American Journal of Analytical Chemistry, 2010, 2, 59-69
doi:10.4236/ajac.2010.12008 Published Online August 2010 (http://www.SciRP.org/journal/ajac)
Quantitative Application to a Polypill by the Development
of Stability Indicating LC Method for the Simultaneous
Estimation of Aspirin, Atorvastatin, Atenolol and
Losartan Potassium
Satheesh K. Shetty1,5*, Koduru V. Surendranath1, Pullapanthula Radhakrishnanand1,
Roshan M. Borkar2,Prashant S. Devrukhakar2, Johnson Jogul3, Upendra M. Tripathi4
1United States Pharmacopeia-India Private Limited, Research and Development Laboratory, ICICI Knowledge Park,
Turkapally, Shameerpet, Hyderabad, India
2National Institute of Pharmaceutical education and Research (NIPER), Hyderabad, India
3Department of Chemistry, St. Kittel Science College, Dharwad, India
4Startech Labs private Limited, SMR Chambers Madinaguda, Hyderabad, India
5Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, India
Received May 21, 2010; revised July 10, 2010; accepted July 23, 2010
Abstract
Polypill is a fixed-dose combination (FDC) containing three or more drugs in a single pill with the intention
of reducing the number of tablets or capsules that need to be taken. Developing a single analytical method
for the estimation of individual drugs in a Polypill is very challenging, due to the formation of drug-drug and
drug-excipients interaction impurities. Here an attempt was made to develop a new, sensitive, single stabil-
ity-indicating HPLC method for the simultaneous quantitative determination of Aspirin (ASP) Atorvastatin
(ATV), Atenolol (ATL) and Losartan potassium (LST) in a polypill form in the presence of degradation
products. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile
phase combination of buffer and Acetonitrile. Buffer consists of 0.1% Orthophosphoric acid (pH 2.9), deliv-
ered in a gradient mode and quantitation was carried out using ultraviolet detection at 230 nm with a flow
rate of 1.0 mL/min. The retention times of Atenolol, Aspirin, Losartan potassium, and Atorvastatin were 3.3,
7.6, 10.7 and 12.9 min respectively. The combination drug product are exposed to thermal, acid/base hydro-
lytic, humidity and oxidative stress conditions, and the stressed samples were analyzed by proposed method.
The method was validated with respect to linearity; the method was linear in the range of 37.5 to 150.0
µg/mL for ASP, 5.0 to 20.0 µg/mL for ATV and 25.0 to 100.0 µg/mL for ATL and LST. Acceptable preci-
sion and accuracy were obtained for concentrations over the standard curve ranges. The validated method
was successfully applied to the analysis of Starpill tablets constituting all the four drugs; the percentage re-
coveries obtained were 99.60% for ASP, 99.30% for ATV, 99.41% for ATL and 99.62% for LST.
Keywords: Liquid Chromatography, Polypill, Aspirin, Atorvastatin, Atenolol and Losartan Potassium,
Forced Degradation, Validation, Stability Indicating
1. Introduction
A Polypill concept to reduce CVD by more than 80% was
firstly given by Wald and Law [1] and has been applied to
pharmaceutical preparations [2-6]. Foreseeing the need of
different analytical methods for estimation of ingredients
of these pills, the ultimate goal of our work was to develop
and validate a single high-performance liquid chroma-
tography method selective for the four main components
of tablets Starpill. Starpill is a fixed dose combination of
Aspirin (ASP), Atorvastatin (ATV), Atenolol (ATL) and
Losartan potassium (LST). Each trilayered tablet contains
Aspirin 75 mg, Atorvastatin 10 mg, Atenolol 50 mg and
Losartan 50 mg. Aspirin (ASP), 2-acetoxybenzoic acid
affects platelet aggregation by irreversibly inhibiting
prostaglandin cyclooxygenase. This effect lasts for the

60 S. K. SHETTY ET AL.
life of the platelet and prevents the formation of the
platelet aggregating factor thromboxane A2 [7-8]. Ator-
vastatin (ATV), (3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl
-4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,
5-dihydroxyheptanoic acid is a selective competitive in-
hibitor of 3-hydroxy-3-methyl-glutarylcoenzyme A (HMG-
CoA) reductase enzyme. This enzyme catalyzes the con-
version of HMG-CoA to mevalonate, an early and rate
limiting step in the synthesis of cholesterol [9]. Atenolol
(ATL), (RS)-2-{4-[2-hydroxy-3-(propan-2-ylamino) pro-
poxy]phenyl} acetamide is a beta1-selective (cardio se-
lective) beta-adrenergic receptor blocking agent without
membrane stabilizing or intrinsic sympathomimetic (par-
tial agonist) activities. This preferential effect is not ab-
solute, however, and at higher doses, Atenolol inhibits
beta2-adrenoreceptors, chiefly located in the bronchial and
vascular musculature [10-11]. Losartan (LST), 2-butyl-
4-chloro-1-{[2’-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1
H-imidazol-5-yl) methanol is an angiotensin II receptor
(type AT1) antagonist. Losartan and its principal active
metabolite block the vasoconstrictor and aldosterone-
secreting effects of angiotensin II by selectively blocking
the binding of angiotensin II to the AT 1 receptor found in
many tissues, (e.g., vascular smooth muscle, adrenal
gland). The active metabolite is 10 to 40 times more po-
tent by weight than Losartan and appears to be a reversi-
ble, non-competitive inhibitor of the AT 1 receptor [12-
13].
