Novel RP-HPLC Method for Simultanious Determination of Sitagliptin and Simvastation in Bulk and Tablet Dosage Form

IOSR Journal Of Pharmacy
(e)-ISSN: 2250-3013, (p)-ISSN: 2319-4219
www.iosrphr.org Volume 5, Issue 8 (August 2015), PP. 34-40
34
Novel RP-HPLC Method for Simultanious Determination of
Sitagliptin and Simvastation in Bulk and Tablet Dosage Form
P. Ravisankar*1
, SK. Hassain1
, Shaik Mohammed Neeha1
*1
Department of Pharmaceutical Analysis and Quality Assurance, Vignan Pharmacy College, Vadlamudi,
Guntur - 522213, A.P., India.
ABSTRACT: A simple, rapid, accurate, precise and novel high-performance liquid chromatographic method
for simultaneous analysis of Sitagliptin (SITA) and Simvastation (SIMV) in pharmaceutical dosage form has
been developed and validated. The chromatographic separation was accomplished on Welchrom RP-C18
Column (250 mm X 4.6 mm; 5µm), Shimadzu LC-20AT Prominence Liquid Chromatograph and with a mixture
of 10 mM Phosphate buffer: acetonitrile and methanol in the range of (45:35:20 v/v/v). The flow rate was fixed
at 1mL/minute and the analysis was performed using Shimadzu SPD-20A Prominence UV-detection was
performed at 255 nm. The SITA and SIMV were separated within seven minutes. The retention time for SITA
and SIMV was found to be 3.352 minutes and 5.402 minutes respectively. The calibration plots were linear over
the concentration range of 10-50 µg/ml for SITA (r2
= 0.9998) and 4-20 µg/ml for SIMV (r2
= 0.9999). There
was no interference due to commonly used excipients. The relative standard deviation for inter-day precision
was lower than 2.0 % which obviously indicates that the present method was said to be highly precise.
Regarding accuracy of the developed method the % RSD were also found less than 2 % which shows the method
is completely accurate. The method was very sensitive with regard to LOD 0.681 µg/ml, 0.116 µg/ml and LOQ
2.250 µg/ml, 0.384 µg/ml respectively. The mean assay values for SITA and SIMV were determined in tablet
dosage form were found to be within limits. The developed RP HPLC method was found to be simple, rapid,
sensitive, highly precise and accurate highly suitable for routine analysis of drug samples containing SITA and
SIMV.
Keywords: Sitagliptin and Simvastatin, RP-HPLC Method, Simultaneous estimation.
I. INTRODUCTION
The combination of SITA and SIMV is used together with a decorous diet and exercise to treat type 2
diabetes. It is also utilized together with a proper diet to treat high cholesterol and triglyceride (fats) levels in the
blood. This medicine may help stop medical problems caused by clogged blood vessels such as heart attacks and
strokes. SITA is a dipeptidyl peptidase - 4 inhibitor utilize to treat type 2 diabetes mellitus i.e., it helps to control
blood sugar levels by increasing substances in the body that make the pancreas release more insulin. It also
signals the liver to stop producing glucose when there is too much sugar in the blood and SIMV belongs to the
group of medicines called statins and is used as HMG-CoA reductase inhibitor to lower the levels of LDL
cholesterol. These two disorders commonly occur in patients at the same time, and have been typically treated
with administration of these two drugs in separate tablets. The combination was approved in 2011 and is
marketed as Juvisync by Merck. Currently, the combination tablet is approved in three doses: SITA/SIMV 100
mg/10 mg, 100 mg/20 mg, and 100 mg/40 mg.
