Design and study of a HPLC method for the

simultaneous estimation of two anti-diabetic drugs

using a statistical approach
Shantikumar Saladi,
a
Sreekanth Gutala,
*
b
Krishnaveni Yadiki,
a
M. V. N. Kumar Talluri
a
and N. Satheeshkumar
*
a
Application of design of experiments to study the robustness of a HPLC method was carried out, where six
factors were selected: the percentage of acetonitrile (ACN) in the mobile phase, the pHof mobile phase, the
detector wavelength, the column temperature, the ow rate and the strength of the buer. These factors
are examined in two-level screening experimental designs created using JMP@(SAS Institute) software. The
systemsuitability parameters like the capacity factor, tailing factor, resolution were calculated and analyzed
using the ANOVA method by least squares tting. The results are within the acceptance criteria showing that
the method is robust within the established limits. Forced degradation studies were also conducted for
sitagliptin and pioglitazone.
Introduction
Developing chromatographic methods involves the utilization
of various approaches such as trial and error with dierent
columns, dierent mobile phases and soware based methods.
The subsequent step aer method development is method
validation which includes several sensitive analytical parame-
ters to ensure that the method is accurate, precise and robust.
Among all of the parameters, the robustness is oen considered
a verication step and plays a key role in successful develop-
ment of an analytical method.
1,2
Robustness is generally studied
through traditional approaches which suer major drawbacks
such as inability to determine complex interactive eects
between method variables like pH, column temperature, ow
rate, buer concentration, mobile phase composition etc. There
are time-consuming processes which take several runs to obtain
data as a single variable is changed for each run; while another
approach is optimization using Quality by Design (QbD), as per
ICH Q8 guidelines, with Design of Experiments (DOE), which
ensures success in nal method validation.
35
The history of
DOE dates back to 1920 when it was originally proposed by
Ronald A. Fischer, a British scientist, in order to maximize the
knowledge gained from experimental data. The approach
proposed by him overcomes the limitations of traditional
approaches by considering all variables simultaneously and
obtaining the most relevant data with minimal eort.
6,7
Generally, DOE is a systematic, rigorous approach that applies
principles and techniques at the data collection stage so as to
ensure the generation of valid, supportable conclusions and
which can be carried out under the constraint of a minimal
amount of runs, time and money. This approach nds its
application in a broad range of elds such as agriculture
(factorial and fractional designs), defense (sequential designs),
chemicals (response surface designs for process optimization),
the pharmaceutical eld and various elds which are concerned
with quality improvement.
8,9
Christine Ye et al.
10
have reported the applicability of DOE
and data treatment using JMP and Brynn Hibbert D
11
has
reviewed the use of DOE in chromatography. In recent years the
applicability of DOE and statistical data treatment to HPLC
methods have greatly increased.
1216
JMP@ (SAS Institute) so-
ware provides dual advantages like ecient data collection and
scientic data interpretation with statistical evaluation of data
from chromatographic analysis. The main aim of the present
paper is to study the robustness parameter in the method vali-
dation of HPLCfor simultaneous estimationof a combination of
two anti-diabetic drugs sitagliptin (SIT) and pioglitazone (PIO)
by application of the statistical method through DOE and
analyzing the data in JMP@ (SAS Institute) soware using the
analysis of variance method. SIT (Fig. 1a) is (R)-4-oxo-4-[3-(tri-
uoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-
1-(2,4,5-triuorophenyl)butan-2-amine and is an anti-diabetic
drug of the dipeptidyl peptidase-4 (DPP-4) inhibitor class. It
works by inhibiting the dipeptidyl peptidase 4 (DPP-4) enzyme
competitively.
17
PIO (Fig. 1b) is [()-5-[[4-[2-(5-ethyl-2-pyridinyl)
ethoxy]phenyl]methyl]2,4]thiazolidinedione mono hydrochlo-
ride. It is a potent agonist for the peroxisome proliferator-
a
Department of Pharmaceutical Analysis, National Institute of Pharmaceutical
Education and Research (NIPER), Balanagar, Hyderabad 500 037, A.P, India.
E-mail: satish@niperhyd.ac.in
b
United States Pharmacopeia India Pvt. Ltd, Reference Standards Laboratory, ICICI
Knowledge Park, Genome Valley, Turkapally, Shameerpet, Hyderabad 500 078, A.P,
India. E-mail: gs1738@yahoo.com
Cite this: DOI: 10.1039/c3ay42330a
Received 31st December 2013
Accepted 10th February 2014
DOI: 10.1039/c3ay42330a
www.rsc.org/methods
This journal is The Royal Society of Chemistry 2014 Anal. Methods
Analytical
Methods
PAPER
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activated receptor gamma (PPARg).
18
SIT and PIO in a combi-
nation pill have complementary eects, when this combination
pill was administered to subjects, results showed that there is
signicant greater reduction inA1c compared to monotherapy.
19
Pharmaceutical formulation for this product is under research
and in our lab we have studied the excipient compatibility with
SIT for a few excipients,
20
which aid in the formulation devel-
opment of this combination. In the present study we have
evaluated the placebo mixtures of SIT and PIO. Among these two
compounds, SIT is soluble in water while PIO is insoluble.
Experimental
Materials
SIT and PIO were kindly provided by Dr. Reddys Ltd. India.
HPLC grade acetonitrile, methanol, acetic acid, hydrochloric
acid and ammonium acetate-GR were purchased from Merck.
Milli-Q water (16.2 MU cm) was obtained using a Milli-Q system
(Millipore, Billerica, MA).
Instrumentation
The HPLC system used for the method development and
degradation study was an Agilent 1100 series consisting of a
quaternary pump plus an auto sampler and diode array detector
(DAD). The output signal was monitored and processed using
Empower soware. The thermal stability study was performed
in a dry air oven (Mack Pharmatech Pvt. Ltd., Mumbai, India). A
photostability chamber (Osworld, Mumbai, India) was used for
the photodegradation study. All pH measurements were done
using a pH-meter (Metrohm SchweizAG, 780 pH-meter, Ger-
many) and weighing was done using a Sartorius balance
(CD225D, 22308105 Germany).
Chromatographic conditions and solution preparation
Chromatographic analysis was carried out at a temperature of
30

