American Journal of Analyt ical Chemistry, 2011, 2, 401-410
doi:10.4236/ajac.2011.24049 Published Online August 2011 (
Copyright © 2011 SciRes. AJAC
Stress Degradation Behavior of a Polypill and Development
of Stability Indicating UHPLC Method for the
Simultaneous Estimation of Aspirin, Atorvastatin,
Ramipril and Metoprolol Succinate
Satheesh Kumar Shetty1,5 , K. V. Surendranath1, P. Radhakrishnanand1, Roshan M. Borkar2,
Prashant S. Devrukhakar2, Johnson J o gul 3, Upendra Mani Tripathi4
1United States Pharmacopeia-India Private Limited, Research and Development Laboratory, ICICI Knowledge Park,
Turkapally, S h ameerpet, Hyderabad, India
2NIPER Instititute of Pharmaceutical sciences, Hyderabad, India
3Department of Che mistry, St. Kittel Science College, Dharwad, Karnataka, India
4Startech Labs privat e Li mi te d, SMR Chambers Madinaguda, Hyderabad, India
5Department of Che mistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, India
Received October 6, 2010; revised November 25, 2010; accepted December 5, 2010
A novel, sensitive and precise UHPLC method has been developed and validated for the simultaneous de-
termination of the all the active components of a Polypill viz Zycad, i.e., Aspirin (ASP) Atorvastatin (ATV),
Ramipril (RMP) and Metoprolol (MTP) in Zycad tablet dosage form in the presence of degradation products.
Forced degradation of individual as well as combination of all the drug substances components of Polypill
was conducted in accordance with ICH guidelines. Acidic, basic, neutral, and oxidative hydrolysis, thermal
stress, and photolytic degradation were used to assess the stability-indicating power of the method. Use of
100 × 2.1 mm, 1.7 µm stationary phases with simple mobile phase combination buffer consisting of 0.1%
Perchloric acid (adjusted to pH 2.5) and Acetonitrile, delivered in a gradient mode and quantitation was car-
ried out using ultraviolet detection at 215 nm with a flow rate of 0.6 mL·min–1. The method was optimized
using samples generated by forced degradation studies. The method was validated for linearity, accuracy
(recovery), precision, Specificity and robustness. The method was linear in the range of 37.5 to 150.0
µg·mL–1 for ASP, 5.0 to 20.0 µg·mL–1 for ATV and 2.5 to 10.0 µg·mL–1 for RMP and 25.0 to 100.0 µg·mL–1
for MTP.
Keywords: Liquid Chromatography; UHPLC, Polypill, Aspirin, Atorvastatin, Ramipril and Metoprolol
Succinate, Forced Degradation, Validation, Stability Indicating
1. Introduction
Ultra-high performance liquid chromatography is a new
category of separation technique based upon well-
established principles of liquid chromatography, which
utilizes sub-2 μm particles for stationary phase. These
particles operate at elevated mobile phase linear veloci-
ties to affect dramatic increase in resolution, sensitivity
& speed of analysis. Polypill is a fixed dose combination,
used as a single daily pill to achieve a large effect in
preventing cardiovascular disease with minimal adverse
effects. The concept has been applied to pharmaceutical
formulations in recent days [1-6]. In the present work,
the UHPLC technology has been applied for the fast es-
timation of the four main components of Polypill viz,
Zycad in a single method.
Zycad is a fixed dose combination of Aspirin (ASP),
Atorvastatin (ATV), Ramipril (RMP) and Metoprolol
Succinate (MTP). Each Zycad Polypill kit contains Aspi-
rin 75 mg, Atorvastatin 10 mg, and Ramipril 5 mg in a
single Capsule form and Metoprolol Succinate 50 mg as
separate tablet. Aspirin (ASP), 2-acetoxybenzoic acid
has an antiplatelet effect by inhibiting the production of
thromboxane, which under normal circumstances binds
platelet molecules together to create a patch over damaged
walls of blood vessels.This effect lasts for the life of the
platelet and prevents the formation of the platelet aggre-
gating factor thromboxane A2 [6,7]. Atorvastatin (ATV),
-tanoic acid is a member of the drug class known as statins,
used for lowering blood cholesterol. It also stabilizes
plaque and prevents strokes through anti-inflammatory
and other mechanisms. Atorvastatin works by inhibiting
HMG-CoA reductase, an enzyme found in liver tissue
that plays a key role in production of cholesterol in the
body. This enzyme catalyzes the conversion of HMG-
CoA to mevalonate, an early and rate limiting step in the
synthesis of cholesterol [8]. Ramipril(RMP), 2S, 3aS,
6aS)-1-[ (2 S)- 2-{[( 2S)-1-ethoxy-1-oxo-4-phenylbutan-2-
carboxylic acid is a prodrug and is converted to the
active metabolite ramiprilat by liver esterase enzymes is
an angiotensin-converting enzyme (ACE) inhibitor, used
to treat hypertension and congestive heart failure [9].
