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African Journal of Pharmacy and Pharmacology Vol. 5(2), pp. 244-251, February 2011
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Rapid and simultaneous determination of aspirin and
dipyridamole in pharmaceutical formulations by
reversed-phase high performance liquid
chromatography (RP-HPLC) method
K. Prakash1, Rama Rao Kalakuntla2,3* and Jayapal Reddy Sama4
1SN Vanitha Mahavidyalaya Pharmacy College for Women, Hyderabad, Andhra Pradesh, India.
2Sree Dattha Institute of Pharmacy, Sheriguda, Hyderabad, Andhra Pradesh, India.
3Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.
4Department of Animal Sciences, School of Life Sciences, University of Hyderabad, India.
Accepted 08 February, 2011
The combination of Dipyridamole and Aspirin and is widely used to reduce thrombosis in patients with
thrombotic diseases. A rapid, simple, precise and cost effective and reversed-phase high performance
liquid chromatography (RP-HPLC) method has been developed and validated for the simultaneous
determination of Aspirin and Dipyridamole in pharmaceutical formulations. Separation of both Aspirin
and Dipyridamole was achieved within 5 min with required resolution, accuracy and precision thus
enabling the utility of the method for routine analysis. Chromatographic separation was achieved on a
waters symmetry C18 3.5 µm, 50 x 4.6 mm using a mobile phase consisting of 0.1% ortho phosphoric
acid and acetonitrile in the ratio of 75:25 at a flow rate of 1.0 ml per minute. The detection was made at
227 nm and the retention time of Aspirin and Dipyridamole were1.5 and 2.8 minutes respectively. The
method was found linear over the range of 4 to 80 µg/ml for Dipyridamole and 0.5 to 10 µg/ml for
Aspirin.
Key words: Aspirin, Dipyridamole, high performance liquid chromatography.
INTRODUCTION
Aspirin (ASP) is 2- (Acetyloxy) benzoic acid, and is cyclo
oxygenase inhibitor which is best known as an anti-
platelet drug (Patel et al., 2010) and is one of the major
antithrombogenic agent widely used for the treatment and
prevention of cerebro and cardiovascular conditions such
as stroke (Purushotam et al., 2009). Dipyridamole is a
platelet inhibitor chemically described as 2,2',2'',2'''-[(4,8-
Dipiperidinopyrimido [5,4-d]pyrimidine-2,6-diyl)dinitrilo]-
tetraethanol. Dipyridamole is widely used as a coronary
vasodilator in patients with high blood pressure, a
prophylactic agent in patients with angina pectoris and an
inhibitor of platelet aggregation in various thromboembotic
*Corresponding author. E-mail: ramarao_2k2@rediffmail.com,
pkatakam9@rediffmail.com. Tel: +91-9642518806.
conditions (Davood et al., 1999).
The combination of Dipyridamole and Aspirin and is
widely used to reduce thrombosis in patients with
thrombotic diseases. This antithrombotic action results
from additive antiplatelet effects of both drugs. Aspirin
inhibits platelet aggregation by irreversible inhibition of
platelet cyclooxygenase and thus inhibiting the
generation of Thromboxane A2. Dipyridamole inhibits the
uptake of adenosine into platelets and endothelial cells,
thus decreasing the adhesion of platelets to
thrombogenic surfaces (Hassan et al., 2008).
Analytical methods based on high performance liquid
chromatography (HPLC), HPTLC, LC-MS (Kachhadia et
al., 2008; Vora et al., 2008; Wada et al., 2007; William et
al., 1983; Rajput et al., 2008; Mishra et al., 2006) and
other methods were reported earlier for the determination
of Aspirin individually and in combination with other drugs.
A few analytical procedures were also proposed for the
determination of Dipyridamole in dosage forms in human
plasma, serum, urine and feces (Zhang et al., 1997; Qin
et al., 2010; Murillo Pulgarín et al., 1997).
