Int J Pharm Pharm Sci, Vol 9, Issue 12, 163-169Original Article


A NOVEL STABILITY-INDICATING REVERSE PHASE LIQUID CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN AND TENELIGLIPTIN IN PURE AND PHARMACEUTICAL FORMULATIONS

A. SWETHA, B. RAMYA KUBER

Department of Pharmaceutical Analysis, Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam, Tirupathi, Andhra Pradesh, India
Email: Swetha.addanki06@gmail.com

Received: 06 Jul 2017 Revised and Accepted: 02 Nov 2017


ABSTRACT

Objective: The present method was proposed to develop a simple, sensitive, rapid, accurate and stability-indicating reverse phase liquid chromatographic method for the simultaneous estimation of metformin and teneligliptin in pure and pharmaceutical formulations.

Methods: The chromatographic separation was done on Discovery [250 mm X 4.6 mm: 5 μm is particle size] using a mobile phase composed of 0.1% orthophosphoric acid buffer: acetonitrile [65:35, v/v], the flow rate is 1 ml/min and the detection was carried out at 260 nm.

Results: The retention time of metformin and teneligliptin were found to be 2.517 min and 3.687 min, respectively. Stability indicating studies were conducting under the guidelines of an international conference on harmonization [ICH] Q1A R2 and the developed method was validated as per guidelines of ICH Q2 RI. The linearity was found in the range of concentration of 125-750 μg/ml and 5-30 μg/ml for metformin and teneligliptin. The detection of limit and quantification of limit was found to be 0.02 μg/ml and 0.07 μg/ml for metformin and 0.19 μg/ml and 0.56 μg/ml for teneligliptin, respectively.

Conclusion: A novel stability-indicating reverse-phase liquid chromatographic method for the simultaneous estimation of metformin and teneligliptin. The proposed method was adopted for the routine estimation of metformin and teneligliptin in bulk and pharmaceutical dosage forms.

Keywords: Method validation, Estimation, Stability indicating, Metformin, Teneligliptin


INTRODUCTION

Metformin (fig. 1) category is biguanide and chemically called as 3-[diaminomethylidene]-1, 1-dimethylguanidine. It is used for the treatment of type 2 diabetics; polycystic syndrome and limited use prevent the (CVD) and cancer complications of diabetes [1].

Teneligliptin (fig. 2) is potent, competitive and long-acting DPP-1V inhibitor and chemically called as {(2S, 4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl) piperazin-1-yl] pyrrolidin-2-yl} (1, 3-thiazolidin-3-yl) methanone. It is used for the treatment of type-2 diabetic mellitus [2-3]. Literature survey revealed that very few analytical method have been reported for the estimation of metformin and teneligliptin by using ultraviolet spectroscopy [4-6], high-performance liquid chromatography [7-9] and liquid chromate-graphy-mass spectroscopy [10] by individually or simultaneously with other drugs. From the literature survey confirms that there is no method has been reported for the stability indicating a simultaneous estimation of metformin and teneligliptin in pure and pharmaceutical dosage form by using RP-HPLC.

The present method has so many advantages like simple standard preparation process, a large range of concentration with high sensitive, low-cost solvent are used in mobile phase preparation and all parameters must be validated as per ICH guidelines [11-12]. Hence, the developed method was used for the simultaneous determination of metformin and teneligliptin in pure and pharmaceutical dosage forms.

Fig. 1: Structure of metformin [1]

Fig. 2: Structure of teneligliptin [2]

MATERIALS AND METHODS

Chemicals and reagents

Metformin and Teneligliptin obtained from Spectrum Research Private limited, [Hyderabad, India]. Orthophosphoric acid purchased from Qualigens fine chemicals limited [Mumbai, India] and acetonitrile [HPLC grade] purchased from Merck chemicals private limited [Mumbai, India].

Instruments

The system composed Waters HPLC 2695 equipped with quaternary pumps with PDA detector. The chromatographic separation was done on Discovery [250 mm X 4.6 mm, 5 μm particle size] column. Empower 2 software was used for the data acquisition and integration purpose.

Methods

Chromatographic conditions

The method development for separation of metformin and teneligliptin by using different solvents finally the separation was achieved with a mobile phase 0.1% orthophosphoric acid buffer: acetonitrile [65:35, v/v], pumped at a flow rate is 1 ml/min. The eluent detection was carried out at 260 nm by the observing of PDA detector. The mobile phase was vacuum filtered through a 0.45 μm membrane filter.