C. The compounds were separated isocratically using a
Hypersil BDS column (C
18
, 250 4.6 mm i.d.; particle size, 5m)
using a mobile phase consisting of acetonitrile : 25 mM
ammonium acetate buer with the pH adjusted to 4.5 with
acetic acid (45 : 55, v/v), at a ow rate of 1 mL min
1
. The eluent
was monitored using UV detection at a wavelength of 267 nm
and an injection volume of 10 mL was used. Sample solutions
were prepared by dissolving appropriate amounts of SIT and
PIO in the ratio (10 : 3) initially in 10 mL of 0.1 N HCl and the
volume was made up to 50 mL with acetonitrile. The analytical
concentrations of SIT and PIO in the stock solution were 2 mg
mL
1
and 0.6 mg mL
1
respectively. For linearity determination
Fig. 1 (a) Structure of sitagliptin. (b) Structure of pioglitazone.
Table 1 PlackettBurman design for the robustness study
Run Pattern
Flow rate (mL
min
1
) pH
Buer strength
(mM)
Wavelength
(nm)
Temperature
(

C) %ACN
1 ++++++ 1.1 4.7 30 269 40 47
2 ++ 0.9 4.3 30 269 30 43
3 ++ 0.9 4.3 20 265 40 47
4 +++ 0.9 4.7 20 269 30 47
5 +++ 0.9 4.7 30 265 40 43
6 +++ 1.1 4.3 20 269 40 43
7 +++ 1.1 4.3 30 265 30 47
8 ++ 1.1 4.7 20 265 30 43
Fig. 2 Chromatogram of standard sitagliptin (R
t
: 3.61) and pioglita-
zone (R
t
9.38), in a ratio of (10 : 3) measured at 267 nm with a mobile
phase of acetonitrile : 25 mM ammonium acetate buer with pH 4.5
(45 : 55, v/v). The inset shows the overlaid spectra of both compounds
(each 10 mg mL
1
) produced on a UV-Vis spectrophotometer.
Anal. Methods This journal is The Royal Society of Chemistry 2014
Analytical Methods Paper
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triplicate samples of the compounds were prepared at 20%,
40%, 60%, 80%, 100%, 120% and 140% of the stock solution
resulting in concentrations between 2001400 mg mL
1
and 60
420 mg mL
1
for SIT and PIO respectively. The detection limit
was 0.2% and 0.38% for SIT and PIO of the analytical
concentration.
Design of the forced degradation study
A forced degradation study was carried out by transferring 50
mg of SIT and 15 mg of PIOinto a 250 mL round bottomed ask.
These proportions were employed under acidic, alkaline,
oxidative, thermal and photolytic conditions. Aer degradation
was complete the samples were collected and allowed to
equilibrate at room temperature, quenching was carried out in
Table 2 Summary of validation parameters: statistical data for the
calibration graphs
Parameter Sitagliptin Pioglitazone
Linearity range (mg mL
1
) 2001400 60420
Correlation coecient 0.9997 0.002 0.9998 0.002
Limit of detection (mg mL
1
) 2.0 1.1
Limit of quantitation (mg mL
1
) 6.5 3.8
Recovery 99.05 2.13 99.5 1.44
Precision (%RSD)
Inter-day (n 6) 1.11 1.12
Intra-day (n 6) 0.21 0.10
Fig. 3 Chromatograms of the forced degradation study, which include (a) acid stressed samples treated with 1 N HCl at 80

