Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301

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Bulletin of Faculty of Pharmacy, Cairo University

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Original Article

Multivariate optimization for simultaneous determination of aspirin and

simvastatin by reverse phase liquid chromatographic method using
AQbD approach
Kalpana G. Patel a,⇑, Apeksha T. Patel a, Purvi A. Shah a, Tejal R. Gandhi b
a
Department of Quality Assurance, Anand Pharmacy College, Opp. Town Hall, Anand, Gujarat 388001, India
b
Department of Pharmacology, Anand Pharmacy College, Opp. Town Hall, Anand, Gujarat 388001, India

a r t i c l e i n f o a b s t r a c t

Article history: The present study describes the development of a robust method for the separation of aspirin and sim-
Received 16 March 2017 vastatin using reverse phase high performance liquid chromatographic method on Kintex reverse phase
Received in revised form 5 August 2017 C18 column (5 lm, 250 mm  4.6 mm) with UV detection at 234 nm. Box-Behnken design was applied for
Accepted 27 August 2017
multivariate optimization of the experimental conditions of RP-HPLC for obtaining desired chromato-
Available online 12 September 2017
graphic resolution with limited number of experiments. Risk assessment was performed to identify
the critical method parameters. Three independent parameters; volume of acetonitrile, molarity of buffer
Keywords:
and flow rate were used to design mathematical models and study the in depth effects of these indepen-
Aspirin
Simvastatin
dent factors on various responses. The optimized and predicted condition consisted of acetonitrile and
Box-Behnken design potassium dihydrogen orthophosphate buffer pH 2.9 adjusted with orthophosphoric acid (83.89:16.11,
HPLC v/v) as mobile phase at a flow rate of 0.93 ml/min. Using these optimum conditions, baseline separation
of both drugs with good resolution and a run time of less than 6 min was achieved. Perturbation plot
revealed that the most important factor affecting the selected responses was volume of acetonitrile.
Percent recoveries in terms of accuracy for both drugs at all three levels was found in the range of
99.15–101.86%. The pooled % relative standard deviation values for repeatability and intermediate preci-
sion studies was found to be less than 2% for both drugs. Hence, a robust, simple, accurate and repro-
ducible high performance liquid chromatographic method was developed and validated and could be
applied for routine quality control testing.
Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).

1. Introduction myocardial infarction. Simvastatin chemically is (1S,3R,7S,8S,8aR)-

Cardiovascular diseases are the major cause of death in devel-
oped countries, projecting almost 1.5 billion people by 2025 [1].
Among these, ischemic heart disease, a major risk factor affects
more than 4.69 million people in India. A clinical study has shown
that concomitant therapy of aspirin and simvastatin led to 15–21%
increase in survival chances of patients as compared to single ther-
apy of either aspirin or simvastatin [2].
Aspirin chemically is 2-(acetyloxy)benzoic acid and irreversibly
inhibits the synthesis of TXA2 by platelets and thus interferes
with platelet aggregation and hence is used in reducing risks of
Peer review under responsibility of Faculty of Pharmacy, Cairo University.
⇑ Corresponding author at: Quality Assurance Department, Anand Pharmacy
College, Opp. Town Hall, Anand, 388 001 Gujarat, India.
E-mail address: kalpanapatel.pharma@gmail.com (K.G. Patel).
8-[2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]ethyl]-3,7-
dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl 2,2-Dimethylbu-
tanoate and decreases cholesterol synthesis by inhibiting rate
limiting step in synthesis of cholesterol [3].
Various analytical methods such as UV–Visible spectrophotom-
etry, high performance liquid chromatography and high perfor-
mance thin layer chromatography have been reported for both
drugs individually as well as with other combinations [4–12]. Prior
art search regarding analytical method for combination of aspirin
and simvastatin depicts that high performance thin layer chro-
matography has been performed [13]. Since, high performance liq-
uid chromatography is more sensitive, the present study focuses
on development of HPLC method using AQbD approach followed
by validation.
Analytical Quality by design (AQbD) is defined as a systematic
approach to development that begins with predefined objective

