International Journal of Pharmacy & Life Sciences

Research Article Nizami et al., 9(7): July, 2018:5860-5865] 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 Ipragliflozin and Sitagliptin in tablet 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 Ipragliflozin and Sitagliptin in pharmaceutical dosage forms has been developed. The method was validated as per ICH guidelines over a range of 50-150 µg/mL for Ipragliflozin and Sitagliptin respectively. Analytical column used was ACE Column C18, (150 mm x 4.6 mm i.d, 5μm) with flow rate of 1.0 mL / min at a temperature of 30°C ± 0.5°C. The separation was carried out using a mobile phase consisting of orthophosphoric acid buffer and methanol in the ratio of 60: 40%v/v. Retention times of 3.092 and 4.549 min were obtained for Ipragliflozin and Sitagliptin respectively. The percentage recoveries of Ipragliflozin and Sitagliptin are 100.12% and 99.42% respectively. The goodness of fit was close to 1 for all the three components. The relative standard deviations are always less than 2%.

Keywords: Ipragliflozin and Sitagliptin, RP -HPLC, Simultaneous analysis, Tablets

Introduction Ipragliflozin (IPRA) a novel SGLT2 selective The empirical formulaC16H15F6N5O and the inhibitor was investigated. In vitro, the potency of molecular mass 523.32. Sitagliptin is an orally-active Ipragliflozin to inhibit SGLT2 and SGLT1 and inhibitor of the dipeptidyl peptidase-4 (DPP-4) stability were assessed. These results suggest that enzyme.The DPP-4 enzyme inactivates incretin Ipragliflozin is an orally active SGLT2 selective hormones, which are involved in the physiologic inhibitor that induces sustained increases in urinary regulation of glucose homeostasis. By inhibiting glucose excretion by inhibiting renal glucose DPP-4, it increases and prolongs active incretin reabsorption, with subsequent ant hyperglycemic levels. This in turn increases insulin release and effect and a low risk of hypoglycemia. Ipragliflozin decreases glucagon levels in the circulation in a has, therefore, the therapeutic potential to treat glucose-dependent manner. hyperglycemia in diabetes by increasing glucose excretion into urine; chemically it is (1S)-1,5- anhydro-1-C-{3-[(1-benzothiophen-2-yl)methyl]-4- fluorophenyl}-D-glucitol Molecular formula is C21H21FO5S, molecular weight 404.45g mol/mol (Fig.2.1). It is a white to off-white crystalline powder and hygroscopic. It is freely soluble in water and slightly soluble in ethanol (95%). Sitagliptin(SITA) is described chemically as7-[(3R)- 3-amino-1-oxo4-(2,4,5 trifluorophenyl) butyl]-5, 6, 7, 8- tetrahydro-3- (trifluoromethyl)-1,2,4-triazolo [4,3- a] pyrazine phosphate (1:1) monohydrate, specifically indicated for the improvement of glycemic control in patients with type II diabetes mellitus as monotheraphy or combination theraphy Structure of Ipragliflozin with metformin.

* Corresponding Author E.mail: [email protected] © Sakun Publishing House (SPH): IJPLS 5860

Research Article Nizami et al., 9(7): July, 2018:5860-5865] CODEN (USA): IJPLCP ISSN: 0976-7126 Volume was made with diluent and mixed well. 5 mL of the above sonicated solution was transferred into 25 mL volumetric flask. Volume was made with diluent and mixed well. Calibration The contents of the drug were determined using a calibration curve established with six dilutions of each standard at concentrations ranging from 50 to 150 mg/mL for IPR and SITA respectively. Each concentration was measured in triplicate. The corresponding peak areas were plotted against the concentrations of the drug sample injected. Peak identification was achieved by comparison of both the retention time and UV absorption spectrum with those obtained for standards. System Suitability Structure of Sitagliptin A standard solution was prepared by using IPRA and Sitagliptin working standards as per test method and Material and Methods was injected six times into the HPLC system. The Materials system suitability parameters were evaluated from Milli-Q Water, Methanol (HPLC Grade), standard chromatograms by calculating the % RSD Orthophosphoric acid (AR Grade), was obtained from ten replicate injections for IPRA and SITA from Merck, Mumbai. All other chemical of retention times and peak areas. Resulted analytical grade were procured from local sources chromatogram was shown in the chromatogram unless specified. All dilutions were performed in Fig.1. Data for system suitability of Ipragliflozin and standard class-A, volumetric glassware. Sitagliptin were given in Table-1 and 2. Instrumentation Validation parameter Chromatographic system consisted of a Waters The method was validated according to ICH Model Alliance 2998 separation module equipped guideline for linearity, precision, accuracy, with auto sampler Photodiode array ultraviolet (UV) selectivity. Selectivity was checked using IPRA and detector. The data recorded using empowers SITA and a mixture of standards in order to optimize software. The separation was carried out using a separation and detection. Linearity of the method was mobile phase consisting of orthophosphoric acid performed by analyzing a standard solution of drugs buffer and methanol in the ratio of 60: 40%v/v. The by the method in the concentration ranging 50-150 column used was ACE Column C18, (150 mm x 4.6 µg/mL for IPRA and SITA. The accuracy of the mm i.d, 5μm) with flow rate of 1.0 mL / min using proposed method was determined by a recovery study PDA detection at 224 nm. carried out by addition method. The samples were Sample Preparation spiked with three different amounts of standard Preparation of Standard Stock Solution compounds. The spiked samples were extracted in 50 mg of IPRA and 50 mg of SITA was accurately triplicate and analyzed under the previously weighed and transferred into a 100 mL volumetric flask established optimal conditions. The obtained average 70 mL of diluent was added and sonicated. Volume contents of the target compounds were used to was made with diluent and mixed well. 5 mL of the calculate the spike recoveries. Precision was above stock solution was diluted to 25 mL with determined by repeatability and inter-day and inter- diluent to produce drug in the Concentration 100 day reproducibility experiments. Standard solution µg/mL of IPRA and SITA respectively. containing IPRA and SITA were injected six times. Preparation of Working Standard Solution Drugs were also extracted six times to evaluate the 10 tablets were weighed accurately and finely repeatability of the extraction process. The mean powdered. Tablet powder equivalent to 50 mg of amount and standard deviation (SD) value of each IPRA and 50 mg of SITA was weighed and constitute were calculated. All the calculated data for transferred into a 100 mL volumetric flask and 70 mL analytical parameters summarized in Table No. 3. of diluent was added. Sonicated for 30 minutes with intermittent shaking and cooled to room temperature. © Sakun Publishing House (SPH): IJPLS 5861