Extensive literature survey did not reveal any simple,
sensitive and stability indicating LC method for the si-
multaneous determination of all the four drugs as a fixed
dose combination. Literature survey reveals that a variety
of spectrophotometric and chromatographic methods,
and a stability indicating LC method, has been reported
for determination of ASP and ATV in pharmaceutical
preparations in combination with other drugs [14-21].
Spectrophotometer and chromatographic methods have
been reported for determination of ATL, in combination
with other drugs, in bulk and pharmaceutical prepara-
tions [22-23].Also there are some papers for the estima-
tion of LST individually and combination with other
drugs [24-27].The present drug stability test guideline
Q1A (R2) [28-29] 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 separation of degradation pro-
ducts and hence supporting the suitability of the pro-
posed analytical procedures.
In the present paper an attempt has been made to de-
velop an accurate, rapid, specific and reproducible me-
thod for the estimation of Aspirin (ASP), Atorvastatin
(ATV), Atenolol (ATL) and Losartan potassium (LST)
in Starpill along with method validation as per ICH
norms.
2. Experimental
2.1. Chemicals
Samples of Aspirin (ASP) Atorvastatin (ATV), Atenolol
(ATL) and Losartan potassium (LST) were procured
from USP India (P) limited, Hyderabad, India (Figure 1).
Market samples of Starpill (Cipla Ltd Mumbai) tablets
were purchased from the retail pharmacy. HPLC grade
Acetonitrile, Analytical reagent grade Orthophosphoric
acid purchased from Merck, Darmstadt, Germany. High
purity water was prepared by using Millipore Milli-Q
plus water purification system. The purity of the all drug
substances and the chemicals used for the experiment
were greater than 99.5% and the purity of the working
standards used for the analysis was 99.9%.
2.2. Equipments
The LC system, used for method development, forced
degradation studies and method validation was Waters
2695 binary pump plus auto sampler and a 2996 photo
diode array detector. The output signal was monitored and
processed using Empower software on Pentium computer
(Digital equipment Co). Photo stability studies were car-
ried out in a photo stability chamber (Mack Pharmatech,
Hyderabad, India). Thermal stability studies were per-
formed in a dry air oven (Mack Pharmatech, Hyderabad,
India).Accelerated stability studies were performed in a
stability Chamber (Thermo Lab Mumbai).
2.3. Chromatographic Conditions
The chromatographic column used was Inertsil ODS C18
(150 × 4.6) mm with 5 µm particles. The mobile phase A
consists of 0.1% Orthophosphoric acid adjusted to pH 2.9
with triethylamine (TEA). The mobile phase B consists of
Acetonitrile. Flow rate of the mobile phase was 1.0
mL/min. The HPLC gradient program was set as: (time
(min)/% solution B: 0/5, 10/60, 15/80, 17/60, 20/5, 25/5.
The column temperature was maintained at 35˚C and the
detection was monitored at a wavelength of 230 nm. The
injection volume was 10 µL. Buffer: Acetonitrile 80:20;
(v/v) was used as diluent.
2.4. Preparation of Standard Solutions
A stock solution of ASP, ATV, ATL,and LST standard
and sample (7.5 mg/mL of ASP, 1 mg/mL of ATV, 5.0
mg/mL each of ATL and LST) were prepared in diluent.
Working solutions 0.075 mg/mL of ASP, 0.01 mg/mL of
ATV, 0.05 mg/mL each of ATL and LST were prepared
from above stock solution in the diluent for assay deter-
mination.

S. K. SHETTY ET AL.
61
O
H
2
N
ONH
OH
(a) Atenolol (ATL): (RS)-2-{4-[2-hydroxy-3-(propan-2-ylamino)pro-
poxy]phenyl}acetamide, M.F: C14H22N2O3, M.W: 266.336 g/mol.
O
HO
O
O
(b) Aspirin (ASP): 2-acetoxybenzoic acid, M.F: C9H8O4, M.W: 180.16
g/mol.
OH
N
N
NH
NN
N
Cl
(c) Losartan (LST): 2-butyl-4-chloro-1-{[2’-(1H-tetrazol-5-yl)biphenyl
-4-yl]methyl}-1H-imidazol-5-yl)methanol, M.F: C22H23ClN6O, M.W:
422.91 g/mol.
O
HO
HO HO
N
HN
O
F
(d) Atorvastatin (ATV): (3R,5R)-7-[2 -(4-fluo rophenyl)-3-ph enyl-4-
(phenylcabamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic
acid, M.F: C33H35FN2O5, M.W: 558.64 g/mol.
Figure 1. Chemical structures and labels of all the drug
substances: ATL, ASP, LST and ATV.