C for 3 h, (b) alkali
stressed samples treated with 1 N NaOH at room temperature for 6 h, (c) peroxide stressed samples treated with 5% H
2
O
2
at 80

C for 3 h, (d)
photo stressed samples and (e) thermally stressed samples.
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Paper Analytical Methods
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the cases of the acid and alkaline samples and nally diluted
with acetonitrile to attain the target concentration. The acid
stressed samples were treated with 1 N HCl at 80

C for 3 h,
alkali stressed samples were treated with 1 N NaOH at room
temperature for 6 h, peroxide stressed samples were treated
with 5% H
2
O
2
at 80

C for 3 h, the photo stressed sample was
exposed as per ICH recommendations and the thermal stressed
sample was kept in a hot air oven at 70

C for 48 h.
Robustness study
Robustness of an analytical procedure is a measure of its
capacity to remain unaected by small, but deliberate variations
in method parameters and it provides an indication of reli-
ability during normal usage. In the present study, six factors
were selected, namely the acetonitrile (ACN) percentage in the
mobile phase, the pH of the mobile phase, the detection
wavelength, the column temperature, the ow rate and the
strength of the buer. Prior knowledge of the physiochemical
properties and chromatographic behaviour of the compounds
suggested that these six factors would not be highly interactive.
Therefore using JMP soware, a fractional factorial design was
generated (Table 1). The eects of variations in the chromato-
graphic parameters were evaluated using system suitability test
results.
Results and discussion
Development and optimization of the HPLC method
The quality of any HPLCmethoddepends onthe proper selection
of stationary and mobile phases whichrelies onthe nature of the
sample and its solubility. As mentioned earlier, the challenge
with these two compounds is their solubility. The compounds
are soluble in 0.1 N HCl, therefore samples were initially solu-
bilized in 0.1 N HCl and the nal volume was made up with
acetonitrile in the composition of 0.1 N HCl and acetonitrile
(1 : 4, v/v). The best results were obtained with this composition.
Aer selecting the proper diluent, preliminary trials were carried
out with a mobile phase consisting of water and methanol or
acetonitrile, but they were lacking in peak characteristics, so
buers like KH
2
PO
4
and ammonium acetate were preferred
instead of water. The best results were obtained with
acetonitrile : 25 mM ammonium acetate buer with the pH
adjusted to 4.5 with acetic acid (45 : 55, v/v). In the present study
forceddegradationwas also included, whereina specic stability
indicating assay method was established. Stability indicating
assay methods are useful for determining the integrity of a drug
substance anddrug product during acceleratedshelf life studies.
It provides informationabout the drug quality. Therefore there is
a need to develop a stability indicating HPLC method.
System suitability
System suitability shows the suitability of a system for that
particular analysis. Five replicate injections of standard prepa-
ration were injected and the tailing factor, capacity factor
theoretical plate, resolution and %RSD of the peak area were
determined. All parameters were within the expected range.
Fig. 2 shows neat separation of compounds along with their
respective UV spectra.
Analytical validation
Linearity. Calibration curves for both compounds in the
laboratory mixture solutions were found to be linear with
Table 3 Summary of the results from the forced degradation study
Samples
No. of degradation products
%degraded
(R
t
values) SIT PIO
Acid (Fig. 3a) 3 (2.72, 3.07, 7.22) 25.12 19.36
Base (Fig. 3b) 4 (2.55, 2.71, 4.38, 6.88) 15.29 10.20
H
2
O
2
(Fig. 3c) 10 (2.62, 2.92, 3.50, 4.33, 4.55, 5.10, 6.16, 7.26, 7.54) 30.50 46.52
Photolytic-UV (Fig. 3d) 4 (1.27, 2.29, 3.32, 7.18) 14.63 20.38
Table 4 Evaluation data for the recovery studies using commonly used excipients
S. no Excipient
Amount
added
Percentage recovery
Sitagliptin Pioglitazone
1 Micro crystalline cellulose 20 mg 99.9 100.1
2 Calcium hydrogen
phosphate
20 mg 95.2 98.8
3 Cross carmellose 20 mg 100.2 97.5
4 Magnesium stearate 20 mg 98.2 101.4
5 Sodium stearate 20 mg 101.3 98.6
6 Lactose monohydrate 20 mg 99.5 100.6
99.05 2.13 99.5 1.44
Anal. Methods This journal is The Royal Society of Chemistry 2014
Analytical Methods Paper
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correlation coecients of 0.9997 and 0.9998 respectively; Table
2 lists a summary of the validation parameters.
Specicity and selectivity. Selectivity was determined by
checking the interference of excipients with the analytes. The
specicity of the proposed method was determined by checking
the peak purity of SIT and PIO during the forced degradation
study. Fig. 3 shows the chromatograms of the acid, alkali,
