Page 1
Research Article Vol: 1; Issue: 2
DEVELOPMENT AND VALIDATION OF HPLC METHOD
FOR THE SIMULTANEOUS ESTIMATION OF
CHLORTHALIDON AND METOPROLOL SUCCINATE IN
BULK AND DOSAGE FORM
Raval Kashyap*1, U Srinivasa
2
1Research Scholar, Department of Pharmacy, Pacific University, Udaipur
2Head of the Department, Srinivas College of Pharmacy, Mangalore
Date Received:
26th Nov 2013
Date of Accepted:
6th Dec 2013
Date Published:
10-Dec-2013
Available Online on: www.ijpda.com Page 1
Abstract: A simple, accurate and precise High Performance Liquid Chromatographic (HPLC) method has been developed for
simultaneous determination of Chlorthalidon and Metoprolol Succinate in bulk and dosage form. The method has been
validated as per the guidelines of ICH. The separation is achieved on 250*4.6mm C18, 5micron (Hypersil BDS) column
with flow rate 1.0 mL per minute in isocratic mode using Buffer pH 4.5: Methanol : acetonitrile (50:25:25) as mobile
phase. Column oven temperature is maintained at 25°C and observations are recorded at 223 nm. The linearity range was
found to be in the range of 5-15 µg/ml for Chlorthalidon and 20-60 µg/ml for Metoprolol Succinate. Correlation co-
efficient for calibration curve of Chlorthalidon and Metoprolol Succinate was found to be 0.998 and 0.999 respectively.
The method is simple, accurate, reproducible and short and can be used for simultaneous analysis of Chlorthalidon and
Metoprolol Succinate.
Keywords: Chlorthalidon, Metoprolol Succinate, Buffer pH 4.5, Methanol and Acetonitrile.
Introduction
A study of the interaction of light (or other
electromagnetic radiation) with matter is an
important and versatile tool for the chemist.
Indeed, much of our knowledge of chemical
substances comes from their specific absorption or
emission of light. In this experiment, we are
interested in analytical procedures based on the
amount of light absorbed (or transmitted) as it
passes through a sample.1 Vinicor-D is available
in 25mg and 50mg. Vinicor-D contains
Metoprolol succinate and Chlorthalidone.
Chlorthalidone is an antihypertensive /diuretic
tablets for oral use. It is a monosulfamyl diuretic
that differs chemically from thiazide diuretics in
that a double ring system is incorporated in its
structure. Its IUPAC nname is 2-chloro-5-(1-
hydroxy-3-oxo-1-isoindolinyl) benzene
sulfonamide. Its Molecular formula and
Molecular weight are C14H11ClN2O4S and
338.766 respectively. Chlorthalidone is practically
insoluble in water, in ether and in chloroform,
soluble in methanol, slightly soluble in alcohol.
Chlorthalidone is a long-acting oral diuretic with
antihypertensive activity. Its diuretic action
commences a mean of 2.6 hours after dosing and
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 2 of 14
continues for up to 72 hours. The drug produces
diuresis with increased excretion of sodium and
chloride. The diuretic effects of Chlorthalidone and
the Benzothiadiazine (thiazide) diuretics appear to
arise from similar mechanisms and the maximal
effect of Chlorthalidone and the thiazides appear to
be similar. The site of the action appears to be the
distal convoluted tubule of the nephron. The diuretic
effects of Chlorthalidone lead to decreased
extracellular fluid volume, plasma volume, cardiac
output, total exchangeable sodium, glomerular
filtration rate, and renal plasma flow.
Metoprolol succinate is a beta1-selective
(cardio selective) adrenoceptor blocking agent, for
oral administration, available as extended-release
tablets. 2 TOPROL-XL has been formulated to
provide a controlled and predictable release of
Metoprolol for once-daily administration. The
tablets comprise a multiple unit system containing
Metoprolol succinate in a multitude of controlled
release pellets. Each pellet acts as a separate drug
delivery unit and is designed to deliver Metoprolol
continuously over the dosage interval. Its chemical
name is (±) 1(isopropyl amino)-3-[p-(2-
methoxyethyl) phenoxy]-2-propanol succinate (2:1)
(salt). Its Molecular formula- and molecular weight
are (C15H25NO3)2 • C4H6O4 and 652.8 respectively.