Although the combinational use of Aspirin and
Dipyridamole is continuously increasing, few methods
were reported for the simultaneous determination of
Aspirin and Dipyridamole using combination of liquid
chromatographic and mass spectrometric detection
(Wang et al., 2008), second-order derivative spectro-
phometry (Periasamy Umapathi, 1994) and by
spectrofluorimetric method (Hassan et al., 2008). Simple
and sensitive HPLC method for the estimation of Aspirin
and Dipyridamole was seldom reported. The objective of
the present work was to develop and validate simple,
robust, sensitive, reproducible and cost effective
analytical method for the simultaneous determination of
Aspirin and Dipyridamole in pharmaceutical dosage
forms. We describe herein a simple, sensitive and
validated HPLC method utilizing isocratic mobile phase
with short retention time for the simultaneous deter-
mination of these two components in pharmaceutical
formulations. The developed method can be successfully
applied to routine quality control and other analytical
purposes.
MATERIALS AND METHODS
Chemicals and reagents
Aspirin and Dipyridamole working standards were procured from
Cipla Labs, and the tested pharmaceutical formulations (Aspirin (25
mg) and Dipyridamole (200 mg) tablets) were procured from
commercial pharmacy. Ortho phosphoric acid, acetonitrile,
methanol and other reagents were of suitable analytical grade.
Apparatus and chromatographic conditions
HPLC analysis was performed on waters HPLC system equipped
with a 2696 separation module and 2996 Photo Diode Array
Detector. Separations were carried on a waters symmetry C18 3.5
µm, 50 x 4.6 mm using isocratic elution. The flow rate was 1.0 ml
min-1. Detection was performed at 227 nm. Injection volume was 10
µl. Peak identities were confirmed by retention time comparison and
the procedures and instrument operation was performed at room
temperature.
Preparation of mobile phase
The mobile phase is composed of a mixture of 0.1% ortho
phosphoric acid and acetonitrile in the ratio of 75:25 (v/v)., filtered
through a 0.45 m nylon filter (Millipore, USA) and degassed by
sonication prior to use.
Preparation of standard solution
The standard stock solution of Aspirin (0.25 mg/ml) and
Dipyridamole (1 mg/ml) was prepared in methanol since both drugs
are soluble in this solvent. The working standard solutions of
Prakash et al. 245
Aspirin (5 µg/ml) and Dipyridamole (40 µg/ml) was prepared by
diluting the stock solution in mobile phase solution.
Preparation of sample solution
Twenty tablets were weighed to get the average weight and the
tablets were grounded and made into powdered form. From the
powdered form amount of powder equivalent to 25 mg of Aspirin
and 200 mg of Dipyridamole was transferred to a 500 ml volumetric
flask and added 150 ml of methanol and kept on rotary shaker for
15 min at 200 RPM and added 200 ml of methanol and sonicated
for 30 min with intermediate shaking. Finally, the volume was made
up with methanol to obtain a solution containing 0.05 mg/ ml Aspirin
and 0.4 mg/ ml Dipyridamole. An aliquot was then removed and
centrifuged at 5000 rpm for 10 min and the centrifuged solution was
filtered using 0.45 µm membrane filter paper. After filtration, the
solutions were diluted with mobile phase to get the final
concentration of Aspirin (5 µg/ml) and Dipyridamole (40 µg/ml).
RESULTS AND DISCUSSION
Method development
Drug quality control, stability, metabolism and pharmaco-
kinetics studies including the toxicity studies necessitate
the determination of drugs in pharmaceutical formulations
and biological samples. Correspondingly efficient and
validated analytical methods are very critical require-
ments for all these investigations. Chromatographic
parameters were preliminary optimized to develop a LC
method for simultaneous determination of Dipyridamole
and Aspirin with short run time (<5 min), and acceptable
resolution (Rs > 2). The polarity of Dipyridamole and
Aspirin differ greatly as Aspirin is more lipophilic than
Dipyridamole. The sample retention increases with
increased column length so a shorter column (50 x 4.6
mm) was selected to have a shortest possible runtime
without compromising on the resolution. Separation of
Aspirin and Dipyridamole were achieved on waters
symmetry C18 3.5 µm, 50 x 4.6 mm column using
isocratic flow. The column was chosen as both the
analytes were separated with acceptable resolution and
with in short run time. Isocratic elution was chosen as the
required resolution was achieved and hence the complex
gradient elution program was not used. Lower particle
size (3.5 µm) column was chosen to increase the
resolution between the Dipyridamole and Aspirin.
In order to identify a suitable organic modifier along
with 0.1% ortho phosphoric acid, various compositions of
acetonitrile and methanol were tested. Methanol
produced a higher retention time for Dipyridamole and
higher column pressures due to the high viscosity.
