard stock solution in a 10ml
volumetric flask and made upto volume with mobile
phase. based up on this 10, 20, 30, 40, 50µg/ml were
prepared and evaluated. Linearity values shown in table -2 and calibration
curves are shown in fig no.3 and 4.
Acceptance criteria:
R2value should not be less than 0.98
Accuracy:
The
closeness of agreement between the true value which is
accepted either conventional new value or an accepted reference value and the
value found.
Preparation of standard stock solution:
Accurately
weigh and transfer 50mg of Omeprazole working
standard into a 10ml clean dry volumetric flask containing mobile phase and
80mg of Domperidone working standard into a 10ml
clean dry volumetric flask containing mobile phase. The solutions were sonicated for about 10mins and then made up to volume with
mobile phase. Chromatograms are shown in fig no. 5 and 6.
Preparation of Intermediate dilution:
2ml
of Omeprazole solution was taken from the prepared
standard stock solution in a 10ml volumetric flask and made upto
volume with mobile phase. 1.30 ml of Domperidone
solution was taken from the prepared standard stock solution in a 10ml
volumetric flask and made upto volume with mobile
phase based up on this 12.5 , 25, 37.5
µg/ml were prepared and evaluated.
Acceptance criteria:
The
% Recovery for each level should be between 98.0 to 102.0%
Precision:
Preparation
of standard stock solution:
Accurately
weigh and transfer 50mg of Omeprazole working
standard into a 10ml clean dry volumetric flask containing mobile phase and
80mg of Domperidone working standard into a 10ml clean dry volumetric flask
containing mobile phase. The standard solution was injected for six times and measured the area
for all six injections in HPLC. The %RSD for the area of six replicate
injections was found to be within the specified limits.
Standard Deviation
S.D =
Where, x = Sample,
Xi = Mean value of samples.
N = number of samples.
Coefficient of variance / Relative standard
deviation:
Standard Deviation
C.V = 
X
100
Mean
Acceptance criteria:
The % RSD
for the area of six standard injections results should not be more than 2%.
Precision values are shown in table no.3.
Assay:
Assay
of different formulations available in the market was carried by injecting
sample corresponding to equivalent weight into HPLC system and percentage
purity was found out by following formulae. Recovery studies were carried out.
Assay values and observations are shown in table no. 4. Calculate the
percentage purity of Omeprazole and Domperidone present in the tablet using the calibration
curve.
Calculation:
Concentration
of tablet = Dilution factor × Sample
concentration
Robustness:
(a) Effect of
variation of flow rate:
A
study was conducted to determine the effect of variation in flow rate by
injecting 0.8and 1.2ml/min. Standard solution was prepared and injected into
the HPLC system. The retention time values were measured and are given in Table
no.5. The chromatograms are shown in Fig.no.7.
Acceptance criteria:
Tailing
factor and %RSD of Omeprazole and Domperidone
standard should not be more than 2.0 for variation in flow rate.
(b) Effect of
variation of Mobile phase composition :
A
study was conducted to determine the effect of variation in mobilephase
composition. Standard solution was prepared and injected into the HPLC system
at 45:55 and 55:45. The effect of variation in mobile phase composition was evaluated.
Acceptance criteria:
Tailing
factor and %RSD of Omeprazole and Domperidone
standard should not be more than 2.0 for variation in mobilephase
composition.
Ruggedness:
Ruggedness
is the degree of reproducibility of results obtained by the analysis of the
same sample under a
variety of normal
test conditions ie
different analysts, laboratories,columns, instruments, reagents,
assay temperatures, , different days etc. (ie from laboratory to laboratory,
from analyst to analyst.).
Acceptance
Criteria:
Overall
RSD should not be more than 2.0 %
Analyst to Analyst variation:
Procedure:
The standard solution is
injected for by different analysts and the area for injections in HPLC was
measured. The %RSD for the area of replicate injections was found to be within
the specified limits. Chromatogramas re
shown in fig.no.8.
Column to Column variation:
Procedure:
The standard solution is
injected for by using different columns and the area for injections in HPLC was
measured. The %RSD for the area of replicate injections was found to be within
the specified limits.
Limit
of detection:
This
is the lowest concentration in a sample that can be detected, but not
necessarily quantitated, under the stated experimental conditions. The limit of detection is important for
impurity tests and the assays of dosages containing low drug levels and
placebos. Based on signal to noise. Chromatogramas re shown in fig.no.9 and 10
and limits in table no.6.
Limit
of quantification:
This is
the lowest concentration
of analyte in
a sample that
can be determined
with acceptable precision and accuracy.
lt is quoted as the concentration yielding a signal-to-noise ratio
of 10: 1. Chromatogramas re shown in
fig. no. 11 and 12 and limits in table no.7
Specificity:
Specificity for an assay
ensures that the signal measured comes from the substance of interest, and
that there is
no interference from excipient
and/or degradation products and/or impurities. The specificity of the method was demonstrated by
establishing a lack of interference from the diluents blank.10µl of the blank
solution was injected into the chromatograph. There should not be any
interference with the Omeprazole and Domperidone peak.
Chromatogramas re shown in fig.no.13.
Degradation studies:
Degradation studies involve exposing the sample to a
variety of stressed conditions to further evaluate the specificity of
degradation products. In this study, the drug substance, drug product, and the
combined excipients (or placebos) are each exposed to
the stressed conditions. These may include, but are not limited to, heat,
light, acidic media, alkaline media, and oxidative environments.
RESULTS AND DISCUSSION:
Development of method for the estimation
of omeprazole and domperidone:16
Several
trails were conducted during the development of a method for the simultaneous
estimation of Omeprazole and Domperidone
in bulk form. The best peak was shown in below chromatograms. based on these trials an optimized method was developed and
the chromatogram was shown below:
Table no 1:
System suitability parameters of Omeprazole and Domperidone
|
Parameters |
Omeprazole |
domperidone |
|
Area |
721992 |
655688 |
|
Retention time |
3.09 |
4.51 |
|
Theoretical plates |
4825 |
4761 |
|
Tailing
factor |
1.58 |
1.46 |
|
Resolution |
20.25 |
|