Metoprolol succinate is a white crystalline
powder. It is freely soluble in water, soluble in
methanol, sparingly soluble in ethanol, slightly
soluble in dichloromethane and 2-propanol,
practically insoluble in ethyl-acetate, acetone,
diethyl ether and heptane. It is indicated for the
treatment of hypertension, to lower blood pressure.
Lowering blood pressure lowers the risk of fatal and
non-fatal cardiovascular events, primarily strokes
and myocardial infarctions. These benefits have
been seen in controlled trials of antihypertensive
drugs from a wide variety of pharmacologic classes
including Metoprolol.3
There are spectroscopic and
Chromatographic methods developed on single
Metoprolol Succinate and Chlorthalidon 4-25
. But up
to now there is no HPLC methods develop for
simultaneous estimation Chlorthalidon and
Metoprolol Succinate Analysis of the drug is
important for development of drugs in their
formulation and their use in therapies, for which we
require standard analytical procedures. The USP has
published specific guidelines for method
validation for compound evaluation. USP defines
eight steps for validation: Accuracy, Precision,
Specificity, Limit of detection, Limit of
quantitation , Linearity and range, Robustness26-27
As quality control process is not static some form
of validation/verification should continue till the
validated procedure is in use. It should not be a
concept that once the method is initially developed
and validated it is forgotten.
Materials and methods:
Chromatographic methods offer an advantage in
terms of sensitivity and selectivity. These methods
can be used for routine analysis of dosage forms
where two or more drugs are present together.
HPLC method was developed for simultaneous
estimation of Chlorthalidon and Metoprolol
Succinate.
Reagents and Material
Metoprolol Succinate and Chlorthalidone were
from Shreeji pharma International, Vadodara.
Acetonitrile for HPLC, Methanol, Water for HPLC,
Potassium Dihydrogen ortho phosphate AR grade,
Marketed formulation
The commercial formulation was purchased from
Local pharmacy. Each Vinicor-D Tablet contains
12.5mg Chlorthalidon and 50mg Metoprolol
Succinate.
Selection of Mobile Phase
After assessing the solubility of drugs in different
solvents as well on the basis of literature survey,
the standard solution of Chlorthalidon and
Metoprolol Succinate were injected into the HPLC
system by using different solvent systems.
Different mobile phases were tried in order to find
the best conditions for the separation of both the
drugs. It was found that Buffer pH 4.5: Methanol:
Acetonitrile give satisfactory results as compared to
other mobile phases. Finally, the optimal
composition of the mobile phase was determined to
be Buffer pH 4.5: Methanol: Acetonitrile
(50:25:25) which show in table no.1
Selection of Detection Wavelength
The Detection of wavelength was done in UV
SIMADZU 1800 instrument. The sensitivity of
HPLC method that uses UV detection depends
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Raval Kashyap
upon proper selection of detection wavelength. An
ideal wavelength is the one that gives good response
for the drugs that are to be detected. In the present
study standard drug solutions of 5µg/ml
Chlorthalidon and 20 µg/ml Metoprolol Succinate
were, therefore, prepared in solvent mixtures of
mixture of Buffer pH 4.5: Methanol : acetonitrile (
50:25:25). This drug solution was than scanned in
the UV region of 200-400 nm and the spec
recorded 223nm which is shown in figure no. 1
Figure 1: Detection of wavelength 223nm
Optimized Chromatographic Conditions
The HPLC used for the method was LC 100 UV
DETECTOR and software used for the data
extracted was WS 100 WORKSTATION
SOFTWARE. To optimize the chromatographic
conditions, the effect of chromatographic variables
such as mobile phase pH, flow rate, and solven
ratio were studied. The resulting chromatograms
were recorded and the chromatographic parameters
such as capacity factor, asymmetric factor and
column efficiency were calculated. The conditions
that gave the best resolution, symmetry and capacity
factor were selected for estimation show in table
no.1 and figure 2.