Acetonitrile was found to display advantageous
separations. Change of percentage of acetonitrile in the
mobile phase provided great influence on retention time
of the two drugs. When the acetonitrile content was lower
than 20%, retention time of Dipyridamole increased
rapidly and when the acetonitrile content was higher than
30%, resolution between Dipyridamole and Aspirin was
246 Afr. J. Pharm. Pharmacol.
Figure 1. Chromatogram of placebo.
reduced. Finally the separation with acceptable resolution
was achieved with the mobile phase consisting of 0.1%
ortho phosphoric acid and acetonitrile in the ratio of
75:25. Effects of the mobile phase pH on retention of the
both drugs were investigated at pH values of 3, 4, 5, 6,
and 7, respectively. It was found that the mobile phase
pH has no effect on the retention of Aspirin and
Dipyridamole. Operating wavelength of 227 nm was
selected based on the absorbance maxima in the
diluents. Flow rate of 1.0 ml per minute was optimized to
yield the shorter retention time with required resolution
and intensity. Lesser flow rates resulted in increased
retention times and higher flow rates of more than 1.0 ml
per minute not provided the required resolution and
intensity.
Finally separation for simultaneous determination of
Dipyridamole and Aspirin was carried out by isocratic
elution using 25% acetonitrile with a flow rate of 1.0 ml
per minute. With the above chromatographic conditions,
retention time Aspirin and Dipyridamole were 1.5 and 2.8
min, respectively.
The above described method is suitable for routine
pharmaceutical applications involving the analysis of
Aspirin and Dipyridamole. The retention time of each
analyte was very reproducible with relative standard
deviations between 0.03 and 0.04% (n = 6) for
Dipyridamole and Aspirin respectively. The peak area
responses were also reproducible with relative standard
deviations of 0.6 and 0.5% (n = 6) for Dipyridamole and
Aspirin respectively.
Method validation
The above method was validated according to ICH and
USP guidelines to establish the performance character-
ristics of a method (expressed in terms of analytical
parameters) to meet the requirements for the intended
application of the method for routine use.
System suitability
In order to determine the adequate resolution and
reproducibility of the proposed methodology, suitability
parameters including retention time, resolution, tailing
factor, %RSD of retention time and peak areas were
investigated and the results were found within the
acceptable specifications.
Specificity
The specificity of an analytical method may be defined as
the ability to unequivocally determine the analyte in the
presence of additional components such as impurities,
degradation products etc. Specificity was evaluated by
preparing the analytical placebo and it was confirmed that
the signal measured was caused only by the analytes. A
solution of analytical placebo (containing all the tablet
excipients except Dipyridamole and Aspirin was prepared
according to the sample preparation procedure and
injected. To identify the interference by these excipients,
a mixture of inactive ingredients (placebo), standard
solutions, and the commercial pharmaceutical pre-
parations including Dipyridamole and Aspirin were
analyzed by the developed method. The representative
chromatograms as shown in Figures 1, 2 and 3 did not
show any other peaks, which confirmed the specificity of
the method. Peak purity of Aspirin and Dipyridamole were
Prakash et al. 247
Figure 2. Chromatogram of standard.
Figure 3. Chromatogram of sample.
also evaluated for confirming the purity of the individual
peaks. The peak purity evaluation profiles indicate that
there were no interference from blank components. The
peak purity profiles of Aspirin and Dipyridamole were
shown in Figures 4 and 5, respectively.
Linearity
The linearity of an analytical method is its ability (within a
given range) to obtain test results which are directly
proportional to the concentration (amount) of analyte in
248 Afr. J. Pharm. Pharmacol.
Figure 4. Peak purity plot of asprin.
Figure 5. Peak purity plot of dipyridamole.
Prakash et al. 249
Figure 6. Linearity graph of aspirin.
Linearity graph
Linear (Linearity graph
)
Figure 7. Linearity graph of dipyridamole.
the sample. Linearity of detector response for
Aspirin/Dipyridamole was established by analyzing serial
dilutions of a stock solution of the working standard.
