STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION OF REMOGLIFLOZIN ETABONATE IN BULK AND PHARMACEUTICAL DOSAGE FORM BY RP-HPLC

STABILITY INDICATING METHOD DEVELOPMENT AND VALIDATION OF REMOGLIFLOZIN ETABONATE IN BULK AND PHARMACEUTICAL DOSAGE FORM BY RP-HPLC

K. Likitha Kanna ^* and Uttam Prasad Panigrahy

Department of Pharmaceutical Analysis, CMR College of Pharmacy, Kandlakoya (V), Medchal (District), Hyderabad - 501401, Telangana, India.

ABSTRACT: An accurate, precise, and simple stability-indicating and RP-HPLC method was developed and validated for the estimation of Remogliflozin Etabonate in bulk and pharmaceutical dosage forms. Primacel C₁₈ (150 × 4.6mm, 5µm) column, with mobile phase Acetonitrile: Water (70:30, v/v) at isocratic mode, was used for the development of this method. At wavelength 280nm, and flow rate of 1ml/min was maintained. The retention time for remogliflozin etabonate was about 2.6 min. This method was validated with respect to ICH guidelines for linearity, the limit of detection, the limit of quantification, precision, accuracy, robustness, solution stability, and forced degradation studies. Linearity was performed in the concentration range of 25µg/ml to 150µg/ml with a correlation coefficient of 0.999. The percentage recovery for Remogliflozin Etabonate was found to be within limits of 98% - 102%. The %RSD was also found to be less than 2% which is within limits. Forced degradation studies result in maximum degradation occurred in alkali, acid, and peroxide degradation studies. There was no degradation occurred in photolytic and thermal degradation studies.

Remogliflozin Etabonate, RP-HPLC, C18 Column, Validation and ICH guidelines

INTRODUCTION: Remogliflozin Etabonate is an anti-diabetic drug, chemically known as 5-Methyl-4-[4-(1-methyl ethoxy) benzyl]-1-(1-methylethyl)-1H-pyrazol-3-yl-6-O-(ethoxycarbonyl)-β-D-glucopyranoside. It inhibits the sodium-glucose transport proteins (SGLT), which are responsible for glucose reabsorption in the kidney. Blocking this transporter causes blood glucose to be eliminated through the urine. The structural formula is as shown in Fig. 1 ^1-3.

FIG. 1: CHEMICAL STRUCTURE OF REMOGLIFLOZIN ETABONATE

In the literature survey, it was found that there is no published literature for Remogliflozin Etabonate, but we found some articles for dapagliflozin, empagliflozin, and canagliflozin, which belong to the family of Remogliflozin ^4-17. In the present work, efforts are made to develop a simple, accurate, and precise RP-HPLC and solution stability method for Remogliflozin Etabonate in bulk and pharmaceutical dosage form and to validate it in accordance with ICH guidelines ^{18, 19}.

MATERIALS AND METHODS: Remogliflozin Etabonate is a gift sample from Metrochem Pvt Ltd, Hyderabad, India. REMO^®-ZEN 100mg (Formulation was manufactured by Glenmark Pharma limited). HPLC grade chemicals were preferred for the development of the method; this was obtained for Merck, Hyderabad, India. The method development conditions are described in Table 1.

Selection of Wavelength: Wavelength was fixed at 280nm by performing UV Spectroscopy Fig. 2.

FIG. 2: WAVELENGTH SPECTRA

Chromatographic Conditions: Shimadzu HPLC with spinchrom software and UV/VIS detector with variable wavelength programme was used for the method development. Primacel C₁₈ (150 × 4.6mm, 5µm) column. Acetonitrile: Water (70:30, v/v) is used as mobile phase at a flow rate of 1 ml/min Rheodyne injector with 25 µl loop was used for injecting the sample, and the sample was analyzed at 280nm.

Preparation of Mobile Phase: 70ml of HPLC grade Acetonitrile is taken into a mobile phase reservoir, and 30ml of HPLC grade water is added to it, and then it is kept for sonication for 15 mins, sonicated, and degassed.

Preparation of Standard Solution of Remogliflozin Etabonate: 100mg API of Remogliflozin Etabonate is dissolved in 100ml solvent (ACN) and the concentration is 1000µg/ml.