Buffer preparation (0.05M KH2PO4 pH4.5), Mobile
phase: Buffer pH 4.5: Methanol: acetonitrile
(50:25:25), Flow rate: 1.0 mL/min, Wavelength:
223nm, Column: 250*4.6mm C18, 5micron
Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
upon proper selection of detection wavelength. An
ideal wavelength is the one that gives good response
for the drugs that are to be detected. In the present
utions of 5µg/ml
Metoprolol Succinate
were, therefore, prepared in solvent mixtures of
mixture of Buffer pH 4.5: Methanol : acetonitrile (
50:25:25). This drug solution was than scanned in
400 nm and the spectrum was
recorded 223nm which is shown in figure no. 1
Detection of wavelength 223nm
Optimized Chromatographic Conditions
The HPLC used for the method was LC 100 UV
DETECTOR and software used for the data
extracted was WS 100 WORKSTATION
SOFTWARE. To optimize the chromatographic
conditions, the effect of chromatographic variables
such as mobile phase pH, flow rate, and solvent
ratio were studied. The resulting chromatograms
were recorded and the chromatographic parameters
such as capacity factor, asymmetric factor and
column efficiency were calculated. The conditions
that gave the best resolution, symmetry and capacity
were selected for estimation show in table
pH4.5), Mobile
phase: Buffer pH 4.5: Methanol: acetonitrile
(50:25:25), Flow rate: 1.0 mL/min, Wavelength:
223nm, Column: 250*4.6mm C18, 5micron
(Hypersil BDS) and Injection volume: 20 micro
liter.
Preparation of standard solutions
Preparation of buffer pH 4.5
Buffer preparation (0.05M Kh2po4 pH4.5):
6.8gm potassium dihydrogen phosphate was taken
into a 1000ml beaker, added 800ml water and
dissolved. Adjusted pH4.5 with 1%
orthophosphoric acid. It was Made up volume with
water upto 1000ml.
Preparation of mobile phase
500 ml of Buffer (pH 4.5), 250ml Methanol and
250 ml of Acetonitrile (HPLC grade) were mixed
and filtered through 0.45µm filter, Sonicated for
10minutes to degas and used as mobile phase. Use
mobile phase as a diluents.
Preparation of STD Stock solution ofChlorthalidon:
Stock solution of Chlorthalidon: 10mg of
Chlorthalidon was taken as working standard into a
100ml volumetric flask. Added 60ml mobile phase
and dissolve, make up volume with mobile phase
(100 µg/ml)
Preparation of STD Stock solution of Metoprolol Succinate:
Stock solution of Metoprolol Succinate
Metoprolol Succinate was taken as working
standard into a 100ml volumetric flask. Add 60ml
mobile phase and dissolve, make up volume with
mobile phase (400 µg/ml)
Calibration curve for
Chlorthalidon and 20-60
Succinate
Appropriate volume of aliquots from standard
Chlorthalidon and Metoprolol Succinate
solutions were transferred to same volumetric
flasks of 10 ml capacity. The volume was adjusted
to the mark with mobile phase give a solution
containing 5, 7.5, 10, 12.5 and 15
Chlorthalidon and 20, 30, 40, 50, 60
Metoprolol Succinate. E
standard solutions was chromatographed for 10
minutes run time using mobile phase at 223nm at
flow rate of 1 ml/min. The graphs were plotted for
Page 3 of 14
(Hypersil BDS) and Injection volume: 20 micro
Preparation of standard solutions
Preparation of buffer pH 4.5
reparation (0.05M Kh2po4 pH4.5):
6.8gm potassium dihydrogen phosphate was taken
into a 1000ml beaker, added 800ml water and
dissolved. Adjusted pH4.5 with 1%
orthophosphoric acid. It was Made up volume with
Preparation of mobile phase
500 ml of Buffer (pH 4.5), 250ml Methanol and
250 ml of Acetonitrile (HPLC grade) were mixed
and filtered through 0.45µm filter, Sonicated for
10minutes to degas and used as mobile phase. Use
Preparation of STD Stock solution of
Stock solution of Chlorthalidon: 10mg of
Chlorthalidon was taken as working standard into a
100ml volumetric flask. Added 60ml mobile phase
and dissolve, make up volume with mobile phase
Preparation of STD Stock solution of
Metoprolol Succinate: 40mg of
was taken as working
standard into a 100ml volumetric flask. Add 60ml
mobile phase and dissolve, make up volume with
Calibration curve for the 5-15 µg/ml
60 µg/ml Metoprolol
Appropriate volume of aliquots from standard
Metoprolol Succinate stock
solutions were transferred to same volumetric
flasks of 10 ml capacity. The volume was adjusted
to the mark with mobile phase give a solution
containing 5, 7.5, 10, 12.5 and 15 µg/ml
and 20, 30, 40, 50, 60 µg/ml
Each of these mixed
standard solutions was chromatographed for 10
minutes run time using mobile phase at 223nm at
flow rate of 1 ml/min. The graphs were plotted for
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 4 of 14
peak area vs. concentration for both the drugs. Data
is recorded in table no. 2 and figure no. 3, 4 and 5.
