Development and Validation of RP-HPLC Method for Simultaneous Estimation of Sitagliptin and Simvastatin in Bulk and Tablet Dosage Form

175
* Corresponding author: B.Shirisha
E-mail address: sirishirisha96@gmail.com
IJPAR |Volume 2 | Issue 4 | Oct-Dec-2013 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
Development and Validation of RP-HPLC Method for
Simultaneous Estimation of Sitagliptin and Simvastatin in Bulk
and Tablet Dosage Form
*B.Shirisha, B.Prathyusha, N.Ramathilagam, J.Priya, N.Sriram.
Department of Pharmaceutical Analysis and Quality Assurance Smt.Sarojini Ramulamma
College of Pharmacy, Sheshadrinagar, Mahabubnagar - 509001, Andhra Pradesh, India.
ABSTRACT
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and
validated for simultaneous determination of Sitagliptin and Simvastatin in bulk and tablet dosage form.
Chromatographic analysis was performed on a Nucleosil C18 (150X4.6 mm, 5µm) column ambient temperature
with a mixture of phosphate buffer and Acetonitrile in the ratio 30:70 (phosphate buffer preparation; 0.01 N
Potassium dihydrogen phosphate, pH 3.5 adjust with triethylamine) as mobile phase, at a flow rate of 1 mL
min-1
. UV detection was performed at 254 nm. The method was validated for accuracy, precision, specificity,
linearity and sensitivity. The retention times of Sitagliptin and Simvastatin were 3.242 min and 6.492 min,
respectively. Calibration plots were linear over the concentration ranges 25-150 μg mL-1
and 5-30 μg mL-1
for
Sitagliptin and Simvastatin respectively. The Limit of detection was 1.305 µg mL-1
and 0.257 µg mL-1
and the
quantification limit was 3.941µg mL-1
and 0.77µg mL-1
Sitagliptin and Simvastatin for respectively. The
accuracy of the proposed method was determined by recovery studies and found to be 99.20% to 100.94%.
Keywords: Sitagliptin, Simvastatin, RP-HPLC, Validation.
INTRODUCTION
Sitagliptin is chemically (R)-4-oxo-4-[3-
(trifluoromethyl)-5,6-dihydro[1,2,4] triazolo [4,3-
a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl) butan
-2-amine (Figure:1). It is a Dipeptidyl peptidase - 4
(DPP-4) inhibitor. This enzyme breaks down the
incretins GLP-1 and GIP, gastrointestinal
hormones released in response to a meal.
Simvastatin is a hypolipidemic drug used to control
elevated cholesterol, or hypercholesterolemia.
(Figure 2),It is chemically(1S,3R,7S,8S,8aR)-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-dimethylbutanoate
.Very few reports are there on simultaneous
estimation of Sitagliptin and Simvastatin. In tablets
they were estimated using spectrophotometry,
HPTLC, and HPLC methods. Till date, to the best
of our knowledge, two method has been reported in
the literature. This manuscript describes the
development and validation, in accordance with
ICH guidelines, of rapid, economical, precise and
accurate isocratic reversed-phase HPLC method for
analysis of Sitagliptin and Simvastatin in bulk and
table dosage form.

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B.Shirisha, et al / Int. J. of Pharmacy and Analytical Research Vol-2(4) 2013 [175-183]
www.ijpar.com
Figure-1 Sitagliptin Figure-2 Simvastatin
MATERIALS AND METHODS
Chemicals
Sitagliptin and Simvastatin obtained from Bio Leo.
lab.Pvt.Ltd, Hyderabad, as a gift samples.
Potassium dihydrogen phosphate & Disodium
hydrogen phosphate (AR Grade), Ortho-phosphoric
acid (AR Grade), Acetonitrile (HPLC Grade), were
purchased from Merck (India) Ltd., Worli,
Mumbai, India. Tablet formulation (Juvisync) was
purchased from local market, containing Sitagliptin
(50 mg), Simvastatin (10 mg). Double distilled
water was used throughout the experiment.
Instruments
Waters HPLC e 2695 series consisting 4 pumps.
Auto sampler with 5 racks, each rack has 24 vials
holding capacity with temperature control. Auto
injector has capacity to inject 5µL to 500µL. UV-
Vis Detector with PDA. Thermostat column
compartment connected it has a capacity to
maintain 5°C to 60°C column temperature.
Waters (alliance) HPLC System is equipped with
Empower-2 software
.
ANALYTICAL METHOD DEVELOPMENT
Optimization of UV conditions
Figure-3 Isobestic point of Sitagliptin and Simvastatin
Chromatographic Conditions
A waters nucleosil C-18 column (150 mm x 4.6
mm i.d.,5-μm) was used for chromatographic
separation. The mobile phase composed of
Acetonitrile and phosphate buffer (70:30 v/v); pH
adjusted to 3.5 with triethylamine at a flow rate of
1 mL min-1 with run time of 20min. Mobile phase
and sample solutions were filtered through a 0.45
μm membrane filter and degassed. The detection of
both drugs was carried out at 254 nm.
Figure-4 Optimized Chromatogram
N
N
O
N
N
F
F
F
NH2
F
F
F