2.5. Preparation of Sample Solutions
Twenty tablets were weighed and their average weight
was calculated. The tablets were crushed to a homoge-
neous powder and a quantity equivalent to one tablet (75
mg ASP, 10 mg ATV, 50 mg: ATL and 50 mg LST) was
weighed in a 100-mL volumetric flask, extracted in dilu-
ents by sonication, and filtered through Whatman no. 41
filter paper. The filtrate (1 mL) was quantitatively trans-
ferred to a 10-mL volumetric flask, and solution was
diluted to volume with the diluent.
2.6. Preparation of System Suitability Solution
System Suitability Solution was prepared by spiking 0.01
mg/mL of Salicylic acid (SA) to a mixture of all the four
drugs at the target test concentration levels (0.075 mg/mL
of ASP, 0.01 mg/mL of ATV, 0.05 mg/mL each of ATL
and LST).
2.7. Analytical Method Validation
The developed chromatographic method was validated
for selectivity, linearity, range, precision, accuracy, sen-
sitivity, robustness and system suitability.
2.7.1. Specificity/Application of Stress (Forced
Degradation Study)
Selectivity of the developed method was assessed by
performing forced degradation studies. The terms selec-
tivity and specificity are often used interchangeably.
Specificity is the ability of the method to measure the
analyte response in the presence of its potential impuri-
ties. Stress testing of the drug substance can help to iden-
tify the likely degradation products, which can in turn
help to establish the degradation pathways and the intrin-
sic stability of the molecule and validate the stability
indicating power of the analytical procedures used.
The specificity of the developed LC method for quan-
tification of all the four drugs was determined in the
presence of its degradation products. Forced degradation
studies were performed on individual as well as mixture
of all the four drugs, to provide an indication of stability
indicating property and specificity of the proposed
method [30-31].
The stress conditions employed for degradation study
includes light (carried out as per ICH Q1B), heat (60˚C),
acid hydrolysis (0.1N HCl), base hydrolysis (0.1N NaOH),
water hydrolysis and oxidation (5% H2O2). For acid
study period was reflux for 1 h, and oxidation it was at
room temperature (RT) for 48 h and for base it was re-
flux for 2 h. Peak purity of stressed samples was checked
by using a 2996 photo diode array detector (PDA) from
Waters. The purity angle within the purity threshold limit
demonstrates the analyte peak homogeneity.
2.7.2. Precision
System Precision was investigated by injecting the 6
replicates of the sample preparations of the commercial
tablet (Starpill).Repeatability (Intra-Day precision) of the
assay method was evaluated by carrying out six inde-

62 S. K. SHETTY ET AL.
pendent assays of the commercial tablets (0.075 mg/mL
of ASP, 0.05 mg/mL of ATL and LST, 0.010 mg/mL of
ATV test concentration) against qualified reference
standard and calculating the % RSD of the assay results.
Intermediate Precision (Inter-Day) was evaluated by
carrying out the experiment with a different analyst, dif-
ferent column on different day and estimating the %
RSD of the result obtained.
2.7.3. Sensitivity
Sensitivity was determined by establishing the Limit of
detection (LOD) and Limit of quantitation (LOQ) for
ASP, ATV, ATL, and LST estimated at a signal-to-noise
ratio of 3:1 and 10:1 respectively, by injecting a series of
dilute solutions with known concentration. The precision
study was also carried out at the LOQ level by injecting
six individual preparations of ASP, ATV, ATL, and LST,
calculated the % RSD for the areas of each drug.
2.7.4. Linearity
Linearity solutions were prepared from stock solution at
five concentration levels from 50 to 200% of analyte
concentrations (37.5 to 150 µg/mL for ASP, 5.0 to 20
µg/mL for ATV and 25.0 to 100 µg/ml for ATL and
LST). The peak area versus concentration data was col-
lected and performed regression analysis by the method
of least squares. The Correlation coefficient, Slope &
y-intercept values were calculated from the calibration
plot obtained.
2.7.5. Accuracy
The accuracy of the method was determined by measur-
ing the recovery of the drugs by the method of standard
additions. Known amounts of each drug corresponding to
50,100, and 150% of the target test concentrations (0.075
mg/mL of ASP, 0.01 mg/mL of ATV, 0.05 mg/mL each
of ATL and LST) were added to a placebo mixture to
determine whether the exccipients present in the formu-
lation led to positive or negative interferences. Each set
of additions was repeated three times at each level. Ex-
traction sample preparation procedure is followed and
assayed against qualified reference standard. The accu-
racy was expressed as the percentage of the analytes re-
covered by the assay.
2.7.6. Robustness
To determine the robustness of the developed method,
experimental conditions were deliberately changed and
the relative standard deviation for replicate injections of
ASP, ATV, ATL and LST peaks and the USP resolution
factor between ASP and SA peaks were evaluated. The
mobile phase flow rate was 1.0 mL/min. This was
changed by 0.1 units to 1.1 and 1.2 mL/min. 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 2.8 and 3.0.