Analysis of marketed formulation:
Solution-1: Sample was taken equivalent to 10mg of
Chlorthalidon (10ml sample) into a 100ml
volumetric flask. Added 60ml of mobile phase and
shaked for 15 minutes to dissolve. Made up with
mobile phase. Filtered this solution with 0.45micron
membrane filter (This solution contains 100 µg/ml
of Chlorthalidon and 400 µg/ml of Metoprolol
Succinate)
Solution-2: 1ml of solution-1 was taken into a 10ml
volumetric flask and make up with mobile phase.
(This solution contains 10 µg/ml of Chlorthalidon
and 40 µg/ml of Metoprolol Succinate)
The prepared sample solution was chromatographed
for 10 minutes run time using mobile phase at
223nm at flow rate of 1 ml/min. From the peak area
obtained in the chromatogram, the amounts of both
the drugs were calculated by fitting peak area
responses into the equation of the straight line
representing the calibration curves for Chlorthalidon
and Metoprolol Succinate. And result shown in table
no 3 and figure no. 6.
Validation of proposed HPLC method
6. System suitability
System suitability testing is an integral part of many
analytical procedures. The tests are based on the
concept that the equipment, electronics, analytical
operations and samples to be analyzed constitute an
integral system that can be evaluated as such.
System suitability test parameters to be established
for a particular procedure depend on the type of
procedure being validated. System suitability test
was carried out to verify that the analytical system is
working properly to give accurate and precise
results. Standard solution (5µg/ml Chlorthalidon and
20µg/ml Metoprolol Succinate) was injected six
times and the chromatograms were recorded in table
no. 4 and figure no. 7.
Acceptance Criteria
The % RSD for area response obtained from six
replicate injections of Standard solution should be ≤
2.0 %, Tailing factor should be ≤ 2.0, Theoretical
should be ≥ 2000 27
and Resolution should be ≥ 2.0
in Standard solution.
Solvent suitability:
Recorded in table no.5
Linearity
The linearity of analytical method is its ability to
elicit test results that are directly proportional to
the concentration of analyte in sample within a
given range. The range of analytical method is the
interval between the upper and lower levels of
analyte that have been demonstrated to be
determined within a suitable level of precision,
accuracy and linearity.
The linearity peak area response was determined
by analyzing solutions having concentrations in
the range of 5-15 µg/ml and 20-60 µg/ml for
Chlorthalidon and Metoprolol Succinate
respectively from same solution. Peak area of
each solution was measured using developed
method. Calibration curve of peak area Vs
concentration was plotted. The correlation
coefficient and regression line equations for
Chlorthalidon and Metoprolol Succinate were
determined. Linearity is recorded in table no.6.
Precision
Repeatability
6 replicates of standard mixture solution having
and Chlorthalidon (5 µg/ml) and Metoprolol
Succinate (20µg/ml) were prepared and
chromatograms were recorded and RSD was
calculated and shown in table no. 7.
