Int J Pharm Pharm Sci, Vol 7, Issue 3, 191-197Original Article


RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION AND STABILITY INDICATING STUDY OF METFORMIN AND LINAGLIPTIN IN PURE AND PHARMACEUTICAL DOSAGE FORMS

MALLIKARJUNA RAO. N*1, GOWRI SANKAR D2

1Research scholar, Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, Kakinada, Andhra Pradesh, India, 2Professor & HOD, Department of Pharmaceutical Analysis & Quality Assurance, University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India.
Email: mallimpharmmba@gail.com

Received: 07 Oct 2014 Revised and Accepted: 29 Oct 2014

ABSTRACT

Objective: The objective of this study was to develop a simple, efficient, specific, precise and accurate Reverse phase High Performance liquid chromatography method for the simultaneous estimation of Metformin and Linagliptin Pharmaceutical Dosage form.

Methods: The separation method was carried out using reverse phase C18 column, Inertsil ODS – 3V (250 mm x 4.6 mm x 5μm). The mobile phase used was a mixture of Phosphate buffer (1.625 g of Potassium Di Hydrogen Ortho Phosphate and 0.3 g of Di Potassium Hydrogen Ortho Phosphate in 550 ml water) pH 4.5 and Acetonitrile in the ratio of 60:40 (v/v) at isocratic mode. The flow rate was 1.0 mL/min, column temperature was 30°C and eluents were monitored at 280 nm using waters 2695 alliance HPLC instrument equipped with the Waters 2998 PDA detector and Empower 2 software.

Results: With the optimized method, the retention times of Metformin and Linagliptinwere found to be 3.048 and 4.457 respectively, with theoretical plate count and asymmetry as per the limits. The method has shown a good linearity in the concentration range of 500-3000µg/ml from Metformin and 2.5-15µg/mL for Linagliptin with Regression coefficient (R2) of 0.99 and 0.99. The percentage assays were found to be 99.28% and 99.54% respectively for Metformin and Linagliptin. The method was found to be accurate (with percentage mean recoveries 100% for Metformin HCl and 100% for Linagliptin), precise, robust, stable and Degradation studies are conducted under various conditions.

Conclusion: The proposed method was validated in accordance with ICH guidelines and hence, can be successfully applied to the simultaneous estimation of Metformin and Linagliptin tablet formulations.

Keywords: Metformin and Linagliptin, Simultaneous estimation, Reverse phase HPLC, Validation, Degradation studies.

INTRODUCTION

Metformin HCl is an oral hypoglycemicdiabetic drug which comes under the class Biguanides. It is chemically 1, 1-Dimethyl biguanide monohydrochloride. It is the first line drug for treating Type-2 Diabetes mellitus. Metformin acts by suppressing hepatic gluconeogenesis and glucose output from the liver. It is official in USP-2010, BP-2012, and IP-2007 [1-3]. It is the first line drug of choice for the treatment of type 2 diabetes, particularly in overweight or obese people and those with normal kidney function. Metformin activates AMP-activated protein kinase (AMPK), a liver enzyme that plays an important role in insulin signaling, whole body energy balance and metabolism of glucose and fats. Metformin is an anti-diabetic agent [4, 5]. Activation of AMPK is required for metformin’s inhibitory effect on the production of glucose by liver cells. The chemical structure of Metformin is shown in fig. 1.

