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ORIGINAL ARTICLE
Year : 2014  |  Volume : 5  |  Issue : 2  |  Page : 153-157

Reverse phase high-performance liquid chromatography method for the simultaneous estimation of sitagliptin with simvasatin in bulk and tablet formulation


Department of Pharmaceutical Chemistry, Government College of Pharmacy, Bengaluru, Karnataka, India

Date of Web Publication27-Aug-2014

Correspondence Address:
G Manikanta
Department of Pharmaceutical Chemistry, Government College of Pharmacy, Bengaluru, Karnataka
India
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Source of Support: None, Conflict of Interest: None


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  Abstract 

Aim: A simple, sensitive, precise, and rapid reverse phase high-performance liquid chromatography method was developed and validated for simultaneous estimation of sitagliptin (SIT) and simvastatin (SIM) in bulk drug and tablet dosage forms. Materials and Methods: The separation was achieved by using Prontosil 120-5-CN (250 mm × 4.6 mm, 5 μm) column with a mobile phase consisting of acetonitrile:methanol:phosphate buffer (45:10:45 v/v). The mobile phase was delivered at a flow rate of 1.0 ml/min. Analysis was performed at ambient temperature with detection at 254 nm. Results: The retention times of SIT and SIM were found to be 4.8 and 6.0 min and the calibration curves were linear (R2 = 0.999) over a concentration range from 1 to 30 μg/ml for SIT and SIM, respectively. Limit of detection and limit of quantitation were 0.6 μg/ml and 0.7 μg/ml for SIT and 0.1 μg/ml and 0.2 μg/ml for SIM, respectively. Conclusion: The developed method was validated for parameters such as system suitability, specificity, linearity, accuracy, precision, ruggedness, and robustness as per International Conference on Harmonization guidelines and the results were found to be within the limits. Hence, it can be used for the routine quality control of SIT and SIM in the bulk sample and tablet dosage forms.

Keywords: Reverse phase high-performance liquid chromatography, simvastatin, sitagliptin


How to cite this article:
Manikanta G, Channabasavaraj K P, Madhusudan Yadav G S. Reverse phase high-performance liquid chromatography method for the simultaneous estimation of sitagliptin with simvasatin in bulk and tablet formulation. Drug Dev Ther 2014;5:153-7

How to cite this URL:
Manikanta G, Channabasavaraj K P, Madhusudan Yadav G S. Reverse phase high-performance liquid chromatography method for the simultaneous estimation of sitagliptin with simvasatin in bulk and tablet formulation. Drug Dev Ther [serial online] 2014 [cited 2017 Mar 24];5:153-7. Available from: http://www.ddtjournal.org/text.asp?2014/5/2/153/139634


  Introduction Top


Sitagliptin (SIT) chemically is (3R)-3-amino-1-[3-(trifluoro methyl)-6,8-dihydro-5h- [1, 2, 4]triazolo [3,4-c]pyrazin-7-yl]-4-(2, 4, 5-trifluorophenyl)butan-1-one, [Figure 1] shows structure of SIT, an oral anti-diabetic agent that blocks dipeptidyl peptidase-4 (DPP-4) activity. Sitagliptin increased incretin levels (glucagon-like peptide-1 and gastric inhibitory polypeptide), which inhibit glucagon release, in turn decreases blood glucose, but more significantly increases insulin secretion. [1],[2],[3] Simvastatin (SIM) chemically is 2,2-dimethyl butanoic acid (1S,3R,7S,8S,8aR)-1, 2, 3, 7, 8, 8ahexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4-hydroxy-6 oxo2H pyran-2yl]ethyl]1-napthalenyl ester; [Figure 2] shows a structure of SIM, used as 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor in the treatment of primary hypercholesterolemia and is effective in reducing total and low-density lipoprotein cholesterol as well as plasma triglycerides and apolipoprotein B. [3],[4],[5],[6] The literature reveals that few methods have been reported for simultaneous estimation of SIT and SIM by ultraviolet (UV), [7],[8],[9] high-performance liquid chromatography (HPLC), [10],[11],[12] and high-performance thin layer chromatography. [13] Hence, attempt was made to develop new versatile, accurate, precise, and economical reverse phase (RP)-HPLC method for the simultaneous estimation of SIT and SIM in bulk and tablet dosage form and method was validated as per International Conference on Harmonization (ICH) guidelines. [14],[15]
Figure 1: Structure of sitagliptin

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Figure 2: Structure of simvastatin

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  Materials and Methods Top


Chemicals and reagents

High-performance liquid chromatography grade methanol, acetonitrile and all other analytical grade reagents were purchased from Merck, India. HPLC grade water was prepared using Milli-Q water purification system. JUVISYNC tablets were purchased from local markets of Bangalore. Class A glassware is used throughout the experiment. Reference standard SIT and SIM gift samples were obtained from Watson Laboratories, Mumbai.

