A REVISED RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF VILDAGLIPTIN AND PIOGLITAZONE HCl – APPLICATION TO COMMERCIALLY AVAILABLE DRUG PRODUCTS

A REVISED RP-HPLC METHOD FOR SIMULTANEOUS DETERMINATION OF VILDAGLIPTIN AND PIOGLITAZONE HCl – APPLICATION TO COMMERCIALLY AVAILABLE DRUG PRODUCTS

Hitesh P. Inamdar *¹, Ashok A. Mhaske ², Shirish P. Sahastrabudhe ²

Getz Pharma Research Pvt. Ltd. ¹, Mumbai, Maharashtra, India

Shri Jagdishprasad Jhabarmal Tiberewala University ², Churu-Bishau Road, Chudela, Dist. Jhunjhunu – 333 001, Rajasthan, India

ABSTRACT

A simple, precise and stability-indicating HPLC method was developed and validated for the simultaneous determination of anti-diabetic drugs. The separation was achieved on ACE 3 150mm*4.6mm, 3.5µm column with gradient flow. The mobile phase at a flow rate of 1.5 mL min⁻¹ consisted of 10mM sodium hexane sulphonate monohydrate and 10mM Potassium dihydrogen phosphate buffer with acetonitrile and methanol in gradient ratio. The UV detection was carried out at 210 nm. The method was successfully validated in accordance to ICH guidelines. Further, the validated method was applied for commercially available pharmaceutical dosage form.

RP-HPLC, Anti-diabetic drugs, Validation

INTRODUCTION: An extensive literature survey was conducted on the analytical method for the simultaneous estimation of Vildagliptin and Pioglitazone HCl by HPLC. The absence of literature provides the need for developing a new method. The available literature studies show various analytical methods reported for the estimation of individual, binary or tertiary combination of anti-diabetic drugs or in combination with diuretics ^1-11.

Recently, a bio-analytical method has been reported for Pioglitazone ¹².However, so far, no method has been reported for the simultaneous estimation of Vildagliptin and Pioglitazone HCl and its application to pharmaceutical samples.

An attempt was made in this study to develop a rapid, economical, precise and accurate stability-indicating assay method for simultaneous estimation of Vildagliptin and Pioglitazone HCl in there drug product. This method can further be used for the simultaneous estimation of Metformin HCl, Rosiglitazone Maleate and Sitagliptin Phosphate in tablet formulation.

The earlier method proposed to estimate Metformin HCl, Rosiglitazone Maleate and Sitagliptin Phosphate ¹³. The same method is further extended for the estimation of Vildagliptin and Pioglitazone HCl in their tablet formulation.The proposed method is rapid, simple, accurate, and reproducible, and can be successfully employed in the routine analysis of both these drugs simultaneously in tablet dosage form.

EXPERIMENTAL:

Chemicals and Reagents: Drug substances were provided by Getz Pharma Research Pvt. Ltd, India. All the chemicals and reagents Ammonium hydroxide, hydrochloric acid, potassium dihydrogen phosphate, hydrogen peroxide (50 %) were used of Analytical grade. While, Acetonitrile and Methanol was procure from Merck (Germany). A Millipore Milli Q plus water purification system (Milford, USA), was used to prepare distilled water (conductivity >18 μΩ). The commercially available drug products were used as Jalara (Vildagliptin tablets, 50mg, Novartis Pharma) and Pioz* 30 (Pioglitazone HCl Tablets, 30mg, USV Limited).

Instruments: Integrated HPLC system, manufactured by Waters (USA) was used for method development and method validation. This system comprised of a quaternary gradient pump, auto sampler, column oven and a photodiode array detector. PC installed Empower was used to record and integrates the chromatograms. The analysis was carried out at ambient temperature. Photostability studies were performed in a photostability chamber, from Thermolab (India).

Chromatographic Conditions: ACE 3 (150 mm ×  4.6 mm, 3.5 μm) analytical column was used as a stationary phase. The flow rate was 1.5 mL min⁻¹ and the detector was set at 210 nm. The volume of the sample solution injected was 20 μL. The gradient mobile phase consisted of 10 mM each of sodium hexane sulphonate monohydrate and Potassium Dihydrogen Phosphate buffer with acetonitrile and methanol with the gradient as mentioned in Table 1. A membrane filter of 0.45 μm porosity was used to filter and degas the mobile phase.