Literature survey revealed that there were few analytical methods have been reported for the
simultaneous estimation of above said drugs individually, tertiary or in combination with some other drugs in
biological samples as well as pharmaceutical dosage forms by UV- spectrophotometry[1-9]
, HPLC[10-18]
,
HPTLC[19]
, LC-MS/MS [20,21]
,Capilary zone electrophoresis[22]
. All the above reported methods were based on
the estimation of SITA/SIMV alone or in combination with other drugs. However most of the available methods
have limitations such as long run time, poor resolution, uneconomical and low sensitivity. So based on the
above mentioned reasons, indeed an attempt has been made to develop a simple, precise, accurate, reproducible
and robust RP-HPLC method for the simultaneous determination of SITA and SIMV in pharmaceutical dosage
form. The structural formulas of SITA and SIMV are shown in Fig. 1 and Fig. 2.

Novel RP-HPLC Method for Simultanious Determination...
35
F
F
FN NH2
O
N
N
N
FF
F
Figure 1: Chemical structure of SITA
O
OHO
O
O
Figure 2: The chemical structure of SIMV
II. MATERIALS AND METHODS
2.1 Chemicals and reagents
Pharmaceutically pure samples of SITA and SIMV were obtained as a gift sample from Hetero Drugs
Ltd., Hyderabad, Andhra Pradesh, India. All the chemicals were analytical grade. Potassium dihydrogen
orthophosphate and phosphoric acid purchased from Rankem Ltd., Mumbai, India, while acetonitrile (HPLC
grade) and triethylamine (HPLC grade) purchased from Merck Pharmaceuticals Private Ltd., Mumbai, India.
Ortho phosphoric acid utilized was of HPLC grade and obtained from Merck Specialties Private Ltd., Mumbai,
India. Glassware used in each procedure were soaked overnight in a mixture of chromic acid and sulphuric acid
mixture, then rinsed thoroughly with triple distilled water and dried in hot air oven. Commercial formulation
was procured from local drug market.
2.2 Instrument specifications
Quantitative HPLC was performed on a isocratic high performance liquid chromatograph (Shimadzu
LC-20AT Prominence Liquid Chromatograph) with a LC-20AT VP pump, manual injector with loop volume of
20 µL (Rheodyne), programmable variable wavelength Shimadzu SPD-20A Prominence UV-Vis detector and
Welchrom C18 Column (4.6 X 250 mm, 5 µm particle size). The HPLC system was equipped with “Spinchrome”
software. In addition an electronic balance (Shimadzu TX223L), digital pH meter (Systronics model 802), a
sonicator (spectra lab, model UCB 40), UV-Visible Spectrophotometer (Systronics model-2203) were used in
this present study.
2.3 Chromatographic conditions
The chromatographic separation was accomplished on Welchrom RP-C18 Column (250 mm X 4.6 mm;
5µm), Shimadzu LC-20AT Prominence Liquid Chromatograph and mobile phase consisted of a mixture of 10
mM Phosphate buffer: acetonitrile and methanol in the range of (45:35:20 v/v/v) and filtered before used
through a 0.45 µ membrane and degassed for 15 minutes. The flow rate was fixed at 1 mL/min, the injection
volume was 20 µl and the analysis was performed using Shimadzu SPD-20A Prominence UV-Visible detector
and the eluents were monitored at 255 nm.
2.4 Preparation of mobile phase
The appropriate composition of the mobile phase prepared contains phosphate buffer, acetonitrile and
methanol in the ratio of 45:35:20 v/v/v. Prior to applying for separation, both the solvent and buffer solutions
were filtered by passing through 0.45 mm nylon filter and sonicated for about 15 min.

36
2.5 Preparation of 0.025 M phosphate buffer solution
0.025 M phosphate buffer solution was prepared by dissolving precisely weighed three of sodium
dihydrogen phosphate in 1000 mL HPLC grade water. The solution was filtered through 0.45 mm membrane
filter and sonicated for about 15 min. The pH was adjusted for 6.3 with triethyl amine.
2.6 Preparation of standard stock solution
Standard Stock solutions of SITA and SIMV pure drugs were separately prepared by dissolving 10 mg
of each in separate 10 ml of volumetric flask with small quantity of mobile phase. The mixture was sonicated
for 10 min and filled up the volume with mobile phase to give a concentration of 1000 μg/ml. These stock
solutions were used for preparing working standards and calibration standards. The standard solution was
filtered through 0.45 µm nylon membrane filter and degassed by sonicator.