peroxide, photolytically and thermally stressed samples where
the method is being used to separate SIT and PIO from their
degradation products. There is no interference from any of the
degradants with the main peak. Thus the proposed HPLC
method is selective for both of the compounds. The results are
tabulated in Tables 3 and 4.
Robustness
System suitability tests were performed, according to the
design, across eight chromatographic runs. Results were
obtained (Table 5) for the area response, retention time, tailing
factors, capacity factor and resolution of the peak of interest,
and the %RSD was calculated and examined for robustness.
JMP soware enables application of the screening design i.e. the
PlackettBurman design through which the main eects can be
confounded and by running the model with data, statistical
Table 5 Results of system suitability parameters
Run Compound
Capacity factor
(mean
S.D)
Tailing factor
(mean
S.D) Resolution (mean S.D)
1 SIT 2.47 0.008 1.42 0.006 1.62 0.004
PIO 7.94 0.071 1.03 0.010
2 SIT 2.53 0.005 1.42 0.004 1.64 0.005
PIO 8.21 0.000 1.02 0.008
3 SIT 2.72 0.024 1.53 0.190 2.05 0.012
PIO 10.6 0.098 1.00 0.008
4 SIT 2.42 0.016 1.50 0.010 2.05 0.000
PIO 9.94 0.004 1.04 0.004
5 SIT 2.65 0.012 1.52 0.012 1.75 0.005
PIO 9.01 0.006 1.02 0.016
6 SIT 2.76 0.011 1.56 0.008 2.02 0.005
PIO 10.52 0.024 1.04 0.013
7 SIT 2.73 0.008 1.54 0.008 1.75 0.080
PIO 9.55 0.012 1.02 0.019
8 SIT 2.22 0.022 1.49 0.004 1.86 0.030
PIO 7.62 0.019 1.02 0.005
Table 6 Results for the variation in retention times and area response
Parameter R
t
of C1 Area of C1 R
t
of C2 Area of C2
Mean 3.58 2 108 923.00 9.45 2 559 697.00
Std dev. 0.16 32 420.67 0.31 33 726.59
%RSD 4.66 1.53 3.31 1.31
Std error mean 0.06 11 462.44 0.11 11 924.15
95% condence level 0.11 22 465.90 0.21 23 370.91
Table 7 Parameter estimates for the capacity factor, tailing factor and resolution
Parameter
Std error t ratio Prob > [t]
T K
0
R
S
T K
0
R
S
T K
0
R
S
SIT PIO SIT PIO SIT PIO SIT PIO SIT PIO SIT PIO
Flow rate (0.9, 1.1) 0.016 0.008 0.018 0.024 0.011 1.31 0.43 6.68 22.42 1.89 0.415 0.742 0.094 0.028 0.310
pH (4.3, 4.7) 0.016 0.008 0.018 0.024 0.011 0.38 0.14 1.23 13.80 2.11 0.766 0.909 0.435 0.056 0.281
Strength of buer (20, 30) 0.016 0.008 0.018 0.024 0.011 0.23 0.14 1.07 0.04 3.89 0.855 0.909 0.479 0.972 0.160
Wavelength (265, 269) 0.016 0.008 0.018 0.024 0.011 0.38 0.43 4.84 3.22 5 0.766 0.742 0.129 0.191 0.122
Column temperature (30, 40) 0.016 0.008 0.018 0.024 0.011 1.15 0.71 1.66 9.53 6.78 0.454 0.605 0.345 0.066 0.093
%ACN (43, 47) 0.016 0.008 0.018 0.024 0.011 1.46 0.14 3.98 34.61 11.22 0.382 0.909 0.156 0.058 0.056
Table 8 Summary of t for the capacity factor, tailing factor and
resolution
Summary of t
T K
0
R
S
SIT PIO SIT PIO
R square 0.8467 0.4842 0.9889 0.9994 0.9954
Root mean square error 0.0459 0.0247 0.0518 0.0689 0.0318
Mean of response 1.50 1.02 2.56 9.17 1.84
Observation 48 48 48 48 48
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evaluation of the data can be done through which the signi-
cant and non-signicant factors can be ascertained.
The factors selected in the robustness test were related to the
analytical procedure like the pH of the mobile phase, the %
ACN, the detection wavelength, the strength of the buer and
the ow rate (operational factors), and to the environmental
conditions like the column temperature (environmental
factors). The operational factors are selected from the descrip-
tion of the analytical method (operating procedure), whereas
the environmental factors are not necessarily specied explicitly
Fig. 4 Prediction proler for the capacity factor (a), the tailing factor (b) and the resolution (c). The desirability was shown for resolution alone
because of its acceptance limits.
Anal. Methods This journal is The Royal Society of Chemistry 2014
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in the analytical method. The levels for these factors were
selected based on systematic determination of the uncer-
tainties.
21
The pH varies with a condence level of 95% in the
interval pH 0.02. The column temperature, %ACN, detection
wavelength, strength of the buer and ow rate low and high
levels were based on the uncertainty. The factors were examined
in an experimental design, which was selected as a function of
the number of factors investigated. The design applied was a
two-level screening design which allowed the screening of a
relatively large number of factors in a relatively small number of
experiments. The design applied was fractional factorial. In this
robustness test we have focused mostly on the main eects of
the factors.
Regarding the screening design, the parameters to be
considered should be selected based on the purpose of the
method and its performance characteristics, and tolerances are
xed by considering the tolerance of the HPLC system. By
application of the design the number of experiments for
robustness testing were decreased as it gives a better under-