Metoprolol (MTP), (RS)-1-(iso-propyl amino)-3-[4-(2-
ethoxyethyl) phenoxy] Propan-2-ol is a selective β1 re-
ceptor blocker used in treatment of the cardiovascular
system, especially hypertension.Due to its selectivity in
blocking the beta receptors in the heart, Metoprolol is
also prescribed for off-label use in performance anxiety,
social anxiety disorder, and other anxiety disorders [10].
Though there were lot of papers published for the in-
dividual estimation of the drug substances ASP, ATV,
RMP and MTP in pharmaceutical preparations, Litera-
ture survey did not reveal any simple, sensitive and sta-
bility indicating LC method for the simultaneous deter-
mination of all the four drugs as a fixed dose combination.
Literature survey reveals that a variety of spectropho-
tometric and chromatographic, stability indicating LC
method has been reported for determination of these indi-
vidual drug components as well as combination of two or
three drugs in pharmaceutical dosage forms [11-16]. The
present drug stability test guideline Q1A (R2) [17,18]
issued by International Conference on Harmonization
(ICH) suggests that stress studies should be carried out on
a drug to establish its inherent stability characteristics,
leading to the development of a separation method for
the degradation products and hence to support the stabil-
ity indicating nature of the method.
This manuscript describes the development and vali-
dation, in accordance with ICH guidelines, of a rapid,
economical, precise, and accurate stability-indicating
reversed-phase UHPLC method for the estimation of
Aspirin (ASP), Atorvastatin (ATV), Ramipril (RMP) and
Metoprolol Succinate (MTP) in Zycad along with
method validation.
2. Experimental
2.1. Chemicals
Pure Samples of Aspirin (ASP) Atorvastatin (ATV),
Ramipril (RMP) and Metoprolol Succinate (MTP) with
purity more than 99.5% were kindly supplied by USP
India (P) limited, Hyderabad, India (Figure 1). Zycad
Polypill kits were purchased from the Market. HPLC
grade Acetonitrile, Perchloric acid and triethylamine was
purchased from Merck Germany. Milli-Q water prepared
by using Millipore purification system.
2.2. Equipment
Waters AQUITY UPLC binary pump plus auto sampler
and an AQUITY photo diode array detector were used
during the method development, Stress studies and for
method validations. Empower software (Digital equip-
ment Co) was used to monitor and process the output
signal. Controlled temperature oven (Mack Pharmatech
Private Ltd., Mumbai, India) was used for solid state
thermal stress studies. Photo stability studies were carried
out in a photo stability chamber (Mack Pharmatech, Hy-
derabad, India.
2.3. Chromatographic Conditions
The Chromatographic separations were achieved on a
Waters Acquity UPLC BEH C18 Column (100 × 2.1) mm
with 1.7 µm particles. 0.1% Perchloric acid (adjusted
pH.2.5) used as solution A & Acetonitrile as solution B in
gradient mode. Flow rate of the mobile phase was 0.6 mL
min-1. The UHPLC gradient program was set as: (time
(min)/% solution B: 0/10, 1.0/60, 1.5/80, 2.0/60, 2.5/10,
3.0/10. The column temperature was maintained at 35˚C
& the detection was monitored at a wavelength of 215 nm.
The injection volume was 2 µL. Buffer: Acetonitrile in the
ratio 80:20 v/v was used as the diluent.
2.4. Preparation of Standard Solutions
Working solutions 0.075 mg·mL–1 of ASP, 0.01 mg·mL–1
of ATV, 0.005 mg·mL–1 of RMP and 0.05 mg·mL–1 of
MTP were prepared from the stock solutions 7.5
mg·mL–1 of ASP, 1 mg·mL–1 of ATV, 0.5 mg·mL–1 of
RMP, and 5 mg·mL–1 of MTP.
