METHOD DEVELOPMENT AND VALIDATION OF VILDAGLIPTIN AND METFORMIN HCl IN PHARMACEUTICAL DOSAGE FORM BY REVERSE PHASE–HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC)

METHOD DEVELOPMENT AND VALIDATION OF VILDAGLIPTIN AND METFORMIN HCl IN PHARMACEUTICAL DOSAGE FORM BY REVERSE PHASE–HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (RP-HPLC)

W. Abu Dayyih ^{* 1}, M. Hamad ², E. Mallah ¹, A. Abu Dayyih ³, R. Awad ¹, Z. Zakaria ¹ and T. Arafat ¹

Faculty of Pharmacy and Medical Sciences ¹, University of Petra, Jordan.

College of Science and Health Professions ², King Saud Bin Abdulaziz, University for Health Sciences, Saudi Arabia.

Hochschule Fresenius ³, Fachbereich Chimie, Biologie and Pharmacy, Germany.

ABSTRACT: Determination of the anti-diabetic drugs vildagliptin and metformin is wildly used to validate drug efficiency in diabetic patients. Here we describe a new and simple chromatographic method to analyze both drugs simultaneously in their commercial pharmaceutical dosage forms. A reverse Phase-high performance liquid chromatography (RP-HPLC) separation method was applied using an Xterra C18 column (250 mmL×4.6 mm I.D × 5µ) with acetonitrile: phosphate buffer (pH 6.0): water (65: 20:15v/v/v) as a mobile phase at a flow rate of 1.0 ml/min. Quantification of these drugs by this method was achieved using an ultra violet detector at λ = 239 nm. The limit of detection (LOD) for vildagliptin was 0.0040 µg/ml and 0.025 µg/ml for metformin using this RP-HPLC method. A linear calibration curves were reached at a concentration range of 4-34 µg/ml and 8-54 µg/ml for vildagliptin and metformin, respectively. The developed technique was validated for concentration linearity, robustness, accuracy and precision, and results were statistically analyzed according to the International Conference on Harmonisation (ICH) guidelines. The results presented in this report revealed the development of simple, rapid, precise and accurate RP-HPLC method for immediate determination and validation of vildagliptin and metformin in their pharmaceutical dosage forms.

Development, Validation, Vildagliptin, Metformin, RP-HPLC

INTRODUCTION: Diabetes mellitus type two (T2DM) is a chronic disease that wants a mixture of anti-diabetic drugs to have different mechanisms of action to succeed glycaemic goals ¹. The widely used of metformin and a sulphonylurea (SU) as dual therapy fails to improve glycaemic control and the adding of a third anti-hyperglycaemic drug is necessary ².

Vildagliptin (VGT) [(S)-1-[N-(3-hydroxy-1-ada-mantyl) glycyl] pyrrolidine-2-carbonitrile], Fig. 1, is a new oral anti-diabetic drug belonging to the class of dipeptidyl peptidase-4 inhibitor (reduces glucose-induced glucagon-like peptide 1 and gastric inhibitory polypeptide secretion) ³ and is used as mono therapy in adults with type 2 diabetes mellitus treatment especially in patients inadequately controlled by diet and exercise alone ^{4, 5}.

Vildagliptin can be used as dual oral therapy in combination with; metformin, in patients with insufficient glycemic control despite maximal tolerated dose of monotherapy with metformin ^{6, 7}. It has similar efficacy as it is used with metformin when compared to sulphonylurea and reduced hypoglycaemia risk with no weight gain ⁸. Vildagliptin has a complement pharmacological effect as metformin, where it improves the glucose-dependent insulin secretion and inhibits glucagon release, thus increasing the glycaemic control and weight control and reduced hypoglycaemia ⁹.

In addition, vildagliptin used with a sulphonylurea, in patients with insufficient glycaemic control despite maximal tolerated dose of a sulphonylurea and for whom metformin is inappropriate due to contraindications or intolerance. Also, used with a thiazolidinedione, in patients with insufficient glycaemic control and for whom the use of a thiazolidinedione is appropriate. Furthermore, it was used with sulphonylurea and metforminas triple oral therapy when diet and exercise plus dual therapy with these medicinal products do not provide adequate glycaemic control ¹⁰. Vildagliptin is also indicated for use in combination with insulin (with or without Metformin) when diet and exercise plus a stable dose of insulin do not provide adequate glycaemic control. The general formula for VGT is C₁₇H₂₅N₃O₂ and the molar mass 303.40 g/mol.