Preparation of solutions

Preparation of mobile of buffer

The buffer solution was prepared by dissolving 1 ml orthophosphoric acid in 1000 ml of water.

Preparation of standard solutions

The powder of 50 mg of metformin and 2 mg of teneligliptin were weighed and transferred into a 100 ml of calibrated volumetric flasks, 70 ml of diluent was added and sonicated for 25 min and makeup to the final volume with diluents. 1 ml was pipetted out from above stock solution and trance vertex into 10 ml volumetric flask and made up to 10 ml with diluent.

Preparation of sample solutions

20 tablets were weighed and calculate the average weight of each tablet then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 70 ml of diluent added and sonicated for 25 min, further the volume made up with diluent and filtered.

From the filtered solution 1 ml was pipetted out into a 10 ml volumetric flask and made up to 10 ml with diluent.

Method validation

The developed method was validated for system suitability, linearity, precision, limit of detection [LOD], limit of quantitation [LOQ] and accuracy under the guideline of ICH Q2R1.

System suitability

Verifying the system suitability parameters like theoretical plate count, tailing factor, percentage relative standard deviation of the peak and retention time.

Linearity

The range of linearity was evaluated between 125-750 μg/ml for metformin and 5-30 μg/ml and for teneligliptin. The calibration curve was a plot between concentration against corresponding peak area and linearity was estimated by least square method.

Precision

The precision of the developed method was carried out for same concentration level, six determinations were established, both intra-day and inter-day precision were conveyed in terms of percent relative standard deviation [% RSD].

LOD and LOQ

Determination value of the limit of detection and quantification by using the following formulas:

Limit of detection= 3.3 α/S

Limit of quantitation= 10 α/S

Where α is the standard deviation of the y-intercept and S is the slope from linearity plot

Accuracy

The accuracy was estimated by using standard addition method at 50 %, 100 % and 150 % levels. The percentage recovery and percentage relative standard deviations [% RSD] were taken into consideration for examine the accuracy.

Stability indicating studies

Acid hydrolysis

1 ml stock solution of metformin and teneligliptin, 1 ml of 2N hydrochloric acid was added and refluxed for 30 min at 60 °C. The solution was diluted to obtain 500 μg/ml for metformin and 20 μg/ml for teneligliptin. 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Base hydrolysis

1 ml stock solution of metformin and teneligliptin, 1 ml of 2N sodium hydroxide was added and refluxed for 30 min at 60 °C. The solution was diluted to obtain 500 μg/ml for metformin and 20 μg/ml for teneligliptin. 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Peroxide hydrolysis

1 ml stock solution of metformin and teneligliptin, 1 ml of 20% hydrogen peroxide was added and refluxed for 30 min at 60 °C. The solution was diluted to obtain 500 μg/ml for metformin and 20 μg/ml for teneligliptin. 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Thermal hydrolysis

1 ml stock solution of metformin and teneligliptin, placed in an oven at 105 °C for 6 h. The solution was diluted to obtain 500 μg/ml for metformin and 20 μg/ml for teneligliptin. 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo hydrolysis

Exposing the 5000 µg/ml for metformin and 200 µg/ml for teneligliptin solution to UV Light by keeping the beaker in UV chamber for 7 d or 200 Watt-hours/m2in photostability chamber. The resultant solution was diluted to obtain 500 µg/ml for metformin and 20 µg/ml for teneligliptin solutions. 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Neutral hydrolysis

Refluxing the drug solutions in water for 6 h at a temperature at 60 °C. The solution was diluted to obtain 500 μg/ml for metformin and 20 μg/ml for teneligliptin. 10 μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

RESULTS AND DISCUSSION

Method development

Optimization of mobile phase

Method development for the simultaneous estimation of metformin and teneligliptin was begins with a different combination of solvents with different ratios like [35:65, 45:55, and 50:50]. Although, finally a combination of 0.1% orthophosphoric acid buffer: acetonitrile [65:35, v/v] has appeared good resolution for metformin and teneligliptin.

Chromatographic conditions

The analytical conditions were selected, keeping in mind the chemical nature of metformin and teneligliptin. The development trails were taken using different conditions. The column selection has been done on the basis of back pressure, peak shape and theoretical plates. After evaluating all these factors, the chromatographic separation was carried out on Discovery column [250 mm X 4.6 mm; 5 μm is particle size] using a mobile phase consisting 0.1% orthophosphoric acid buffer: acetonitrile [65:35 v/v], the flow rate 1 ml/min and the injection volume were 10 μl, the detection was carried out at 260 nm. The peak retention time of metformin and teneligliptin were found to be 2.517 min and 3.687 min respectively. Hence this method was finalised as an optimized method for the simultaneous estimation of metformin and teneligliptin. The optimised chromatographic condition was shown in table 1 and the typical HPLC chromatogram of standard and sample were shown in fig. 3 and 4.

