SIMULTANEOUS ESTIMATION OF CLOPIDOGREL BISULPHATE AND
ATORVASTATIN CALCIUM IN CAPSULE FORMULATION BY
ABSORBANCE RATIO METHOD
Neeharika.M*, K. Shantha kumari2, S.A .Rahaman3, Maheswari.G4, Revathi.S5
Department of pharmaceutical Analysis, Nirmala College of pharmaceutical sciences,
Atmakuru (vill), Mangalagiri (Mndl)-522503, Andhra Pradesh.
E-mail: neeharika.mj@gmail.com
ABSTRACT:
A simple UV absorbance ratio method (Q-method) has been developed for the
simultaneous analysis of Clopidogrel bisulphate & Atorvastatin Ca in a capsule dosage
form using methanol as solvent. The two wavelengths selected for the analysis are 234nm
(iso-absorptive point) & 245 nm (absorption maxima of Atorvastatin Ca). Clopidogrel
bisulphate (CLO) & Atorvastatin Ca (ATR) obeyed Beer‐Lambert’s law in the
concentration range of 10-50μg/ml and 2-10μg/ml with coefficient of correlation 0.999
and 0.999 at 234 nm respectively. This proposed method was statistically validated in
accordance with ICH guidelines. This method was found to be accurate, precise as
indicated by the values (<2%) of % RSD hence it can be successfully be applied for the
routine analysis of combined dosage form.
Keywords: Clopidogrel bisulphate, Atorvastatin calcium, Q- Absorbance ratio method.
Introduction:
Clopidogrel bisulfate is a thienopyridine class inhibitor of P2Y12-ADP platelet receptors
used to inhibit blood clots in coronary artery disease, peripheral vascular disease,
and cerebrovascular disease. Chemically it is methyl (+)-(S)-α-(2-chlorophenyl)-6, 7dihydrothieno [3, 2-c] pyridine-5(4H) acetate sulfate. Atorvastatin Ca is a synthetic lipidlowering agent. Atorvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to
mevalonate, an early and rate-limiting step in cholesterol biosynthesis. Atorvastatin
calcium is [R-(R*, R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4[(phenylamino)carbonyl]-1Hpyrrole- 1-heptanoic acid, calcium salt (2:1) trihydrate .This
combination of CLO &ATR is prescribed in treatment of cardiovascular disease like
atherosclerosis. Literature survey revealed that many methods are available for
estimations of these drugs singly or in combination with aspirin [1] or with other drugs [2, 3]
or with the related substances
[4]
but only few methods
[5, 6, 7, 8]
were found for the
combination of CLO & ATR alone .This paper presents a simple, precise and accurate
method for the estimation of the two components in a capsule dosage form.
Clopidogrel bisulphate
.H2SO4
Atorvastatin Ca
The objective of this work was to develop efficient UV spectroscopic method for
simultaneous determination of Clopidogrel bisulphate (CLO) & Atorvastatin calcium
(ATR) in a capsule and validate as per the guidelines of International Conference on
Harmonization.
MATERIALS AND METHOD
Instrument
A Single beam UV/Visible spectrophotometer (Thermo Scientific Aquamate plus) was
used to measure absorbances of solutions. An Electronic analytical balance (Shimadzu)
and an ultrasonic bath sonicator (Cyber labs) were used in the study.
Reagents and chemicals
Analytical pure drugs of CLO and ATR were obtained as gift samples from
Dr.Reddy’s laboratories, Hyderabad, India. The combined tablet formulation (ATORFITCV) with a label claim of CLO 75mg & ATR 10mg respectively, were obtained from
local drug store. Methanol of analytical grade purchased from Merck, Mumbai.
Preparation of Standard Stock Solutions
Clopidogrel bisulphate:
An accurately weighed quantity of Clopidogrel bisulphate (25 mg) was dissolved in
few ml of Methanol in 25 mL volumetric flask and volume was made up to the mark
using Methanol to get final concentration (1000 µg/ mL).From this 10 ml was pipetted to
100ml vol.flask and volume was made up to the mark with methanol to get the
concentration of 100 µg/ mL
Atorvastatin Ca:
An accurately weighed Atorvastatin Ca (10 mg) was dissolved in few ml of Methanol
in 10 mL vol. flask and volume was made up to the mark using Methanol to get final
concentration (1000µg/ ml).From this 2ml was pipetted to a 100ml vol.flask and volume
was made up to the mark with the solvent to get concentration of 20 µg/ mL
Study of Spectra and Selection of Wavelength:
The aliquot portions of standard stock solutions of CLO and ATR were diluted
appropriately with Methanol to obtain concentration 15 µg/ mL of both drugs. The
solutions of both drugs were scanned separately in the range of 200 – 400 nm. The
overlain UV absorbance spectrum of CLO and ATR is shown in Fig. 3. From the overlain
spectrum the wavelengths selected for estimation of drugs were 234 nm as iso absorptive
point and 245nm as λ max of ATR.
Overlain spectra of CLO & ATR
Preparation of sample solution
Twenty capsules containing 75mg CLO and 10mg ATR were weighed and average
weight was calculated. The contents of capsules were crushed and powdered in glass
mortar. Quantity of powder equivalent one capsule was transferred to 50mL volumetric
flask, dissolved in sufficient quantity of methanol, sonicated up to 10min and volume was
adjusted up to mark using methanol. From this 1mL was transferred to 10ml vol.flask to
get concentrations of 150µg/ml CLO & 20 µg/ml ATR.
Analysis of tablet dosage form
1.5ml of above sample stock solution was diluted with methanol to 10ml and the
absorbance was measured at the selected wavelengths i.e., 234 & 245nm and the
concentrations of the two drugs were estimated using the following equations.
