RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium and Ramipril from Capsule Dosage Form

Abstract

INTRODUCTION Atorvastatin [R-(R*, R*)]-2-(4-fluorophenyl)-β,∂ dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenyl amino) carbonyl]-1H-pyrrole-1-heptanoic acid calcium salt for trihydrate] is Anti-Lipidemic agent act by inhibiting HMG CoA reductase enzyme. [1] Ramipril [2S-[1[R*(R*)], 2α, 3αβ, 6αβ]]-1-[2[[1(Ethoxycarbonyl)-3-phenylpropyl] amino]-1-oxon propyl] octahydro cyclopenta [b] pyrrole-2-carboxylic acid.] is ACE inhibitor. [2-3] The combination of both drugs is used in treatment of Hypertension. Several HPLC [4-6] , HPLC [7] , Liquid Chromatography– electro Spray Ionization Tandem Mass Spectrometry [8] methods have been reported for atorvastatin alone or in combination with other drugs. [9-11] Only one UV spectroscopy i.e. derivative spectroscopy method has been reported for simultaneous estimation of atorvastatin (ATR) and ramipril (RAM). [12] The present method (Internal Standard RP-HPLC method) quantitates both drugs simultaneously without exipients interference in analysis from capsule dosage form [13] MATERIALS AND METHODS: Instrumentation: The separation of ATR and RAM was carried out on an isocratic JASCO RP-HPLC system using C18 column (150x4.6mm internal diameter, particle size 5µm).The pump used in this HPLC system was PU 2080 pump (Dual piston with gear driven pump). The 20 µl sample solutions were injected to chromatographic system using Rheodyne

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INTRODUCTION
Atorvastatin [R-(R*, R*)]-2-(4-fluorophenyl)-
β,∂ dihydroxy-5-(1-methylethyl)-3-phenyl-4-
[(phenyl amino) carbonyl]-1H-pyrrole-1-
heptanoic acid calcium salt for trihydrate] is
Anti-Lipidemic agent act by inhibiting HMG
CoA reductase enzyme.[1] Ramipril [2S-
[1[R*(R*)], 2α,
[2[[1(Ethoxycarbonyl)-3-phenylpropyl]
amino]-1-oxon propyl] octahydro cyclopenta
[b] pyrrole-2-carboxylic
inhibitor.[2-3] The combination of both drugs is
used in treatment of Hypertension. Several
HPLC[4-6] , HPLC[7], Liquid Chromatography–
electro Spray Ionization
Spectrometry[8] methods have been reported
for atorvastatin alone or in combination with
other drugs.[9-11] Only one UV spectroscopy i.e.
derivative spectroscopy method has been
reported for simultaneous estimation of
atorvastatin (ATR) and ramipril (RAM).[12] The
present method (Internal Standard RP-HPLC
method) quantitates
simultaneously without exipients interference
in analysis from capsule dosage form [13]
MATERIALS AND METHODS:
Instrumentation:
The separation of ATR and RAM was carried
out on an isocratic JASCO RP-HPLC system
using C18
column (150x4.6mm
diameter, particle size 5µm).The pump used in
this HPLC system was PU 2080 pump (Dual
piston with gear driven pump). The 20 µl
sample solutions
chromatographic system using Rheodyne
3αβ, 6αβ]]-1-
acid.] is ACE
Tandem Mass
both drugs
internal
were injected to