Method validation

System suitability

The developed method has produced theoretical plate above 2000 for metformin and teneligliptin with tailing factor less than 2. Similarly, the percent relative standard deviation [% RSD] of metformin and teneligliptin were less than 2, which ensure the suitability of the developed method. The results of the system suitability study were summarised in table 2.

Acceptance criteria

1. The relative standard deviation of six replicate injections for peak area should not be more than 2.0%.

2. The tailing factor should not be more than 2.

3. The theoretical plates should not be less than 2000.

Linearity

For linearity of six point’s calibration curve were obtained in concentration ranges from 125-750 μg/ml for metformin and 5-30 μg/ml for teneligliptin.

The response of the drug was found to be linear in the selected concentration range the correlation coefficient for metformin and teneligliptin were 0.9993 and 0.9991 respectively. The results of linearity of v metformin and teneligliptin were summarised in table 3.

Table 1: Optimised chromatographic condition for the estimation of metformin and teneligliptin

Parameter Condition
Mobile phase 0.1 % ortho phosphoric acid buffer: acetonitrile (65:35, v/v)
Diluent Water: acetonitrile
Column Discovery (250 mm X 4.6 mm, 5μm is particle size)
Column temperature 30 °C
Detection wavelength 260 nm
Injection volume 10 μl
Flow rate 1 ml/min
Run time 6 min

Fig. 3: Typical HPLC chromatogram of standard

Fig. 4: Typical HPLC chromatogram of a sample

Table 2: System suitability of the developed method

Parameters Metformin Teneligliptin Acceptance criteria
Retention time 2.517 3.687 …….
Theoretical plates 9788 6734 >2000
Tailing factor 1.22 1.40 <2
Asymmetry factor 1.68 1.75 >1<10
Resolution 8.0 8.0 >2

Table 3: Linearity and range of the developed method

S. NO Metformin Teneligliptin
Concentration (μg/ml) Peak area Concentration (μg/ml) Peak area
1 125 1092284 5 106601
2 250 2040782 10 205080
3 375 3006306 15 294146
4 500 4217649 20 380439
5 625 5222174 25 473114
6 750 6198935 30 569050
Slope 8295.3 18628
Y-intercept 434.31 9539.8
Correlation coefficient 0.9993 0.9991

n is the number of experiments (n=6)

Precision

The developed method has shown percent relative standard deviation [% RSD] less than 2 for both intra-day and inter-day precision study, which ensures precision of the developed method. The results of the precision study were summarised in table 4 and 5.

Limit of detection and limit of quantification

Limit of detection [LOD] and limit of quantification [LOQ] was estimated from the standard deviation of the y-intercepts and slope of the calibration curve of metformin and teneligliptin. The LOD and LOQ were found to be 0.02 and 0.07 μg/ml for metformin and 0.19 and 0.56 μg/ml for teneligliptin. This showed that the developed method can detect and quantify at lower concentration was highly sensitive whereas other methods is less sensitive.

Accuracy

The percentage recovery of the spiked sample was within 99±2% which ensures the accuracy of the developed method. The results of recovery studies were summarised in table 6 and 7.

Table 4: Intra-day and inter-day precision of the developed method for metformin

S. No. Intra-day precision Inter-day precision
Assay-1 4262777 4202650
Assay-2 4201714 4180943
Assay-3 4223667 4207508
Assay-4 4241537 4219171
Assay-5 4280186 4204943
Assay-6 4283314 4204943
Mean 4248866 4203360
S. D 32475 12451.8
%RSD 0.8 0.3

SD: standard deviation, RSD%: relative standard deviation, n = number of experiments.

Table 5: Intra-day and inter-day precision of the developed method for teneligliptin

S. No. Intra-day precision Inter-day precision
Assay-1 389369 333029
Assay-2 384696 334982
Assay-3 384297 335332
Assay-4 389597 336546
Assay-5 389035 335049
Assay-6 384171 334049
Mean 386861 334998
S. D 2720 1130.5
%RSD 0.7 0.3

SD: standard deviation, RSD%: relative standard deviation, n = number of experiments.