Metformin (MTF) is chemically known as [1-carbamimidamido-N, N-dimethylmethanimidamide] (Fig. 2) is an oral anti-diabetic drug in the class of biguanides. It is used as the first-line drug for noninsulin-dependent diabetes mellitus treatment ¹¹. It works as improving glycemic control factor through decreasing hepatic glucose production, decreasing glucose absorption, and increasing the insulin-mediated uptake of glucose. Therapeutic indications of metformin competent is indicated as second line treatment of type 2 diabetes mellitus adult patients, particularly overweight patients, who are unable to achieve sufficient glycaemic control at their maximally tolerated dose of oral metformin alone ¹². The mechanism through which metformin HCl decreases blood glucose and lipid concentrations is by activation of the enzyme AMP-activated protein kinase (AMK) and the Peutz-Jeghers protein, LKB1, to regulate AMPK ¹³.

Therefore, a complementary mechanism of action in treatment of patients with type 2 diabetes was achieved by using a decreasing hepatic glucose production. The possible relation between metformin and sugar was studied in rats’ plasma ¹⁴. The general formula for MTF is C₄H₁₁N₅ and the molar mass 129.16 g/mol. Many studies used HPLC to prepare an easy, rapid, precise and accurate method to evaluate compounds in different pharmaceutical dosage forms ^{15 - 17}.

Several methods were developed for the analysis of both vildagliptin and metformin in combination such as UV-Vis spectroscopies, HPLC and LCMS/ MS methods. Instantaneous estimation of these compounds by RP-HPLC methods were showing more time of analysis and complicated procedures; hence the present study was focused on chromatographic analysis of vildagliptin and metformin in a less time consuming simultaneous analysis of these compounds inactive ingredient (API) and Pharmaceutical dosage form which found in the pharmaceutical market ^{18 - 21}.

FIG. 1: STRUCTURE OF VILDAGLIPTIN                     

FIG. 2: STRUCTURE OF METFORMIN HCl

MATERIALS AND METHODS:

Chemicals Reagents and Instrumentation:  Chemicals and reagents used in this experiment, Active Pharmaceutical Ingredients (API) of vildagliptin and metformin HCl (TQ Pharma Pharmaceutical Technology), HPLC grade acetonitrile (Merck), potassium dehydrogenate orthophosphate (sigma chemicals) Thomas Baker, Ortho phosphoric acid (sigma chemicals), HPLC grad water (Hikma - Pharma) and marketed formulation obtained from local Jordanian Market.

HPLC (WATERS EQ, Aliance 2796 Model, and Detector 2678 with Em.power 2.3 software), UV spectrophotometer (Make: Labindia, Model: UV- 3000 with UV win 5 software). Weighing balance (Make: Ascoset, Model: ER.200A), Sonicator (Make: Enertech, Model: SE60.US), pH Meter (Make: ADWA, Model: AD102U), nylon filter Paper 0.45 microns (Make: Milli Pore), and column used was XTerra c18 (150×4.6mm, 5µ).

Chromatographic Conditions: HPLC (WATERS EQ.: Aliance 2796 Model, Detector 2678 with Em. power 2.3 software). The column used was symmetry C18 (4.6 x 250mm, 5 µm, Make: X. Terra 1). The mobile phase consisting of phosphate buffer (6.0pH), acetonitrile and water (65:20:15v/v/v), with flow rate 1.0 ml/min, the run time (min) was6.6 min, columns temperature was maintained at room temperature in normal laboratory condition, injection volume 20 µl and detection wavelength 239 nm.

Buffer Solution Preparation: 6.75gms of KH₂PO₄ was dissolved with water in 1000 ml beaker, then the volume was adjusted with HPLC grade water. After sonication and filtration using nylon G filter the pH was adjusted to 6.0 ± 0.01 with 1M sodium hydroxide (NaOH). The mobile phase was prepared by mixing 15% of above salt KH₂PO₄ prepared buffer (150 ml), 20 % of water (200 ml) and 65 % of acetonitrile (650ml).