1 Department of Pharmaceutical Chemistry,
Tatyasaheb Kore College
Warananagar-416113(M.S.) India.
swapnil.mpharm@gmail.com
2 Department of Pharmaceutical Chemistry,
Bharati Vidyapeeth College
Kolhapur-416013(M.S.) India.
of Pharmacy,
Of Pharmacy,
Injector. The UV detector used in this HPLC
system was Czerny
monochromater with deuterium lamp as light
source. The chromatographic
integrated data were recorded using Hercule
2000 (interface) computer system. Data
processing was carried out using Borwin®
Version 1.5software.
Reagents and Chemicals:
The solvents used were of HPLC/AR grade.
Pure drug samples of ATR, RAM and Valsartan
(VAL) are obtained as gift samples from Cipla
India Pvt. Ltd. Kurkumbh, Mumbai. Marketed
formulation ATOCORR(Company Name) was
procured from the local market.
Chromatographic Conditions:
The analysis was carried out by HPLC using
Acetonitrile: 0.02 M Potassium dihydrogen
phosphate (pH 3.2) [75:25 %V/V] as a mobile
phase and HIQ SII C18 column -10 (4.5 mm x
250 mm) as a stationary phase at a flow rate of
1ml /minute in an isocratic elution mode.
Before delivering the mobile phase in to the
system, it was degassed and filtered through
0.20µm syringe filter. The injection volume
was 20 µl and the detection was performed at
217 nm.
Standard Stock Solution:
About 20 mg of each of reference standard of
ATR, RAM and VAL (Internal Standard) was
weighed accurately and transferred to three
separate 100 ml volumetric flasks. Both drugs
were dissolved in 50 ml of mobile phase with
shaking and volume was made up to the mark
with mobile phase to get 200 μg/ml of
standard stock solution of each drug. These
stock solutions were then sonicated and
filtered through 0.20μ syringe filter.
Calibration Curves:
For each drug, appropriate aliquots were
pipetted out from each standard stock solution
into a series of 10 ml volumetric flasks. The
volume was made up to mark with mobile
turners mount
and the
phase to get set of solutions having
concentrations 10-90 μg/ml , 20-180 μg/ml
and 50 μg/ml in each flask for RAM, ATR and
VAL respectively. Triplicate dilutions of each
concentration of each drug were prepared
separately. From these triplicate solutions, 20
μl injections of each concentration of each
drug were injected into the RP-HPLC system
separately and chromatographed under the
conditions as described above. Evaluation of
both drugs was performed with UV detector at
217 nm. Peak areas were recorded for all the
peaks. Retention factors were calculated and
plotted against the concentrations to obtain
the standard calibration curves (Figure 1).
Analysis of capsule Formulation:
Twenty capsules of ATOCORR were weighed
and powder separated from capsule shells.
From the content of 20 capsules, an amount
equivalent to 20 mg of ATR and 10 mg of RAM
was weighed and dissolved in 50 ml of mobile
phase and sonicated for 20 minutes. The
solution was filtered through 0.2 μm Nylon 6,
6(N66) membrane filter and then final volume
of the solution was made up to 100 ml with
acetonitrile to get stock solution containing
200 μg ml -1 of ATR and RAM 100 μg ml -1 in
100 ml volumetric flask. After appropriate
dilutions, the solutions were run on HPLC
system and the concentration of each analyte
was determined using calibration curve
data.[14] The statistical data obtained after
replicate determinations (n = 6) is shown in
Table 1 and chromatogram is shown in Figure
2.
Validation of HPLC Method:
The proposed RP-HPLC method was validated
as per ICH guidelines.[15]
Specificity
The specificity of the RP-HPLC method was
determined by comparison
chromatogram of mixed standards and sample
solutions. The parameters like retention time,
resolution and tailing factor were calculated.
Good correlation was found between the
results of mixed standards and sample
solutions.
Precision
Precision study was performed to find out
intra-day and inter-day variations. The
%relative standard deviation (RSD) for intra-
day precision was 0.993 % for ATR and 0.877
% for RAM and for inter-day precision was
1.025% for ATR and 0.743% for RAM
respectively which is less than 2% indicating
high degree of precision.
Accuracy (Recovery studies)
Recovery studies were performed by standard
addition method at three levels i.e., 80%,
100% and 120%. Known amounts of standard
ATR and RAM were added to preanalyzed
samples and they were subjected to proposed
HPLC. Results of recovery studies are shown in
Table 1.