Ten concentrations ranging from 10 to 200% of the test
concentrations were prepared and analyzed. The final
concentration of each solution in µg/ml was plotted
against peak area response. The method was found
linear over the range of 4 to 80 µg/ml for Dipyridamole
and 0.5 to 10 µg/ml for Aspirin. Correlation coefficient (R)
was found to be greater than 0.999 for both Aspirin and
Dipyridamole. The linear graphs of Aspirin and
Dipyridamole were shown in Figures 6 and 7,
respectively.
Precision
The precision of an analytical procedure expresses the
closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple sampling
of the same homogeneous sample under the prescribed
conditions. Six replicate samples were prepared and
analyzed as per the sample preparation procedure and
the assay values were calculated.
The precision (n = 6) of the assay values for Aspirin
and Dipyridamole was found to be 0.5 and 0.4% for
Aspirin and Dipyridamole, respectively. The precision
values are shown in Table 1.
250 Afr. J. Pharm. Pharmacol.
Table 1. Precision of aspirin and dipyridamole.
Assay
Sample No. Aspirin Dipyridamole
1 10.2 80.2
2 10.1 80.1
3 10.3 80.8
4 10.2 79.7
5 10.3 80.5
6 10.1 80.6
Mean (
X
) 10.20 80.32
S.D. 0.089 0.397
%RSD 0.9 0.5
Table 2. Recovery for aspirin and dipyridamole.
Dipyridamole Aspirin
Sample No. Spike level Amount
recovered (mg) Sample No. Spike level Amount
recovered (mg)
1 40.23 1 4.87
2 38.75 2 5.29
3 40.32 3 5.21
4 39.35 4 5.17
5 40.15 5 4.94
6
50% (40 mg)
41.05 6
50% (5 mg)
5.03
Mean recovery 39.975 Mean recovery 5.085
Mean % recovery 99.9 Mean % recovery 101.7
1 78.67 1 10.36
2 79.45 2 9.79
3 82.67 3 9.48
4 80.32 4 9.75
5 79.24 5 10.47
6
100% (80 mg)
83.24 6
100% (10 mg)
10.14
Mean Recovery 80.598 Mean Recovery 9.998
Mean % Recovery 100.7 Mean % Recovery 100.0
1 123.56 1 19.42
2 126.38 2 20.35
3 116.29 3 19.61
4 118.89 4 20.87
5 124.67 5 20.94
6
150% (120 mg)
117.98 6
150% (20 mg)
19.13
Mean recovery 121.295 Mean recovery 20.053
Mean % recovery 101.1 Mean % recovery 100.3
Recovery
Recovery studies for dipyridamole and aspirin were
performed at 50, 10%, and 150% of the highest
concentration of the linearity range (80 µg/ml for
dipyridamole 10 µg/ml for Aspirin) by spiking placebo
blend with the drug substance. Six replicates each were
spiked and analyzed after extraction. The amount spiked,
amount recovered and mean percent recovery were
calculated and reported in Table 2.
Range
The range of an analytical procedure is the interval
Prakash et al. 251
Table 3. Range for aspirin and dipyridamole.
Result
Parameter Acceptance criteria Dipyridamole Aspirin
Linearity R 0.999 0.9999 0.9998
Precision %RSD of 6 replicates NMT 2.0% 0.9% 0.5%
Accuracy Recovery 97.0 to 103.0% 99.9-101.1% 100.3 to 101.7
between the upper and lower concentration (amounts) of
analyte in the sample (including these concentrations) for
which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and
linearity. The results are shown in Table 3.
Robustness
The robustness of an analytical procedure is a measure
of its capacity to remain unaffected by small, but
deliberate variations in method parameters and provides
an indication of its reliability during normal usage. The
variations like flow rate of mobile phase, column
temperature and ratio of organic content in the mobile
phase etc does not have any significant effect on the
method performance.
Conclusions
A simple, rapid, reproducible and cost effective RP-HPLC
method was developed for the simultaneous determina-
tion of Aspirin and Dipyridamole in pharmaceutical
formulations by isocratic mode elution. The analytical
conditions and the solvent system developed provided
good resolution for Aspirin and Dipyridamole within a
short run time. The HPLC method was validated and
demonstrated good linearity, precision, accuracy and
specificity. Thus, the developed HPLC method can be
utilized for routine analysis during the analysis of Aspirin
and/or Dipyridamole.
ACKNOWLEDGEMENTS
The authors are thankful to Cipla labs for providing the
working standards of Aspirin and Dipyridamole.
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