2.5. Preparation of Sample Solutions
The contents of twenty Capsules and MTP tablets were
weighed to determine the average weight of the single
Copyright © 2011 SciRes. AJAC
Figure 1. Chemical structures and labels of all the drug
substances: ASP, ATV, RMP and MTP (a) Aspirin (ASP):
2-acetoxybenzoic acidM.F: C9H8O4 M.W: 180.16 g·mol–1;
(b) Atorvastatin (ATV): (3R, 5R)-7-[2-(4-fluorophenyl)-
-yl]-3,5-dihydroxyheptanoic acid. M.F:C33H35 F N2O5 M.W:
558.64 g·mol–1; (c) Ramipril (RMP): (2S,3aS,6aS)-1-
propanoyl]-octahydrocyclopenta[b] pyrrole-2-carboxylic
acid M.F: C23H32N2O5 M. W: 416.51 g·mol–1; 9d) Meto-
prolol (MTP): (RS)-1-(isopropyl amino)-3-[4-(2-ethoxyethyl)
phenoxy] Propan-2-ol C15H25NO3.W: 267.34 g·mol–1.
dosage unit. The contents of the Capsules and tablets
were crushed to a homogeneous powder and a quantity
equivalent to one dosage form (i.e. Equivalent to 75 mg
ASP, 10 mg ATV, 5 mg RMP and 50 mg MTP) was
weighed in to a 100-mL volumetric flask, extracted in
diluents by sonicating for 10 mins, and filtered through
Whatman no. 41 filter paper. The 1 ml of the clear fil-
trate) was quantitatively transferred to a 10-mL volumet-
ric flask, and solution was diluted to volume with the
2.6. Analytical Method Validation
The method was validated for specificity, precision, sen-
sitivity, linearity, accuracy, robustness, and system suit-
Forced degradation studies were performed on indi-
vidual as well as mixture of all the four drugs, to provide
an indication of stability indicating property and speci-
ficity of the proposed method [19,20].
Acid hydrolysis was performed in 0.1 N HCl at reflux
condition for 1 h. Basic hydrolysis was performed in 0.1N
NaOH at reflux condition for 0.5 h.
For studies in oxidation condition, the drug combina-
tions were exposed to 5% H2O2. RT for 8 h. Photolytic
degradation studies were performed carried out as per
ICH Q1B.The dug samples was exposed to light for
overall illumination of 1.2 × 106 lux h and an integrated
near ultraviolet energy of 200 W h m2.
To study the thermal degradation the drug combinations
were exposed to dry heat at 60˚C. Peak purity of stressed
samples was checked by using a Acquity photo diode
array detector (PDA). All stressed samples were analyzed
for extended run time (30 min) to check for late-eluting
degradation products.
Initially, system suitability was prepared, by dissolv-
ing appropriate amounts of all the four standard drug sub-
stances in the diluents to get a final concentration of 75
µg·mL–1 ASP, 10 µg·mL–1 ATV, 5 µg·mL–1 RMP, and 50
µg·mL–1 MTP, and injecting into the LC system and
evaluating the criteria like Resolution, USP Tailing fac-
tor and No. of theoretical plates for each drug component.
The optimized method was validated with respect to
various parameters summarized in the ICH guideline Q2
The precision of the assay method was checked by in-
jecting the 6 replicates of the sample preparations of the
commercial tablet (Zycad).Repeatability (Intra-Day pre-
cision) of the assay method was evaluated by carrying
out six independent assays of the commercial tablets
with concentrations 0.075 mg·mL–1 of ASP, 0.01
mg·mL–1 of ATV, 0.005 mg·mL–1 of RMP and 0.05
mg·mL–1 of MTP, against qualified reference standard
Copyright © 2011 SciRes. AJAC
3. Results and Discussions
and calculating the % RSD of the assay results. Interme-
diate precision (ruggedness) was evaluated by conduct-
ing precision studies using different analysts and differ-
ent columns in the same laboratory.
LOD and LOQ for ASP, ATV, RMP, and MTP were
estimated by injecting a series of dilute solutions with
known concentrations. The precision study was also car-
ried out at the LOQ level by injecting six individual
preparations and calculating the RSD (%) of peak area
for each drug components.