Fig. 1: Structure of Metformin

Linagliptin is described chemically as 1H-Purine- 2,6-Dione, 8-[(3R) -3-amino-1-piperidinyl] -7-(2-butyn-1- yl) -3,7-dihydro-3-methyl-1-[(4-methyl-2-quinazolinyl) methyl] -The empirical formula is C ₂₅H₂₈N₈O₂. The structural formula is shown in fig (2). Linagliptin is a white to yellowish or only slightly hygroscopic solid substance. It is very slightly soluble in water (0.9 mg mL-1). Linagliptin is soluble in methanol (CA. 60 mg mL-1), sparingly soluble in ethanol (CA. 10 mg mL-1), very slightly soluble in isopropanol (<1 mg mL-1), and very slightly soluble in acetone (CA. 1 mg mL-1). Linagliptin is an oral drug that reduces blood sugar (glucose) levels in patients with type 2 diabetes. Linagliptin is a member of a class of drugs that inhibit the enzyme, DI peptidyl peptidase-4 (DPP-4). Following a meal, insertion hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulin tropic polypeptide (GIP) are released from the intestine, and their levels increase in the blood. GLP-1 and GIP reduce blood glucose by increasing the production and release of insulin from the pancreas. GLP-1 also reduces blood glucose by reducing the secretion by the pancreas of the hormone, glucagon, a hormone that increases the production of glucose by the liver and raises the blood level of glucose. The net effect of increased release of GLP-1 and GIP is to reduce blood glucose levels. Linagliptin inhibits the enzyme, DPP-4, that destroys GLP-1 and GIP and thereby increases the levels and activity of both hormones. As a result, levels of GLP-1 and GIP in the blood remain higher, and blood glucose levels fall. Linagliptin reduces blood glucose levels by inhibiting DPP-4 and increasing the levels of GLP-1 and GIP [6-8]. The chemical structure of Linagliptin is shown in fig. 2.

Fig. 2: Structure of Linagliptin

For the simultaneous estimation of drugs present in multicomponent dosage forms, HPLC method is considered to be most suitable for this is a powerful and rugged method. Many Methods have been reported in the literature for the estimation of Metformin Hydrochloride [9-19] and Linagliptin [7, 8, 20-23] individually and in combination. However, there is no simple method with shorter run times has been reported for the simultaneous estimation of Metformin Hydrochloride with Linagliptin. The present investigation was aimed at developing a fully validated RP-HPLC method for the simultaneous estimation of Metformin and Linagliptin in pure and pharmaceutical dosage forms that is more economical, simple and accurate than the previous methods.

MATRIALS AND METHODS

Instruments used

The chromatographic determination was performed on waters 2695 alliance HPLC instrument equipped with the waters 2998 PDA detector and Empower 2 software. The different columns were used during method trials such as Inertsil ODS-3V C18column (250 mm×4.6 mm, 5μ particle size), Boston C18 (150 mmX4.6 mm, 5μ), Zodiac C18 (250 mm×4.6 mm, 5μ), etc. Other equipment used were Schimadzu electronic balance AY220, Global Digital pH meter DPH 500, ultrasonic cleaner (Frontline FS 4, Mumbai, India).

Chemicals and reagents

Standard gift samples of Metformin Hydrochloride and Linagliptinwere obtained from Lara drugs Pvt. Ltd., Hyderabad, India. Marketed formulation of combination was purchased from local markets. Acetonitrile, Methanol and water were purchased from HPLC grade was purchased from E. Merck (India) Ltd., Mumbai. Potassium Di Hydrogen Phosphate and Di PotassiumHydrogen Phosphate were purchased from E. Merck, Mumbai, India. All the solvents and reagents were of HPLC grade.

Preparation of standard solution

Accurately weighed standards of Metformin HCl (2000 mg) andLinagliptin (10 mg) were weighed accurately 2000mg of Metformin and 10 mg of Linagliptinwas transferred into 50 ml of volumetric flask dissolved and diluted to volume with the mobile phase and sonicated for 15 min. Pipette out 5 ml of this solution into 25 ml volumetric flasks and make up the volume with the mobile phase.

Preparation of sample solution

Twenty tablets were weighed (average weight 2000 mg) and powdered using mortar and pestle. The quantity of powder equivalent to 500 mg of Metformin HCl and Linagliptini. e., 2000 mg was transferred to a 50 ml volumetric flask. The content was dissolved in the mobile phase, sonicated for15 minutes to dissolve the drug as completely as possible. The solution was then filtered through 0.45μNylon disposable Syringe filter. The volume was then made to mark with the mobile phase. This is the standard stock solution. From the standard stock solution, an aliquot of 5 ml solution was transferred to a 25 ml volumetric flask and diluted to mark with the mobile phase

RESULTS AND DISCUSSION

Method development

Initially, many method trials were performed using different mobile phases, different columns, and varying chromatographic conditions in an attempt to obtain the best separation and resolution between Metformin HCl and Linagliptinas shown in fig. 3. The finalized method involved the use of a mixture of Phosphate buffer (1.625 g. of Potassium Di Hydrogen Ortho Phosphate and 0.3 g of Di Potassium Hydrogen Ortho Phosphate in 550 ml water); pH 4.5 and Acetonitrile in the ratio of 60:40 (v/v) as the mobile phase at isocratic mode and eluents were monitored at 280 nm using UV-Visible spectrophotometer as the detector allowing the adequate separation of both the compounds using the column Inertsil ODS - 3V C18 (250 mm x 4.6 mm x 5μm particle size) at a flow rate of 1.0 ml/min and column temperature 30°C. Sample injection volume was 1.0 ml/minas shown in fig. 4.