TABLE 1: GRADIENT FOR CHROMATOGRAPHIC METHOD

 Standard and Test solutions: Weighed accurately about 50 mg of each Vildagliptin and Pioglitazone HCl, reference standard in 100 ml volumetric flask. Added to it 70 ml diluents (Water:Aetonitrile::70:30, pH 3.0) and sonicated to dissolve. Diluted this solution up to volume with diluents. Pipette out 5.0 ml of this solution into 50 ml volumetric flask and diluted to volume with diluent. (50 μg/mLeach of Vildagliptin and Pioglitazone HCl). Similarly, the test solutions were prepared at same concentration using same diluents. (50 μg/mLof each).

MethodDevelopment: A variety of mobile phases were investigated in the development of a stability-indicating LC method for the analysis of Vildagliptin and Pioglitazone HCl drug substances. The suitability of mobile phase was decided on the basis of selectivity and sensitivity of the assay, stability studies and separation among impurities formed during forced degradation studies.

1. Wavelength Selection: The individual drug substance solution at concentration of 50μg mL⁻¹ in diluent was scanned on PDA from 200nm to 400nm. The maximum wavelength was observed for Pioglitazone HCl is (222.8 and 269.0) and for Vildagliptin no as such maxima was found. However, detection was carried out at 210 nm on basis of higher response (Fig. 1 and Table 2).

FIG 1: UV SPECTRA FOR ALL DRUG SUBSTANCES

TABLE 2: AREA RESPONSE OF PEAKS AT DIFFERENT WAVELENGTHS

 Method validation: The optimized chromatographic conditions were validated by evaluating specificity-Forced degradation,  linearity, precision, accuracy, robustness and system suitability parameters in accordance with the ICH guideline Q2 (R1) [2].

Specificity- Forced Degradation study:

1. Acid and Base Hydrolysis: Forced degradation study was conducted on 50 mg/mL of drug solution. The solution mixture was prepared by mixing 50mg of each drug in 100 mL flask. This was considered as a stock solution. To 5mL of stock solution 5mL of 1N hydrochloric acid was exposed at 60ºC for 30 minutes and same was followed in case of Ammonium hydroxide and was exposed at 60ºC for 10 minutes. Then, neutralized with acid or base (when necessary) and dilute up to 50 ml with diluent.
2. Oxidation: Forced degradation study was conducted on drug substances by exposing with 50% H₂O₂ and dilute up to 50 mL with diluent.
3. Thermal Degradation: 5mL stock solution was kept in dry oven at 105°C for 24 hours.
4. Photolysis: Photolysis studies were carried out on stock solution in 50mL volumetric flask. The sample was exposed to light in a photo-stability chamber. The study was carried out on transparent and Amber color flask. The method's analytical data were collected at a single wavelength of 210 nm. Additional PDA detector data were collected for the peak purity evaluation.

FIG 2: OVERLAY AND INDIVIDUAL CHROMATOGRAM FOR STRESSED CONDITION

Force Degradation study was conducted by varying various parameters. No degradation was observed. A peak was observed in alkaline hydrolysis adjacent to metformin peak. The resolution between the respective peaks was found to be greater than 2. i.e. 2.3. The overlay chromatogram specifies each parameter of force degradation study. Peak Purity assessment is given in Table 3.

TABLE 3: PEAK PURITY ASSESSMENTS

     Note:  Peak Purity: Purity angle should be less than purity threshold

2. Linearity: Standard stock solution of the drug was diluted to prepare linearity standard solutions in the concentration range of 12–100 μg mL⁻¹ for all Vildagliptin and Pioglitazone HCl. Three sets of such solutions were prepared. Each set was analyzed to plot a calibration curve. Slope, intercept and coefficient of determination (r²) of the calibration curves were calculated to ascertain linearity of the method.

FIG. 3: LINEARITY CURVE WITH CORRELATION CO-EFFICIENT

3. Recovery: Recovery of the method was determined by analyzed the drug products and synthetic mixture of drug products with 50%, 100% and 150% levels. These mixtures were analyzed by the proposed method. The experiment was performed in triplicate and recovery (%); RSD (%) were calculated (Table 4).