To establish linearity and range, a stock solution con-
taining 150 µg·mL–1 ASP, 20 µg·mL–1 ATV, 10 µg·mL–1
RMP, 100 µg·mL–1 MTP in methanol was diluted to
yield solutions in the concentration range of 37.5 to
150.0 µg·mL–1 for ASP, 5.0 to 20.0 µg·mL–1 for ATV
and 2.5 to 10.0 µg·mL–1 for RMP and 25.0 to 100.0
µg·mL–1 for MTP (i.e. 50 to 200% of analyte concentra-
tions). The solutions were prepared and analyzed in trip-
licate. Peak-area and concentration data were used to plot
the calibration graph and by least squares linear regres-
sion analysis, the correlation coefficients, slope and in-
tercept values were calculated.
For determination of accuracy, recovery studies were
carried out by spiking analysis. Known amounts of each
drug corresponding to 50%, 100%, and 150% of the tar-
get test concentrations i.e. 75 µg·mL–1 ASP, 10 µg·mL–1
ATV, 5 µg·mL–1 RMP, and 50 µg·mL–1 MTP were added
to a placebo mixture the sample was extracted, and the
assay was evaluated against the standard and percentage
recovery was calculated.
Robustness was tested using the so-called “one factor
at a time” method. The factors evaluated were flow rate,
column temperature and pH. The experimental condi-
tions were deliberately changed and the relative standard
deviation for replicate injections of ATL, ASP, RMP
and MTP peaks and the USP resolution factor between
MTP and ASP peaks were evaluated. The mobile phase
flow rate was 0.6 mL·min–1. This was changed by 0.05
units to 0.55 and 0.65 mL·min–1. The effect of column
temperature was studied at 40˚C and 30˚C instead of
35˚C. The effect of buffer pH was studied at pH 2.4 and
The solution stability of ASP, ATV, RMP and MTP
was carried out by leaving the test solution in tightly
capped volumetric flasks at room temperature for 48 h
and assayed at 6 h interval, against the freshly prepared
standard solution. The mobile phase stability was car-
ried out by assaying the freshly prepared sample solu-
tion against the freshly prepared standard at 6 h interval
up to 48 h. The percentage of RSD of assay of ASP,
ATV, RMP and MTP was calculated for the study pe-
riod during mobile phase and solution stability experi-
men ts.
3.1. Method Development and Optimization
A detection wavelength of 215 nm was used for method
development work. This was established by preparing
100 µg·mL–1 solutions of each individual drug compo-
nents of the formulation and scanning in UV-Visible
Initial experiments during the analytical method de-
velopment reveals that the critical challenge is to obtain
adequate retention for the polar parent compound, MTP
and ASP, while maintaining a reasonable elution time for
the less-polar Atorvastatin (ATV) and to separate one of
the degradation impurity Salicylic acid (SA) from the
ASP peak.
The ACQUTY UPLC BEH C18 (50 × 2.1) mm 1.7 um
Column was chosen for initial trial with 0.1% Formic
acid and Acetonitrile as the mobile phase. Different mo-
bile phase compositions containing Formic acid and
Acetonitrile (50:50–20:80 v/v) were tried. But was not
successful in getting the good peak shapes for the Rami-
pril (RMP). There were no improvements in the peak
shape of the RMP even under different pH of the buffer.
Using of 0.1% Orthophosphoric acid as buffer also un-
successful in obtaining good peak shape for RMP. Al-
though good separation was achieved with 25 mM So-
dium perchlorate: Acetonitrile in the gradient mode,
Atorvastatin and Ramipril peak symmetry was found to
be greater than 2.0. The asymmetries of the peaks were
improved by addition of Triethylamine (TEA) and ad-
justing the mobile phase pH to 2.5 in the aqueous
The chromatographic separation with better peak
shape was achieved using a mixture of aqueous 0.1%
Perchloric acid and Acetonitrile in the ratio of 40:60
(v/v). However to improve the resolution between the
closely eluting peaks, the method was changed to gradi-
ent mode and optimized the gradient conditions. But
when stressed samples injected with this optimized con-
ditions, it was found that some of the degradants, co
eluted with the principal peaks. So the length of the
column is increased to 100 mm and the method was op-
timized to separate all the degradants from the main
peaks by changing to Gradient mode. Several gradient
conditions were tried before optimizing the final gradient
programme as: time (min)/% solution B: 0/10, 1.0/60,
1.5/80, 2.0/60, 2.5/10, 3.0/10.