Absorbance-ratio method (Q-method)
Q-method uses the ratio of absorbances at two selected wavelengths, one at
isoabsorptive point and other being the λ max of one of the two drugs. CLO and ATR
have λ max at 220 nm and 245nm respectively and isoabsorptive point is at 234 nm. The
wavelengths selected for analysis were 234 and 245nm. Absorptivity values of CLO and
ATR were determined at 234 and 245nm. The concentration of each drug in the sample
containing mixture of both was calculated by using following equations:
𝐂𝐗 =
QM − QY A1
×
QX − QY ax1
Where;
𝐐𝐌 =
𝐂𝐘 =
A2
A1
𝐐𝐗 =
QM − QX A1
×
QY − QX ay1
ax2
ax1
𝐐𝐘 =
ay2
ay1
A1- Absorbance of sample at 234nm
A2- Absorbance of sample at 245nm
aX1 and aX2 are absorptivities of CLO at 234nm and 245nm respectively.
aY1 and aY2 are absorptivities of ATR at 234 nm and 245nm respectively.
CX and CY are concentrations of CLO and ATR respectively.
Absorptivity values of Clopidogrel bisulphate and Atorvastatin Ca
Absorptivity of CLO
Absorptivity ATR
at 234nm
at 245 nm
at 234 nm
at 245 nm
ax1
ax2
aY1
aY2
0.0335
0.0182
0.0335
0.077
Absorbance values & ratios
Variable
A1
A2
QM
QX
QY
Value
0.855
0.642
0.752
0.55
2.29
Assay results
Drug
Label claim(mg)
Amount present (mg)
% Assay
CLO
75
75.03
100.04
ATR
10
9.866
98.6
METHOD VALIDATION
Linearity
A Series of solutions were prepared using CLO and ATR standard stock solution at
concentration levels from 10-50µg/mL and 2-10 µg/mL respectively. The absorbances of
these solutions were measured at both 234 and 245 nm against methanol as blank. The
calibration curves were constructed by plotting concentrations on X-axis and absorbance
on Y-axis. Also; mixed standard solutions were prepared by taking 10ml volumetric flask
and pipetting out the required aliquots from the standard stock solutions of CLO & ATR
into the same flask (in the linearity concentrations of the drugs). A solution containing
mixture of both drugs in the same concentration as that of sample solution was also
prepared. The absorbance of all these solutions was measured at 234 and 245nm
respectively.
Acceptance criteria
Correlation Coefficient should be not less than 0.998.
Linearity of Clopidogrel bisulphate and Atorvastatin Ca
Conc. of CLO
(μg/mL)
Abs(234nm)
Conc. of ATR
Abs(234nm)
(μg/mL)
10
0.354
2
0.206
20
0.561
4
0.281
30
0.786
6
0.365
40
0.981
8
0.446
50
1.204
10
0.53
R2=0.999
R2=0.999
Calibration curve of Clopidogrel bisulphate (234nm)
1.4
Absorbance
1.2
1
y = 0.0212x + 0.1412
R² = 0.9996
0.8
0.6
0.4
0.2
0
0
20
40
60
Concentration(µg/mL)
Calibration curve of AtorvastatinCa(234nm)
0.6
y = 0.040x + 0.121
R² = 0.999
Aborbanc
e
0.5
0.4
0.3
0.2
0.1
0
0
2
4
6
8
Concentration(µg/mL)
PRECISION
10
12
Precision was studied by preparing six replicates of mixed standard solution
containing 20µg/mL CLO & 4µg/mL ATR & determining their absorbances at 234
&245nm.
Acceptance criteria
The % relative standard deviation should be not more than 2.0%.
Results of precision studies
ACCURACY
S.No.
Abs at 234nm
Abs at 245nm
1
0.534
0.733
2
0.533
0.732
3
0.533
0.734
4
0.535
0.733
5
0.534
0.732
6
0.532
0.733
Mean
0.5335
0.7328
S.D
0.00104
0.00075
%RSD
0.19
0.10
The accuracy of the developed method was determined by recovery studies. Recovery
studies were carried out at three different levels (50%, 100% and 150% of target
concentration). The preanalysed samples were spiked with CLO & ATR stock solutions.
The mixtures were analyzed and the recoveries were determined. The study was carried
out in triplicate.
Accuracy data of Clopidogrel bisulphate
S. no
1
2
3
Amount
Amount
%
Statistical
(mg)
(mg)
Recovery
analysis
added
Found
50 %
11.25
11.1
98.66
50 %
11.25
11.15
99.11
50 %
11.25
11.12
98.84
%RSD 0.22
100 %
22.5
22.3
99.11
Mean 98.63
100%
22.5
22.18
98.57
100%
22.5
22.1
98.22
%RSD
150 %
33.75
33.23
98.51
Mean 98.25
150 %
33.75
33.16
98.25
150 %
33.75
33.08
98.01
Spike
Level
Mean 98.87
SD
SD
SD
0.226
0.448
0.45
0.25
%RSD 0.25
Accuracy data of Atorvastatin Calcium
S.no
1
2
3
Amount
Amount
%
Statistical
(mg)
(mg)
Recovery
analysis
added
Found
50 %
1.5
1.48
98.6
50 %
1.5
1.47
98
50 %
1.5
1.47
98
100 %
3
2.96
98.9
Mean
98.41
100%
3
2.95
98.33
SD
0.454
100%
3
2.94
98
150 %
4.5
4.42
98.2
Mean
98.06
150 %
4.5
4.41
98.1
SD
0.152
150 %
4.5
4.40
97.9
Spike
Level
Mean 98.2
SD
0.346
%RSD 0.35
%RSD
0.46
%RSD 0.15
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