Instrument and chromatographic conditions

The chromatographic system consists of LC-20AT SHIMADZU-SPD-M20A equipped with photodiode array (PDA) detector and rheodyne injector with 20 μl loop volume. Chromatographic separations were carried out using Prontosil 120-5-CN (E) column (250 mm × 4.6 mm i.d.) packed with 5μm diameter particles. The mobile phase consisting of acetonitrile methanol and 10 mM phosphate buffer (1.36 g of potassium dihydrogen orthophosphate in 1000 ml of HPLC water) in the ratio of 45:10:45 v/v. The flow rate was 1 ml/min and detection was carried out at 254 nm using PDA detector.

Preparation of standard stock solutions of sitagliptin and simvastatin

Accurately 10 mg of SIT and SIM were weighed separately into a clean and dry 10 ml volumetric flasks, dissolved with sufficient volume of diluent. The volumes were made up to 10 ml with diluent to get a concentration of 1000 μg/ml each drug (stock I). About 1 ml of each resulting stock solution was further diluted separately into 10 ml volumetric flasks with diluent to get a concentration of 100 μg/ml for both SIT and SIM, respectively (stock II). From stock II, 2 ml of SIT and 1 ml of SIM were transferred together into a 10 ml volumetric flask and final volume was then made up to 10 ml with diluent to get a concentration of 20 μg/ml and 10 μg/ml of SIT and SIM, respectively.

Preparation of sample stock solution

Twenty tablets of JUVISYNC each containing 100 mg of SIT and 40 mg of SIM were weighed and finely powered. Powder equivalent to 100 mg of SIT and 40 mg of SIM was taken together and transferred into a clean, dry 100 ml volumetric flask. The powder was first dissolved in methanol and sonicated for 10 min. The resulting mixture was then filtered through Whatmann Filter No. 0.45 μ. The final volume of filtrate was made up to 100 ml with diluent. About 1 ml of the resulting solution was diluted to 10 ml with diluent to get a concentration of 100 μg/ml and 40 μg/ml of SIT and SIM, respectively.


  Method Validation Top


The developed method has been validated with respect to various parameters, in accordance with the ICH guidelines. [14],[15]

Linearity

The various concentration of working standard solutions of SIT and SIM were made by pipetting 0.1-3.0 ml from stock II separately into a series of 10 ml volumetric flask and diluted to 10 ml to get the final concentration of 1-30 μg/ml solutions. About 20 μl of each of these working standard solutions of SIT and SIM ranging from 1 to 30 μg/ml were injected into a chromatograph at flow rate of 1 ml/min. Retention time and peak area obtained were recorded and standard calibration curve was plotted for SIT and SIM and linearity equations were derived. The calibration curves were shown in [Figure 3] and [Figure 4].
Figure 3: Linearity plot for sitagliptin

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Figure 4: Linearity plot for simvastatin

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Specificity

The specificity of the proposed method was demonstrated that the excipients from sample and diluents do not interfere in the drug peak. The chromatogram was presented in [Figure 5].
Figure 5: Chromatogram for specificity

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System suitability parameter

This test ensures that the analytical system is working properly and can give accurate and precise results. [Figure 6] and [Table 1] shows the results.
Figure 6: Chromatogram of sitagliptin and simvastatin

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Table 1: System suitability parameters

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Precision

System precision


Successive six injections of standard solution (six replicates) were injected into a HPLC chromatograph, the peak area, and chromatograms obtained were recorded. The percentage relative standard deviation was calculated for peak areas of replicates. The results are shown in [Table 2].
Table 2: Results of precision parameters

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Method precision (repeatability)

Method precision was assessed by replicate injections and measurement of peak area for SIT and SIM in six replicates. The amounts of SIT and SIM in sample were calculated from their respective standard linearity equations. The results obtained are presented in [Table 2].

Intermediate precision (ruggedness)

Intermediate precision was determined by the assay of sample sets by different analysts, the peak area and the chromatograms were recorded. The percentage assay of standard drug was calculated from the peak areas of replicates. The results obtained are mentioned in [Table 3].
Table 3: Results of intermediate precision (ruggedness)

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Accuracy

Accuracy is expressed as percentage recovery by the assay of known added amounts of analyte carried out at three different levels (80%, 100%, and 120%). The results are tabulated in [Table 4].
Table 4: Results of accuracy of SIT and SIM

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Limit of detection and limit of quantification

For estimation of limit of detection (LOD) and limit of quantification (LOQ), visualization method was followed. In visualization method lower dilutions of the standard drugs of SIT and SIM were successively prepared, injected in to the chromatograph and response obtained was recorded and presented in [Table 5].
Table 5: Results of LOD and LOQ

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Robustness

The robustness of the method was carried out by small, but deliberate variations in the flow rate, mobile phase ratio and detection wavelength. Results are presented in [Table 6].
Table 6: Results of robustness

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Assay

The proposed method was also evaluated by the assay of SIT and SIM in their combined dosage formulation. The results are mentioned in [Table 7].
Table 7: Assay results of tablet formulation

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  Results and Discussion Top


