International Journal of Pharmacy & Life Sciences

Research Article Nizami et al., 9(7): July, 2018:5854-5859] CODEN (USA): IJPLCP ISSN: 0976-7126 INTERNATIONAL JOURNAL OF PHARMACY & LIFE SCIENCES (Int. J. of Pharm. Life Sci.) Analytical method development and validation for simultaneous estimation of Ertugliflozin and Metformin in tablet dosage form by RP-HPLC method Tahir Nizami*, Birendra Shrivastava and Pankaj Sharma School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur, (RJ) - India

Abstract An economical RP-HPLC method using a PDA detector at 224 nm wavelength for simultaneous estimation of Ertugliflozin and Metformin in pharmaceutical dosage forms has been developed. The method was validated as per ICH guidelines over a range of 50-150 µg/mL for ERTU and METF respectively. The column used was ACE Column C18, (150 mm x 4.6 mm i.d, 5μm) and samples were eluted using Buffer (Potassium dihydrogen Ortho Phosphate) : Acetonitrile (70:30v/v) as the mobile phase at a flow rate of 1ml/min the mobile phase and samples were degassed by ultrasonication for 30 min and filtered through 0.45µm Nylon (N66) 47mm membrane filter. The measurements were carried out with an injection volume of 10μL, flow rate was set to 1 mL/min, and PDA detection was carried out at 240 nm. All determinations were done at ambient column temperature (30°C). The method was linear at the concentration range 3-15 μg/ml for ERTU and 12.5-125 μg/ml for METF respectively. The method has been validated according to ICH guidelines with respect to system suitability, specificity, precision, accuracy and robustness. The LOD value for ERTU and METF was found to be 0.43μg/mL and 0.74 μg/mL respectively and the LOQ value 1.30μg/mL and 2.24μg/mL respectively.

Keywords: Ertugliflozin and Metformin, RP -HPLC, Simultaneous analysis, Tablets

Introduction Ertugliflozin (ERTU) is (1S,2S,3S,4R,5S)-5-(4- It is the first-line drug of choice for the treatment of chloro-3-(4ethoxybenzyl) phenyl)-1- type 2 diabetes, in particular, in overweight and (hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane- obese people and those with normal kidney function. 2,3,4-triol, compound with (2S)-5oxopyrrolidine-2- Its use in gestational diabetes has been limited by carboxylic acid Fig 2.3 is potent and selective safety concerns. It is also used in the treatment of inhibitors of the sodium-dependent glucose co polycystic ovary syndrome, and has been investigated transporters (SGLT). The molecular formula is for other diseases where insulin resistance may be an C22H35ClO7 and the molecular weight is important factor. Metformin hydrochloride works by 436.89 g·mol−1. Ertugliflozin as a monotherapy is suppressing glucose production by the liver. indicated to improve the glycaemic control in adult Metformin hydrochloride is the only antidiabetic patients with type 2 diabetes. Ertugliflozin, in drug that has been conclusively shown to prevent the combination with Metformin hydrochloride, is cardiovascular complications of diabetes. It helps indicated to improve glycaemic control in patients reduce LDL cholesterol and triglyceride levels, and is with diabetes type 2 who are not controlled on a not associated with weight gain. regimen of ERTU and METF or in patients who are already treated with both ERTU and METF

Metformin( METF) has been shown to be effective in combination with insulin, sulfonylureas and thiazolidinediones. Metformin (N, N-dimethyl imido di carbonimidic diamide) is an oral antidiabetic drug in the biguanide class.