Intraday precision
Standard solutions containing 5, 10, 15 µg/ml
Chlorthalidon and 20, 40, 60 µg/ml Metoprolol
Succinate were analyzed 3 times on the same day.
Chromatogram of each sample was recorded. SD
and RSD were calculated and shown in table no.
8.
Interday precision
Standard solutions containing 5, 10, 15 µg/ml
Chlorthalidon and 20, 40, 60 µg/ml Metoprolol
Succinate were analyzed on three different days.
Chromatogram of each sample was recorded. SD
and RSD were calculated and shown in table no.
9.
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 5 of 14
Accuracy
Accuracy is the closeness of the test results obtained
by the method to the true value. Recovery studies
were carried out by addition of standard drug to the
pre analysed sample at 3 different concentration
levels (80, 100 and 120 %) taking into consideration
percentage purity of added bulk drug samples. It
was determined by calculating the recovery
Chlorthalidon and Metoprolol Succinate by standard
addition method.
Preparation of sample solution for % recovery:
An accurately weighed powder equivalent to about
10 mg of Chlorthalidon and 4mg of Metoprolol
Succinate was transferred to 100 ml volumetric
flask; dissolved and the volume was made up to the
mark using mobile phase. The solution was
sonicated for 20 minutes. The solution was filtered
through whatman Filter Paper No.42. First few ml
of filtrate were discarded. 1 ml of the solution from
above filtrate was diluted to 10 ml with mobile
phase. The prepared sample solution was
chromatographed for 10 minutes using mobile phase
at flow rate of 1 ml/min. concentration of
Chlorthalidon and Metoprolol Succinate is
calculated which is known as pre-analyzed sample.
In pre-analyzed sample 80, 100 and 120% of
Chlorthalidon and Metoprolol Succinate was spiked.
Chromatogram of each spiked solutions was taken
and total amount of drug was calculated and from
which % recovery was calculated. This is shown in
table no. 10 and 11.
Limit of Detection (LOD)
The LOD is estimated from the set of 6 calibration
curves used to determine method linearity. The
LOD may be calculated as;
LOD = 3.3 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept
of 6 calibration curves.
Slope = the mean slope of the 6 calibration curves.
This is shown in table no. 12.
Limit of Quantification (LOQ)
The LOQ is estimated from the set of 6 calibration
curves used to determine method linearity. The
LOQ may be calculated as;
LOQ = 10 x (SD / Slope)
Where, SD = the standard deviation of Y- intercept
of 6 calibration curves.
Slope = the mean slope of the 6 calibration curves.
This is shown in table no. 12.
Robustness
The robustness of an analytical method was carried
out to confirm that the method remained unaffected
by small but deliberate variations in method
parameters and provides an indication of its
reliability during normal usage. The standard
solution was injected five times for each varied
conditions of flow, column temperature, pH, and
mobile phase ratio and chromatograms were
recorded in table no. 13 & 14. Change in
Conditions for Robustness like Change in flow rate,
M.P. and pH.
RESULT AND DISCUSSION:
High Performance Liquid Chromatographic
(HPLC) method has been developed for
simultaneous determination of Chlorthalidon and
Metoprolol Succinate in bulk and dosage form. The
linearity range was found to be in the range of 5-15
µg/ml for Chlorthalidon and 20-60 µg/ml for
Metoprolol Succinate with using mobile phase
Buffer pH 4.5: Methanol : acetonitrile (
50:25:25).Correlation co-efficient for calibration
curve Chlorthalidon and Metoprolol Succinate was
found to be 0.998 and 0.999 respectively. The
method is simple, accurate, reproducible and short
and can be used for simultaneous analysis of
Chlorthalidon and Metoprolol Succinate
Optimization of Mobile phase
Conclusion Different mobile phases were tried in
order to find the best conditions for the separation
of both the drugs. It was found that Buffer pH 4.5:
Methanol: acetonitrile (50:25:25). Gives
satisfactory results as compared to other mobile
phases. This is shown in table no.1 and best
resolution shown in figure 2.