http://dx.doi.org/10.1016/j.bfopcu.2017.08.003
1110-0931/Ó 2017 Publishing services provided by Elsevier B.V. on behalf of Faculty of Pharmacy, Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
294 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301
and emphasis method understanding and control based on sound
science and quality risk management. The AQbD plays an impor-
tant role in developing a robust method as early risk assessment
helps to identify critical analytical parameters and to focus on
these factors in method development. Quality of the method is
ensured throughout the product lifecycle due to AQbD principles
[14,15]. Traditional method of optimization study is time consum-
ing, requires large number of runs and further modification is
required during its lifecycle. Hence, QbD in analytical methodology
has gained increased attention since the application of QbD in
pharmaceutical product development, including analytical meth-
ods such as RP-HPLC, used for quality control and analysis of active
pharmaceutical ingredients (API) and drug products for product
quality and thereby assuring patient safety [16].
Method transfer and reproducibility in inter-laboratory studies
are the potential benefits of application of AQbD. Furthermore,
experimental design is a good alternative than traditional
approach for proper planning and conducting of optimization
study [17,18]. For optimization of the analytical methodology in
terms of design space where the response exhibits the optimum,
various experimental design have gained considerable attention.
The most convenient three-level factorial designs applied are cen-
tral composite design, Box–Behnken (BBD) design or Doehlert
matrix design [19,20].
This research article focuses on the optimization of HPLC chro-
matographic conditions by BBD. BBD is a response surface design
for multivariate optimization characterized by set of points lying
at the midpoint of each edge of a multidimensional cube and cen-
tre replicates. Selection of critical parameters and responses is an
important aspect for the development of HPLC method. In line with
this, preliminary experiments and risk assessment was performed
for identification of critical parameters based on its effect on
response. Besides preliminary experiments, Ishikawa (fishbone)
diagram was used for the identification and assessment of critical
parameters posing overall risk to the performance of the method.
The factors such as flow rate, molarity of buffer and volume of ace-
tonitrile were found to have significant effect on chromatographic
separation and hence were optimized using BBD. These three influ-
ent chromatographic parameters; flow rate, molarity of buffer and
volume of acetonitrile, on the basis of optimized BBD experimental
domain were varied within a real range, and their quantitative
influence on the response variable, retention time of aspirin, reso-
lution of simvastatin, theoretical plates of aspirin, retention time of
simvastatin and tailing factor of simvastatin was determined. So,
the main objective of present research study was to develop a sim-
ple, precise, accurate, robust and specific method suitable for the
quality control testing of aspirin and simvastatin combination
using BBD.
2. Experimental
2.1. Materials
Pure aspirin and simvastatin were obtained as a gratis sample
from Baroque Pharmaceuticals Pvt. Ltd., Khambhat, India and
Zydus Cadila Pvt. Ltd, Ahmedabad, India, respectively. All solvents
and chemicals used were of HPLC grade, purchased from Merck
Specialities Pvt. Ltd., Mumbai, India.
2.2. Instrumentation
A HPLC instrument (LC-2010C HT, Shimadzu, Japan) was
used. The system also includes photodiode array (Shimadzu
SPD- M20A) detector. Data were acquired and processed using LC
solution software version 1.25. Analytical balance AUW 220D (Shi-
madzu, Japan) with minimum 1 mg sensitivity was used.
2.3. Chromatographic conditions
Chromatographic separation was performed using Kintex
reverse phase C18 column (5 lm, 250 mm  4.6 mm). The mobile
phase consisted of acetonitrile and potassium dihydrogen
orthophosphate buffer pH 2.9 adjusted with orthophosphoric acid
(83.89:16.11, v/v). The mobile phase was prepared daily followed
by filtration through 0.45 lm nylon membrane filter and sonicated
for 15 min. The flow rate of the mobile phase was optimized as
0.93 ml/min and the column temperature was maintained at ambi-
ent conditions. The injection volume was 10 ml. The chromatogram
was monitored at detection wavelength of 234 nm. Mobile phase
was used as a diluent.
2.4. Preparation of standard solutions
A standard stock solution of aspirin and simvastatin (1000 lg/
ml) was prepared individually by dissolving accurately weighed,
10 mg of drug in 10 ml volumetric flask in some quantity of mobile
phase and final dilution up to the mark with the mobile phase.
From this stock solution, 1 ml aliquot was transferred and diluted
up to the mark with methanol in 10 ml volumetric flask to obtain a
final concentration of 100 lg/ml for both, aspirin and simvastatin.
2.5. Preparation of mobile phase
The mobile phase was prepared by mixing acetonitrile and
potassium dihydrogen orthophosphate buffer followed by adjust-
ment of pH to 2.9 with orthophosphoric acid (83.89:16.11, v/v).
2.6. AQbD assisted method development
The development of HPLC method for the simultaneous estima-
tion of aspirin and simvastatin in the newer developed capsule
dosage form was performed by applying AQbD principles to
decrease variability in performance of the method. The AQbD
approach involves definition of method goals, risk assessment,
selection of critical parameters and response, experimental design,
design space, working point selection and verification and method
control strategy (Fig. 1).
2.7. Optimization of chromatographic conditions using BBD
Various preliminary experiments were conducted for increasing
the understanding about method performance and identification of
critical independent parameters and its effect on dependent vari-
ables, by trial and error. The chromatogram was recorded in vari-
ous composition and ratios of mobile phase using solvents such
as acetonitrile, methanol and different buffers like potassium dihy-
drogen orthophosphate buffer and di-sodium hydrogen orthophos-
phate buffer of different pH to achieve proper separation of both
aspirin and simvastatin drugs with acceptable system suitability
parameters in terms of retention time, theoretical plates, resolu-
tion and asymmetric factor. Selection of critical parameters and
responses is an important aspect for the development of HPLC
method using AQbD approach with the help of preliminary exper-
iments and risk assessment. Besides preliminary experiments, Ishi-
kawa (fishbone) diagram was used for the identification and
assessment of critical parameters posing overall risk to the perfor-
mance of the method. Further, Box–Behnken design (BBD) was
used to optimize the chromatographic conditions. BBD is a
response surface design for multivariate optimization character-
ized by set of points lying at the midpoint of each edge of a multi-
 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301 295