2.7 Preparation of calibration standards
Aliquots stock solutions of SITA and SIMV were accurately transferred in to 10 ml volumetric flasks
and diluted to mark with mobile phase to yield a concentration range of 10 - 50 μg/ml for SITA and 4 - 20
μg/ml for SIMV. The calibration line was obtained by plotting the mean peak area (n = 3) against concentrations
of drug.
2.8 Preparation of test sample solution
20 tablets of juvisync containing SITA 100 mg and SIMV 20 mg were weighed and finely powdered.
The powder equivalent to 100 mg of SITA and 20 mg of SIMV was weighed and transferred to a 100 ml
volumetric flask containing 60 mL mobile phase as a diluent. After sonication for complete solubility, the
volume was made up to the mark with mobile phase to obtain concentration of 1000 μg/ml. Resultant solution
was filtered through whatman filter paper followed by 0.45 μ nylon filter. 1 mL of filtrate was taken in 10 mL
volumetric flask and was made up to 10 mL with diluent to obtain a concentration of 100 μg/ml for SITA and 20
μg/ml for SIMV. From these solution aliquots concentrations are prepared and utilized for the sample analysis.
2.9 Selection of analytical wave length
The solution of SITA and SIMV was prepared with the diluents and the UV spectrum was recorded by
scanning the sample in the UV region (200 - 400) in spectrum mode. The results showed that the absorption
maximum for the drug in the given solvent was at 255 nm and hence the detector was set at 255 nm for
monitoring the eluents.
2.10 METHODOLOGY
To optimize the RP-HPLC chromatographic parameters several mobile phase compositions (several
trials) were tried. A satisfactory separation and excellent peak symmetry for SITA and SIMV was obtained with
a mobile phase mixture of 10 mM Phosphate buffer: acetonitrile and methanol in the range of (45:35:20 v/v) at
a flow rate of 1ml/min to get better reproducibility and repeatability. Quantification was carried out at 255 nm
based on peak area. Complete resolution of the peak with clear baseline was obtained (Fig 3). system suitability
test parameters and optimized chromatographic conditions for SITA and SIMV for the proposed method are
reported in Table 1.
Table 1: Optimized chromatographic conditions and system suitability parameters[

37
Figure 3: Chromatogram of the SITA and SIMV synthetic drug
2.11 Method validation[23,24]
The method was validated for Specificity, linearity, precision, accuracy, limit of detection, limit of
quantitation, robustness and stability of the analytical solution as per the international conference on
harmonization ICH Q2(R1)[25]
guidelines.
2.11.1 Specificity
Specificity of the method is performed by separate injections of SITA and SIMV standard and sample.
Specificity is the ability to assess unequivocally the analyte in the presence of components which may be
expected to be present. Typically this might include impurities, degradants, matrix, etc., Specificity of the
method was performed by comparing the chromatograms of blank, standard and sample. It was found that there
is no interference due to excipients in the tablet formulation and also that there is good correlation between the
retention times of standard and sample. The specificity results are tabulated in Table 2.
Table 2: Specificity study
Name of the solution Retention time, (tR)min.
Mobile phase No peaks
Placebo No peaks
Solution containing a concentration of SITA and , 50
µg/ml and SIMV, 20 µg/ml.
Peaks at 3.352 min and 5.402 min for SITA and
SIMV respectively.
2.11.2 Precision
The precision (repeatability and intermediate precision) experiments were conducted by determining
the intra-day precision and inter-day precision of the method. The intra-day precision was examined by
replicating the assay thrice for the three levels in the same day where as the inter-day precision was studied by
taking over the assay on three dissimilar days, three times on each day for the three concentration levels. The
results of precision study were stated in terms of % RSD and it is presented in Table 3.