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 6 of 14
Table 1: Mobile Phase trial for Sample analysis
Trial
no.
Mobile phase Ratio Retention time (min.) Remark
Chlorthalidon Metoprolol
Succinate
Trial 1 Water : Methanol 50:50 4.043 _ Metoprolol Succinate Peak was not Found
Trial 2 Water : Methanol 30:70 2.697 _ Metoprolol Succinate Peak was not Found
Trial 3 Water : Methanol:
Acetonitrile
30:30:40 2.527 8.520 Metoprolol Succinate Peak was found
broad
Trial 4 Water : Acetonitrile 30:70 2.250 7.767 Metoprolol Succinate Peak was found
broad with tailing
Trial 5 Water : Methanol:
Acetonitrile
20:30:50 2.530 7.567 Metoprolol Succinate Peak was found
broad
Trial 6 Buffer pH 4.5: Methanol:
Acetonitrile
30:30:40 2.540 3.107 Peak was found but no god resolution
Trial 7 Buffer pH 4.5: Methanol:
Acetonitrile
35:30:35 2.703 3.230 Peaks were resolved but tailing was
observed
Trial 8 Buffer pH 4.5: Methanol:
Acetonitrile
50:25:25 4.383 6.120 Both peak separates with good resolution
Figure 2: Best result Trial
Figure 4: Calibration curve for Chlorthalidone
Calibration curve for the Chlorthalidon (5-15
μg/ml) and Metoprolol Succinate (20-
60μg/ml)
Five concentrations were taken for STD curve.
But for linearity samples were analyzed 6 times
as per ICH guideline.
Conclusion: The linearity range was found to be
in the range of Chlorthalidon (5-15μg/ml).
Metoprolol Succinate (20-60μg/ml). Correlation
co-efficient for calibration curve Chlorthalidon
and Metoprolol Succinate was found to be 0.998
and 0.9996 respectively. Data is recorded in
table no. 2 and figure no. 3, 4, 5.
y = 72.632x + 19.1
R² = 0.999
0
1000
2000
3000
4000
5000
0 20 40 60 80
a
r
e
a
Conc.(ppm)
Metoprolol Succinate
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 7 of 14
Fig-5: Linear calibration curve of Metoprolol
Analysis of marketed formulation:
Result shown in table no 3 and figure no. 6.
Table 3: Analysis of marketed formulation
Vinicor-D
Drugs
Label
Claim
(mg)
Amount
Found(mg)
%
Recovery
Chlorthalidon
12.5mg
12.613
100.90
Metoprolol
Succinate
50mg
50.193
100.38
System suitability:
Conclusion: The column efficiency was more than
2000 theoretical plates and the tailing factor was
less than 2.0 for Chlorthalidon and Metoprolol
Succinate drugs. Resolution is 5.196. The study
concludes the suitability of the HPLC system being
use. And data was recorded in table no.4 and figure
no.7
Figure 3: STD curve linearity
Table 2: STD curve data for Chlorthalidon and Metoprolol Succinate
Chlorthalidon Metoprolol Succinate
Concentration
(µg/ml)
Concentration
(µg/ml)
Concentration
(µg/ml)
Peak Area*
(mAU*S) 5 967.967 20 1472.488
7.5 1458.624 30 2201.129
10 1947.099 40 2938.241
12.5 2349.151 50 3610.859
15 2915.285 60 4399.236
y = 191.4x + 13.56
R² = 0.998
0
500
1000
1500
2000
2500
3000
3500
0 5 10 15 20
a
r
e
a
Conc.(ppm)
Chlorthalidon
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 8 of 14
Figure 6: Chromatogram of marketed formulation
Table 4: System suitability data for Chlorthalidon and Metoprolol Succinate
Sr No. Standard Response (mAU*S)
Chlorthalidon (5 µg/ml) Metoprolol Succinate (20µg/ml)
Average ± SD 971.23 ± 9.31 1471.33±6.49
%RSD 0.9588 0.4412
Retention Time 4.390 6.127
Theoretical plates 3295 4569
Tailing Factor 1.57 1.50
Resolution 5.196
Figure 7: Chromatogram of Standards for System Suitability
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Raval Kashyap et al; Vol: 1, Issue: 2, Page: 1 – 14
Page 9 of 14
Solvent suitability:
Conclusion: The % RSD of response for both
drugs was found to be less than 2. So, it was
concluded that proposed mobile phase Buffer pH
4.5: Methanol: acetonitrile (50:25:25) is suitable for
estimation of Chlorthalidon and Metoprolol
Succinate in combined dosage form and shown in
table no 5.