Fig. 1. Graphical representation of method development flow using AQbD approach.

dimensional cube and centre replicates. A BBD design comprising
of a standard set of 3 factors and 3 levels was developed resulting
in total of 17 chromatographic conditions, with 5 centre points
(Table 1). Bias effect of uncontrolled factors was minimized due
to randomization of experimental runs. Hence, systematic scouting
resulted in selection of three key critical parameters viz. flow rate,
molarity of buffer and volume of acetonitrile. For separative ana-
lytical methods like chromatography, the CQAs can be related to
the system suitability parameters in terms of retention time, theo-
retical plates, resolution and asymmetric factor along with accu-
racy and robustness of method.
The nominal value for all these three factors, flow rate, molar-
ity of buffer and volume of acetonitrile, were 1 ml/min, 15 mM,
75 ml, respectively. In context to this, flow rate (A) was kept
between 0.8 ml/min and 1.2 ml/min. Similarly, minimum and
maximum content of molarity of buffer (B) were fixed as 10
and 20 mM, respectively. Likewise, minimum and maximum val-
ues for volume of acetonitrile (C) were selected as 65 and 85 ml,
respectively. The data generated were analyzed using Design
Expert (Version 9.0.1, Stat-Ease Inc., Minneapolis, MN, USA) sta-
tistical software.
2.8. Validation of method
The method was validated with respect to various parameters
including system suitability testing, linearity, limit of detection,
limit of quantitation, precision, accuracy, specificity and robust-
ness according to ICH Q2 (R1) guidelines [21].
2.8.1. System suitability test
System suitability was determined by six replicate injections of
the standard solution of aspirin and simvastatin (100 mg/ml) before
the sample analysis. The results of system suitability parameters;
resolution, theoretical plate (N), tailing factor (T) were evaluated
in terms of %RSD for six replicate injections of the drugs.
2.8.2. Linearity and range
Linear relationship between peak area and concentration of
both drugs was evaluated over the concentration range of 50–
300 lg/ml for aspirin and 20–120 lg/ml for simvastatin by per-
forming five replicate measurements. The analytical range was
selected by highest and lowest concentration of the analyte where
acceptable linearity, accuracy and precision were obtained. More-
over, Bartlett lest was used to check the heteroscedasticity of lin-
earity data.
2.8.3. Limit of detection (LOD) and limit of quantitation (LOQ)
As per ICH guideline, LOD and LOQ of the developed method
were calculated from the standard deviation of the response and
slope of the calibration curve of each drug using the formulae, limit
of detection = 3.3  r/S and limit of quantitation = 10  r/S,
where, r is standard deviation of response and S is the slope of cal-
ibration curve.
2.8.4. Precision
The precision of proposed method was studied by performing
repeatability and intermediate precision. Three replicates of three

Table 1
Experimental design domain (BBD) for each run with responses.