standing of the process, rather than studying extremities like
which factors are more or less inuential on the results.
Variation in the retention time and area response. The %
RSD for the retention time of eight experimental runs for both
of the compounds was between 3.33 and 4.66% on average,
which is within the proposed criterion of 5%. The %RSD for the
area response was between 1.3 and 1.5%, for which the
proposed acceptance criterion of <2% was passed. The results
are shown in Table 6 and the contour plots are shown in Fig. 5
which shows the relationship between the results. These
contour plots represent three numerical variables like the area
and retention time in two dimensions. These aid in explaining
how the retention time and peak areas changed as a function of
X and Y. The center lines are iso-response values.
Tailing factor. The tailing factor (T) for each of the 8 injec-
tions was entered in the JMP soware and analyzed using the
ANOVA method by least squares tting. The tting results
revealed that Prob > [t] was greater than 0.05 for the ow rate,
the pH of the mobile phase, the strength of the buer, the
detection wavelength, the column temperature and the ACN
percentage. It demonstrated that no signicant dierences were
observed when changing the above factors within the tested
ranges. The prediction prole on the 95% condence interval
showed that all T values would be within the acceptance crite-
rion, 1.0 # T # 1.56, if parameters were changed within their
testing ranges. Therefore, the conclusion was made that the
tailing factor was acceptable when chromatographic parameters
were changed within the experimental range. These results are
shown in Tables 7 and 8.
Capacity factor. The capacity factor (K
0
) for each of the 8
injections for both of the compounds was also analyzed using
the ANOVA methods by least squares tting. The tting revealed
that each of the six factors showed no signicant dierences
between the two levels tested. However, parameter estimates for
the ow rate, the pH of the mobile phase, the strength of the
Fig. 5 Contour proles of the retention times and peak areas of sitagliptin (C1) and pioglitazone (C2).
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buer, the detection wavelength, the column temperature and
the ACN percentage were 0.122, 0.022, 0.019, 0.088, 0.0304
and 0.0729 respectively, which were very small compared with
the mean value of 2.56, therefore these factors will not be
considered as primary factors in changing the capacity factor.
The results are shown in Tables 7 and 8.
Resolution. The resolution (R
S
) is the separation between two
chromatographic peaks. It is not only the measure of the
separation of peaks but also the eciency of the column. For
reliable quantication, well-separated peaks are essential. A
resolution of >1.5 between the peaks of interest is desirable. The
resolution (R
S
) for each of the 8 injections was also analyzed
using the ANOVA methods by least squares tting. The tting
revealed that each of the six factors showed no signicant
dierences with the set minimum. However, parameter esti-
mates for the ow rate, the pH of the mobile phase, the strength
of the buer, the detection wavelength, the column temperature
and the ACN percentage were 0.02125, 0.02375, 0.04375,
0.05625, 0.07625 and 0.12625 respectively, which were very
small compared with the mean value of 1.84, therefore these
factors will not be considered as primary factors in changing the
resolution and method parameters. These results are shown in
Tables 7 and 8. Fig. 4 is the prediction proler for all of the
system suitability parameters, where parameter estimates and
the respective proles of each run with respect to all of the
selected factors are depicted.
Fig. 6 Contour proler with response surface plots which show the impact of various parameters, against a xed ow rate as a variable, on the
capacity factor, the tailing factor and the resolution for both sitagliptin and pioglitazone. (a) Flowrate vs. buer strength, (b) owrate vs. detection
wavelength, (c) ow rate vs. pH, (d) ow rate vs. temperature and (e) ow rate vs. %ACN.
Anal. Methods This journal is The Royal Society of Chemistry 2014
Analytical Methods Paper
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Contour proler and the impact of various factors
The contour proler is useful for studying the response
surfaces graphically, the shaded regions show the unaccept-
able regions and the unshaded regions (white) represent the
acceptable regions in the contour plots. Contour plots were
plotted for responses like the tailing factor, the resolution, the
capacity factor and the eects of the various variables selected
for the robustness study were evaluated. Fig. 6ae show the
contour proles. Flow rate was xed as a horizontal factor
against which vertical factors like pH (X 4.5), buer
strength (X 25 mM), wavelength (X 267 nm), temperature
(X 35