VALIDATION
Linearity: 6 times chromatogram were taken for all
five concentration and data were recorded table
no.6
Precision
Repeatability:
Discussion: The % RSD for Repeatability of both
the drugs was found to be less than 2. So, it was
concluded that proposed method for estimation of
Chlorthalidon and Metoprolol Succinate is précised
in nature and shown in table no.7.
Intraday precision:
Discussion: The % RSD for Repeatability of both
the drugs was found to be less than 2. So, it was
concluded that proposed method for estimation of
Chlorthalidon and Metoprolol Succinate is précised
in nature and shown in table no.8.
Interday precision:
Discussion: The % RSD for Repeatability of both
the drugs was found to be less than 2 so, it was
concluded that proposed method for estimation of
Chlorthalidon and Metoprolol Succinate is précised
in nature and shown in table no.9.
Accuracy:
Discussion: Result obtained reveals that %
recovery of Chlorthalidon and Metoprolol
Succinate was within acceptance criteria given in
ICH guideline. And data for Accuracy
Chlorthalidon and Metoprolol Succinate were
recorded in table no. 10 and 11.
LOD and LOQ: Discussion: The proposed method can detect
Chlorthalidon and Metoprolol Succinate at very low
level .So, it was concluded that the proposed
method is very sensitive in nature which shown in
table no. 12.
Robustness:
Discussion: The %RSD in both cases is less than
2.0. The study proves the reliability of test method
for minor changes in chromatographic condition
and which shown in table no. 13 and 14.
ACKNOWLEDGEMENT:
The author wishes to thanks mates who helped me
lot for my work. And how can I forget U.Srinivas,
my guide who suggests me in all way.
Table 5: Solvent suitability
Time Standard Response (mAU*S)
Chlorthalidon (5 µg/ml) Metoprolol Succinate (20µg/ml)
0 hrs 970.75 1469.45
6 hrs 968.56 1468.58
12 hrs 962.25 1463.56
18 hrs 960.88 1460.87
24 hrs 955.78 1458.25
Average 963.65 1464.14
SD 6.04 4.83
% RSD 0.62 0.33
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Table 6: Linearity data for Chlorthalidon and Metoprolol Succinate
Chlorthalidon Metoprolol Succinate
Concentration
(µg/ml)
Mean Peak Area [n=6]
(mAU*S) ± SD
Concentration
(µg/ml)
Mean Peak Area[n=6]
(mAU*S) ± SD
5 964.99 ± 11.05 20 1467.10 ± 8.31
7.5 1460.70± 7.86 30 2202.26± 6.19
10 1951.68± 8.97 40 2941.87± 9.82
12.5 2349.70± 6.94 50 3614.77 ± 9.81
15 2916.48 ± 5.08 60 4394.30 ± 6.50
Table 7: Repeatability data for Chlorthalidon and Metoprolol Succinate
Chlorthalidon Metoprolol Succinate
Concentration
( µg/ml)
Peak Area
(mAU*S)
Concentration
(µg/ml)
Peak Area
(mAU*S)
10 1947.09 40 2938.24
10 1950.12 40 2940.53
10 1966.69 40 2930.87
10 1950.84 40 2951.36
10 1960.47 40 2944.65
10 1938.74 40 2930.89