Run Factors Responses

Flow rate (A) Molarity of buffer Volume of Rt of Resolution of Theoretical plates of Rt of Tailing factor of
(ml/min) (B) (mM) acetonitrile (C) (ml) Aspirin Simvastatin (R2) Aspirin (R3) Simvastatin Simvastatin (R5)
(R1) (R4)
1 0.8 20 85 3.078 27.876 5595.11 8.510 1.173
2 1.0 15 75 2.428 27.907 5254.83 7.041 1.181
3 1.2 10 65 2.116 25.414 4067.05 6.098 1.169
4 1.0 15 75 2.414 27.650 5096.23 6.986 1.182
5 1.0 20 65 2.472 44.533 4898.28 12.331 1.123
6 1.0 20 75 2.422 17.023 5785.31 4.858 1.173
7 1.0 10 75 2.433 38.323 3648.63 10.622 1.134
8 1.2 15 65 1.944 38.432 3557.18 9.162 1.14
9 0.8 10 75 3.137 29.918 5666.55 9.164 1.173
10 1.0 15 75 2.411 27.696 5254.82 7.035 1.181
11 1.0 15 75 2.400 27.771 5087.71 6.997 1.181
12 0.8 15 85 3.113 18.349 6578.09 6.233 1.221
13 1.0 10 85 2.452 16.486 6429.34 4.749 1.251
14 1.0 15 85 2.410 27.920 5045.08 7.028 1.181
15 0.8 15 65 2.985 42.760 4540.43 13.681 1.112
16 1.2 15 75 2.025 15.549 4722.07 4.029 1.275
17 1.2 20 75 2.040 24.124 4133.05 5.633 1.217
296 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301
different concentrations (50, 150 and 300 lg/ml of aspirin and 20,
60 and 120 lg/ml of simvastatin) were analyzed on the same day
for repeatability and on different days to ascertain intermediate
precision. Over all mean, % RSD was calculated.
2.8.5. Accuracy
The accuracy was assessed by the methodological recovery
studies to check the recovery of both drugs at different levels by
optimized method. It was carried out by adding known amount
of standard to developed formulation for each drug at 50, 100
and 150% level and analyzed by the proposed method, in triplicate.
Recovery studies for aspirin were carried out by spiking three dif-
ferent amount of aspirin standard (50, 100 and 150 lg/ml) to the
developed formulation (100 lg/ml) by standard addition method.
Similarly, recovery studies for simvastatin were carried out by
spiking three different amounts of simvastatin standard (20, 40
and 60 lg/ml) to the synthetic mixture (40 lg/ml) by standard
addition method. % Recovery was then calculated for both drug.
2.8.6. Robustness
To test the capacity of this newly developed analytical proce-
dure to withstand small deliberate changes in the method, various
factors were deliberately changed like change in volume of ace-
tonitrile (85.89 and 81.89 ml); change in wavelengths (232 nm
and 236 nm); and change in flow rates (0.98 and 0.88 ml/min).
2.8.7. Specificity
Chromatogram of synthetic mixture representing formulation
was compared with the blank and standard solution of same drugs
to find interference of excipients. Chromatogram of excipients
must not show any significant interference at the retention time
of both aspirin and simvastatin.
2.9. Analysis of developed formulation
The developed HPLC method was used to quantify aspirin and
simvastatin in the developed capsule dosage form. Analysis of pre-
pared formulation containing 75 mg aspirin and 20 mg simvastatin
was carried out. All the contents of the capsule were triturated in
mortar pestle and then carefully transferred in a 100 ml volumetric
flask containing mobile phase and sonicated for 5 min followed by
dilution up to the mark with the same diluent, and filtered using a
0.45 lm nylon membrane filter. The final concentration obtained
was 750 and 200 lg/ml for aspirin and simvastatin respectively.
Working sample solutions were freshly prepared by appropriate
dilution of stock solution with mobile phase to obtain final concen-
tration of 150 and 40 lg/ml for aspirin and simvastatin respec-
tively. This solution was injected in HPLC system and the method
described above was then applied for the determination of peak
area and triplicate analysis was performed by following the same
procedure.
2.10. Statistical analysis
Statistical analysis was performed using Microsoft excel 2007
software, and included computation of linear regression analysis,
standard deviation, mean, relative standard deviation. Design
expert software version 9.0.1. was used for response surface opti-
mization with ANOVA and construction of Perturbation plot, 3D
response surface plot. Bartlett’s test and test for Lack of fit were