2.7.7. Solution Stability and Mobile Phase Stability
The solution stability of ASP, ATV, ATL and LST was
carried out by leaving the test solution in tightly capped
volumetric flasks at room temperature for 48 h and as-
sayed at 6 h interval, against the freshly prepared stan-
dard solution. The mobile phase stability was carried out
by assaying the freshly prepared sample solution against
the freshly prepared standard at 6 h interval up to 48 h.
The percentage of RSD of assay of ASP, ATV, ATL and
LST was calculated for the study period during mobile
phase and solution stability experiments.
3. Results and Discussions
3.1. Method Development and Optimization
All the four drug solutions were prepared in diluent at a
concentration of 100 µg/mL and scanned in UV-Visible
spectrometer; all the drugs were having UV maxima at
around 230 nm. Hence detection at 230 nm was selected
for method development purpose.
The main analytical challenge during development of
a new method was obtaining adequate retention of the
polar parent compound, Atenolol (ATL) while maintain-
ing a reasonable elution time for the less-polar Atorvas-
tatin (ATV) and to separate one of the degradation impu-
rity Salicylic acid (SA) from the ASP peak.
To achieve this, Water and Acetonitrile (50:50 v/v)
mobile phase, on a C18 stationary phase with a 25 cm
length, 4.6 mm ID and 5µm particle size were investi-
gated. Experiments were also performed using the col-
umns of varying lengths from 250 to 100 mm. Different
mobile phase compositions containing phosphate buffer
and Acetonitrile (50:50-20:80 v/v) were also tried. But
was unsuccessful in getting good peak shapes for all the
peaks. Although good separation was achieved with 0.1%
phosphoric acid: Acetonitrile in the ratio of 50:50 (v/v),
Atorvastatin peak symmetry was found to be greater than
2.0. The asymmetry of the Atorvastatin peak was im-
proved by addition of Triethylamine (TEA) and adjusting
the mobile phase pH to 2.9 in the aqueous phase. The
chromatographic separation with better peak shape was
achieved using a mixture of aqueous 0.1% Orthophos-
phoric acid and Acetonitrile in the ratio of 50:50 (v/v).
The columns, 250, 150 and 125 mm × 4.6 mm, 5 μm of
varied lengths were tried but 150 × 4.6 mm, 5 μm C18
Column gave reasonable retention for all the peaks. But
when degradation samples were injected with these con-
ditions, one of the degradation impurity of ASP namely
salicylic acid (SA) was not separated from the ASP Peak.
Then method was optimized to separate all the de-
gradants from the main peaks by changing to Gradient
mode. Several gradient conditions were tried before op-
timizing the final gradient programme as: time (min)/%
solution B: 0/5, 10/60, 15/80, 17/60, 20/5, 25/5 Effect of

S. K. SHETTY ET AL.
63
the diluent on the peak shapes was studied. The ATL
peak was observed as split in most of the compositions
of the buffer and Acetonitrile. Finally Buffer: Acetoni-
trile (80:20, v/v) was optimized as the diluent to obtain
good peak shapes.
The satisfactory chromatographic separation, with
good peak shapes were achieved on Inertsil ODS-C18
(150 × 4.6) mm with 5 µm particles, using 0.1 % Or-
thophosphoric acid (adjusted to pH 2.9 with TEA) as
mobile phase A and Acetonitrile as solution B with a
flow rate of 1.0 mL/min. The HPLC gradient program
was optimized as: (time (min)/% solution B: 0/5, 10/60,
15/80, 17/60, 20/5, 25/5. The column temperature as
maintained at 35˚C and the detection was monitored at
a wavelength of 230 nm. The injection volume was 10
µL. Buffer: Acetonitrile (80: 20, v/v) was used as diluent.
In the optimized gradient conditions ATL, ASP, SA,
LST and ATV were well separated with a resolution
(Rs) of greater than 2 and the typical retention times of
ATL, ASP, SA, LST and ATV were about 3.3, 7.6, 8.1,
10.7 and 12.9 respectively, the typical chromatogram
of System suitability shown in Figure 2.
Peak purity of stressed samples of all the four drug sub-
stances were checked by using 2996 Photo diode array
detector of Waters (PDA). The purity angle within the pu-
rity threshold limit obtained in all stressed samples demon-
strates the analyte peak homogeneity. All stressed samples
of the drug product (heat (60˚C), acid hydrolysis (0.1N
HCl), base hydrolysis (0.1N NaOH), water hydrolysis and
oxidation (5% H2O2)) were analyzed for extended run
time of 60 min to check the late eluting degradants.The
System suitability results were given in (Table 1).
The proposed method is applied for the assay analysis
of 3 different batches of the polypills. The assay results
obtained were within the specification limit. The assay of
polypill is unaffected in the presence of excipients con-
firming the stability indicating power of the developed
method.
3.2. Method Validation
3.2.1. Precision
The percentage RSD values for the assays in precision
study were 0.4, 0.8, 0.4, 0.5% (intra-day precision) and
0.5, 0.8, 0.6, 0.7% (inter-day precision) for ASP, ATV,
ATL, and LST confirming a good precision and the rug-
gedness of the method ,The % RSD obtained for the
Ruggedness study were as shown in (Table 2).