As peak shape of Atenolol (ATL) was one of the issue
even after optimizing the LC parameters, effect of the
diluent on the peak shapes was studied by changing so
many combinations of the diluents. The ATL peak was
observed as split in most of the compositions of the buffer
Copyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
3.3. Results of Forced Degradation Study and Acetonitrile. Finally Buffer: Acetonitrile in the ratio
80:20, v/v, was optimized as the diluent to obtain good
peak shapes.
Specificity/Application of Stress (Forced
Degradation Study)
3.2. Optimization of Chromatographic Singh and Bakshi suggested target degradation of 20% -
30% when establishing the stability-indicating properties
of analytical methods, because even intermediate degra-
dation products should not interfere with any stage of
drug analysis. Stress studies on combination of all the
four drugs under different stress conditions suggested the
following degradation behavior. The combination of the
drugs showed considerable stability under thermal condi-
Conditions for UHPLC
The satisfactory chromatographic separation, with good
peak shapes were achieved on ACQUTY UPLC BEH
C18 ( 100 × 2.1) mm with 1.7 µm particles, using 0.1%
Perchloric acid (adjusted to pH 2.5 with Triethylamine)
as solution A and Acetonitrile as solution B with a flow
rate of 0.6 mL·min–1. The UHPLC gradient program was
optimized as: (time (min)/% solution B: 0/10, 1.0/60,
1.5/80, 2.0/60, 2.5/10, 3.0/10. The Column temperature
as maintained at 35˚C and the detection was monitored at
a wavelength of 215 nm. The injection volume was 2 µL.
Buffer: Acetonitrile (80:20, v/v) was used as diluent.
The method was found to be high degree of specificity
to the drugs viz ASP, ATV, RMP, and MTP. All drugs
were well separated from one another as well as resolved
from degraded impurities. The specificity of the method
was confirmed by the separation of all the peaks in the
degraded samples obtained under different conditions.
In the optimized gradient conditions MTP, ASP, SA,
RMP and ATV were well separated with a resolution (Rs)
of greater than 2 and the typical retention times of MTP,
ASP, SA, RMP and ATV 1.12, 1.16, 1.27, 1.42 and 1.78
respectively .Peak purity of stressed samples of all the
four drug substances were checked by using Acquity
Photo diode array detector. All stressed samples of the
drug product (heat (100˚C), acid hydrolysis (0.1 N HCl),
base hydrolysis (0.1 N NaOH), water hydrolysis and
oxidation (5% H2O2) were analyzed for extended run
time of 60 min to check the late eluting degradants.
The mixture of all the four drug components was ex-
posed to 0.1 N HCl at 100˚C for 1 h. ASP and ATV
Showed considerable degradation with time in 0.1 N HCl
with the formation of Salicylic acid (SA) as the major
degradant Figure 2.
When treated under basic conditions, ASP and RMP
has shown significant degradation immediately in 0.1 N
NaOH with ASP is converted into salicylic acid (SA)
(Figure 3).
Under oxidation conditions, ASP and ATV has
0. 38 8
0. 49 2
A spirin - 1. 14 6
1.205 S alicylic acid - 1.253
Ramipril - 1. 400
A torvas tatin - 1. 7 55
1. 865
1. 963
0.00 0.20 0.40 0.600.80 1.00 1.201.40 1.60 1.80 2.002.20 2.40 2.60 2.80 3. 0
Figure 2. Typical chromatogram of acid degradation, 0.1 N HCl (100˚C, 1 h).
0. 422
1. 082
A spi ri n - 1.146
1.205 S al icyl i c acid - 1.253
Rami pri l - 1. 404
A t o rv astati n - 1.753
1. 891
1. 963
2. 116
0.00 0.200.40 0.60 0.80 1.00 1.201.40 1.601.80 2.00 2.20 2.402.602.803.0
Figure 3. Typical chromatogram of base degradation, 0.1 N NaOH (under reflux, 0.5 h).
showed considerable degradation by the treatment of 5
hydrogen peroxide. Typical chromatogram is as shown
in Figure 4.
All the four drug combinations were exposed to light
for an overall illumination of 1.2 million lux hours and an
integrated near ultraviolet energy of 200-watt hours/
square meter (w/m hr) (in photo stability chamber. Major
degradation observed with ATV (Figure 5).