applied on the data of areas of linearity for evaluation of
homoscedasticity of variance and deviation from linearity [22].
3. Results and discussion
3.1. AQbD assisted method development
The primary goal of analytical quality by design (AQbD) was to
separate both peaks in less run time with good peak shape, satisfy-
ing parameters. Secondary goals were accuracy and precision of
HPLC method with % RSD less than 2, resulting in a robust method
with reduced variability. The mobile phase conditions were opti-
mized so that both the drugs would be separated in short run time.
Based on literature review, several mobile phase and their different
ratios were tried like acetonitrile, methanol, di-sodium hydrogen
orthophosphate buffer, potassium dihydrogen orthophosphate
buffer along with different pH and their combinations with the
objective of satisfying system suitability parameter. Various ratio
of di-sodium hydrogen orthophosphate buffer pH 2.9: acetonitrile
were tried, which illustrates that high concentration of buffer lead
to increased retention time of simvastatin. The ratio di-sodium
hydrogen orthophosphate buffer pH 2.9:acetonitrile 70:30 was
better in all aspects except that the solvent interference was found.
Potassium dihydrogen orthophosphate buffer with different pH
was tried but as pH of buffer increased, it was observed that peak
of simvastatin was broadened. The sample was then prepared
using mobile phase as diluent that removed solvent interference.
Hence, preliminary experiments revealed that mobile phase com-
position, potassium dihydrogen orthophosphate buffer: acetoni-
trile was suitable due to acceptable system suitability parameters
experiment. Moreover, volume of acetonitrile led a large change
in retention time in preliminary experiments. From Ishikawa dia-
gram and preliminary experiments that were conducted, critical
parameters selected for the further study were molarity of buffer
(mM), volume of acetonitrile (ml), as method variables and flow
rate (ml/min) as instrumental parameter, that were found to have
most influential effect on system suitability parameters (Fig. 2).
BBD is a response surface design for multivariate optimization
characterized by set of points lying at the midpoint of each edge
of a multidimensional cube and centre replicates. BBD was used
to optimize chromatographic conditions by considering the effects
of different factors such as flow rate (A), molarity of buffer (B) and
volume of acetonitrile (C) on retention time of aspirin (R1), resolu-
tion of simvastatin (R2), theoretical plates of aspirin (R3), retention
time of simvastatin (R4) and tailing factor of simvastatin (R5).
Table 1 represents the effect of all these three variables on
responses like retention time of aspirin (R1), resolution of simvas-
tatin (R2), theoretical plates of aspirin (R3), retention time of sim-
vastatin (R4) and tailing factor of simvastatin (R5) were selected
as responses/CQAs.
The model was also validated by analysis of variance (ANOVA)
using Design Expert software and the results are as presented in
Table 2. Based on PRESS value, quadratic model was selected for
resolution of simvastatin (R2) and 2FI model was selected for
retention time of aspirin (R1), theoretical plates of aspirin (R3),
retention time of simvastatin (R4) and tailing factor of simvastatin
(R5). Significant effects showed p value less than 0.05. Adequate
Precision defined as a signal-to-noise ratio greater than 4 is
desirable, and the obtained ratio for all the responses indicated
an adequate signal (Table 2). The low standard deviation (% CV)
and high adjusted R-square value indicated a good relationship
between the experimental data and those of the fitted models.
The predicted R-square value was in acceptable concordance with
the adjusted R-square value for all responses. The final equation, in
terms of actual components and factors, which can be used to
make predictions about the response for given levels of each factor,
are for retention time of Aspirin (R1), 10.265+18.58 * A-0.204 *
B+0.114 * C-0.280 * A * B-0.18 * A * C+6.15500E 003 * B * C; for
 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301 297