Standard Stock Solution Preparation: Stock solution of 500µg/ml and 1000 µg/ml of vildagliptin and metformin, respectively was prepared by taking accurately weighing 5 mg of Vildagliptin (VGT) and 10 mg of Metformin (MTF) working standard in to a clean 10ml volumetric flask individually. 2, 5 -3.0 ml of mobile phase was used for dissolving completely and then the volume was made up to the mark with mobile phase. More dilutions were prepared with mobile phase. 0.3 ml of both the standard stock was diluted with mobile phase up to 10ml to get mixed standards of 15µg/ml vildagliptin and 30µg/ml metformin.   Analysis of marketed formulation weigh an equivalent to 10 mg of marketed formulation was transferred to a clean 10ml volumetric flask and added with 3 ml of mobile phase for dissolving the content and the volume was made up to 10 ml with mobile phase. Further 0.3 ml of the solution was taken into a clean conical flask and volume was made to 10ml with mobile phase. The resulting solution was sonicated and filtered through nylon filter 0.45µ membrane filter. This solution was injected into the HPLC; the chromatograms are shown in Fig. 3.

Validation Method: The validation method was developed as per the ICH guidelines and accordingly the parameters evaluated were specificity, pre-cision, accuracy, linearity, ruggedness, robustness and system suitability studies. For all the parameters %RSD were calculated ^{22 - 25}.

System Suitability Parameters: System suitability test is commonly applied to validate resolution, column efficiency, and repeatability of a chromato-graphic system to ensure its capability for a specific analysis. The stock solution was injected into the chromatographic system and system suitability parameters were determined Table 1.

Specificity: Specificity of the pharmaceutical analysis is the ability to measure accurately and specifically the concentration of API, without interference from other active ingredients, diluents, mobile phase. Solutions of mobile phase, sample solution, standard solution were injected into liquid chromatography. Retention times of samples and standard were compared.

Linearity: Linearity of an analytical procedure is its ability to elicit test results that are directly proportional to the concentration of analyte in samples within given range.

TABLE 1: PARAMETERS OF SYSTEM SUITABILITY

Linearity of the method was studied by analyzing five analyte concentrations of drug ranging from 5-25ppm for VGT and 9.99-49.99 ppm MTF are tabulated in Table 2 and linearity plot is given in the Fig. 3A and 3B.

Accuracy: Accuracy is the closeness of a measured value to a standard value. Accuracy was studied by means of recovery experiments for 50%, 100% and 150 %. Each level was injected three times. The accuracy was calculated in the form of percentage the test of the analyte recovered by the assay and the dated are shown in Table 3.

Precision: The precision expresses the closeness of agreement between a series of measurement obtained from multiple sampling of same homogenous sample under prescribed conditions. This experiment was conducted to prove the repeatability of the assay results obtained by quantification methodology. System precision, method precision and intermediate precision was performed for the homogeneous sample, according tom ICH guideline.

20 µl of standard solution was injected for six times and measured the peak area for all six injections in HPLC. The % RSD for the area of six replicate injections was calculated for system precision and shown in Table 4. 20 µl of sample solution was injected for six times and the peak area of the resulting chromatogram was used for the calculation of standard deviation and relative standard deviation for method precision.

Limit of Detection (LOD) and Limit of Quanti-fication (LOQ): The detection and quantification limits for the VGT and MTF were performed and calculated using S/N ratio method.

Robustness: Robustness measures the lack of internal influences on the test results. As part of the robustness, deliberate change in the flow rate and mobile phase composition was made to evaluate the impact on the method.

Change in flow rate the flow rate was varied at 0.80 ml/min to 1.00ml/min.  Standard solution 15µg/ml of VGT and 30µg/ml of MTF were prepared and analyzed using the varied flow rates along with method flow rate and tailing around 1.60 Table 5 and 6.

Change in organic composition: the organic composition in the mobile phase was varied to ± 5 %. Standard solution 15 µg/ml of VGT and 30 µg/ml of MTF were prepared and analyzed using the varied mobile phase composition along with the actual mobile phase composition in the method Table 7.

RESULTS AND DISCUSSION: Vildagliptin and metformin can be effectively analyzed by the RP- HPLC method with phosphate Buffer, pH around 6.0 and composition of acetonitrile: water (20: 65: 15v/v/v) at a flow rate of 1.0 ml/minute and detection wavelength of 239 nm.

The retention time of the drugs was 2.32 and 4.29 minute for vildagliptin and metformin respectively. The assay limits for vildagliptin and metformin was 92-109% and the results were obtained for vildagliptin and metformin was found to be 99.66%, 101.44% hence the results were within the limits.