Assay procedure

With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected five times and the chromatograms were recorded. This procedure was repeated for the sample solution too. The averages of peak areas were determined for standard and sample solutions. The concentration of the drug was calculated using the following formula

The results are described in table 1.

Fig. 3: Typical chromatogram for the trial


Fig. 4: Typical chromatogram for the standard


Table 1: Assay results of Metformin and Linagliptin

Drug % Assay
Metformin 99.28
Linagliptin 99.54

Method validation [20-23]

System suitability

The suitability of the chromatography system was tested before each stage of validation. Six replicates of working standard solution are injected and the chromatograms are recorded. The % Relative Standard Deviation (%RSD) of retention times, asymmetry, theoretical plate count and of peak areas (should not be more than 2%) was determined as shown in table 2 and fig. 5.

Fig. 5: Typical chromatogram for the standard


Table 2: System suitability parameters

Parameters Metformin Linagliptin
Retention time 3.048 4.457
Resolution - 7.970
Theoretical plates 6782 8231
Tailing 1.147 1.096

Accuracy

To the pre-analyzed sample solution, a known amount of standard solution (usually 5-20%) was spiked at three different levels (50%, 100%, and 150%). These solutions were injected in three replicates and Percentage Mean Recoveries are determined for Metformin and Linagliptin which should lie between 98-102%. The results are described in table 3, 4 and fig. 6, 7 and 8.

Table 3: Accuracy for Metformin

Spiked level Sample weight Sample area µg/ml added µg/ml found % recovery Mean
50% 2045.00 4520363 3958.065 3958.42 100 100
50% 2045.00 4525808 3958.065 3958.42 100
50% 2045.00 4524672 3958.065 3958.42 100
100% 4090.88 9055238 7917.832 7930.00 100 100
100% 4090.88 9056005 7917.832 7930.00 100
100% 4090.88 9053824 7917.832 7930.00 100
150% 6135.0 13518335 11874.194 11838.50 100 100
150% 6135.00 13516919 11874.194 11838.50 99
150% 6135.00 13568589 11874.194 11838.50 100

Table 4: Accuracy for Linagliptin

Spiked level Sample weight Sample area µg/ml added µg/ml found % recovery Mean
50% 2045.00 4358197 19.990 19.96 100 100
50% 2045.00 4356218 19.990 19.95 100
50% 2045.00 4352141 19.990 19.93 100
100% 4090.88 8707432 39.989 39.88 100 100
100% 4090.88 8700880 39.989 39.85 100
100% 4090.88 8706209 39.989 39.87 100
150% 6135.0 13056602 59.971 59.80 100 100
150% 6135.00 13022412 59.971 59.64 99
150% 6135.00 13048584 59.971 59.76 100

Fig. 6: chromatogram of 50% accuracy level

Fig. 7: chromatogram of 100% accuracy level

Fig. 8: chromatogram of 150% accuracy level

Precision

The precision of the method (Intra-day variation) was determined by repeatedly injecting the sample solution (8mg/ml of Metformin HCl and 0.5mg/ml Linagliptin) six times. The retention times and peak areas of six replicates are recorded. The precision is expressed as the % RSD of Peak areas and it should not be more than 2%. The results are described in table 5 and 6.