Fig. 2. The Ishikawa (fishbone) diagram to identify potential variables in HPLC method development.

Table 2
ANOVA regression analysis for models and responses.

Response Standard Mean % C.V. PRESS r2 Adjusted Predicted Adequate p value

deviation R-square R-square Precision
Retention time of Aspirin (R1) 0.039 2.49 1.58 0.17 0.995 0.989 0.927 37.90 0.015
Resolution of Simvastatin (R2) 1.64 28.1 5.83 72.44 0.97 0.965 0.94 35.25 0.016
Theoretical plates of Aspirin (R3) 130.73 5021.16 2.60 13,4564 0.99 0.970 0.98 31.84 0.017
Retention time of Simvastatin (R4) 0.42 7.66 5.47 19.64 0.98 0.970 0.91 30.00 0.017
Tailing factor of Simvastatin (R5) 0.021 1.18 1.79 0.012 0.9 0.950 0.98 13.01 0.009

resolution of Simvastatin (R2), 105.72+557.02 * A+2.66 * B
3.45 * C-0.92 * A * B-3.59 * B * C-0.01 * B * C-134.78 * A2-0.048680 *
B2+0.043 * C2; for theoretical plates of Aspirin (R3), +12,540.005+
2558.15 * A-714.59 * B-178.35 * C-368.74 * A * B+45.10 * A * C+14.37 *
B * C; for Retention time of Simvastatin (R4), 81.57+120.64 *
A-0.43 * B+1.05 * C-0.73 * A * B-1.41 * A * C+0.01 * B * C; for Tailing
factor of Simvastatin (R5), +2.59–1.60 * A-0.03 * B-0.01 * C+0.02 *
A * B+0.016 * A * C+1.15000E 004 * B * C. A positive value represents
an effect that favours the optimization, while a negative value indi-
cates an inverse relationship between the factor and the response.
Response surface and perturbation plots were constructed to
evaluate the effect of the factors on selected responses. Perturba-
tion plots reveal the change in response from its nominal value
and curvature indicates magnitude to factor. The Fig. 3b shows that
molarity of buffer showed most prominent effect on resolution of
simvastatin. Change in flow rate resulted in decrease in resolution
of simvastatin at both lower level and higher level from reference
point. Change in volume of acetonitrile resulted in increase in res-
olution of simvastatin at lower level and decrease in resolution of
simvastatin at higher level from reference point. Similarly, change
in molarity of buffer revealed increased resolution at lower level
and decrease in resolution up to certain point at higher level from
reference point.
As shown in Fig. 4(a–e), the three- dimensional graphs of RSM
are generated as a function of the significant variables while the
third variable was held constant at a specified level, usually the
proposed optimum. The representative plots for responses are pre-
sented in Fig. 4(a–e) in which the interaction between the variables
(factors A and B) and their mutual dependence is clearly observed,
while factor C is kept constant. Fig. 4(a) and (b) indicates that flow
rate showed prominent effect on retention time of aspirin and res-
olution of simvastatin. The retention time of aspirin increased with
decrease in flow rate and molarity of buffer (Fig. 4(a). The resolu-
tion of simvastatin increased with increase in flow rate up to a
certain value and then decreased gradually while molarity of buffer
showed insignificant effect on resolution of simvastatin Fig. 4(b).
Fig. 4(c) demonstrates that the theoretical plates of aspirin
decreased with decrease in flow rate and increase in molarity of
buffer. Fig. 4(d) represents that retention time of simvastatin
increased with decrease in flow rate while molarity of buffer
showed insignificant effect on retention of simvastatin. Fig. 4(e)
depicts that the flow rate showed prominent effect on tailing factor
of simvastatin, where tailing factor of simvastatin decreased with
decrease in flow rate whereas molarity of buffer showed insignifi-
cant effect.
During numerical optimization, the design offered 30 solutions
for optimized chromatographic condition, but the solutions were
reduced by setting the goals. Various constraints for factor and
responses were set and three solutions were selected for optimiza-
tion of chromatographic conditions. It was demonstrated that the
observed value of optimized chromatographic condition was quite
closer to the predicted value, resulting in less % error and high
desirability. Hence, the optimized condition; flow rate (0.93 ml/
min), molarity of buffer (15.81 mM) and volume of acetonitrile
(83.89 ml) resulted in retention time of aspirin and simvastatin
at 2.4 and 4.6 min, respectively (Fig. 5). Design space is used for
establishment of a multidimensional space based on method fac-
tors and settings; design space hence can provide suitable method
performance in terms of system suitability and retention time.
3.2. Validation of method
System suitability testing is used as method control strategy.
System suitability tests are an integral part of method develop-
ment and were performed to evaluate the behaviour of the chro-
matographic system. The results of system suitability testing,
retention time (2.34 ± 0.635 for ASP and 4.64 ± 0.686 for SIM), res-
olution (16.3 ± 0.877), theoretical plate (2393.432 ± 0.147 for ASP
298 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301

Fig. 3. (a–e) Perturbation plot showing effect of factors on (a) retention time of aspirin, (b) resolution of simvastatin, (c) theoretical plates of aspirin, (d) retention time of
simvastatin and (e) tailing factor of simvastatin.
 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301 299

Fig. 4. (a–e) 3 dimensional plot of RSM showing effect of factors (a) retention time of aspirin, (b) resolution of simvastatin, (c) theoretical plates of aspirin, (d) retention time
of simvastatin and (e) tailing factor of simvastatin.
300 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301

Fig. 5. Optimized HPLC chromatogram for aspirin and simvastatin at 234 nm.