Table 6: Accuracy of the developed method for met for min

Drug name Level of addition (%) Amount added (mg) Drug found (mg/ml) % recovery Average % recovery
Metformin 50 250 249.83 98.73
50 250 245.96 98.38
50 250 245.98 98.39
100 500 491.39 98.28 98.78±0.9
100 500 497.25 99.45
100 500 490.65 98.13
150 750 736.86 98.25
150 750 744.38 99.25
150 750 750.78 100.10

Table 7: Accuracy of the developed method for teneligliptin

Drug name Level of addition (%) Amount added (mg) Drug found (mg/ml) % recovery Average % recovery
Teneligliptin 50 10 9.94 99.45
50 10 9.87 98.73
50 10 9.91 99.15
100 20 19.75 98.79 99.46±0.89
100 20 20.10 100.51
100 20 20.04 100.20
150 30 29.82 99.42
150 30 29.42 98.09
150 30 30.22 10077

Stability-indicating studies

Stability indicating studies were carried under a condition of acid/base/neutral hydrolysis, oxidation, dry heat and photolysis. For each study, samples were prepared. The blank subjected to stress in the same manner for the drug solution, working standard solution of metformin and teneligliptin subjected to stress degradation. Dry heat and photolytic degradation were carried out in a solid state. The concentration of degrading reagent and time of exposure was optimised to degradation within the range of 10%. During optimisation of degradation conditions, if the higher percentage of degradation was observed, milder conditions were used for the lesser duration of exposure. Although percent assay reduced under all conditions; the separate peak for degradation product was observed only under acid and alkali conditions fig 5 and 6. Summary of stress degradation results is given in table 9 and 10.

In order to develop a suitable RP-HPLC method for the estimation of metformin and teneligliptin, different buffer ratios at different flow rate were applied. Some of the reported methods were costly due to the use of expensive solvents and it was replaced by buffer and acetonitrile in this study. The LOD and LOQ were found to be 0.02 and 0.07 μg/ml for metformin and 0.19 and 0.56 μg/ml for teneligliptin which indicates that the method was sensitive, and can detect and quantify at lower levels metformin and teneligliptin. Linearity range was from 125-750 μg/ml for metformin and 5-30 μg/ml for teneligliptin. The response of the drug was found to be linear in the selected concentration range the correlation coefficient for metformin.

The LOD and LOQ were found to be 0.02 and 0.07 μg/ml for metformin and 0.19 and 0.56 μg/ml were 0.9993 and 0.9991 for teneligliptin, respectively. Which indicates that at this concentration range both were highly linear. Present assay the amount of both the drugs recovered was found to be 98.78% for metformin and 99.46% for teneligliptin. The developed RP-HPLC stability indicating assay method was found to be appropriate for the analysis of drug in their pharmaceutical dosage form.

Fig. 5: Chromatogram of acid hydrolysis

Fig. 6: Chromatogram of alkali hydrolysis

Fig. 7: Overlay of linearity chromatograms

Fig. 8: Overlay of precision chromatograms

Table 9: Stability-indicating data of met for min

Degradation parameter Peak area of sample Peak area of standard % recovery % degradation
Acid degradation 4059092 4267272 95.03 4.97
Alkali degradation 4155479 4267272 97.28 2.72
Oxidative degradation 4191118 4267272 98.12 1.88
Dry heat degradation 4248957 4267272 99.47 0.53
Photo stability degradation 4247378 4267272 99.43 0.57
Neutral degradation 4236053 4267272 99.90 0.10

Table 10: Stability-indicating data of teneligliptin

Degradation Parameter Peak area of sample Peak area of standard % recovery % degradation
Acid degradation 366712 385565 95.02 4.98
Alkali degradation 375116 385565 97.19 2.81
Oxidative degradation 379152 385565 98.24 1.76
Dry heat degradation 382367 365565 99.07 0.93
Photo stability degradation 383633 385565 99.40 0.60
Neutral degradation 382781 385565 99.18 0.82

CONCLUSION

In pharmaceutical industry settings, recent studies that quantify two drugs reported a simple, sensitive and more precise spectrophotometric method with UV-Visible [4-6]. Another study quantify more than on drug, used PDA as analyte detector [7-9]. In this study, we used PDA detector to prove the selectivity of the method. Another study was reported for teneligliptin in human plasma and its application to a pharmacokinetic study by LC-MS-MS [10]. In this method to identify a wide range of linearity, recovery, rapid extraction and shorter run time. It concludes that the present method can be useful for pharmacokinetic/bioequivalence studies with desired precision and accuracy. The newly developed RP-HPLC method for simultaneous determination of metformin and teneligliptin in pure and in the pharmaceutical formulation was found to be simple, sensitive, rapid, precise and accurate. The proposed method was completely validated as per ICH guidelines. The method validation data showing satisfactory results for all the method parameters tested. The stability-indicating nature of the proposed method was established by performing forced degradation, which provided degradation behaviour of metformin and teneligliptin under various conditions. Hence the developed RP-HPLC method is stability-indicating and can be used for routine analysis of production samples and also to check the stability of bulk samples of metformin and teneligliptin.