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B.Shirisha et al / Int. J. of Pharmacy and Analytical Research Vol-2(4) 2013 [175-183]
METHODOLOGY
Mobile phase preparation
Buffer preparation
0.01 N Potassium dihydrogen phosphate adjust pH
to 3.5 with triethylamine. Mix buffer and
Acetonitrile at 30:70 ratio sonicate the resulting
solution and degauss it using vacuum filtration
through 0.45 micron membrane filter.
Standard stock solution preparation
Weigh and transfer50 mg of Sitagliptin working
standard and 10 mg of Simvastatin working
standard into 50 mL volumetric flask, add 50 mL
of diluent and sonicate to dissolve and dilute to
volume with diluent.
Standard preparation
Transfer10 mL of standard stock solution into 100
mL volumetric flask and dilute to volume with
diluent.
Sample Preparation
Finely grind pre weighed 20 tablets. Transfer
grinded sample quantitatively equivalent to 50 mg
of Sitagliptin and 10 mg Simvastatin of in to 100
mL volumetric flask add 50 mL of diluent,
sonicate to dissolve for 10 minutes and dilute to
volume with diluent. Further filter the solution
through filter paper. Dilute 10 ml of filtrate to 100
ml with mobile phase.
Procedure
Inject 20 µL of blank solution, placebo solution,
Standard solution, Disregard peaks due to blank and
placebo if any.
VALIDATION OF METHOD:
The HPLC method was validated in accordance
with ICH guidelines.
Precision
The system precision of the method was verified by
six replicate injections of standard solution
containing Sitagliptin and Simvastatin. The method
precision was carried out the analyte six times
using the proposed method. Repeatability was
measured by multiple injections of a homogenous
sample of Sitagliptin and Simvastatin
Accuracy
Accuracy was carried out by % recovery studies at
three different concentration levels. To the pre-
analyzed sample solution of Sitagliptin and
Simvastatin; a known amount of standard drug
powder of Sitagliptin and Simvastatin were added
at 80, 100 and 120 % level.
Specificity and Selectivity
Specificity of the method was determined through
study of resolution factor of drug peak from the
nearest resolving peak. Specificity is a procedure to
detect quantitatively the analyte in presence of
component that may be expected to be present in
the sample matrix, while selectivity is the
procedure to detect qualitatively the analyte in
presence of components that may be expected to be
present in the sample matrix.
Limit of detection and Limit of quantitation
Sensitivity of the proposed method was estimated in
terms of Limit of Detection (LOD) and Limit of
Quantitation (LOQ). LOD = 3.3 x ASD/S and LOQ =
10 x ASD/S, Where, ‘ASD’ is the average standard
deviation and ‘S’ is the slope of the line.
Robustness
Robustness was evaluated by making deliberate
variations in few method parameters such as variation
of wave length; flow rate and variations in
temperature. The robustness of the method was studied
for Sitagliptin and Simvastatin
RESULTS AND DISCUSSION
Selection of Chromatographic Conditions
and Optimization of Mobile Phase:
Mobile phase was optimized to separate Sitagliptin
and Simvastatin using nucleosil C-18 column (150
mm x 4.6 mm i.d., 5μm). Initially, ACN and phosphate
buffer in the ratio of (70:30) were tried as mobile
phase but the splitting of the peaks for both these
drugs was observed. Therefore, after adjustment of pH
of mixed phosphate buffer to 3.5 with Triethyle amine,
and mobile phase composition (ACN and phosphate
buffer in 70:30 % v/v) was tried for resolution of both
drugs. Good resolution and symmetric peaks were
obtained. The flow rate of the mobile phase was 1 mL
min-1. Under optimum chromatographic conditions,
the retention time for Sitagliptin and Simvastatin were
found to be 3.242 and 6.497 min, respectively when
the detection was carried out at 254 nm. A typical
chromatogram of two drugs is shown in (Figure 3).