and 14,660.23 ± 0.499 for SIM), tailing factor (1.467 ± 0.826 for ASP ity was also confirmed by Lack of Fit [18], where the deviation of
and 1.228 ± 0.952 for SIM), and % RSD evaluated for six replicate the value of response when computed in terms of F ratio for both
injections were found to be acceptable. Aspirin and simvastatin the drugs was less than the tabulated one (Table 3). The LOD and
showed a good correlation coefficient (r2 = 0.9999 for aspirin and LOQ were found to be 1.282 and 3.885 mg/ml, respectively for
0.9991 for simvastatin) in the given concentration range 50– aspirin and 0.991 and 3.003 lg/ml respectively for simvastatin.
300 lg/ml for aspirin and 20–120 lg/ml for simvastatin respec- Repeatability and intermediate precision was carried out by per-
tively (Table 3 and Fig. 6). Homoscedasticity of variance was con- forming three replicates of three different concentration (50, 150
firmed by Bartlett’s test and the response of peak area for both and 300 lg/ml of aspirin and 20, 60 and 120 lg/ml of simvastatin)
drugs showed homogenous variance that was exemplified by the showed % RSD less than 2% (Table 3), indicating acceptable preci-
v2 value less than the tabulated value (Table 3). Moreover, linear- sion in terms of repeatability of peak area measurement and sam-
ple application. The percentage recovery for both the drugs at three
concentration levels (50, 100, 150%) was found in the range of
99.15–101.86%, suggesting suitability of method to perform rou-
Table 3
Analytical parameters of proposed HPLC method. tine drug analysis (Table 3). The robustness of the proposed
method was evaluated by making small deliberate variations in
Parameters Aspirin Simvastatin
liquid chromatography conditions. Moreover, the method was
Linearitya found to be specific as the excipients did not produce any interfer-
Range (lg/ml) 50–300 20–150
ence at the retention time of both drugs. In this study, the chro-
Correlation coefficient (r2) 0.9999 0.9991
SD of slope 96.044 136.964
matographic parameters monitored were retention time and
Confidence interval of slope 19,983.991– 33,759.64– area. The results of analysis of robustness study are as shown in
19,826.009 33,534.36 Table 3, where % RSD less than 2 indicated that the method is
SD of intercept 7733.103 10,105.22 robust, and is not affected by small changes in routine use.
Confidence interval of intercept 19,012.2–6292.04 57,125.38
Bartlett’s testb (v2) 0.0008 0.0007
Lack of fitc 0.0001 0.0002 3.3. Analysis of developed formulation
Sensitivity
Limit of detection (lg/ml) 1.282 0.991 The developed HPLC method in the present study was used to
Limit of quantitation (lg/ml) 3.885 3.003
quantify aspirin and simvastatin in the newer developed capsule
Precisiond dosage form. Percentage amount of drug found on triplicate analy-
Repeatability (% RSD) 0.460–1.029 0.398–0.751
sis for both aspirin and simvastatin was found to be 99.782 ± 0.699
Intermediate precision (% RSD) 1.066–1.282 0.792–1.029
and 99.958 ± 1.1354% w/w, respectively and hence the developed
Accuracye
method can be routinely used for analysis of aspirin and
Recovery studies 99.19–101.57 99.15–101.86
simvastatin.
Robustness
Change of flow rate (% RSD) 1.571 1.522
Change in mobile phase 1.652 1.932
4. Conclusion
composition (% RSD)
Change in wavelength (% RSD) 1.362 1.251
A simple, sensitive, accurate, robust, economical and precise
a
Mean of five replicates.
HPLC analytical method using AQbD approach has been developed
b
v2 (0.05, 5) value <11.070 at 95% confidence interval.
c
Critical value = 2.759 at a = 0.05.
for the simultaneous determination of aspirin and simvastatin in
d
Mean of three concentration/three replicates. newer developed capsule dosage form. Using AQbD approach,
e
Average of three determinations at each level. experimental design allowed evaluating the selected factors simul-
 K.G. Patel et al. / Bulletin of Faculty of Pharmacy, Cairo University 55 (2017) 293–301
Fig. 6. Overlay chromatogram showing linearity of aspirin (50–300 mg/ml) and simvastatin (20–120 mg/ml).
taneously, including interactions between factors, by means of a
rational approach in order to reach the optimum conditions.
Flow rate was found to be the most significant factor for reten-
tion time of aspirin and simvastatin, as well as for resolution of
simvastatin by BBD. Molarity of buffer and volume of acetonitrile
both were found to have profound effect on theoretical plates of
aspirin while volume of acetonitrile was the most important factor
for tailing factor of simvastatin. It is concluded that the use of
experimental design and multi-criteria decision making approach
is a flexible procedure, able to reduce the number of the needed
experiments for the development and optimization of HPLC
method can generate maximum information in a less time with
small number of experiments. The good % recovery in formulation
suggests that the excipients present in the formulation have no
interference in the determination. The validation study supported
the selection of the best conditions by confirming that the method