ACKNOWLEDGMENT

Thanks to Prof. Ramya kuber as the first author had done almost all of the steps in this study, Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam, Tirupathi, Andhra Pradesh, India, for providing the research facilities and Spectrum laboratories limited, Hyderabad, Telangana, India, for providing drug samples.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Maruthur NM, Tseng E, Hutfless S, Wilson LM, Suarez CC, Berger Z, et al. Diabetes medications as monotherapy or metformin-based combination therapy for type 2 diabetes: a systematic review and meta-analysis. AIM 2016;6:164-74.

  2. Eto T, Inoue S, Kadowaki T. Effects of once-daily teneligliptin on 24-h blood glucose control and safety in Japanese patients with type 2 diabetes mellitus: a 4-week, randomized, double-blind, placebo-controlled trial. Diabetes Obes Metab 2012;14:1040-6.

  3. Yoshida T, Akahoshi F, Sakashita H. Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2,ylcarbonyl] thiazolidine, A highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes. Bioorg Med Chem 2012;1:5705-19.

  4. Kaushelendra M, Himesh S, Nayak G, Patel SS, Singhai AK. Method development and validation of metformin hydrochloride in tablet dosage form. Eur J Cancer 2011; 8:1309-13.

  5. Chirag V, Amrita P. Development and validation of UV spectro-photometric method for simultaneous estimation of metformin hydrochloride and alogliptin benzoate in bulk drugs and combined dosage forms. Pharm Chem 2014;6:303-11.

  6. Ashim KS, Denish NH, Dhanya BS, Aarti SZ, Rajesh AM. Analytical method development and validation for simultaneous estimation of teneligliptin hydrobromide hydrate and metformin hydrochloride from its pharmaceutical dosage form by three different UV spectrophotometric methods. J Appl Pharmacol Sci 2016;6:157-65.

  7. Murthy TGK, Geethanjali J. Development of a validated rp-hplc method for simultaneous estimation of metformin hydrochloride and rosuvastatin calcium in bulk and in-house formulation. J Chromatogr Sep Tech 2014;5:252.

  8. Doredla NR, Mannepalli C. Method development and validation of an rp-hplc method for simultaneous analysis of three component tablet formulation containing metformin hydrochloride, pioglitazone hydrochloride and glibenclamide. IJPRIF 2012;4:948-56.

  9. Kumar TNVG, Vidyadhara S, Ashok NN, Sai SY, Lakshmi MR. Method development, validation, and stability studies of teneligliptin by rp-hplc. J Anal Sci Technol 2016;7:18.

  10. Raja HBC, Gowri SD. Development and validation of lc-ms/ms method for quantification of teneligliptin in human plasma and its application to a pharmacokinetic study. World J Pharm Pharm Sci 2016;5:838-50.

  11. International conference on harmonization tripartite guideline on validation of analytical procedures text and methodology: Q2(R1); 2005.

  12. International conference on harmonization tripartite guideline on stability testing of new drug substances and products text and methodology: Q1A(R2); 2003.



About this article

Title

A NOVEL STABILITY-INDICATING REVERSE PHASE LIQUID CHROMATOGRAPHIC METHOD FOR THE SIMULTANEOUS ESTIMATION OF METFORMIN AND TENELIGLIPTIN IN PURE AND PHARMACEUTICAL FORMULATIONS

Keywords

Method validation, Estimation, Stability indicating, Metformin, Teneligliptin

DOI

10.22159/ijpps.2017v9i12.21151

Date

01-12-2017

Additional Links

Manuscript Submission

Journal

International Journal of Pharmacy and Pharmaceutical Sciences
Vol 9, Issue 12, 2017 Page: 163-169

Online ISSN

0975-1491

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Authors & Affiliations

A. Swetha
Department of Pharmaceutical Analysis, Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam, Tirupathi, Andhra Pradesh, India
India

B. Ramya Kuber
Department of Pharmaceutical Analysis, Institute of Pharmaceutical Technology, Sri Padmavathi Mahila Visvavidyalayam, Tirupathi, Andhra Pradesh, India
India


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