Preparation of Sample Solution: Estimation of Remogliflozin Etabonate (REMO^®-ZEN) in dosage form, 20 tablets were weighed individually, and an average weight of the tablets was calculated and triturated into fine powder. 100mg equivalent powder is weighed and transferred it into a 100ml volumetric flask, then dissolve it using 30ml of a suitable solvent (ACN) and sonicated it for 15 min, then make up the volume of the contents in volumetric flask up to the mark using same solvent (ACN) and filter it using Whatman filter paper (No. 1) the concentration obtained is 1000µg/ml. from this solution various sample solutions are prepared and validated according to ICH guidelines.

Preparation of Calibration Curve: From the standard solution pipette out 0.25ml, 0.5ml, 0.75ml, 1ml, 1.25ml & 1.5ml into respective 10ml volumetric flasks and make up the volume up to the mark using suitable solvent (ACN) the concentrations are 25µg/ml, 50µg/ml, 75µg/ml, 100µg/ml, 125µg/ml and 150µg/ml. then the solutions were injected thrice using 25µl injector at the flow rate 1ml/min, at 280nm the chromatograms are recorded and calibration curve was plotted by taking concentration on X-axis and peak area on Y-axis.

Method Development:

Trail 1: Theoretical plates are very less.

Trail 2: Theoretical plates are not within the limits.

Optimized chromatographic conditions: Theoretical plates are more than 2000 which are acceptable and within the limits and the retention time is 2.6 min.

TABLE 1: CHROMATOGRAPHIC CONDITIONS FOR METHOD DEVELOPMENT

Assay Procedure: Remogliflozin Etabonate is available in the local pharmacy with the brand name of REMO^®-ZEN (100mg, Glenmark Pharma limited). 1000µg/ml of sample solution was prepared and diluted to 50µg/ml and was injected into the HPLC system, and its obtained peak areas were noted down, and its % Assay was calculated as shown in Table 2.

TABLE 2: ASSAY OF FORMULATION

RESULTS AND DISCUSSION:

Specificity: The blank preparation from the formulation, which consists of excipients, was injected. Peaks were not detected in the retention time of analyte peak; this indicates that there is no interference of excipients of the formulation with the pure drug (API); this indicates that this method has specificity. The chromatogram is given in Fig. 3.

FIG. 3: BLANK AND STANDARD CHROMATOGRAM OF REMOGLIFLOZIN ETABONATE

Linearity: From the standard solution, a series of concentrations in the range of 25-125 µg/ml was prepared and injected thrice. The calibration curve was plotted by taking concentration on X Peak area on Y-axis. The obtained linearity has r²=0.999, as shown in Table 3 and Fig. 4.

TABLE 3: PEAK AREAS OF REMOGLIFLOZIN ETABONATE

FIG. 4: CALIBRATION CURVE OF REMOGLIFLOZIN ETABONATE

LOD: The limit of detection (LOD) was calculated according to the following formula results are discussed below.

LOD = 3.3 × Standard deviation /Slope

LOD = 0.12µg/ml

LOQ: The limit of quantification was calculated according to the following formula results are discussed below.

LOQ = 10 ×Standard deviation / Slope

LOQ = 0.35µg/ml

Accuracy: Accuracy is described as an aggregate between the true value and the measured value. Accuracy was performed by injecting 50%, 100% & 150% levels thrice, and then its Amount found, Amount added, %Recovery, Mean recovery, and %RSD were calculated and are discussed in Table 4.

TABLE 4: ACCURACY RESULTS OF REMOGLIFLOZIN ETABONATE

Precision: Repeatability:

System Precision: It was performed by injecting 50µg/ml of standard solution six times and its peak areas were noted and its average, SD, % RSD was calculated as shown in Fig. 5 and Table 5.

FIG. 5: CHROMATOGRAM FOR SYSTEM PRECISION OF REMOGLIFLOZIN ETABONATE

TABLE 5: RESULTS FOR SYSTEM PRECISION OF REMOGLIFLOZIN ETABONATE

FIG. 6: CHROMATOGRAM FOR METHOD PRECISION OF REMOGLIFLOZIN ETABONATE

Method Precision: It was performed by injecting 50µg/ml of sample solution six times, and its peak areas were noted, and its % Assay, Average, S. D & %RSD were calculated as shown in Fig. 6 and Table 6.