The proposed method is applied for the assay analysis
of 3 different batches of the Zycad. The assay results
obtained were within the specification limit. The assay of
Polypill is unaffected in the presence of degradation im-
purities confirming the stability indicating power of the
developed method. The stability indicating nature of the
method was further confirmed by injecting three month
accelerated stability sample and observed that all the
degradants were well separated from the main compo-
For all the stressed sample injections, peak purity was
checked by using a Acquity photo diode array detector
(PDA). Peak purity test results derived from PDA detector,
confirmed that the all the four drug components were
homogeneous and pure in all the analyzed stress samples.
3.4. Analytical Method Validation:
The developed chromatographic method was validated
for selectivity, linearity, range, precision, accuracy, sen-
sitivity, robustness and system suitability. The typical
chromatogram of System suitability shown in Figure 6.
The results of system suitability were depicted in Table
3.4.1. Precision
The percentage RSD values for the assays in precision
study were 0.4%, 0.8%, 0.4%, 0.5% (inter-day precision)
and 0.5%, 0.8%, 0.6%, 0.7% (intra-day precision) for
ASP, ATV, RMP, and MTP confirming a good precision
and the ruggedness of the method.
3.4.2. Sensiti vity
The limits of detection (LOD) and quantitation (LOQ)
were established at signal-to-noise ratios of 3:1 and 10:1,
respectively. The LOD and LOQ of ASP, ATV, RMP
and MTP were determined experimentally by injecting
each drug six times. The LOD for ASP, ATV, RMP and
MTP were 0.1, 0.2, 0.25 and 0.12 µg·mL–1 respectively.
The LOQ for ASP, ATV, RMP and MTP were 0.3, 0.7,
0.8 and 0.4 µg·mL–1, respectively.
3.4.3. Linearity
The linear ranges were from ((37.5 to 150.0 µg·mL–1 for
ASP, 5.0 to 20.0 µg·mL–1 for ATV and 2.5 to 10.0
µg·mL–1 for RMP and 25.0 to 100.0 µg·mL–1for MTP).
The correlation coefficient Obtained was greater than
The Slope and the Intercept value obtained from the
linear regression graph is as shown in (Table 2). The
result shows an excellent correlation existed between the
peak area and concentration of the analyte in the range
50% - 200% of analyte concentration
3.4.4. Accu racy
The percentage recovery obtained was in the range of
99.02% to 100.8%. The results indicate the method en-
ables highly accurate simultaneous determination of the
Copyright © 2011 SciRes. AJAC
P eroxide - 0.409
0. 670
0. 942
0. 96 7
A spi ri n - 1.15 0
1.197 S ali cylic acid - 1.258
Ramipril - 1. 404
1. 479
1. 520
1. 56 4
1. 58 7
1. 613
1. 636
1. 65 3
1. 79 0
1. 96 6
2. 09 3
2. 218
2. 796
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.002.20 2.40 2.60 2.803.0
Figure 4. Typical chromatogram of Peroxide degradation, 5% hydrogen peroxide (RT, 8 h).
Aspirin - 1. 155
1. 198
S al i cylic aci d - 1.264
Ram i p ril - 1. 411
1. 564
1. 619
A torvastatin - 1.761
1. 820
1. 871
1. 897
1. 970
2. 040
2. 098
0.00 0.200.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.0
Figure 5. Typical chromatogram of photo stability degradation.
all the four drugs in the Polypill combination.
3.4.5. Robustness
The results obtained by deliberate variation in method
parameters and data are summarized in Table 3. The
data revealed that the resolution between closely eluting
peaks, namely ASP and MTP was always greater than
2.0 and also there was not much effect on the peak
shapes, illustrating the robustness of the method
3.4.6. Solution Stability and Mobile Phase Stability
The % RSD of assay of Polypill during solution stability
and mobile phase stability experiments was less than 1.0.
No significant changes were observed in the content of
ASP, ATV, RMP, and MTP during the study. The solu-
tion stability and mobile phase stability experiments data
confirms that sample solutions and mobile phase used
during assay determination were stable up to the study
period of 48 h.