TABLE 4: RECOVERY FROM COMMERCIALLY AVAILABLE SAMPLES

  Precision: The precision of the proposed method was evaluated by carrying out six independent assays of test samples. RSD (%) of six assay values obtained was calculated. intermediate precision was carried out by analyzing the samples by a different analyst on another instrument (Table 5).

TABLE 5: PRECESSION

5. Robustness: The robustness was studied by evaluating the effect of small but deliberate variations in the chromatographic conditions. The conditions studied were flow rate (altered by ±0.2 mL/min), wavelength (altered by ±0.2 nm), and pH of buffer in mobile phase (altered by ± 0.2). These chromatographic variations were evaluated for resolution between all drug substances.

TABLE 6: CHANGE IN FLOW RATE (1.5 ML/MIN ± 0.2 ML/MIN)

 TABLE 7: CHANGE IN WAVELENGTH (210NM ± 2NM)

 TABLE 8: CHANGE IN PH OF BUFFER SOLUTION IN MOBILE PHASE (PH 3.0 ± 0.2)

TABLE 9: RESULT OF ROBUSTNESS ON THE RESOLUTION BETWEEN THE DRUGS

6. Solution Stability: To assess the solution stability, standard and test solutions were kept at 25°C (laboratory temperature) for 24 hrs.

TABLE 10: RESULTS FOR SOLUTION STABILITY (SAMPLE SOLUTION)

 System suitability: The system suitability parameters with respect to theoretical plates, tailing factor, repeatability and resolution between peaks of all drug substances were defined.

TABLE 11: CHROMATOGRAPHIC PARAMETERS OF SYSTEM SUITABILITY

 RESULTS AND DISCUSSION:

HPLC method development: The maximum absorption wavelength of the reference drug solution and of the forcefully degraded drug solution was found to be 210 nm. This was observed from the UV absorption spectra (Fig. 1) and was selected as detection wavelength for LC analysis. The main objective of this chromatographic method was separation of degraded impurities from all drugs. Forced degradation study revealed a critical separation of closely eluting impurity formed from the Pioglitazone HCl and Vildagliptin peaks.

An additional peak was also observed at the retention time of about 1.5 minutes, which was supposed to be due to placebo. The resolution between the metformin peak and the placebo peak was more than 2. i.e. 2.30. This effect was observed by using the mobile phase 10mM sodium hexane sulphonate monohydrate and 10mM potassium dihydrogen phosphate buffer (pH 3.0) and acetonitrile and methanol in the gradient ratio.

Summary of Validation parameters: The assay test method is validated for Specificity, Linearity, Precision, Accuracy (Recovery), Stability of Analytical Solution and Robustness and was found to be meeting the predetermined acceptance criteria. The validated method is Specific, Linear, Precise, Accurate and Robust for determination of assay of Vildagliptin and Pioglitazone HCl drug substances and drug products. Hence, this method can be introduced into routine and stability analysis for the assay of Vildagliptin and Pioglitazone HCl drug substances and their product.

CONCLUSION: The stability indicating RP-HPLC assay method was developed and validated for simultaneous determination of Vildagliptin and Pioglitazone HCl and some commercial drug products.

The method was found to be simple, specific, Precise and Robust and can be applied for the routine and stability analysis for commercially available formulation. The earlier method consisting of three compounds that are Metformin HCl, Rosiglitazone Maleate and Sitagliptin Phosphate. This method further extended for the simultaneous estimation of Vildagliptin and Pioglitazone HCl in tablet formulation. So, this method is applicable for the simultaneous estimation of Metformin HCl, Rosiglitazone Maleate, Sitagliptin Phosphate, Vildagliptin and Pioglitazone HCl in the combination as well as in their respective drug product.

ACKNOWLEDGEMENTS: The authors are thankful to entire team of JJT University and Getz Pharma Research Pvt. Ltd. for their encouragement and support during the work.