Literature review reveals only few UV and HPLC methods have been reported for the simultaneous estimation of SIT and SIM in bulk and tablet dosage formulation till date. So, new simple, rapid, and accurate RP-HPLC method has been developed and validated for the simultaneous estimation of SIT and SIM in bulk and tablet dosage formulation. The separation was carried out by using Prontosil 120-5-CN (250 mm × 4.6 mm, 5 μm) column with a mobile phase consisting of acetonitrile:methanol:phosphate buffer (45:10:45 v/v). The mobile phase was delivered at a flow rate of 1.0 ml/min with detection at 254 nm. The retention times of SIT and SIM were found to be 4.8 and 6.0 min and the calibration curves were linear (R2 = 0.999) over a concentration range from 1 to 30 μg/ml for SIT and SIM, respectively. LOD and LOQ were 0.6 μg/ml and 0.7 μg/ml for SIT and 0.1 μg/ml and 0.2 μg/ml for SIM, respectively. The developed method was validated for parameters such as system suitability, specificity, linearity, accuracy, precision, ruggedness, and robustness as per ICH guidelines and the results were found to be within the limits.


  Conclusion Top


Good agreement was seen in the assay results of tablet formulation as well as in bulk by developed method. It can be concluded that the proposed method was good approach for obtaining reliable results and were found to be suitable for the routine estimation of SIT and SIM in bulk and tablet formulation.


  Acknowledgment Top


The authors are thankful to Watson Laboratories, Mumbai, India, for providing gift sample of SIT and SIM for the research work.

 
  References Top

1.Available from: http://www.en.wikipedia.org/wiki/sitagliptin.svs. [Last accessed on 2011 Dec 19].  Back to cited text no. 1
    
2.Available from: http://www.drugbank.ca/drugs/DB0126. [Last accessed on 2011 Dec 19].  Back to cited text no. 2
    
3.Available from: http://www.merck.com/product/usa/pi_circulars/j/juvisync/juvisync_pi.pdf. [Last accessed on 2011 Dec 19].  Back to cited text no. 3
    
4.Indian Pharmacopoeia. Vol. 3. Ghaziabad: The Indian Pharmacopoeia Commission; 2010. p. 2103-5.  Back to cited text no. 4
    
5.United States of Pharmacopoeia and National Formulary. United States Pharmacopeial; 26 th edition, November, 2002 p. 1571-2.  Back to cited text no. 5
    
6.Harry GB. Analytical Profiles of Drug Substances and Excipients. Vol. 22. Academic Press, USA. The Academic Press; 2005. p. 359-88.  Back to cited text no. 6
    
7.Phaneemdra D, Venkatesh V, Ramarao N. Simultaneous estimation of simvastatin and sitagliptin by using different analytical methods. Int J Adv Pharm Anal 2012;2:19-23.  Back to cited text no. 7
    
8.Sonali DR, Pallavi MP, Vitthal VC, Pravin DC. Q-absorbance ration spectrophotometric method for the simultaneous estimation of sitagliptin phosphate and simvasatatin in their combined dosage form. Asian J Biochem Pharm Res 2012;2:162-9.  Back to cited text no. 8
    
9.Sheetal S, Nimita M, Priya B, Sohail H, Prabhat J. Development of UV-spectrophotometry and RP-HPLC method and its validation for simultaneous estimation of sitagliptin phosphate and simvastatin in marketed formulation. Int J Pharm Bio Arch 2012;3:673-8.  Back to cited text no. 9
    
10.Anilkumar V, Arunakumari V, Saikumar VS, Srinivasarao A, Santoshkumar T. Validated RP-HPLC method for the simultaneous estimation of sitagliptin and simvastatin in dosage forms. Int J Chem Anal Sci 2012;3:1611-4.  Back to cited text no. 10
    
11.Kavitha KY, Geetha G, Hariprasad R, Venkatanarayana R, Subramanian G. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of sitagliptin and simvastatin. Int Res J Pharm 2012;3:123-7.  Back to cited text no. 11
    
12.Burugula L, Mullangi R, Pilli NR, Makula A, Lodagala DS, Kandhagatla R. Simultaneous determination of sitagliptin and simvastatin in human plasma by LC-MS/MS and its application to a human pharmacokinetic study. Biomed Chromatogr 2013;27:80-7.  Back to cited text no. 12
    
13.Rathod S, Patil P, Chopade V. Development and validation of HPTLC method for the estimation of sitagliptin phosphate and simvastatin in bulk and marketed formulation. Int J Drug Dev Res 2012;4:292-7.  Back to cited text no. 13
    
14.ICH Guidance. Validation of Analytical Methods: Definition and Terminology. Geneva: International Conference on Harmonization, Q2A; Nov., 2005.  Back to cited text no. 14
    
15.ICH Guidance. Validation of Analytical Procedures: Methodology. Geneva: International Conference on Harmonization, Q2B; Nov., 2005.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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Abstract
Introduction
Materials and Me...
Method Validation
Results and Disc...
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Acknowledgment
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