3.2.2. Sensitivity
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, ATL
and LST were determined experimentally by injecting
Atenolol - 3.497
Aspirin - 7.674
Salicylic Acid - 8.090
Losartan - 10.722
Atorvastatin - 12.966
AU
0.00
0.05
0.10
0.15
0.20
Time (min)
2.00 4.00 6.008.0010.00 12.00 14.00 16.00 18.00 20.00
Figure 2. Typical chromatogram of system suitability.
Table 1. System suitability report.
Compound Retention time
USP Resolution
(RS)
USP Tailing factor
(T)
No of theoretical plates USP
tangent method (N)
Atenolol (ATL) 3.5 1.03 16743
Aspirin (ASP) 7.6 34.1 1.02 53435
Salicylic acid (SA) 8.1 2.8 1.1 46436
Losartan (LST) 10.7 16.6 0.99 76004
Atorvastatin (ATV) 12,9 13.4 0.97 91198

64 S. K. SHETTY ET AL.
Table 2. Results of intermediate precision.
S. No Parameter Variation % RSD for Assay
ASP ATV ATL LST
1 Different System
(a) Waters 2695 Alliance system
(b) Agilent 1100 series VWD system
0.7%
0.6%
0.3%
0.8%
0.5%
0.6%
0.7%
0.9%
2 Different Column
(a) B.No: 0014
(b)B.No:00118
0.4%
0.5%
0.5%
0.4%
0.7%
0.6%
0.6%
0.6%
3 Different Analyst
(a) Analyst-1
(b) Analyst-2
0.5%
0.4%
0.9%
0.7%
0.6%
0.6%
0.6%
0.8%
4 Different Days
(Interday precision)
(a) Day-1
(b) Day-2
(c) Day-3
0.4%
0.5%
0.6%
0.9%
0.8%
0.7%
0.3%
0.5%
0.4%
0.5%
0.6%
0.4%
each drug six times. The LOD for ASP, ATV, ATL and
LST were 0.1, 0.2, 0.15 and 0.02 µg/mL respectively.
The LOQ for ASP, ATV, ATL and LST were 0.3, 0.7,
0.4 and 0.06 µg/mL, respectively.
3.2.3. Linearity
The linear ranges were from (37.5 to 150 µg/mL for ASP,
5.0 to 20 µg/mL for ATV and 25.0 to 100 µg/mL for ATL
and LST).The correlation coefficient obtained was greater
than 0.999. The Slope and the Intercept value obtained
from the linear regression graph is as shown in (Table 3).
The result shows an excellent correlation existed between
the peak area and concentration of the analyte in the range
50-200% of analyte concentration.
3.2.4. Accuracy
The percentage recovery of the results obtained is listed
in Table 4, the results indicate the method enables highly
accurate simultaneous determination of the all the four
drugs in the polypill combination.
3.2.5. Robustness
Close observation of analysis results for deliberately
changed chromatographic conditions (flow rate, column
temperature and pH of the mobile phase) revealed that
the resolution between closely eluting peaks, namely
ASP and SA was always greater than 2.0 and also there
was not much effect on the peak shapes, illustrating the
robustness of the method (Table 5).
3.2.6. Solution Stability and Mobile Phase Stability
The % RSD of assay of Polypill during solution stability
and mobile phase stability experiments was within 1.0.
No significant changes were observed in the content of
ATL, ASP, LST and ATV during the study. The solution
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.
Table 3. Results of Linearity study for drug substance.
Atenolol (ATL) Aspirin (ASP) Losartan (LST) Atorvastatin (ATV)
Calibration Equation Y=8027X-28993 Y=18480X-65531 Y=17299X-38592 Y=1584X-6390
Linearity Range 50-200 % 50-200 % 50-200 % 50-200 %
Regression coefficient 0.999 0.999 0.999 0.999
Slope 8027 18480 17299 1584
Intercept -28993 -65531 -38592 -6390

S. K. SHETTY ET AL.
65
Table 4. Results of accuracy study for drug substance.
Analyte Initial Added Concentration RSD Recovery
concentration (%) concentration (mg)found (mg) (%) (%)
Atenolol (ATL) 50 24.9 24.8 0.23 99.60
100 50.1 49.8 0.28 99.41
150 74.8 74.9 0.22 100.13
Aspirin(ASP) 0 0 0
50 37.4 37.2 0.36 99.46
100 75.1 74.8 0.29 99.60
150 112.3 112.1 0.37 99.82
Losartan(LST) 0 0 0
50 25.2 25.1 0.40 99.60
100 50.1 49.9 0.38 99.62
150 74.8 74.9 0.34 100.13
Atorvastatin(ATV) 0 0 0
50 5.02 5.05 0.40 100.60
100 10.05 9.98 0.28 99.30
150 14.85 14.89 0.18 100.27
Table 5. Results of robustness study.