TABLE 6: RESULTS FOR METHOD PRECISION OF REMOGLIFLOZIN ETABONATE

Ruggedness / Intermediate Precision: Ruggedness was performed on two different days by two different analysts in two different labs by maintained the same laboratory conditions. On Day 1, Analyst 1 and Analyst 2 injected 100% concentration of the sample (50µg/ml) six times and noted its peak area values, and calculated its S. D & % RSD. Similarly, on Day 2, Analyst 1 and Analyst 2 injected 100% concentration of the sample (50µg/ml) for six times, and its peak areas were noted and calculated for S. D & %RSD. The results are discussed in Table 7 and Fig. 7.

FIG. 7: CHROMATOGRAM FOR INTERMEDIATE PRECISION OF REMOGLIFLOZIN ETABONATE

TABLE 7: RESULTS FOR INTERMEDIATE PRECISION OF REMOGLIFLOZIN ETABONATE

Reproducibility: Precision between the laboratories is known as Reproducibility. The procedure was carried out by injecting the 100% concentration of the sample (50µg/ml) for six times in two different laboratories into two different HPLC system. The peak areas were noted and its SD & %RSD was calculated which is discussed in Table 8 and Fig. 8.

FIG. 8: CHROMATOGRAM FOR REPRODUCIBILITY OF REMOGLIFLOZIN ETABONATE

TABLE 8: RESULTS FOR REPRODUCIBILITY OF REMOGLIFLOZIN ETABONATE

Robustness: Robustness is known as changes in conditions of the parameters that should not affect any method. So, robustness was performed by small changes in the parameter conditions of the mobile phase (±10%) and Flow rate (±10%).

Change in Mobile Phase: This procedure was carried out by changing the Mobile phase proportion ±10% (i.e., 77:23, v/v and 63:37, v/v). 100% concentration (50µg/ml) of sample solution was injected six times by varying the mobile phase proportions of ACN: water, i.e., 77:23, v/v and 63:37, v/v, its peak areas were noted and calculated for its % Assay, Average, SD & %RSD as shown in Fig. 9 and 10 and Table 9 and 10.

FIG. 9: CHROMATOGRAM FOR ROBUSTNESS AT +10% OF MOBILE PHASE

TABLE 9: RESULTS FOR ROBUSTNESS +10% OF MOBILE PHASE

FIG. 10: CHROMATOGRAM FOR ROBUSTNESS AT -10% OF MOBILE PHASE

TABLE 10: RESULTS FOR ROBUSTNESS AT -10% OF MOBILE PHASE

Change in Flow Rate: This procedure was carried out by changing the flow rate proportion ±10% (0.9ml/min & 1.1ml/min) 100% concentration (50µg/ml) of sample solution was injected six times by varying the Flow rate conditions i.e., 0.9ml/min and 1.1ml/min, its peak areas were noted and calculated for its %Assay, Average, SD & %RSD as shown in Fig. 11 and 12, Table 11 and 12.

FIG. 11: PEAK ELUTED FOR ROBUSTNESS AT 1.1ml/min OF FLOW RATE

TABLE 11: RESULTS FOR ROBUSTNESS AT 1.1ml/min OF FLOW RATE

FIG. 12: PEAK ELUTED FOR ROBUSTNESS AT 0.9ml/min OF FLOW RATE

TABLE 12: RESULTS FOR ROBUSTNESS AT 1.1ml/min OF FLOW RATE

Solution Stability: Solution stability was performed for 0 hr, 24 h and 48 h. 100% concentration (50µg/ml) of sample solution was injected for six times at 0 h, 24 h and 48 h. Peak areas obtained were noted and further calculation was done for % Assay, SD and %RSD was shown in Table 13 and Fig. 13, 14 and 15.

FIG. 13: PEAK ELUTED AT 0 h

FIG. 14: PEAK ELUTED AT 24 h

FIG. 15: PEAK ELUTED AT 48 h

TABLE 13: RESULTS FOR SOLUTION STABILITY AT 0 h, 24 h & 48 h FOR REMOGLIFLOZIN ETABONATE

Forced Degradation Studies: It was performed for Acid Degradation (0.1N HCl), Alkali Degradation (0.1N NaOH), Oxidative Degradation (3% H₂O₂), Thermolytic Degradation (60°C Heating) & Photolytic Degradation (sunlight).

Solutions Preparation:

0.1N HCl Preparation: 0.812 ml of HCl is taken into a 100ml volumetric flask, and volume it made up to the mark using distilled water.