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LINEARITY DATA
The Linear detector response for Sitagliptin and
Simvastatin is demonstrated by concentration
versus Area. Over the range of 25 to 150% with
respect to the target concentration (Dosage).
Table-1 For Peak Area of Sitagliptin
% Linearity Conc(mcg) Area
25 25 766916
50 50 1531453
75 75 2287590
100 100 3048305
125 125 3786765
150 150 4579050
Figure- 5 Calibration curve for sitagliptin
Table-2 For Peak Area of Simvastatin
% Linearity Conc(mcg) Area
25 5 310546
50 10 624181
75 15 939359
100 20 1248779
125 25 1563474
150 30 1890091
Figure-6 Calibration curve for Simvastatin
R² = 0.9999
0.000
1000000.000
2000000.000
3000000.000
4000000.000
5000000.000
1 2 3 4 5 6
R² = 0.9999
0
500000
1000000
1500000
2000000
2500000
1 2 3 4 5 6 7

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Table-3 PRECISION
S No Name Sita Simva
RT Area RT Area
1 S-Precision-1 3.245 3088245 6.504 1270021
2 S-Precision-2 3.243 3091365 6.501 1267588
3 S-Precision-3 3.244 3100702 6.497 1272347
4 S-Precision-4 3.242 3082899 6.492 1258526
5 S-Precision-5 3.242 3096365 6.488 1275423
6 S-Precision-6 3.242 3100568 6.488 1273320
Average 3.243 3093357 6.495 1269538
Standard Deviation 0.0013 7133.9 0.007 6035.35
RSD 0.0390 0.231 0.10 0.48
Table-4 Method Precision
S No Name Sita Simva
RT Area RT Area
1 M-Precision-1 3.242 3092232 6.500 1264535
2 M-Precision-2 3.246 3091365 6.502 1265545
3 M-Precision-3 3.241 3092623 6.502 1271365
4 M-Precision-4 3.245 3095865 6.492 1264531
5 M-Precision-5 3.244 3096445 6.502 1268435
6 M-Precision-6 3.241 3091545 6.495 1263452
Average 3.243 3093346 6.499 1266311
Standard Deviation 0.0021 2230.7 0.004 3004.58
RSD 0.0659 0.072 0.066 0.237
Acceptance criteria
The % of RSD for Area and RT from Repeated
injections should not be more than 2.0%.
ACCURACY
The accuracy of the test method is demonstrated by
% of recovery. The sample preparations are spiked
with known amount of standard at three
concentration levels and injected three times (Like
80% 100% and 120%).
Accuracy data.
Table-4 Recovery studies of Sitagliptin by RP-HPLC method
S.No Spike
level
Peak
area
Amount
Added
(µg/ml)
Amount
Recovered
(µg/ml)
%Recovery Avg %
RSD
1 80%
2484463 80 79.47 99.34 99.24 0.76
2457436 80 78.64 98.30
2497082 80 80.12 100.16
2s 100%
3127747 100 100.01 100.01 99.48 0.38
3093509 100 99.1 99.1
3106925 100 99.3 99.35
3 120%
3786765 120 121.38 101.15 100.9 0.17
3774773 120 120.86 100.72
3784305 120 121.08 100.86