Table 5: Intraday precision

S. No. Sample weight Metformin Linagliptin % Assay (metformin) % Assay (linagliptin)
1 4090.88 9058615 8707008 99 100
2 4090.88 9055892 8706154 99 100
3 4090.88 9058196 8705139 99 100
4 4091.88 9056969 8701982 99 100
5 4090.88 9053353 8700572 99 100
6 4090.88 9054070 8703774 99 100
Average Assay:       99 100
SD       0.02 0.03
%RSD       0.02 0.03

Table 6: Interday Precision

S. No. Sample weight Metformin Linagliptin % Assay (metformin) % Assay (linagliptin)
1 4091 9058245 8702651 100 100
2 4091 9057486 8703148 100 100
3 4091 9058954 8705246 100 100
4 4091 9056847 8701431 100 100
5 4091 9055078 8706054 100 100
6 4091 9059834 8704297 100 100
Average Assay:   9057740.7  8704297  100 100
SD    1677  2234 0.02 0.03
%RSD       0.02 0.03

Linearity

The calibration curve was constructed by plotting peak area against concentration of solutions. Metformin and Linagliptin were found to be linear in the concentration range of 500-3000µg/mL (25 % to 150%) and 2.5-15µg/mL (25% to 150%) respectively. The results show that an excellent correlation exists between areas and concentration of drugs within the concentration range indicated above. The results of linearity were represented in tables 7. And the results for calibration curves are given in fig. 9 & 10.

Table 7: Linearity of Metformin and Linagliptin

Linagliptin and metformin Con. c% Metformin area Metformin µg/ml Linagliptin area Linagliptin µg/ml
25 2336359 500 2148043 2.5
50 4524579 1000 4354630 5
75 6788434 1500 6536100 7.5
100 9057249 2000 8704856 10
125 11379261 2500 10866198 12.5
150 13583129 3000 13086472 15.00

Fig. 9: Linearity Curve for Metformin

Robustness

The robustness of the method is determined under normal operating conditions different conditions such as change in flow rate and detection wave length. 10 μl of standard and sample solutions are injected by varying wavelength (282, 284, 286 nm) and the flow rate (0.8 ml/min, 1.0 ml/min, 1.2 ml/min) and the chromatograms are recorded and changes in parameters are observed. The results are shown in table 8 and 9

Fig. 10: Linearity curve for Linagliptin


Table 8: Robustness for Metformin

S. No. Sample name Change RT Area Tailing Plate count
1 Flow1 0.8 ml 3.810 11279449 1.211 7134
2 Flow 1 0.8 ml 3.805 11359450 1.210 6800
3 Flow 1 0.8 ml 3.804 11325956 1.230 6935
4 Flow 2 1.2 ml 2.546 7368090 1.158 5916
5 Flow 2 1.2 ml 2.544 7343308 1.179 5989
6 Flow 2 1.2 ml 2.542 7329988 1.191 6128
7 Temp 1 +5°C 3.048 8967960 1.194 6494
8 Temp 1 +5°C 3.052 8962415 1.173 6423
9 Temp 1 +5°C 3.047 8971075 1.256 6581
10 Temp 2 -5°C 3.041 8956081 1.158 6602
11 Temp 2 -5°C 3.034 8922707 1.198 6732
12 Temp 2 -5°C 3.045 9003965 1.155 6679

Forced degradation studies

Degradation studied is performed under different conditions like acid, base, peroxide, photo and Thermal. In each degradation study for both Metformin and Linagliptin it was observed that purity angle is less than the threshold value, it indicated the no interference of degradants with the drug peaks so the peak was said to be pure. Degradation studies reveal that the developed method was stability indicating hence, this method can easily and conveniently adopt for routine quality control analysis of Metformin and Linagliptin in pure and its pharmaceutical dosage forms. It was observed that there was marked degradation in the chromatograms, and the data given in Tables 10&11. Purity plots for Metformin (Fig.11a-11e) and Linagliptin (Fig.12a-12e) were shown.

Table 9: Robustness for Linagliptin

S. No. Sample name Change RT Area Tailing Plate count
1 Flow 1 0.8 ml 5.519 11117649 1.169 9036
2 Flow 1 0.8 ml 5.504 11133699 1.186 9236
3 Flow 1 0.8 ml 5.501 11149431 1.208 8958
4 Flow 2 1.2 ml 3.694 7214335 1.134 7607
5 Flow 2 1.2 ml 3.682 7215805 1.144 7584
6 Flow 2 1.2 ml 3.678 7191727 1.154 7446
7 Temp 1 +5°C 4.403 8828575 1.143 8352
8 Temp 1 +5°C 4.419 8802088 1.148 8290
9 Temp 1 +5°C 4.417 8821620 1.182 8012
10 Temp 2 -5°C 4.363 8832817 1.185 8753
11 Temp 2 -5°C 4.343 8813283 1.177 8447
12 Temp 2 -5°C 4.369 8872802 1.146 8560