Specificity: The method was found to be specific since there was no interference of mobile phase or placebo in the retention time of the analyte peak Fig. 3.

Linearity: The linearity range was found to be 5-25 µg/ml for vildagliptin and 10-50 µg/ml for metformin. Calibration curves were plotted between the peak area and the concentrations and the linear regression coefficients for both drugs VGT and MTF were found to be 0.999 and 0.998 respectively (Table 2, Fig. 4 and 5). Hence the results obtained within the limits.

TABLE 2: LINEARITY MEASUREMENTS OF VILDAGLIPTIN AND METFORMIN

FIG. 3:  CHROMATOGRAMS OF (3-A) VILDAGLIPTIN, (3-B) METFORMIN AND (3-C) MIXED STANDARD VILDAGLIPTIN AND METFORMIN

FIG. 4: CALIBRATION CURVE OF PEAK AREA VERSUS CONCENTRATION (PPM) FOR VILDAGLIPTIN       

FIG. 5: CALIBRATION CURVE OF PEAK AREA VERSUS CONCENTRATION (PPM) FOR METFORMIN

Accuracy: The accuracy studies were shown as % recovery for Vildagliptin (VGT) and Metformin (MTF) at three levels; 50 %, 100 % and 150 % (Table 3).  The mean % recovery of the vildagliptin was 99.6% and of the metformin was 99.8 %.

The limits of % recovery of drugs were 98-102 % and the above results which indicates that the method was accurate the limits of % recovered should be in range of 98-102 %.

TABLE (3) ACCURACY VALUES OF VILDAGLIPTIN AND METFORMIN

Precision: In the System precision study, %RSD was found to be less than 2%. For VGT 1.03% and MTF 0.32% which system precision indicates has good reproducibility.

TABLE 4: PRECISION VALUES OF VILDAGLIPTIN AND METFORMIN

In the method precision study % RSD was found to be less than 2 %. For VGT 1.19 % and MTF 0.171 % which indicates that the method has good repeatability. In the Intermediate System precision study, % RSD was found to be less than 2 %. For VGT 0.67 % and MTF 0.92 % which indicates that the system has good reproducibility.

The results obtained for precision (RSDs) for both vildagliptin and metformin are shown in the Table 4.

Limit of Detection and Limit of Quantification: The limit of detections (LOD) was 0.0040 µg/ml for vildagliptin and 0.025 µg/ml for metformin.

TABLE 5: ROBUSTNESS VALUES OF VILDAGLIPTIN (CHANGE IN FLOW RATE)

TABLE 6: ROBUSTNESS VALUES OF METFORMIN (CHANGE IN FLOW RATE)

TABLE 7: ROBUSTNESS VALUES OF VILDAGLIPTIN (CHANGE IN ORGANIC PHASE)

TABLE 8: ROBUSTNESS VALUES OF METFORMIN (CHANGE IN ORGANIC PHASE)

Robustness: The effect of changes in flow rate and mobile phase composition were studied for both drugs and are shown in the Table 5 and 7 for vildagliptin and Table 6 and 8 for metformin.

CONCLUSION: The developed and validated proposed HPLC method was found to be simple, specific, precise, accurate, rapid and economical for simultaneous determination of vildagliptin and metformin in pharmaceutical dosage form in Jordanian Market.

Moreover, this method introduces a simple extraction procedure with a little chromatographic run time, that make this method appropriate for the analysis of large number of samples of the pharmacokinetic, bioavailability or bioequivalent studies of vildagliptin and metformin hydrochloride. This developed method was validated as per ICH guidelines. All sample recoveries in all formulations were in good agreement with their respective label claims.

ACKNOWLEDGEMENT: The authors would like to thank Faculty of Pharmacy and Medical Sciences, University of Petra for their support and assistance with this project and for their appreciation of the science benefits to be gained from this research.

CONFLICT OF INTEREST: The authors declare that no conflict of interest for this research.

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    How to cite this article:

    Dayyih WA, Hamad M, Mallah E, Dayyih AA, Awad R, Zakaria Z and Arafat T: Method development and validation of vildagliptin and metformin HCl in pharmaceutical dosage form by Reverse Phase–High Performance Liquid Chromatography (RP-HPLC). Int J Pharm Sci Res 2018; 9(7): 2965-72. doi: 10.13040/IJPSR.0975-8232.9(7).2965-72.

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