of the
RP-HPLC Method for Simultaneous Estimation of
Atorvastatin Calcium and Ramipril from Capsule
Dosage Form.
Jadhav S. D.1*, Thamake S. L.2
Abstract: A reverse phase high performance liquid chromatography (RP-HPLC) method for the
simultaneous estimation of atorvastatin and ramipril in marketed formulation is developed. The
determination was carried out on a HIQ SII C18 column -10 (4.5 mm x 250 mm).column using a
mobile phase of acetonitrile: 0.02 M Potassium dihydrogen phosphate (pH 3.2) [75: 25%v/v].
The flow rate was 1 ml/min with detection at 217 nm. The Valsartan is used as internal standard
in this method. The retention time for atorvastatin was 6.1 min, for ramipril 3.43 min.
Atorvastatin and ramipril showed a linear response in the concentration range of 20-180 μg/ml
and 10-90 μg/ml respectively. The correlation co-efficient (‘r’ value) for atorvastatin and ramipril
was 0.9999 for both drugs. The results of analysis have been validated statistically and by
recovery studies. The percentage recoveries obtained for atorvastatin and ramipril ranges from
99.04 to 101.15 %.

Keywords: Atorvastatin, Ramipril, RP-HPLC.
Pishawikar S. A.2

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Table 1: Results of Marketed Formulation Analysis.
Drug Label claim (mg/capsule) Estimated % of Label claim ±S.D.*
% Recovery ± S.D.*
80 100 120
ATR 10 99.73±0.568 100.13±0.853 99.47±0.752 100.90±0.905
RAM 5 99.97±1.069 99.13±1.053 100.07±0.619 99.23±1.072
*Average of six determinations; S.D., standard deviation.

Table 2: System Suitability Parameters.
Sr. No. Parameters ATR RAM
1. Theoretical Plates 4813 4781
2. Tailing Factor 1.21 1.17
3 Separation Factor 1.78 0.00
4. Resolution 10.90 0.00
5. Limit of Detection (µg ml -1) 0.1712 0.2423
6. Limit of Quantitation (µg ml-1) 0.5189 0.7342



Figure1: Overlain Chromatogram of Mixed Standards.



Figure 2: Chromatogram of Marketed Formulation Analysis

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Limit of detection (LOD) and limit of
quantitation (LOQ)
The LOD and LOQ were separately determined
based on the calibration curves. The standard
deviation of the y-intercepts and slope of the
regression lines were used. Results of LOD and
LOQ are given in Table 2.
Robustness
The robustness study was done by making
small changes in the optimized method
parameters like ±0.1 change in pH ±1% change
in mobile phase ratio and column temperature.
There was no significant impact on the
retention time and tailing factor.
Ruggedness
The ruggedness study was done by the two
analysts. The %RSD for analyst-I was 0.8526%
for ATR and 0.6390% for RAM and for analyst-
II was 0.5941% for ATR and 0.6829 % for RAM
respectively.
System Suitability Parameters
System suitability parameters were analyzed
on freshly prepared standard stock solutions
of ATR and RAM. Both the drugs were injected
into the chromatographic system under the
optimized chromatographic
Parameters that were studied to evaluate the
suitability of the system were number of
theoretical plates, tailing factor, resolution,
separation factor etc.[16]

RESULT AND DISCUSSION
The proposed method describes a RP-HPLC
procedure employing a C18 column and a
mobile phase water and acetonitrile in the
ratio 25:75 (pH 3.2). The two drug solutions
having concentration of 10 µg ml-1 were
scanned in range of 200 nm to 400 nm on a
UV-Visible spectrophotometer for selection of
sampling wavelength. After recording the
spectra of the two drugs, 217 nm was selected
as suitable wavelength for estimation. The best
resolution of the two drugs and internal
standard eluting within 7.5 minutes was
achieved with a flow rate of 1 ml min-1.
The linearity response of the HPLC
system for ATR and RAM were obtained over
the range of 20-180 µg ml-1 and 10-90 µg ml-1
respectively. Accuracy of the method was
checked by adding known amounts of pure


conditions.
drug to each known concentration of the
capsule formulations. The %RSD for the tablet
analysis and recovery studies was less than
2% indicating high degree of accuracy. The
other validation parameters have proved
accuracy, precision, sensitivity and selectivity
of the proposed method as number of
theoretical plates, tailing factor, resolution,
separation factor, results of analysis and
statistical parameters were found be within
limits.

CONCLUSION
The statistical data
developed HPLC method for simultaneous
estimation of ATR and RAM was found to be
more accurate, precise, sensitive and selective.
Therefore the proposed method could be
applied for routine analysis in quality control
laboratories for both in bulk and multi-
component formulations.