2.11.3 Accuracy (Recovery studies)
Accuracy of the method was determined by standard addition method. Recovery tests were carried
out by analyzing mixtures of SITA and SIMV different compositions. Known amount of standard drugs were
added to a pre-analyzed sample at three different levels (80 %, 100 % and 120 %) and the mixed standard
solutions were analyzed in triplicate at every level as per the suggested method. The percent of individual
recovery and % RSD at each level for the all drugs are given in Table 3.
2.11.4 Robustness
The method of robustness known as its capacity to remain unaltered due to the deliberate changes in
optimized conditions set for the method. To ascertain the robustness of the method, the conditions of experiment
were deliberately changed for their effect on the assay, peak tailing factor and number of theoretical plates. In

38
the present study robustness of the method was examined by carrying out slight but deliberate modifications in
chromatographic conditions namely flow rate (± 0.1 ml/min), detection wavelength (± 5 nm) and Mobile phase
composition (± 2 %). The effect of these variables on the developed method was determined. The results
obtained are summarized in Table 3.
2.11.5 LOD and LOQ
As per ICH guideline both LOD and LOQ were ascertained by computing the standard deviation of the
response of the working standard solution at lowest concentration near the y-intercept divided by the average of
the slope. For each drug six replicates of the analyte at lowest concentration within the calibration range were
measured and quantified. The LOD was decided by multiplying the ratio between the standard deviation to the
slope with 3.3. The LOQ was assessed by multiplying the same ratio with 10. The results of LOD and LOQ
obtained for the drugs are exhibited bellow in Table 3.
Table 3: Summary of validation parameters
Parameters SITA SIMV
Retention time (min) 3.352 5.402
LOD (µg/ml) 0.6819 0.1165
LOQ (µg/ml) 2.250 0.384
Linearity (µg/ml) 10-50 µg/ml 4-20 µg/ml
Correlation coefficient 0.9998 0.9999
Precision (inter-day) % RSD (n=6)
Precision (intra-day) % RSD (n=6)
0.280* 0.290*
0.320* 0.330*
Recovery (%)
80 % analyte added 98.0 0.35*
100 % analyte added 99.9 0.54*
120 % analyte added 98.95 0.42*
Robustness Robust Robust
Assay % (n = 6)
Bulk drug 98.42 0.32* 99.32 0.65*
Tablets 99.82 0.12* 99.55 0.32*
RSD = Relative standard deviation, * = % RSD
2.12 Analysis of marketed formulation
20 µl of above prepared sample solution was injected into HPLC system and peak areas were measured
under optimized chromatographic conditions. The result formulation analysis and its chromatogram is given in
Table 4 and Fig.4.
Figure 3: Chromatogram of Juvisync tablet formulation

39
Table 4: Assay results for SITA and SIMV
Formulation Label claim Amount found (mg)
Mean±SD (n=3)
Assay (%) RSD (%)
Juvisync SITA SIMV SITA SIMV SITA SIMV SITA SIMV
50 mg 20 mg 49.91 19.91 99.82 99.55 0.12 0.32
RSD= Relative standard deviation
III. RESULTS AND DISCUSSION
The mobile phase and stationary phase play a prominent role on resolution, peak shape, theoretical
plates, and asymmetry. To attain symmetrical peaks with decorous resolution, various chromatographic
conditions was investigated and optimized for the estimation of SITA and SIMV, such as mobile phase with
different composition, pH, and various stationary phases. For obtaining appropriate mobile phase for the
analysis, various mobile phase consisting of mixtures of acetonitrile, methanol HPLC grade water, phosphate
buffer were tested. Eventually it was demonstrated that highly symmetrical, sharp, well resolved SITA and
SIMV peaks were obtained in a short time when utilized Welchrom C18 column with mobile phase consisted of
a mixture of 10 mM Phosphate buffer (pH 6.3): acetonitrile: methanol (45:35:20 v/v/v). with flow rate of
1mL/minute at the analytical wavelength 255 nm.