Research Article Nizami et al., 9(7): July, 2018:5860-5865] CODEN (USA): IJPLCP ISSN: 0976-7126 Tablet studies simultaneous analysis of IPRA and SITA. The The proposed method was successfully applied to the advantages of proposed method are its short analysis analysis of marketed products and the results time and a simple procedure for sample preparation. obtained are given in Table 4. The RP-HPLC method developed for simultaneous Results and Conclusion analysis of IPRA and SITA can be used for routine The results showed that the method provided quality control of their bulk drug mixture and their adequate accuracy, precision, sensitivity, combined dosage form. reproducibility with better resolution for the

Fig. 1: Chromatogram of standard Table 1: Data for system suitability Ipragliflozin S.No. Name of the Retention Theoretical Tailing Resolution Compound Time plates factor 1 Ipragliflozin 3.094 11034 1.197 -

2 Ipragliflozin 3.091 10650 1.204 -

3 Ipragliflozin 3.092 10529 1.224 -

4 Ipragliflozin 3.089 10096 1.221 -

5 Ipragliflozin 3.089 9279 1.218 -

Table 2: Data for system suitability Sitagliptin S.No. Name of the Retention Theoretical Tailing factor Resolution Compound Time plates 1 Sitagliptin 4.555 8485 1.269 9.032

2 Sitagliptin 4.549 8012 1.277 8.932

Research Article Nizami et al., 9(7): July, 2018:5860-5865] CODEN (USA): IJPLCP ISSN: 0976-7126 3 Sitagliptin 4.549 8034 1.253 8.863

4 Sitagliptin 4.543 7964 1.265 8.759

5 Sitagliptin 4.541 7816 1.267 8.547

Table 3: Validation Parameters S.No Validation Specification Results parameters

System suitability Ipragliflozin Sitagliptin

Retention time Not applicable 3.092 4.549

Tailing NMT 2 1.193 1.296 Resolution NLT 2 9.156 1 Theoretical NLT 2500 11677 8485 plates Similarity factor 0-98 to 1.02 1.00 %RSD NMT 2.0% 0.4 1.1 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 0.05 0.061 assay results NMT 2.0% The value should be Accuracy (50%) 101 101 between 97% to 103%

The value should be Accuracy (100%) 101 101 between 97% to 103% 4 The value should be Accuracy (150%) 101 101 between 97% to 103%

5 Linearity Correlation coefficient NLT 0.999 0.99 0.99

6 LOD Not applicable 2.86 0.55 7 LOQ Not applicable 9.55 1.84

Research Article Nizami et al., 9(7): July, 2018:5860-5865] CODEN (USA): IJPLCP ISSN: 0976-7126

500µg to 1500 50µg to 8 Range Not applicable µg 150 µg Robustness(Flow-1) Tailing NMT 2 1.231 1.301 Resolution NMT 2 8.162 Theoretical NLT 2500 7652 plates Robustness(Flow-2) Tailing NMT 2 1.197 1.277 Resolution NMT 2 8.923

Theoretical NLT 2500 10650 plates Robustness(Temp-1)

Tailing NMT 2 1.224 1.253

Resolution NMT 2 8.863 9 Theoretical NLT 2500 10529 plates Robustness(Temp-1) Tailing NMT 2 1.221 1.265 Resolution NMT 2 8.759 Theoretical NLT 2500 10096 plates

Table :4 Result of analysis of Formulation Label claim Assay% of label claim ± %RSD Formulation IPRA SITA IPRA SITA

Formulation-1 50 mg 50 mg 99.86+0.53 100.14+0.93

Formulation-2 50 mg 50 mg 98.92+0.81 99.71+0.71 2. ICH-Q2A, Text on Validation of Analytical Acknowledgement Procedures, ICH Harmonised Tripartite The authors wish to thanks Principal and Guideline, Geneva, 2-3, A-1 to A-3, (1995). Management of Smriti College of Pharmaceutical 3. Indian Pharmacopoeias Vol II, New Delhi, Education, Indore for providing required lab facilities The controller of Publications. Govt of with enthusiastic environment. India, p.554, (1996). References 4. Meher Vijay Dalawai et al, Development 1. El-Bagary RI, Elkady EF and Ayoub BM. and validation of stability indicating assay Spectrophotometric methods for the method by HPLC for the analysis of determination of Sitagliptin and Vildagliptin sitagliptin phospahte in bulk drug in bulk and dosage forms. International substances, Journal of Chemical and Journal of Biomedical Science. Pharmaceutical Research, 2015, 7(10):781- 2011;7(1):55- 61. 787.

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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 Ipragliflozin and Sitagliptin in tablet form by RP-HPLC method. Int. J. Pharm. Life Sci., 9(7):5860- 5865. Source of Support: Nil; Conflict of Interest: None declared

Received: 20.05.18; Revised: 18.06.18; Accepted: 20.07.18