* Corresponding Author E.mail: [email protected] Structure of Ertugliflozin © Sakun Publishing House (SPH): IJPLS 5854

Research Article Nizami et al., 9(7): July, 2018:5854-5859] CODEN (USA): IJPLCP ISSN: 0976-7126 taking 25 mg of METF to produce standard stock solution of 1000μg/ml of METF. The above standard stock solutions suitably diluted with diluents to obtain various concentrations of ERTU and METF. Preparation of Working Standard Solution 10 tablets were weighed accurately and finely powdered. Tablet powder equivalent to 2.5 mg of ERTU and 50 mg of METF was weighed and transferred into a 100 mL volumetric flask and 70 mL of diluent was added. Sonicated for 30 minutes with intermittent shaking and cooled to room temperature. Structure of Metformin Volume was made with diluent and mixed well. 5 mL of the above sonicated solution was transferred into Material and Methods 25 mL volumetric flask. Volume was made with Materials diluent and mixed well. Milli-Q Water, Methanol (HPLC Grade), Calibration Orthophosphoric acid (AR Grade), was obtained The contents of the drug were determined using a from Merck, Mumbai. Gift samples of ERTU and calibration curve established with six dilutions of METF were obtained from Merck & Co., Inc. each standard at concentration range 3-15 μg/ml for Bengaluru. All other chemical of analytical grade ERTU and 12.5-125 μg/ml for METF respectively. were procured from local sources unless specified. The corresponding peak areas were plotted against All dilutions were performed in standard class-A, the concentrations of the drug sample injected. Peak volumetric glassware. identification was achieved by comparison of both Instrumentation the retention time and UV absorption spectrum with Chromatographic system consisted of a Waters those obtained for standards. Model Alliance 2998 separation module equipped System Suitability with auto sampler Photodiode array ultraviolet (UV) A standard solution was prepared by using detector. The data recorded using empowers Ipragliflozin and Sitagliptin working standards as per software. The column used was ACE Column C18, test method and was injected six times into the HPLC (150 mm x 4.6 mm i.d, 5μm) and samples were system. The system suitability parameters were eluted using Buffer (Potassium dihydrogen Ortho evaluated from standard chromatograms by Phosphate) : Acetonitrile (70:30v/v) as the mobile calculating the % RSD from ten replicate injections phase at a flow rate of 1ml/min the mobile phase and for Ipragliflozin and Sitagliptin retention times and samples were degassed by ultrasonication for 30 min peak areas. Resulted chromatogram was shown in the and filtered through 0.45µm Nylon (N66) 47mm chromatogram Fig.1. Data for system suitability of membrane filter. The measurements were carried out Ipragliflozin and Sitagliptin were given in Table-1. with an injection volume of 10μL, flow rate was set to Validation parameter 1 mL/min, and PDA detection was carried out at 240 The method was validated according to ICH nm. All determinations were done at ambient column guideline for linearity, precision, accuracy, temperature (30°C). The chromatograms of the selectivity. Selectivity was checked using IPRA and prepared standard stock solutions of Ertugliflozin and SITA and a mixture of standards in order to optimize Metformin were recorded under optimized separation and detection. Linearity of the method was chromatographic conditions performed by analyzing a standard solution of drugs Sample Preparation by the method in the concentration range 3-15 μg/ml Preparation of Standard Stock Solution for ERTU and 12.5-125 μg/ml for METF Standard stock solution of ERTU was prepared by respectively. The accuracy of the proposed method dissolving 2.5 mg of ERTU in 25 ml of diluent was determined by a recovery study carried out by (Acetonitrile and Water, 50:50v/v) in a 25 ml clean addition method. The samples were spiked with three dry volumetric flask separately and the standard different amounts of standard compounds. The spiked solutions was filtered through 0.45μm nylon samples were extracted in triplicate and analyzed membrane filter and degassed by sonicator to get the under the previously established optimal conditions. concentration of 100μg/ml of ERTU. In separate The obtained average contents of the target volumetric flask same procedure was followed by compounds were used to calculate the spike © Sakun Publishing House (SPH): IJPLS 5855

Research Article Nizami et al., 9(7): July, 2018:5854-5859] CODEN (USA): IJPLCP ISSN: 0976-7126 recoveries. Precision was determined by repeatability Results and Conclusion and inter-day and inter-day reproducibility The results showed that the method provided experiments. Standard solution containing ERTU and adequate accuracy, precision, sensitivity, METF were injected six times. Drugs were also reproducibility with better resolution for the extracted six times to evaluate the repeatability of the simultaneous analysis of ERTU and METF. The extraction process. The mean amount and standard advantages of proposed method are its short analysis deviation (SD) value of each constitute were time and a simple procedure for sample preparation. calculated. All the calculated data for analytical The RP-HPLC method developed for simultaneous parameters summarized in Table No. 2. analysis of IPRA and SITA can be used for routine Tablet studies quality control of their bulk drug mixture and their The proposed method was successfully applied to the combined dosage form. analysis of marketed products and the results obtained are given in Table 3.