Mean 1952.32 Mean 2939.42
SD 9.91 SD 7.98
%RSD 0.50 %RSD 0.27
Table 8: Intraday precision data for estimation of Chlorthalidon and Metoprolol Succinate
Chlorthalidon
Concentration
(µg/ml)
Peak
Area(mAU*S)
±
S.D [n=3]
%RSD
Metoprolol
Succinate
Concentration
(µg/ml)
Peak
Area(mAU*S)
±
S.D [n=3]
%RSD
5 967.95 ± 7.94 0.82 20 1472.25±12.15 0.82
10 1947.78 ±18 .90 0.97 40 2934.75±31.10 1.05
15 2902.38±22.80 0.78 60 4379.17±34.06 0.77
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Table 9: Interday precision data for estimation of Chlorthalidon and Metoprolol Succinate
Chlorthalidon
Concentration
(µg/ml)
Peak Area
(mAU*S)* ±
S.D [n=3]
%RSD
Metoprolol
Succinate
Concentration
(µg/ml)
Peak Area
(mAU*S)*
±
S.D [n=3]
%RSD
5 968.94±12.12 1.25 20 1472.48±18.43 1.25
10 1950.38±18.90 0.96 40 2942.76±28.41 0.96
15 2919.46±27.22 0.93 60 4406.10±41.33 0.93
Table 10: Accuracy for Chlorthalidon
Sample
amount
amount
added
amount
recovered % recovery AVG SD %RSD
80% 10 8 7.93 99.22 99.69 0.49 0.49
80% 10 8 8.01 100.20
80% 10 8 7.97 99.66
100% 10 10 9.89 98.90 99.89 1.02 1.02
100% 10 10 10.09 100.93
100% 10 10 9.985 99.85
120% 10 12 11.88 99.07 99.75 0.90 0.90
120% 10 12 12.09 100.77
120% 10 12 11.92 99.40
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Table 11: Accuracy for Metoprolol Succinate
Sample
amount
amount
added
amount
recovered % recovery AVG SD %RSD
80% 40 32 33.83 105.74 105.70 1.074 1.01
80% 40 32 34.16 106.75
80% 40 32 33.47 104.60
100% 40 40 42.13 105.33 105.41 0.95 0.90
100% 40 40 41.80 104.50
100% 40 40 42.56 106.40
120% 40 48 51.29 106.86 106.73 0.53 0.50
120% 40 48 50.95 106.14
120% 40 48 51.45 107.19
Table 12: LOD & LOQ data for Chlorthalidon and Metoprolol Succinate
Parameters Chlorthalidon Metoprolol Succinate
Mean Slope (n=6) 191.67 92.41
SD (n=6) 12.97 11.11
LOD (μg/ml) 0.22 0.39
LOQ (μg/ml) 0.67 1.20
Table 13: Robustness for Chlorthalidon (10µg/ml)
Sr no. Flow rate
(+0.2 ml/min)
Flow rate
(- 0.2ml/min) M.P. +0.2 M.P. -0.2 pH +0.2 pH -0.2
1 1952.95 1914.28 1960.73 1958.78 1962.70 1958.77
2 1916.14 1964.57 1929.62 1927.68 1918.04 1925.81
3 1931.62 1939.371 1939.34 1947.12 1935.48 1947.12
avg.area 1933.57 1939.41 1943.23 1944.53 1938.74 1943.89
SD 18.48 25.14 15.91 15.71 22.50 16.71
%RSD 0.95 1.29 0.81 0.80 1.16 0.85
Table 14: Robustness for Metoprolol Succinate (40µg/ml)
Sr no. Flow rate
(+ 0.2ml/min)
Flow rate
(-0.2ml/min) M.P. +0.2 M.P. -0.2 pH +0.2 pH -0.2
1 2946.97 2888.63 2958.72 2955.78 2961.60 2955.67
2 2891.46 2964.46 2911.85 2908.93 2894.30 2905.98
3 2913.91 2922.08 2925.55 2937.10 2930.31 2930.83
avg.area 2917.45 2925.06 2932.04 2933.94 2928.74 2935.26
SD 27.92 38.00 24.09 23.58 33.67 26.00
%RSD 0.95 1.29 0.82 0.80 1.14 0.88
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