Table 10: degradation studies for metformin

Mode of degradation Conditions Sample weight Area % Assay Purity Angle % Deg. Purity Threshold
Control No treatment - - - - - -
Acid degradation (5N HCl) 40°C/5 minutes 4091 7287975 80 -19 0.746 0.891
Alkali degradation (1N NaOH) 80°C/1 hours 4091 7681508 84 -15 0.818 1.062
Peroxide (30%W/V H2O2) 80°C/10 minutes 4091 7790730 85 -14 0.702 0.994
Light (200watts hrs/min) 105°C/72 hours 4091 8593479 94 -5 0.961 1.085
Heat (105°C/72hr) 25°C/72 hours 4091 8544169 94 -5 0.743 0.910

Table 11: Degradation studies for Linagliptin

Mode of degradation Conditions Sample weight Area % Assay % Deg.

Purity

Angle

Purity

Threshold
Control No treatment - - - - - -
Acid degradation (5N HCl) 40°C/5 minutes 4091 6973352 80 -20 9.048 14.724
Alkali degradation (1N NaOH) 80°C/1hour 4091 7367552 84 -16 9.563 16.016
Peroxide (30%W/V H2O2) 80°C/10 minutes 4091 7666328 88 -12 9.328 13.864
Light (200watts hrs/min) 105°C/72 hours 4091 8068628 92 -8 9.109 13.038
Heat (105°C/72hr) 25°C/72 hours 4091 8031372 92 -8 8.275 15.559

Purity Plots for Metformin
Fig. 11a: Acid degradation Fig.11 b: Alkali degradation
Fig. 11c: thermal degradation Fig. 11d: Peroxide degradation
Fig. 11e: Photolytic degradation

Purity Plots for Linagliptin
Fig. 12a: Acid degradation Fig. 12b: Alkali degradation
Fig. 12c: photolytic degradation Fig. 12d: Thermal degradation
Fig. 12e: Peroxide degradation

CONCLUSION

The developed RP-HPLC method was developed and validated as per ICH guidelines in terms of specificity, accuracy, precision, linearity, robustness, limit of detection and limit of quantitation for the simultaneous quantitative estimation of Metformin and Linagliptin. The correlation coefficients were greater than 0.99 for both the drugs. The precision results were good enough to say that the method developed is precise and reproducible.

Accuracy studies revealed that mean recoveries after spiking experiments were between 99 and 101%, indicative of accurate method. Degradation studies reveal that the developed method was stability indicating hence, this method can easily and conveniently adopt for routine quality control analysis of Metformin and Linagliptin in pure and its pharmaceutical dosage forms.

ACKNOWLEDGEMENT

Authors are thankful to the Department of Pharmaceutical Sciences, Jawaharlal Nehru Technological University, and Kakinada. And Department of Pharmaceutical Sciences, Andhra University, Visakhapatnam for providing instrumental and analytical support.