S.No Parameter Variation
Resolution (Rs)
between ASP
and salicylic acid
1
Temperature (±
5˚C of set
temperature)
(a) At 30°C
(b) At 40°C
2.9
2.7
2
Flow rate (±
20% of the set
flow)
(a) At
0.8 mL min-1
(b) At
1.2 mL min-1
2.9
2.8
3 pH Buffer
pH 2.8
pH 3.0
2.9
2.5
3.2.7. Results of Forced Degradation Studies
1) Degradation Behavior
Stress studies on combination of all the four drugs
under different stress conditions suggested the following
degradation behavior.
2) Degradation in Acidic solution
The combination of all the four drugs was exposed to
0.1 N HCl at 100°C for 1 h. ATL, ASP and ATV showed
considerable degradation. The drugs gradually under-
gone degradation with time in 0.1 N HCl and prominent
degradation was observed (Figure 3(a)).
3) Degradation in Basic solution
The combination of all the four drugs was exposed to
0.1 N NaOH under reflux for 2 h. ASP and ATV has
shown significant sensitivity towards the treatment of 0.1
N NaOH. The drug undergone degradation immediately in
0.1 N NaOH and prominent degradation was observed for
ASP with the conversion to SA (Figure 3(b)).
4) Oxidative Conditions
The combination of all the four drugs was exposed to
5% hydrogen peroxide at room temperature for 48 h. ASP,
LST and ATV has shown significant sensitivity towards
the treatment of 5% hydrogen peroxide and the drugs
gradually undergone oxidative degradation with time to
yield prominent degradation products (Figure 3(c)).
5) Photolytic Conditions
When the combination of all the four drugs was ex-
posed 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/mhr) (in photo stability
chamber). Major degradation observed with LST and
ATV (Figure 3(d)).
6) Thermal Degradation
When the drug product was exposed to dry heat at
60˚C for 8 h, Considerable degradation was observed
with ATL and ASP (Figure 3(e)).
Accelerated Stability sample:
The stability indicating nature of the method was fur-
ther confirmed by injecting three month accelerated sta-
bility sample and observed that all the degradants were
well separated from the main components (Figure 3(f)).
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.
No degradants were observed after 30 min in the ex-
tended runtime of 60 min of all the samples.
6) Assay analysis
Assay analysis was performed for different batches of
the drug product in tablets (n=3), with the targeted ana-
lyte concentrations. The assay results obtained for the
three Starpill tablets were, STP/002 (99.7% ATL, 99.8%
ASP, 100.25 LST and 99.6% ATV), STP/005 (99.67%
ATL, 100.3% ASP, 99.4% LST and 99.8% ATV) and
STP/011 (100.1% ATL, 99.9% ASP, 99.7% LST and
00.3% ATV) depicted in (Table 6). 1

S. K. SHETTY ET AL.
66
1.752
Atenolol - 3.372
3.834
4.035 4.374
4.780
5.590
Aspirin - 7.563
Salicylic Acid - 7.864
9.162
9.695
10.268 Losartan - 10.678
12.564
Atorvastatin - 12.900
13.927
14.311
14.930
15.717
17.029
AU
0.00
0.05
0.10
0.15
0.20
Time
(
min
)
2.004.00 6.008.00 10.00 12.0014.00 16.0018.00 20.0022.0024.00
(a)
Atenolol - 3.50
8
4.028 4.142
4.490
6.751
Salicylic Acid - 8.089
8.662
9.185
10.056
Losartan - 10.730
11.271
Atorvastatin - 12.972
13.280 13.789 14.076
14.279
AU
0.00
0.05
0.10
0.15
0.20
Time (min)
2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00
(b)
Atenolol - 3.516
4.352
4.491
5.134
6.387
Salicylic Acid - 7.946
8.622 8.927
9.128 9.278
9.967
10.349 Losartan - 10.702
11.218
11.338
11.462 11.872
12.106
Atorvastatin - 12.852
13.233
14.952
AU
0.00
0.05
0.10
0.15
0.20
Time (min)
2.004.006.008.00 10.00 12.00 14.0016.00 18.00 20.00 22.00 24.00
(c)

S. K. SHETTY ET AL.
67
Atenolol - 3.461
3.992 4. 321 4. 801
5. 091
6. 073 6. 248
6.709
Aspirin - 7.63
2
Salicylic Acid - 8.063
8. 606
Losartan - 10.663
11. 192
12. 409
Atorvastatin - 12.882
AU
0.00
0.05
0.10
0.15
0.20
Time
(
Min
)
2.00 4.006.00 8.0010.0012.00 14.00 16.0018.00 20.00 22.0024.00
(d)
Atenolol - 3.486
4.124 4.34 6
6.723
Aspirin - 7.633
Salicylic Acid - 8.025
10.136
Losartan - 10.685
11.187
12.411
Atorvastatin - 12.880
13.471
13.888
14.879
15.354
15.612
15.901
16.186
16.901
AU
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Time
(
min
)
2.00 4.00 6.00 8.0010.00 12.00 14.0016.0018.0020.00
(e)
Atenolol - 3.463
4.094 4.321
6. 699
Aspirin - 7.612
Salicylic Acid - 7.975
10. 128
Losartan - 10.667
12. 010
12. 401
Atorvastatin - 12.870
13. 884
14. 877
16. 898
AU
-0.02
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Time (min)
2.00 4.00 6.00 8.00 10.0012.00 14.0016.00 18.0020.00
(f)
Figure 3. (a) Acid Degradation sample 0.1N HCl (1h, 100˚C); (b) Base Degradation sample: 0.1N NaOH (2h, Reflux);
(c) Peroxide Dégradation sample: 5% Peroxide (48h, RT); (d) Photo stability degradation; (e) Thermal degradation:
60˚C for 8 h; (f) Accelerated 40˚C/75% RH degradation.

