Simultaneous Estimation of Metformin HCl and Sitagliptin Phosphate in Tablet Dosage Forms by RP-HPLC.
Ravi Pratap Pulla1, B.S. Sastry2, Y. Rajendra Prasad3 and N. Appala Raju4
1Department of Pharmaceutical Chemistry, SSJ College of Pharmacy, V.N. Pally, Gandipet, Hyderabad-500 075. 2Department of Pharmaceutical Chemistry, Principal and Professor, Medak Institute of Technology-Pharmacy, Kothapet (V), Near Narsapur, Shivampet, Medak District- 502 334. 3Department of Pharmaceutical Chemistry, Associate Professor, Andhra University – College of Pharmaceutical Sciences, Peda Waltair, Visakhapatnam- 530 003. 4Department of Pharmaceutical Chemistry, Sultan-Ul-Uloom College of Pharmacy, Mount Pleasant, Road No# 3, Banjara Hills, Hyderabad-500 034.
*Corresponding Author E-mail: ravi_477@rediffmail.com
ABSTRACT:
A simple, precise, rapid and accurate reverse phase HPLC method was developed for the estimation of Metformin HCl and Sitagliptin PO4 in tablet dosage forms. An Xterra C18, 250x4.6 mm, column with 5 μm particle size and the mobile phase consisting of 0.03M K2HPO4 in water pH-3.2 adjusted with o-Phosphoric Acid: Acetonitrile in the gradient mode was used. The flow rate was 1.0 ml/min and the effluents were monitored at 207 nm. The retention times were 2.510 min for Metformin HCl and 7.673 min for Sitagliptin PO4. The detector response was linear in the concentration of 14-168 mcg/mL for Metformin HCl and 1.3-15.6 mcg/mL for Sitagliptin PO4. The respective linear regression equation being Y= 48822.5x+10329 for Sitagliptin PO4 and Y= 70435x+238894 for Metformin HCl. The Limit of Detection (LOD) is 0.13 mcg and 0.14 mcg for Sitagliptin PO4 and Metformin HCl respectively. The Limit of Quantification (LOQ) is 0.39 mcg for Sitagliptin PO4 and 0.42 mcg for Metformin HCl. The percentage assay of Metformin HCl and Sitagliptin PO4 was 99.46 % and 99.19% respectively. The method was validated by determining its accuracy, precision and system suitability. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Metformin HCl and Sitagliptin PO4 in bulk drug and in its pharmaceutical dosage forms.
KEYWORDS: Metformin HCl, Sitagliptin PO4, RP-HPLC, Estimation, and Tablets.
INTRODUCTION:
Metformin Hydrochloride, chemically is (N, N-Dimethylimidodicarbonimidic Diamide Hydrochloride (Figure: 1). The empirical formula is C4H11N5.HCl and the molecular weight is 165.63gms/mol. It is an oral antihyperglycemic drug used in the management of Type II Diabetes. It is a biguanide that reduces glycemia, improves insulin action and lowers blood glucose. Sitagliptin Phosphate, chemically is, 7-[(3R)-3-Amino-1-oxo-4-(2,4,5 Trifluorophenyl) butyl]-5,6,7,8-Tetrahydro-3-(Trifluoro methyl)-1,2,4-Triazolo [4,3-a] pyrazine phosphate (1:1) monohydrate (Figure:2). The empirical formula is C16H15F6N5O.H3PO4.H2O and the molecular weight is 523.32 gms/mol.
It is an orally active, potent and selective inhibitor of Dipetidyl Peptidase-IV (DPP-IV) enzyme for treatment of Type II Diabetes1-3. It is the first and only prescribed medication in a new class of oral antihyperglycemic agents, which enhances the body’s own ability to lower blood glucose when it is elevated. Literature survey reveals a few chromatographic methods 4-8 to determine the sitagliptin phosphate in tablet dosage form and in biological fluids. So far, no assay methods by liquid chromatography were reported for the simultaneous estimation of Metformin HCl and Sitagliptin PO4 in pharmaceutical dosage forms. The availability of an HPLC method with high sensitivity and selectivity will be very useful for the determination of Metformin HCl and Sitagliptin PO4 in pharmaceutical formulations. The aim of the study was to develop a simple, precise and accurate reverse-phase HPLC method for the estimation of Metformin HCl and Sitagliptin PO4 in bulk drug samples and in pharmaceutical dosage form.
Figure: 1 Metformin HCl
Figure: 2 Sitagliptin PO4
EXPERIMENTAL:
Materials and Methods:
Metformin HCl and Sitagliptin PO4 were obtained as a gift samples from MSN Pharmachem Pvt.Ltd, Hyderabad. Acetonitrile and water used were of HPLC grade (Qualigens). Commercially available tablets (Janumet®-Novartis) were procured from local market.