CONFLICT OF INTEREST

Declared None

REFERENCES

  1. The united states pharmacopeia by united state pharmacopeial convention INC. Rock hill: MD; 2010.
  2. Indian pharmacopoeia published by ministry of health and family welfare. Government of India; 2007.
  3. British Pharmacopoeia, Medicinal and pharmaceutical substances. Stationary Office London; 2012.
  4. Klepser TB, Kelly MW. Metformin hydrochloridean anti-hypoglycemic agent. Am J Health System Pharm 1997;54:893–903.
  5. Archana M, Sriram N, Gayasuddin MD. Method development and validation of RP-HPLC method for determination of new ant diabetic agent linagliptin in bulk and in pharmaceutical formulation. IJMCA 2013;3(1):1-5.
  6. Lakshmi B, Reddy TV. A Novel RP–HPLC method for the quantification of linagliptin in formulations. J Am Online 2012;2(2):155–64.
  7. Chandra K Sekhar, Sudhakar P, Tammisetty Mohan Rao, VijayaBabu P, Kumar Manikanta A. A new UV-method for determination of linagliptin in bulk and pharmaceutical dosage form. Int J Uni Pharm Bio Sci 2013;2(4):1-6.
  8. Narendra Nyola, Govinda SamyJeyabalan. Method development of simultaneous estimation of Sitagliptin and Metformin hydrochloride in pure and Tablet dosage form by UV-Vis spectroscopy. World J Pharm Pharm Sci 2012;1(4):1392-401.
  9. Nilam P, Pinkal, Khushbu S. Development and validation of analytical method for simultaneous estimation of miglitoland metformin hydrochloride in tablet dosage form. IJPR 2014;5(11):4820-4.
  10. Madhukar A, Prince, Vijay Kumar R, Sanjeev Y, Jagadeeshwar K, Raghupratap. Simple and sensitive analytical method development and validation of Metformin hydrochloride by RP-HPLC. Int J Pharm Pharm Sci 2011;3(3):117-20.
  11. Nazar Mustafa mansoory, Anurekha Jain. Simultaneous estimation of Metformin hydrochloride, Pioglitazone hydrochloride and Gliclazide by validated RP-HPLC method in solid dosage form. Int J Pharm Pharm Sci 2012;4(5):72-6.
  12. Bonde S, Bhadane RP, Avinash Gaikwad, Deepak Katale, Sumit Gavali S Narendiran. A simple and sensitive method for determination of Metformin and Sitagliptin in human plasma using Liquid chromatography and tandem Mass spectrometry. Int J Pharm Pharm Sci 2013;5(3):463-70.
  13. Anushaakula N, Prajwala, Sandhya M, Uma maheswararao. Development and validation of RP-HPLC method for simultaneous estimation of Metformin hydrochloride and Gliclazide in bulk and combined dosage form. Int J Pharm Sci 2013;5(4):511-7.
  14. Bhoomaiah B, Jaya shree. Development and validation of RP-HPLC method for simultaneous determination of Metformin and Miglitol in bulk and pharmaceutical formulation. Int J Pharm Pharm Sci 2014;6(6):135-41.
  15. Nashwahgadallah M. Validated HPLC method for simultaneous determination of Sitagliptin, Metformin and Atorvastatin in pure form and in pharmaceutical formulations. Int J Pharm Pharm Sci 2014;6(5):665-70.
  16. Srilekha K, Rajiv Dinakar B, Jyotsna Reddy B, Devala Rao G. Method development and validation of metformin and gliclazide by RP-HPLC-PDA method. Int J Inv Pharm Sci 2013;1(4):319-28.
  17. Loni AB, Ghante MR, Sawant SD. Method development and validation for simultaneous determination of Sitagliptin phosphate and Metformin hydrochloride by RP-HPLC in bulk and tablet dosage form. AJPSR 2012;2(8):24-37.
  18. Vani R, VijayaKumer B, Krishna Mohan G. Analytical method development and validation for the determination of sitagliptin and metformin using Reverse phase HPLC method in bulk and tablet dosage form. World J Pharm Pharm Sci 2014;3(3):1803-11.
  19. Edigasasikirangoud, Krishna Reddy V, Chandra K Sekhar. A new simple RP – HPLC method for simultaneous estimation of metformin hcland gliclazide tablet dosage form. IJPBS 2012;2(4):277-83.
  20. Ramzia I, Bagary E, Ehab F, Elkady, Bassam M, Ayoub. Spectrophotometric methods for the determination of linagliptin in binary mixture with metformin hydrochloride and simultaneous determination of linagliptin and metformin hydrochloride using high performance liquid chromatography. Int J Biomed Sci 2013;9(1):41-7.
  21. Kavitha KY, Geetha G, Hariprasad R, Kaviarasu M, Venkatnarayanan R. Development and validation of stability indicating RP-HPLC method for the simultaneous estimation of Linagliptin and Metformin in pure and pharmaceutical dosage form. J Chem Pharm Res 2013;5(1):230-5.
  22. Janardhan Swamy A, Harinadha Baba K. Analytical method development and method validation for the simultaneous estimation of Metformin HCL and linagliptin in bulk and tablet dosage form by RP-HPLC Method. Int J Pharm 2013;3(3):594-600.
  23. Rajasekaran A, Kavitha K, Arivukkarasu R. Development and validation of HPTLC method for simultaneous estimation and stability indicating study of Metformin HCl and Linagliptin in pharmaceutical formulation. World J Pharm Sci 2014;2(4):317-27.