C) and %ACN (X 45), were plotted. Within the
tested ranges the method didn't show any unacceptable areas.
The lines on the graphs are contours for responses set by Y
slider controls; there are separately coloured contours for each
response.
Desirability function
These are smooth piecewise functions which are designed to t
control points. In Fig. 4 the minimum desirability function is
shown; this associates high response values with low desir-
ability and low response values with high desirability. The top
function handle is positioned at the minimum desirability i.e.
Y 2.1 with a desirability of 0 and Y 1.6 with a desirability of
1. The desirability traces are also shown for each factor.
Conclusion
A new HPLC method has been developed to be routinely applied
to determine SIT and PIO in pharmaceutical dosage form. The
method was validated by employing ICH recommended stress
conditions. The method has been proven to be specic, linear,
precise, accurate, robust and an indicator of stability. Hence the
method can be recommended for routine quality control anal-
ysis. The proposed method was robust within the specied limits
which can be assured by the statistical data provided. The
acceptance criteria for the system suitability were %RSD for R
t
#
5.0% and %RSD for area #2.0%, 1.0 # T # 1.56, K
0
$ 1.5. The
control limits for the HPLC method parameters tested in this
robustness study are as follows: owrate 1 0.1 mL min
1
, pH
of the mobile phase 4.5 0.2, buer strength 25 5 mM,
detectionwavelength267 2 nm, temperature 35 5

Cand
%ACN45 2. To conclude, approaches like DOE ts well with
these applications which is economical and reduces the total
number of experiments tobe carriedout whichinturnsaves time.
Acknowledgements
The authors wish to thank Dr K. V. Surendranath, Sr Vice
President and other scientic sta of United States
Pharmacopeia (USP)-India Private Limited for supporting this
work. The authors are indebted to NIPER-Hyderabad, for
providing the support and encouragement to carry out this work
and S. Shanti Kumar is grateful to NIPER for providing
fellowship.
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