S. K. SHETTY ET AL.
68
Table 6. Batch analysis for starpill drug product.
Ba
N y)
tch
o:
ATL
(%assay)
ASP
(%assay)
LST
(%assay)
ATV
(%assa
S TP/002 99.7 99.8 100.2 99.6
ST5 P/0099.6 100.3 99.4 99.8
STP/011 100.1 99.9 99.7 100.3
3licaof ththod tabilityy
stablish the stability indicating nature of the method.
RP-LC method developed for simul-
neous quantitative assay of ASP, ATV, ATL and LST
the management of Un
tates Pharmacopeia laboratory-India for supporting th
erences
d M. R. Law, “A Strategy to Reduce Car-
diovascular Disease by More Than 80 Percent,” British
Risk Factors in Middle-Aged Individuals
[4] M. R. Law and N. J. Wald, “Risk factor thresholds: their
existence under scrutiny,” British Medical Journal, Vol.
d S. Singh, “LC and LC-MS
Davidson, R. J. Goldstein,
7, 2005, pp. 2571-2574.
.3. Apption e Meo St Stud
Accelerated conditions stability studies are performed to
e
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 LC
method for period of 3 month both initially and after
intervals of 1, 2, and 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.
3.4. Conclusions
The single gradient
ta
in Polypill is precise, accurate and specific. The method
was completely validated showing satisfactory data for
all the method validation parameters tested. The devel-
oped method is stability indicating and can be used for
the routine analysis of production samples and also to
check the stability of Polypill tablets.
4. Acknowledgements
The authors wish to thank ited
is [13] 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
S
work.
5. Ref
[1] N. J. Wald an
of Ph
Medical Journal, Vol. 326, No. 7404, 2003, p. 1419.
[2] G. Sanz and V. Fuster, “Fixed-Dose Combination Therapy
and Secondary Cardiovascular Prevention: Rationale, [15] 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, 2003,
Selection of Drugs and Target Population,” Nature Clinical
Practice Cardiovascular Medicine, Vol. 6, No. 2, 2009,
pp. 101-110.
[3] S. Yusuf, P. Pais, R. Afzal, et al., “Effects of a Polypill
(Polycap) on
without Cardiovascular Disease (TIPS): A Phase II,
Double-Blind, Randomised Trial,” Lancet, Vol. 373, No.
9672, 2009, pp. 341-351.
324, No. 7353, 2002, pp. 570-576.
[5] V. Kumar, R. P. Shah an
Methods for the Investigation of Polypills for the Treat-
ment of Cardiovascular Diseases: Part.1Separation of Ac-
tive Compo,” Journal of Pharmaceutical and Biomedical
Analysis, Vol. 47, 2008, pp. 508-515.
[6] V. Kumar, S. Malik and S. Singh, “Polypill for the treat-
ment of cardiovascular diseases: Part 2. LC-MS/TOF
characterization of interaction/degradation products of
atenolol/lisinopril,” Journal of Pharmaceutical and Bio-
medical Analysis, Vol. 48, No. 3, 2008, pp. 619-628.
[7] M. C. Koester, “An Overview of the Physiology and Phar-
macology of Aspirin and Nonsteroidal Anti-Inflammatory
Drugs,” Journal of Athletic Training, Vol. 28, No. 3,
1993, pp. 252-254, 256-259.
[8] S. P. Clissold, “Aspirin and Related Derivatives of Sali-
cylic Acid,” Drugs, Vol. 32, Supplement 4, 1986, pp. 8-
26.
[9] R. G. Bakker-Arkema, M. H.
“Efficacy and Safety of a New HMG-CoA Reductase
Inhibitor, Atorvastatin, in Patients with Hypertriglyceri-
demia,” Journal of the American Medical Association,
Vol. 275, No. 2, 1996, pp. 128-133.
[10] A. N. Wadworth, D. Murdoch, R. N. Brogden, “Atenolol:
A Reappraisal of its Pharmacological Properties and
Therapeutic Use in Cardiovascular Disorders,” Drugs,
Vol. 42, No. 3, 1991, pp. 468-510.
[11] B. M. Psaty, T. D. Koepsell, J. P. LoGerfo, et al.,
“B-Blockers and Primary Prevention of Coronary Heart
Disease in Patients with High Blood Pressure,” Journal of
the American Medical Association, Vol. 261, No. 14,
1989, pp. 2087-2094.
[12] K. L. Goa and A. J. Wag, “Losartan Potassium: A Re-
view of its Pharmacology, Clinical Efficacy and Toler-
ability in the Management of Hypertension,” Drugs, Vol.