Assay analysis was performed for different batches of
the drug product in tablets (n = 3), with the targeted ana-
lyte concentration. The assay results obtained for the
three Zycad Polypill tablets were, ZYD/001 (99.7% ASP,
99.8% ATV, 100.2% RMP and 99.6% MTP), ZYD/002
(99.6% ASP, 100.3% ATV, 99.4% RMP and 99.8%
MTP) and ZYD/003 (100.1% ASP, 99.9% ATV, 99.7%.
opyright © 2011 SciRes. AJAC
Met oprol ol - 1. 105
A spirin - 1.14 2
Salicylic acid - 1.250
Ramipri l - 1. 397
Atorvastatin - 1.749
) 0.600.80 1.00 1.201.40 1.60 1.802.002.20 2.402.60 2.80 3.0
Figure 6. Typical chromatogram of System suitability.
Table 1. System suitability results.
Compound Retention time
USP Resolution (RS ) USP
Tailing factor (T) USP Plate count(N)
MTP 1.12 1.14 58245
ASP 1.16 2.08 1.11 60939
SA 1.27 5.42 1.14 64823
RMP 1.42 6.68 1.15 60868
ATV 1.78 16.86 1.05 137740
Table 2. Result of Linearity study.
Calibration Equn. Y = 4690X + 3976 Y = 907X – 6273.8 Y = 292X – 4881.7 Y = 200X – 710.3
Linearity Range 50% - 200% 50% - 200% 50% - 200% 50% - 200%
R2 0.999 0.999 0.997 0.999
Slope 4689.9 907 292.4 1909.9
Intercept 3976 –6273.8 –4881.7 –710.3
Table 3. Result of Robustness study.
S.No Parameter Variation Resolution (Rs) between MTP and ASP
1 Temperature 5˚C
of set temperature)
(a) At 30˚C
(b) At 40˚C
2 Flow rate 20% of the set flow) (a) At 0.55 mL·min–1
(b) At 0.65 mL·min–1
3 pH Buffer (a) pH 2.4
(b) pH 2.6
Table 4. Batch analysis for Zycad drug product.
Batch No: ASP
ZYD/001 99.7 99.8 100.2 99.6
ZYD/002 99.6 100.3 99.4 99.8
ZYD/003 100.1 99.9 99.7 100.3
opyright © 2011 SciRes. AJAC
RMP and 100.3% MTP) depicted in Table 4.
3.5. Application of the Method to Stability Study
Accelerated conditions stability studies are performed to
establish the stability indicating nature of the method.
Accelerated conditions (temperature 40 ± 2˚C, relative
humidity 75 ± 5%) stored sample of the four drug com-
binations were analyzed by use of the developed UHPLC
method after the period of 3 months. The results obtained
clearly indicates that the method is able to separate all
the drug-drug interaction impurities or any other degra-
dation impurities formed during the storage conditions,
indicating the method was stability-indicating and highly
suitable for drug stability studies and for monitoring the
quality of the Polypill.
4. Acknowledgements
The authors wish to thank the management of United
States Pharmacopeia laboratory- India for supporting this
5. References
[1] G. Sanz and V. Fuster, “Fixed-Dose Combination Ther-
apy and Secondary Cardiovascular Prevention: Rationale,
Selection of Drugs and Target Population,” Nature
Clinical Practice Cardiovascular Medicine, Vol. 6, No. 2,
2009), pp. 101-110. doi:10.1038/ncpcardio1419
[2] “Global Burden of Disease 2004 Update,” World Health
Organization, 2008.
[3] C. D. Mathers and D. Loncar,Projections of Global
Mortality and Burden of Disease from 2002 to 2030,”
PLoS Medicine, Vol. 3, No. 11, 2006, p. e442.
[4] M. R. Law and N. J. Wald, “Risk Factor Thresholds:
Their Existence under Scrutiny,” British Medical Journal,
Vol. 324, No. 7353, 2002, pp. 570-576.
[5] V. Kumar, R. P. Shah and S. Singh, “LC and LC–MS
Methods for the Investigation of Polypills for the Treat-
ment of Cardiovascular Diseases: Part.1 Separation of
Active Compo,” Journal of Pharmaceutical and Bio-
medical Analysis, Vol. 47, 2008, pp. 508-515.
[6] S. P. Clissold, “Aspirin and Related Derivatives of Sali-
cylic Acid,” Drugs, Vol. 32, 1986, pp. 8-26.
[7] D. G. Julian, D. A. Chamberlain and S. J. Pocock, “A
Comparison of Aspirin and Anticoagulation Following
Thrombolysis for Myocardial Infarction (the AFTER
Study): A Multicentre Unblinded Randomized Clinical
Trial,” BMJ (British Medical Journal), Vol. 313, No.