REFERENCES AND NOTES
1. Budawari S, The Merck Index, 23rd Edn, Merck
and Co. Inc.; Whitehouse Station, NJ, 1996, 893;
8280.
2. Ministry of Health and Family Welfare;
Government of India, Indian Pharmacopoeia, 5th
Edn, Vol. II, The Controller of Publication, New
Delhi, 2007, 749.
3. Florey K, Analytical Profiles of Drug Substances,
Vol-10, Elsevier India Private Limited, New
Delhi, 2005, 405-461.
4. Mishra P, Gupta A and Shah K, Simultaneous
Estimation of Atorvastatin
Amlodipine Besylate from Tablets, Indian
Journal of Pharmaceutical Sciences, 69(6),
2007,831-833.
5. Jain N, Raghuwanshi R and Jain D, Development
and Validation of
Simultaneous Estimation
Calcium and Fenofibrate in Tablet Dosage
Forms, Indian Journal of Pharmaceutical
Sciences, 70(2), 2008, 263-265.
6. Maheshwari K R, Sankar G G and Rao A L, HPLC
Method for the Simultaneous Determination of
Atorvastatin and Amlodipine in Tablet Dosage
Forms, Indian Journal of Pharmaceutical
Sciences, 68(2), 2006, 275-277.
7. Patel N M, et al., Development and Validation of
HPTLC Method for the Simultaneous Estimation
of Atorvastatin Calcium and Ezetimibe, Indian
have proven that
Calcium and
RP-HPLC Method
Atorvastatin
for
of
Journal of Pharmaceutical Sciences, 68, 2006,
793-796.
8. Miao X S and Metcalfe C D, Determination of
Cholesterol-lowering Statin Drugs in Aqueous
Samples Using Liquid Chromatography–electro
Spray Ionization Tandem Mass Spectrometry,
Journal of Chromatography A, 998, 2003, 133–
141.
9. Bonazzi D and Cavrini V, Analysis of ACE
inhibitors in Pharmaceutical Dosage Forms by
Derivative UV Spectroscopy
Chromatography (HPLC),
Pharmaceutical and Biomedical Analysis, 16,
1997, 431-438.
10. Mohammadia A and Rezanour N, A stability-
indicating high
chromatographic (HPLC)
simultaneous determination of atorvastatin and
amlodipine in commercial tablets, Journal of
Chromatography B, 846, 2007, 215–221.
11. Zaheer Z and Farooqui M N, Stability-indicating
High Performance Liquid Chromatographic
Determination of Atorvastatin Calcium in
Pharmaceutical Dosage Form, African Journal of
Pharmacy and Pharmacology, 2, 2008, 204-210.
12. Thamake S L, Jadhav S D and Pishawikar S A,
Development and Validation of Method for
Simultaneous Estimation
Calcium and Ramipril from Capsule Dosage form
by First Order Derivative Spectroscopy Asian
Journal of Research in Chemistry, 2 (1), 2009,
52-53.
13. Chitlange S S, Bagri K, Stability Indicating RP-
HPLC Method for Simultaneous Estimation of
Valsartan and Amlodipine
Formulation, Asian Journal of Research in
Chemistry, 1, 2008, 15-18.
14. Shethi P D, Quantitative Analysis of Drugs in
Pharmaceutical Formulations, 3rd Edn, CBS
Publishers and Distributors, New Delhi, 1997,
17.
15. ICH Q2B, Text on Validation of Analytical
Procedures, Geneva, 1994, 1–5.
16. Snyder L R, Kirkland J J and Glajch J L, Practical
HPLC method Development, 2nd Edn, A Wiley-
Interscience Publication, 1997, 250-747.

ACKNOWLEDGEMENT: The authors are very
thankful to Cipla India Pvt. Ltd. for providing gift
sample of Atorvastatin Calcium and Ramipril.
The authors are also thankful to Tatyasaheb
Kore College of Pharmacy, Warananagar and
Bharati Vidyapeeth
Morewadi, Kolhapur for provision of facilities for
this research work.
and
Journal
Liquid
of
performance
assay
liquid
the for
of Atorvastatin
in Capsule
College of Pharmacy,