0.1N NaOH Preparation: 0.4 gm of Sodium Hydroxide pellets are taken into a 100ml volumetric flask, and volume is made up to the mark using distilled water.

3% H₂O₂ Preparation: 3ml of H₂O₂ is pipetted out into a 100ml volumetric flask, and volume is made up to the mark using distilled water.

Procedure:

Acid Degradation Studies: From the stock solution (100µg/ml), pipette out 5ml and add into a 10ml volumetric flask, to it add 1ml of the prepared 0.1N HCl and then make up the volume up to the mark using Acetonitrile then kept for 60 mins and inject the prepared sample for six times and check the peak area at optimized conditions shown in Fig. 16 and Table 14.

FIG. 16: PEAK ELUTED FOR ACID DEGRADATION

Alkali Degradation Studies: From the stock solution (100µg/ml) pipette out 5ml and add into a 10ml volumetric flask, to it add 1ml of the prepared 0.1N NaOH and then make up the volume up to the mark using Acetonitrile then kept for 60 min and inject the prepared sample for six times and check the peak area at optimized conditions shown in Fig. 17 and Table 14.

FIG. 17: PEAK ELUTED FOR ALKALI DEGRADATION

FIG 18: PEAK ELUTED FOR OXIDATIVE DEGRADATION

Oxidative Degradation Studies: From the stock solution (100µg/ml) pipette out 5ml and add into a 10ml volumetric flask, to it add 1ml of the prepared 3% H₂O₂ and then make up the volume up to the mark using Acetonitrile then kept for 60 min and inject the prepared sample for six times and check the peak area at optimized conditions shown in Fig. 18 and Table 14.

Thermal Degradation Studies: From the stock solution (100µg/ml) pipette out 5ml and into a 10ml volumetric flask and then make up the volume up to the mark using Acetonitrile and kept the solution in Hot air oven at 60°C for 60 min and inject the sample for six times and check the peak area at optimized conditions shown in Fig. 19 and Table 14.

FIG. 19: PEAK ELUTED FOR THERMAL DEGRADATION

Photolytic Degradation Studies: From the stock solution (100µg/ml) pipette out 5ml and add into a 10ml volumetric flask and then make up the volume up to the mark using Acetonitrile and kept the solution in UV chamber for 60 min and then inject the sample for six times and check the peak area at optimized conditions shown in Fig. 20 and Table 14.

FIG. 20: PEAK ELUTED FOR PHOTOLYTIC DEGRADATION

TABLE 14: RESULTS FOR FORCED DEGRADATION STUDIES OF REMOGLIFLOZIN ETABONATE

From the above values, we can explain clearly that maximum degradation occurred in Alkali, Acid, and Peroxide. There was no degradation that occurred in Photolytic and Thermal.

CONCLUSION: From the obtained result, it was concluded that a simple, rapid, sensitive, linear, accurate, rugged, robust, and precise method was developed for the estimation of Remogliflozin Etabonate by HPLC. Various validation parameters like Specificity, Linearity, Precision, LOD, LOQ, Accuracy, Solution Stability, and Forced Degradation Studies were carried out. The linearity was obtained in the concentration range of 25µg/ml - 150µg/ml with correlation factor (r²) 0.999. The %RSD for all the parameters was found to within limits i.e., less than 2%, and % recovery was also found to within limits i.e., 98% - 102%. Hence, it was concluded that the projected method can be used for the determination of Remogliflozin Etabonate by Stability indicating and RP-HPLC method.

ACKNOWLEDGEMENT: I would like to thank our Principal, Dr. K. Abbulu, CMR College of Pharmacy, Hyderabad, for providing all the necessary facilities. I express my deepest gratitude to Metrochem labs Pvt, Hyderabad, for providing the API of Remogliflozin Etabonate to facilitate the work. I take my pleasure to thank all the teaching and non-teaching staff members for their valuable support during my academics and project.

CONFLICTS OF INTEREST: The author declared that they have no conflicts of interest.

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    How to cite this article:

    Kanna KL and Panigrahy UP: Stability indicating method development and validation of remogliflozin etabonate in bulk and pharmaceutical dosage form by RP-HPLC. Int J Pharm Sci & Res 2021; 12(8): 4197-07. doi: 10.13040/IJPSR.0975-8232.12(8).4197-07.

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