51, No. 5, 1996, pp. 820-845.
armaceutical and Biomedical Analysis, Vol. 15, No.
1, 1996, pp. 73-82.
[14] D. G. Sankar, M. S. M. Raju, K. Sumanth and P. V. M.
Latha, “Asian HPLC Method for Estimation of Atorvas-
tatin in Pure and Pharmaceutical Dosage Form,” Journal
of Chemistry, Vol. 1
pp. 1017-1023.
[16] A. Puratchikody, R. Valarmathy, P. Shiju, “RP-HPLC
Determination of Atorvastatin Calcium in Solid Dosage
Forms,” Journal of Pharmaceutical Reviews, 2003, pp.
79-80.

S. K. SHETTY ET AL.
69
l. 63, No. 6, 2006, pp. 471-476.
ography B:
d Communications in Mass
Chemistry
005, pp. 182-186.
mination of
“Simultaneous Spectrophotometric Determi-
ultaneous Determina-
ous Determination of Losar-
[17] B. Stanisz and L. Kania, “Validation of HPLC Method
for Determination of Atorvastatin in Tablets and for
Monitoring in Solid Phase,” Acta Poloniae Pharmaceu-
tica, Vo
[18] G. Bahrami, B. Mohammadi, S. Mirzaeei and A. Kiani,
“Determination of Atorvastatin in Human Serum by Re-
verse Phase High Performance Liquid Chromatography
with UV Detection,” Journal of ChromatAna-
lytical Technologies in the Biomedical and Life Sciences,
Vol. 826, 2005, pp. 41-45.
[19] M. Jemal, Z. Ouyang, B. C. Chen and D. Teitz, “Quanti-
tation of Atorvastatin and its Bio-Transformation Products
in Human Serum by HPLC with Electro Spray Tandem
Mass Spectrometry,” Rapi
Spectrometry, Vol. 13, 1999, pp. 1003-1015.
[20] M. Hermann, H. Christensen and J. L. Reubsaet, “Deter-
mination of Atorvastatin and Metabolites in Human
Plasma with Solid Phase Extraction Followed by LC-
Tandem MS,” Analytical and Bioanalytical,
ta
Vol. 382, No. 5, 2005, pp. 1242-1249.
[21] S. R. Dhaneshwar, S. Yadav, A. Mhaske and S. Kadam,
“HPTLC Method for Determination of Content Uni-
formity of Atorvastatin Calcium Tablets,” Indian Journal
of Pharmaceutical Sciences, Vol. 67, 2
[22] C. V. N. Prasad, C. Parihar, K. Sunil and P. Parimoo,
“Simultaneous Determination of Amiloride, Hydrochloro-
thiazide and Atenolol in Combined Formulation by
Derivative Spectroscopy,” Journal of Pharmaceutical
and Biomedical Analysis, Vol. 17, 1998, pp. 877-884.
[23] S. M. Al-Ghannam, “A Simple Spectrophotometric
Method for the Determination of B-Blockers in Dosage
Forms,” Journal of Pharmaceutical and Biomedical
Analysis, Vol. 40, No. 1, 2006, pp. 151-156.
[24] A. P. Agrekar and S. G. Powar, “Reverse Phase High
Performance Liquid Chromatographic Deter
Ramipril and Amlodipine in Tablets,” Journal of Phar-
maceutical and Biomedical Analysis, Vol. 21, 2000, pp.
1137-1142.
[25] M. B. Shankar, F. A. Metha, K. K. Bhatt, R. S. Metha and
M. Geetha,
nation of Losartan Potassium and Hydrochlorothiazide in
Tablets,” Indian Journal of Pharmaceutical Sciences,
Vol. 65, No. 2, 2003, pp. 167-170.
[26] C. F. Pedroso, J. G. de Oliveira, F. R. Campos, et al., “A
Validated RP-LC Method for Sim
tion of Losartan Potassium and Amlodipine Besilate in
Pharmaceutical Preparations,” Chromatography, Vol. 69,
Supplement 2, pp. 201-206.
[27] D. D. Rao, N. V. Satyanarayana, S. S. Sait, Y. R. Reddy
and K. Mukkanti, “Simultane
n Potassium, Atenolol and Hydrochlorothiazide in Phar-
maceutical Preparations by Stability-Indicating UPLC,”
Chromatography, Vol. 70, No. 4, 2009, pp. 647-651.
[28] “ICH Stability Testing of New Drug Substances and
Products Q1A (R2),” International Conference on Har-
monization, IFPMA, Geneva, 2003.
[29] “ICH guidelines on Validation of Analytical procedures,
Text and Methodology Q2 (R1),” FDA, Published in the
ention, Rockville, 2009.
Federal Register 60, 1995.
[30] “United States Pharmacopoeia,” 32nd Edition, United
States Pharmacopeial Conv
[31] M. Bakshi and S. Singh, “Development of Validated
Stability-Indicating Assay Methods-Critical Review,”
Journal of Pharmaceutical and Biomedical Analysis, Vol.
28, No. 6, 2002, pp. 1011-1040.