7070, 1996, pp. 1429-1431.
[8] J. W. Nawrocki, S. R. Weiss, M. H. Davidson, D. L.
Sprecher, S. L. Schwartz, P. J. Lupien, P. H. Jones, H. E.
Haber, et al., “Reduction of LDL Cholesterol by 25% to
60% in Patients with Primary Hypercholesterolemia by
Atorvastatin, a New HMG-CoA Reductase Inhibitor,”
Arteriosclerosis, Thrombosis, and Vascular Biology, Vol.
15, No. 5, May 1995, pp. 678-682.
[9] L. Pilot, M. Abrahamowicz, M. Eisenberg, K. Humphries,
H. Behlouli and J. V. Tu, “Effect of Different Angio-
tensin-Converting-Enzyme Inhibitors on Mortality among
Elderly Patients with Congestive Heart Failure,” Cana-
dian Medical Association Journal, Vol. 178, No. 10,
2008, pp. 1303-1311. doi:10.1503/cmaj.060068
[10] Q. Zhang, H. Jin, L. Wang, J. Chen, C. Tang and J. Du,
“Randomized Comparison of Metoprolol Versus Con-
ventional Treatment in Preventing Recurrence of Vas-
ovagal Syncope in Children and Adolescents,” Interna-
tional Medical Journal of Experimental and Clinical Re-
search, Vol. 14, No. 4, pp. 199-203.
[11] E. R. Montgomery, S. Taylor, J. Segretario, et al., “De-
velopment and Validation of a Reversed-Phase Liquid
Chromatographic Method for Analysis of Aspirin and
Warfarin in a Combination Tablet Formulation,” Journal
of Pharmaceutical and Biomedical Analysis, Vol. 15, No.
1, 1996, pp. 73-82.
[12] S. Erturk, A. E. Sevinc, L. Ersoy and S. Ficicioglu,
“HPLC Method for the Determination of Atorvastatin and
Its Impurities in Bulk Drug and Tablets,” Journal of
Pharmaceutical and Biomedical Analysis, Vol. 33, No. 5,
2003, pp. 1017-1023.
[13] A. Puratchikody, R. Valarmathy and P. Shiju, “RP-HPLC
Determination of Atorvastatin Calcium in Solid Dosage
Forms,” Journal of Pharmacological Reviews, 2003, pp.
[14] H. J. Panchal, B. N. Suhagia, N. J. Patel, I. S. Rathod, B.
H. Patel, “Development and Validation of a HPTLC
Method for the Simultaneous Estimation of Atorvastatin
Calcium and Ezetimibe,” Chromatographia, Vol. 69, No.
1-2, 2009, pp. 91-95. doi:10.1365/s10337-008-0831-z
[15] F. Belal, I. A. Al-Zaagi, E. A. Gadkariem and M. A.
Abounassif, “A Stability-Indicating LC Method for the
Simultaneous Determination of Ramipril and Hydro-
chlorothiazide in Dosage Forms,” Journal of Pharmaceu-
tical and Biomedical Analysis, Vol. 24, No. 3, 2001, pp.
335-342. doi:10.1016/S0731-7085(00)00474-X
[16] M. B. Shankar, F. A. Metha, K. K. Bhatt, R. S. Metha and
M. Geetha,Simultaneous spectrophotometric determi-
nation of losartan potassium and hydrochlorothiazide in
tablets,” Indian Journal of Pharmaceutical Sciences, Vol.
65, No. 2, 2003, pp. 167-170.
[17] “ICH Stability Testing of New Drug Substances and
Products Q1A (R2),” International Conference on Har-
monization, IFPMA, Geneva, 2003.
[18] “ICH guidelines on Validation of Analytical Procedures,
Text and Methodology Q2 (R1),” FDA, Published in the
Federal Register 60, 1995.
[19] “United States Pharmacopoeia,” 32nd Edition, United
opyright © 2011 SciRes. AJAC
Copyright © 2011 SciRes. AJAC
States Pharmacopeial Convention, Rockville, USP 32,
[20] M. Bakshi and S. Singh, “Development of Validated
Stability-Indicating Assay Methods-Critical Review,”
Journal of Pharmaceutical and Biomedical Analysis, Vol.
28, 2002, pp. 1011-1040.