REFERENCES:

1. K.S Lakshmi, T. Rajesh, Shrinivas Sharma, “Simultaneous determination of metformin and pioglitazone by reversed phase HPLC in pharmaceutical dosage forms”. International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 1, Issue 2, Oct-Dec. 2009.
2. Adukondalu D, P S Malathy, J Venkateshwar rao, Y Madhusudan Rao. Development and Validation of HPLC method for detection of pioglitazone hydrochloride in dosage forms. IJPBS, Volume 1, Issue 4, oct-dec 2011. 474-478.
3. Sahoo P. K., R. Sharma, and S. C. Chaturvedi. Simultaneous Estimation of Metformin Hydrochloride and Pioglitazone Hydrochloride by RP-HPLC Method from Combined Tablet Dosage Form Indian J Pharm Sci. 2008 May-Jun; 70(3): 383–386.
4. Anand Prem D.C., K.L. Senthil kumar, B. Senthil kumar, M. Saravana kumar, R.Thirumurthy. A New RP-HPLC Method Development and Validation for Simultaneous estimation of Telmisartan and Pioglitazone in Pharmaceutical Dosage Form. International Journal of Chem Tech Research, Vol. 3, No.1, 448-454, Jan-Mar 2011.
5. Ramzia I. El-Bagary, Ehab F. Elkady, Bassam M. Ayoub. Liquid Chromatographic Methods for the Determination of Vildagliptin in the Presence of its Synthetic Intermediate and the Simultaneous Determination of Pioglitazone Hydrochloride and Metformin Hydrochloride. International Journal of Biomedical Science 7(3), 201-208, Sep 15, 201.
6. Rashmitha N., Hiriyanna S.G, C H. Sreenivasa Rao, K. Chandra Sekhar Reddy, M. Hari Kiran, Hemant Kumar Sharmaa, K. Mukkanti, A validated stability indicating HPLC method for the determination of impurities in pioglitazone hydrochloride. Der Pharma Chemica, 2010, 2(5): 426-433.
7. AMR Lotfy Saber, Determination of Pioglitazone Hydrochloride in Tablets by High-Performance Liquid Chromatography, Pak. J. Anal. Environ. Chem. Vol. 9, No. 2 (2008) 118. 121.
8. Srinivasulu D., B.S.Sastry, G. Omprakash, Development and Validation of new RP-HPLC method for determination of pioglitazone HCl in pharmaceutical dosage forms Int J Chem Res, Vol 1, issue 1 (2010), 1820.
9. Ravikanth CH, A. Anil Kumar, V Uday Kiran, S Prashanth, B Madhu, Y Narsimha Reddy. Sensitive and Rapid HPLC Method for the Determination of Pioglitazone in Rat Serum. International Journal of Pharmaceutical Sciences and Drug Research 2011; 3(1): 38-41.
10. Jedlicka A, Klimes J, Grafnetterová T. Reversed-phase HPLC methods for purity test and assay of pioglitazone hydrochloride in tablets. Pharmazie. 2004 Mar; 59 (3):178-82.
11. Rashmitha N., Hariyanna S. G, C H Sreenivasa Rao, K. Chandra Sekhar Reddy, M. Hari Kiran, Hemant Kumar Sharma, K. Mukkanti. A Validates Stability indicating method for the Determination of Impurities in Pioglitazone Hydrochloride. De Pharma Chemica 2010, 2(5): 426-433.
12. Pattana Sripalakit, Penporn Neamhom, Aurasorn Saraphanchotiwitthaya. High Performance Liquid chromatographic method for the determination of Pioglitazone in human plasma using ultraviolet detection and its application to a pharmacokinetic study. Journal of chromatography B. Volume 843, Issue 2, 7 November 2006, Page 164-169.
13. Inamdar HP and Mhaske AA. RP-HPLC method for simultaneous determination of Metformin Hydrochloride, Rosiglitazone and Sitagliptin – application to commercially available Drug Products. Int J Pharm Sci Res, 2012; Vol. 3(9): 3267-3276.
    

    ---

    How to cite this article:

    Prashanth FMB, Kannan I, Sambandam C, Jayalakshmi M, Premavathy RK and Shantha S: A study on In vitro Antibacterial activity of Ficus bengalensis Linn. on Dental caries pathogens Streptococcus mutans and Actinomyces viscosus. Int J Pharm Sci Res. 2013; 4(2); 847-855.

    ---