Instrument:
Quantitative HPLC was performed on liquid Chromatograph, Waters separation 2996, PDA detector module equipped with automatic injector with injection volume 20 µl, and 2693 pump. An X-terra RP-C18 column (250x4.6 mm i.d; particle size 5 μm) was used. The HPLC system was equipped with Empower Software.
HPLC Conditions:
The contents of the mobile phase were 0.03M K2HPO4 in water pH-3.2 adjusted with o-Phosphoric Acid: Acetonitrile in the gradient mode has been used. They were filtered before use, through a 0.45 μm membrane filter, and pumped from the respective solvent reservoirs to the column at a flow rate of 1.0 ml/min. The run time was set at 25.0 min and the column temperature was ambient. Prior to the injection of the drug solution, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. The eluents were monitored at 207 nm.
Preparation of Standard Stock solution:
A standard stock solution of the drug was prepared by dissolving 13 mg of Sitagliptin PO4 and 140 mg of Metformin HCl in 100 ml volumetric flask containing 30 ml of diluents (Acetonitrile: HPLC Grade Water 50:50), sonicated for about 15 min and then made up to 100 ml with methanol to get standard stock solution of 1.4 mg/mL of Metformin HCl and 0.13 mg/mL Sitagliptin PO4.
Working Standard solution:
5ml of the above stock solution was taken in 50 ml volumetric flask and made up to 50 ml with diluents (Acetonitrile: HPLC Grade Water-50: 50v/v) to get a concentration of each 140µg/ml of Metformin HCl and 0.13 mcg/mL Sitagliptin PO4.
Preparation of Sample solution:
Twenty tablets (Janumet® Novartis) were weighed, and then powdered. A sample of the powdered tablets, equivalent to mixture containing concentration of each 140 mg/ml of Metformin HCl and 13 mg/mL Sitagliptin PO4 active ingredients, was mixed with 30 ml of Acetonitrile: HPLC Grade Water-50: 50v/v as diluent in 50 ml volumetric flask. The mixture was allowed to stand for 1 hr with intermittent sonication to ensure complete solubility of the drug, and then filtered through a 0.45 μm membrane filter, followed by adding methanol up to100 ml to obtain a stock solution each of 1.4mg/ml of Metformin HCl and 0.13 mg/mL. Sitagliptin PO4. 5ml of the above sample stock solution was taken in 50 ml volumetric flask and made up to 50 ml with diluent to get a concentration of each 140 µg/ml of Metformin HCl and 13 mcg/mL of Sitagliptin PO4.
Linearity:
Aliquots of standard Metformin HCl and Sitagliptin PO4 stock solution were taken in different 10 ml volumetric flasks and diluted up to the mark with the mobile phase such that the final concentrations of Metformin HCl and Sitagliptin PO4 are in the range of 7-182 μg/ml and 0.65-16.9 mcg/mL respectively. Each of these drug solutions (20 μL) was injected three times into the column, and the peak areas and retention times were recorded. Evaluation was performed with PDA detector at 207 nm and a Calibration graphs were obtained by plotting peak area versus concentration of Metformin HCl and Sitagliptin PO4 (Figure: 3). The plot of peak areas of each sample against respective concentration of Metformin HCl and Sitagliptin PO4 were found to be linear in the range of 14-168 μg/ml and 1.3-15.6mcg/mL with correlation coefficient of 0.9998. Linear regression least square fit data obtained from the measurements are given in Table I. The respective linear regression equation being Y= Y= 48822.5x+10329 for Sitagliptin PO4 and Y= 70435x+238894 for Metformin HCl. The regression characteristics, such as slope, intercept, and %RSD were calculated for this method and given in Table I.
Table I: Linear Regression Data of Calibration curves.
|
Parameter |
Metformin |
Sitagliptin |
|
Conc.range (µg/ml) Slope (m) Intercept (b) Correlation coeff. % RSD |
14-168 70435.88 238894.23 0.9998 0.5 |
1.3-15.6 48822.5 10329.99 0.9998 0.2 |
Table II: Assay and Recovery of Metformin HCl and Sitagliptin in Tablet dosage form.
|
Tablet formulation |
Amount claim (mg/tablet) |
Amount claim (mg/tablet) |
Amount Obtained (mg)* by proposed method |
** % Recovery by the Proposed method |
||
|
Metformin HCl |
Sitagliptin PO4 |
Metformin HCl |
Sitagliptin PO4 |
Metformin HCl |
Sitagliptin PO4 |
|
|
1 |
500 |
50 |
497.3 |
49.58 |
99.08 |
101.2 |
|
2 |
500 |
50 |
498.5 |
49.22 |
99.19 |
99.79 |
|
3 |
500 |
50 |
499.1 |
49.76 |
99.73 |
99.24 |
*Average of three determinations. ** After spiking the sample.