Fig 1: Chromatogram of standard

Table 1: Data for system suitability Ertuflozin and Metformin Ertugliflozin Metformin

USP RT RT USP Plate Resoluti Inj Plate Tailing Area Tailing Area (min) (min) Count on Count

1 3.188 6022 1.09 2.100 5371 1.20 377112 7.5 4294213

2 3.188 6051 1.07 2.102 5365 1.20 377288 7.4 4297170 3 3.197 5944 1.12 4297220 2.104 5123 1.20 374762 7.5 4 3.201 5867 1.08 4292142 2.105 5061 1.19 375094 7.6 5 3.204 5974 1.08 4297103 2.112 5024 1.17 378661 7.7 6 3.210 6032 1.06 4278079 2.115 5273 1.18 377650 7.7 MEAN 4292655 MEAN 376761 standard deviation 7431.8 standard deviation 1521.6 %RSD 0.2 %RSD 0.4

Research Article Nizami et al., 9(7): July, 2018:5854-5859] CODEN (USA): IJPLCP ISSN: 0976-7126 Table 2: Validation Parameters Validation S.No Specification Results parameters

System suitability ERTU METF

Retention time Not applicable 3.203 and 2.106

Tailing NMT 2 1.08 1.19 Resolution NLT 2 7.56 1 Theoretical NLT 2500 5982 5203 plates Similarity factor 0-98 to 1.02 1.00 %RSD NMT 2.0% 0.2 0.4 There is no peak in blank at Nil Nil the Rt of analyte 2 Specificity There is no peak in placebo Nil Nil at the Rt of analyte 100 100 100 100

100 100

The value should be between 100 100 97% to 103% 100 100 3 Precision 100 100 The %RSD of six replicate 0.1 0.2 assay results NMT 2.0% The value should be Accuracy (50%) 99.87 99.13 between 97% to 103%

The value should be Accuracy (100%) 99.29 99.78 between 97% to 103% 4 The value should be Accuracy (150%) 99.94 99.76 between 97% to 103%

5 Linearity Correlation coefficient NLT 0.999 0.999 0.999

6 LOD Not applicable 0.43 0.74 7 LOQ Not applicable 1.30 2.24 12.5-125 8 Range Not applicable 3-15µg/ml µg/ml

Robustness(Flow-1) Tailing NMT 2 1.131 1.211 Resolution NMT 2 1.008

Research Article Nizami et al., 9(7): July, 2018:5854-5859] CODEN (USA): IJPLCP ISSN: 0976-7126

Theoretical NLT 2500 5944 5365 plates

Robustness(Flow-2) Tailing NMT 2 1.182 1.275 Resolution NMT 2 1.117 9 Theoretical NLT 2500 5817 5061 plates Robustness(Temp-1) Tailing NMT 2 1.012 1.164 Resolution NMT 2 1.215 Theoretical NLT 2500 5974 5273 plates Robustness(Temp-1) Tailing NMT 2 1.221 1.265 Resolution NMT 2 1.102 Theoretical NLT 2500 5982 5127 plates

Table 3: Result of analysis of Formulation Label claim Assay% of label claim ± %RSD Formulation ERTU METF ERTU METF

Formulation-1 2.5 mg 50 mg 99.89+0.63 99.77+0.63

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How to cite this article Nizami T., Shrivastava B. and Sharma P. (2018). Analytical method development and validation for simultaneous estimation of Ertugliflozin and Metformin in tablet dosage form by RP-HPLC method. Int. J. Pharm. Life Sci., 9(7):5854-5859. Source of Support: Nil; Conflict of Interest: None declared

Received: 22.05.18; Revised: 22.06.18; Accepted: 18.07.18