was specific, accurate, linear, precise, and robust. Therefore, the
same methodology can be applied for quality control testing and
estimation of aspirin and simvastatin.
Acknowledgement
We are highly grateful to Gujarat Council of Science and Tech-
nology, Gujarat for providing funds for this research study.
Compliance with ethical standards
Funding: This study was funded by Gujarat Council of Science
and Technology, Gujarat as minor research project grant.
Conflict of Interest: The authors declare that they have no con-
flict of interest.
References
[1] P.M. Kearney, M. Whelton, K. Reynolds, P. Muntner, P.K. Whelton, Global
burden of hypertension: analysis of world wide data, Lancet 365 (2005) 217–
223.
[2] C. Lewinter, J.M. Bland, S. Crouch, J.G.F. Cleland, P. Doherty, M.M. Lewinter,
Impact of aspirin and statins on long-term survival in patients hospitalized
with acute myocardial infarction complicated by heart failure: an analysis of
1706 patients, Eur. J. Heart Failure 16 (2014) 95–102.
[3] K.D. Tripathi, Essentials of Medical Pharmacology, sixth ed., Jaypee Brothers
Medical Publishers Ltd., New Delhi, 2008.
[4] G. Murtaza, S.A. Khan, A. Shabbir, A. Mahmood, H. Hassan, B. Asad,
Development of a UV-spectrophotometric method for the simultaneous
determination of aspirin and paracetamol in tablets, Sci. Res. Essays 6 (2011)
2417–2421.
301
[5] S.J. Rajput, H.J. Raj, Development of UV spectrophotometric method for the
simultaneous estimation of simvastatin and ezetimibe in tablet dosage form
by simultaneous equation and absorbance ratio method, Indian J. Pharm. Sci.
69 (2007) 6759–6762.
[6] A. Panchal, G. Sanghvi, A. Vachhani, N. Sheth, D. Vaishnav, Simultaneous
determination of aspirin and rosuvastatin calcium in capsules by using RP-
HPLC coupled with photo diode array detection, Int. Lett. Chem. Phys. Astron.
14 (2014) 2218–2230.
[7] E.S.M. Abu-Nameh, R. Shawabkeh, A. Ali, High-performance liquid
chromatographic determination of simvastatin in medical drugs, Am. J. Anal.
Chem. 61 (2006) 163–166.
[8] B.A. Patel, A.D. Captain, Development and validation of HPTLC method for
simultaneous estimation of atorvastatin calcium and aspirin in bulk and
dosage form, Am. J. Pharm. Health Res. 2 (2014) 119–129.
[9] R.P. Dixit, C.R. Barhate, M.S. Nagarsenker, Stability-indicating HPTLC method
for simultaneous determination of ezetimibe and simvastatin,
Chromatographia 67 (2008) 101–107.
[10] F. Sultan, M.H. Shoaib, R.I. Yousuf, F.R. Ahmed, F.A. Salam, M.I. Nasiri,
Simultaneous quantitation of aspirin, amlodipine and simvastatin in a fixed
dose combination of encapsulated tablet formulation by HPLC-UV method,
Pak. J. Pharm. Sci. 27 (2014) 1553–1558.
[11] A.K.M. Pawar, K. Sreekanth, N.A.B.N. Rao, S.D. Gowri, An isocratic method for
the simultaneous estimation of aspirin, ramipril and simvastatin by RP-HPLC,
Int. J. Pharm. Pharm. Sci. 4 (2012) 1425–1428.
[12] S.S. Yadav, J.R. Rao, RP-HPLC method for simultaneous estimation of aspirin,
ramipril, hydrochlorothiazde, simvastatin and atenolol from pharmaceutical
dosage form, Int. J. Pharm. Pharm. Sci. 6 (2014) 9443–9448.
[13] M.R. Desai, D.R. Marfatia, Z.R. Dedania, V. Swamy, HPTLC method development
and validation of simvastatin and aspirin in combine dosage form, Pharm. Sci.
Monit. 5 (2014) 215–220.
[14] D.A. Bhatt, S.I. Rane, QbD approach to analytical RP- HPLC method
development and its validation, Int. J. Pharm. Pharm. Sci. 3 (2011) 1179–1187.
[15] H. Bhutani, M. Kurmi, S. Singh, S. Beg, B. Singh, Quality by design (QbD) in
analytical sciences: an overview, Pharm. Times 46 (2014) 71–75.
[16] M.L. Jadhav, S.R. Tambe, Implementation of QbD approach to the analytical
method development and validation for estimation of propafenone
hydrochloride in tablet dosage form, Chromatogr. Res. Int. (2013), Article ID
676501.
[17] A.H. Schmidt, M. Stanic, I. Molnar, In silico robustness testing of a compendial
HPLC purity method by using a multidimensional design space build by
chromatography modeling- case study pramipexole, J. Pharm. Biomed. Anal.
91 (2014) 97–107.
[18] A.H. Schmidt, I. Molnar, Using an innovative quality- by- design approach for
development of a stability indicating UHPLC method for ebastine in the API
and pharmaceutical formulations, J. Pharm. Biomed. Anal. 78 (2013) 7965–
7974.
[19] K.G. Patel, P.M. Shah, P.A. Shah, T.R. Gandhi, Validated high-performance thin-
layer chromatographic (HPTLC) method for simultaneous determination of
nadifloxacin, mometasone furoate, and miconazole nitrate cream using
fractional factorial design, J. Food Drug Anal. 24 (2016) 610–619.
[20] T.B. Solanki, P.A. Shah, K.G. Patel, Central composite design for validation of
HPTLC method for simultaneous estimation of olmesartan medoxomil,
amlodipine besylate and hydrochlorothiazide in tablets, Indian J. Pharm. Sci.
76 (2014) 179–187.
[21] ICH harmonized tripartite guideline, validation of analytical procedures: text
and methodology Q2 (R1), in: International Conference on Harmonization,
Geneva, Switzerland, 2005.
[22] J.H. Zar, Biostatistical Analysis, fifth ed., Pearson Education Inc, New Jersey,
2010.