Chromatograms showed a peak of SITA at retention time of 3.352 min and peak of SIMV at retention
time of 5.402 min respectively. The specificity was determined to test the interference of commonly used
excipients. The comparison of standard and blank chromatograms indicates no co-eluting peak between the two
main peaks in the chromatograms as well as it was properly resolved, well shaped peaks also shows the
specificity of the method. Therefore, it was concluded that the method is specific. The specificity results are
summarized in Table 2. All the system suitability parameters were evaluated. Since there are no marked
changes in the system suitability parameters, the method was capable to remain unaffected by small variation of
tested variables for robustness study. The calibration curve was obtained for a series of concentration in the
range of 10-50 μg/ml for SITA and 4 - 20 μg/ml for SIMV respectively and it was found to be linear. The
regression equation obtained from linearity plot for SITA was Y = 27.239 x -6.4173 with R2
=0.9998 and for
SIMV was Y = 58.463 x + 0.5685 with R2
=0.9999 which shows that this method had good linearity. The
standard chromatograms of SITA and SIMV are shown in Figure 3. Recovery studies of the drug were carried
out for the accuracy parameter at three different concentrations levels i.e. multiple level recovery studies. A
known amount of standard drug was added into pre-analyzed sample and subjected them to the proposed HPLC
method. The % recovery was found to be within the limits. Precision was studied to find out intra and inter day
variations in the test methods of SITA and SIMV for the six times on the same day and on different days. The
values of % RSD (< 2.0) indicate that the proposed method is quite precise and reproducible and results are
shown in Tables 3. Robustness was done by small changes in the chromatographic conditions like mobile phase
flow rate, temperature, mobile phase composition etc., It was observed that there were no marked changes in the
chromatograms. Infact the parameters are within the limit which indicates that the method has robustness and
suitable for routine use. The calculated LOD and LOQ are found to be 0.681 µg/ml and 2.250 µg/ml for SITA
and 0.116 µg/ml and 0.384 µg/ml for SIMV showed that the method is specific and sensitive to estimate these
drugs at low concentration level. Finally the developed validated method was applied for quantitative estimation
of the marketed formulation tablets. 20 μL of sample solution was injected into liquid chromatograph and
chromatogrm was recorded and the mean assay value was found to be 99.82 ± 0.12 % for SITA and 99.55 ±
0.32 % for SIMV. Satisfactory results were achieved. The mean % found for all two drugs were in good
agreement with the label claim and results are presented in Table 4. The sample chromatogram of SITA and
SIMV is shown in Figure 4. Bench top solution stability was tested to the working solutions of the drugs and the
results suggest that the solutions are stable up to 48 hours in the routine and operational and environmental
conditions.
IV. CONCLUSION
The present proposed research study by the author describes the estimation of SITA and SIMV
available as combination tablet dosage forms and was carried out by utilizing RP-HPLC. The linearity of the
proposed method was in the range of 10-50 μg/ml for SITA and 4-20 μg/ml for SIMV respectively. The LOD
and LOQ of SITA were 0.681μg/ml and 2.250 μg/ml and for the estimation of SIMV were 0.116 μg/ml and
0.384 μg/ml respectively. The total run time of the above titled drugs were 8 minutes. The developed method
has several advantages like good linearity, decrease the time needed for analysis and low flow rare used in this

40
method consumes less solvent consumption, improved resolution as compared with the previous methods which
makes the method more economical than the existing methods in practice. The developed RP-HPLC method for
the quantification of SITA and SIMV was found to be simple, specific, highly sensitive, fast, economical,
precise and extremely accurate with robustness. Therefore this method can be recommended for the routine
analysis of SITA and SIMV in quality control and clinical laboratories.
V. ACKNWOLEDGEMENTS
The authors are thank to Hetero Labs, Hyderabad, India for getting the samples of Sitagliptin and
Simvastatin. We are highly thankful to Dr. L. Rathaiah, honourable Chairman, Vignan group of institutions,
Vadlamudi, Guntur, for providing the necessary facilities to carry out this research work.
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Novel RP-HPLC Method for Simultanious Determination of Sitagliptin and Simvastation in Bulk and Tablet Dosage Form