Figure 3: Typical Chromatogram of Metformin HCl and Sitagliptin PO4 by RP-HPLC.
Mobile phase composition in gradient mode for RP-HPLC:
|
Time in Minutes |
% of Mobile phase-A |
% of Mobile phase-B |
|
0.01 |
85 |
15 |
|
11.0 |
30 |
70 |
|
25.0 |
30 |
70 |
|
26.0 |
85 |
15 |
|
30.0 |
85 |
15 |
|
Mobile Phase-A= 0.03M K2HPO4 in water pH-3.2 adjusted with o-Phosphoric Acid and Mobile Phase-B=Acetonitrile |
||
Figure 3: Calibration curves of the Metformin HCl and Sitagliptin PO4 by RP-HPLC.
Metformin HCl:
Sitagliptin PO4:
Assay:
20 µl of sample solution (Janumet® Tablets, Novarstis) was injected into the injector of liquid chromatograph. The retention times were found to be 7.673 min for Sitagliptin PO4 and 2.510 for Metformin HCl. The amount of drug present per tablet was calculated by comparing the peak area of the sample solution with that of the standard solution. The data are presented in Table II.
Recovery Studies:
Accuracy was determined by recovery studies of Metformin HCl and Sitagliptin PO4; known amount of standard was added to the preanalysed sample and subjected to the proposed HPLC analysis. Results of recovery study are shown in Table II. The study was done at three different concentration levels.
RESULTS AND DISCUSSION:
The system suitability tests were carried out on freshly prepared standard stock solutions of Metformin HCl and Sitagliptin PO4. Parameters that were studied to evaluate the suitability of the system are given in Table III.
Table III: Validation Summary:
|
Parameter |
Metformin HCl |
Sitagliptin PO4 |
|
System Suitability Theoretical Plates(N) Tailing factor Retention time(min) Resolution % Peak Area |
10841.99 1.23 2.504 --- 93.93 |
81992.29 1.29 7.673 43.81 6.03 |
|
LOD (µg/ml) LOQ (µg/ml) |
0.14 0.42 |
0.13 0.39 |
Limit of Detection (LOD) and Limit of Quantification (LOQ) :
The Limit of Detection (LOD) is 0.13 mcg and 0.14 mcg for Sitagliptin PO4 and Metformin HCl respectively. The Limit of Quantification (LOQ) is 0.39 mcg for Sitagliptin PO4 and 0.42 mcg for Metformin HCl. From the typical chromatogram of Metformin HCl and Sitagliptin PO4 as shown in Figure: 3, it was found that the retention times were found to be 7.673 min for Sitagliptin PO4 and 2.510 for Metformin HCl. A mixture of 0.03M K2HPO4 in water pH-3.2 adjusted with o-Phosphoric Acid: Acetonitrile in the gradient mode was found to be the most suitable as mobile phase to obtain the peaks well defined and free from tailing. In the present developed HPLC method, the standard and sample preparation required less time and no tedious extractions were involved. A good linear relationship (r=0.9998) was observed between the concentration range of 14-168 μg/ml and 1.3-15.6mcg/mL for Metformin HCl and Sitagliptin PO4 respectively. Low values of standard deviation are indicative of the high precision of the method. The assay of Metformin HCl and Sitagliptin PO4 tablets was found to be 99.46% and 99.19% respectively. From the recovery studies it was found that about 99.08% of Metformin HCl and 101.2% of Sitagliptin PO4 were recovered which indicates high accuracy of the method. The absence of additional peaks in the chromatogram indicates non-interference of the common excipients used in the tablets. This demonstrates that the developed HPLC method is simple, linear, accurate, sensitive and reproducible. Thus, the developed method can be easily used for the routine quality control of bulk and tablet dosage forms of Metformin HCl and Sitagliptin PO4 within a short analysis time.
ACKNOWLEDGEMENTS:
The authors are grateful to M/s MSN Pharmachem Pvt. Ltd, Hyderabad for the supply of the gift samples of Metformin HCl and Sitagliptin PO4.
REFERENCES:
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Received on 30.01.2011 Modified on 20.02.2011
Accepted on 06.03.2011 © RJPT All right reserved
Research J. Pharm. and Tech. 4(4): April 2011; page 646-649