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Table-5 Recovery studies of Simvastatin by RP-HPLC method
S.No Spike
level
Peak
area
Amount
Added
(µg/ml)
Amount
Recovered
(µg/ml)
%Recovery Avg %
RSD
1 80%
1056502 16 16.05 100.35 99.4 0.71
1037119 16 15.78 98.65
1047401 16 15.87 99.20
2 100%
1282182 20 19.83 99.15 99.2 0.69
1282777 20 19.68 98.42
1305643 20 20.02 100.1
3 120%
1563474 24 23.78 99.10 99.2 0.16
1570685 24 23.86 99.45
1570685 24 23.86 99.45
Table-5 Results of global % recovery studies
Different level in % Sitagliptin Simvastatin
80 99.24 99.43
100 99.48 99.20
120 100.9 99.29
Average 99.87 99.30
SD 0.732 0.094
%RSD 0.74 0.095
Acceptance criteria
The % of recovery should be between 98 to 102%.
LIMIT OF DETECTION (LOD)
Table-6 Limit Of detection results.
S.NO Name LOD Value (µg/ml)
1. Sitagliptin 1.305
2. Simvastatin 0.257
Table-7 Limit of Quantitation (LOQ) results.
S.NO Name LOQ Value( µg/ml)
1. Sitagliptin 3.941
2. Simvastatin 0.77
ROBUSTNESS
Robustness for Sitagliptin and Simvastatin
The robustness of test method is demonstrated by
carrying out intentional method variations like
mobile phase flow changes, mobile phase
compositions and column oven temperature
variations etc...

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Table-8 Robustness for Sitagliptin and Simvastatin
S No Sitagliptin Simvastatin
RT Area RT Area
1 Standard 3.245 3095888 6.492 1264555
2 Robustness-Flow Change-1 2.89 2863684 5.777 1197338
3 Robustness-Flow Change-2 3.7 3667137 7.4 1530950
4 Robustness-Column Oven Temperature-1 3.23 3208756 6.084 1353173
5 Robustness-Column Oven Temperature-2 3.253 6.908 3204189 1329066
ASSAY
Assay for Sitagliptin and Simvastatin
Standard preparation
Transfer 10 ml of standard stock solution in to 100 mL
volumetric flask and make up to volume with diluent.
Sample Preparation
Transfer sample quantitatively equivalent to 50
mg of Sitagliptin and 10 mg of Simvastatin in to
50 mL volumetric flask add 50 mL of diluent,
sonicate to dissolve for 10 minutes and dilute to
volume with diluent. Further filter the solution
through filter paper. Dilute 10 ml of filtrate to 100
ml with mobile phase.
Procedure
Inject 20 µL of blank solution, standard solution, and
sample solution record the chromatogram. And
calculate percentage of assay.
Table-9 Assay
Sitagliptin Simvastatin
S No Name RT Area RT Area
1 Standard-1 3.256 3133162 6.491 1296594
2 Standard-2 3.250 3127115 6.489 1285416
Avg 3.253 3130139 6.490 1291005
3 Sample-1 3.254 3099385 6.498 1278425
4 Sample-2 3.253 3128408 6.492 1287541
Avg 3.254 3113897 6.495 1282983
Table-10Results for Sitagliptin
3113897 50 10 50 100 99.82 360 mg/Tab %Assay
3130139 50 100 360 10 100 49.74 99.48
Table- 11Results forSimvastatin
1282983 10 10 50 100 99.75 360 mg/tab %Assay
1291005 50 100 360 10 100 9.94 99.38
SYSTEM SUITABILITY PARAMETERS
Table-12 System suitability parameters results for Sitagliptin and Simvastatin
Parameters Results
Sitagliptin Simvastatin
Tailing factor 0.61 1.45
Theoretical plates per column 0.79 0.4835

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CONCLUSION
The developed RP-HPLC method is simple,
precise, accurate, selective and reproducible. The
method has been found to be adequately robust and
can be used for simultaneous determination of
Sitagliptin and Simvastatin in bulk and tablet
formulation. The method was validated as per ICH
guidelines.
ACKNOWLEDGEMENT
The authors are thankful to Bio Leo lab. Pvt. Ltd,
Hyderabad for providing a gift samples, the authors
are also thankful to Department of pharmaceutical
analysis, Smt.Sarojini Ramulamma college of
pharmacy, Palamuru University, Mahaboobnagar,
Andhra Pradesh for encouragement
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Development and Validation of RP-HPLC Method for Simultaneous Estimation of Sitagliptin and Simvastatin in Bulk and Tablet Dosage Form

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