Development and Validation of New Analytical RP-HPLC Method for the Estimation of Antidiabetic Drugs Metformin Hydrochloride and Ertugliflozin in Combined Pharmaceutical Dosage Form

Metformin Hydrochloride and Ertugliflozin is combination of Antidiabetic drug in tablet Segluromet ® Tablet (500/7.5 mg), a member Antidiabetic drug, is a recent drug developed by Merck Sharp & Dohme Company for the treatment of Type 2 diabetes. A new sensitive and rapid HPLC method was developed for the determination of Metformin Hydrochloride and Ertugliflozin in pharmaceutical dosage forms; it was validated according to International Conference on Harmonization and Food and Drug Administration guidelines. The analysis was performed on the HPLC system equipped with a using Kromasil C18 (5 μm ), (250 mm x 4.6 mm column, with of Buffer (0.1 % v/v Phosphoric acid in water ) and Acetonitrile(ACN) in the ratio 60 : 40 v/v with at a flow rate of 1 mL/min , column temperature 25°C , total run time was 20 min, injection volume 20 μl, and detection was performed at the a wavelength (λ) of 235 nm. the calibration plot gave linear relationship over the concentration range of Metformin Hydrochloride 350, 450, 500, 550 and 600 μg/ml, and Ertugliflozin 6.80, 8.74, 9.72, 10.69 and 11.66 μg/ml, respectively. The accuracy of the proposed method was determined by recovery studies and was found to be Metformin Hydrochloride 98.5 % to 101.3 % and Ertugliflozin 98.2 % to 100.3%. The Limit of Detection were 0.0003 and 0.0009 μg/ml for Metformin Hydrochloride and Ertugliflozin, respectively and the Limit of Quantitation were 0.0037 and 0.0112 μg/ml for Metformin Hydrochloride and Ertugliflozin, respectively. % Relative Standard Deviation of the determination of precision was <2%. The results of robustness and solutions stability studies were within the acceptable limits as well the main features of the developed method are low run time and retention time of around 1.8 min for Metformin Hydrochloride (Met) and 3.8 min for Ertugliflozin (Ertu). © 2020 iGlobal Research and Publishing Foundation. All rights reserved. Cite this article as: Khoja, S.S.; Patel, L.J. Development and Validation of new analytical RP-HPLC method for the estimation of Antidiabetic Drugs Metformin hydrochloride and Ertugliflozin in combined pharmaceutical dosage form. Indo Global J. Pharm. Sci., 2021; 11(1): 62-69. DOI: http://doi.org/10.35652/IGJPS.2021.111009 . Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 62-69 63 Figure. 1 Metformin Hydrochloride Ertugliflozin: Ertugliflozin is an oral, selective inhibitor of sodium glucose co-transporter-2 (SGLT2) which inhibits renal glucose reabsorption and results in urinary glucose excretion (UGE) and reductions in plasma glucose and haemoglobin A1c (Estimated Blood Sugar) in patients with type 2 diabetes mellitus (T2DM). It possesses a high selectivity for SGLT2 versus SGLT1 and other glucose transporters (GLUT1-4). Ertugliflozin is a new chemical entity with a chemical name of (1S,2S,3S,4R,5S)-5-[4-Chloro-3(4-ethoxybenzyl)phenyl]-1hydroxymethyl-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol (Figure 2). Ertugliflozin is included in the drug product as a co crystal with L-pyroglutamic acid (L-PGA), known as Ertugliflozin L-PGA. Molecular formula: C27H32ClNO10. Ertugliflozin L-PGA Relative molecular mass: 566.00 g/mol. Ertugliflozin molecular mass: 436.9 g/mol [1-3]. Figure 2. Ertugliflozin and Ertugliflozin L-PGA Analytical method validation ensures that various HPLC analytical techniques shall give reliable and repeatable results; it is a crucial step in developing new dosage forms as it provides information about accuracy, linearity, precision, detection, and quantitation limits. According to the International conference harmonization guideline, “the objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose.” It is now obligatory in the process of drug development to supply the validation data for the responsible authorities. Guidelines for analysis method validation include ICH and USP guidelines [4–9]. Literature survey revealed a few methods reported for determination for Ertugliflozin and Metformin in Pharmaceutical preparation [14-21]. In this research, a new sensitive and rapid HPLC method was developed for the determination of Ertugliflozin and Metformin Hydrochloride in pharmaceutical dosage forms, and this method was validated according to ICH and FDA guidelines. MATERIALS AND METHODS Instrumentation Chromatographic HPLC system equipped with a Kromasil C18 (5 μm), 250 mm x 4.6 mm column. Chemicals and Reagents Acetonitrile, Phosphoric acid, Methanol, Water were of HPLC Grade. Chromatographic Conditions Mobile Phase (Buffer (0.1 % v/v Phosphoric acid in water) and Acetonitrile (ACN) in the ratio 60: 40 v/v with a flow rate of 1 mL/min. the detection was performed at the wavelength (λ) of 235 nm, injection volume 20 μl, run time 20 min, and column temperature 25°C Diluent –HPLC Grade water. Preparation of Standard Solution Weigh accurately and transfer about 9.72 mg of Ertugliflozin L-PGA (Equivalent to 7.5 mg Ertugliflozin) and 500 mg of Metformin Hydrochloride standard into 100 ml volumetric flask, add 70 ml of diluent and sonicate to dissolve, cool. Dilute to volume with diluent and mix. Transfer 5 ml of this solution to a 50 ml volumetric flask and dilute with diluent to volume and mix well. Preparation of Sample Solution Weigh accurately and transfer Approx. 595 mg of synthetic mixture Equivalent to 9.72 mg of Ertugliflozin L-PGA (Equivalent to 7.5 mg Ertugliflozin) and 500 mg of Metformin Hydrochloride standard into 100 ml volumetric flask, add 50 ml of diluent and sonicate for 15 min with intermittent shaking. Dilute to volume with diluent and mix. Filter a portion of this solution using 0.45 μ PVDF Syringe filter, transfer 5 ml of this solution to a 50 ml volumetric flask and dilute with diluent to volume and mix well. Method Validation The method was validated as per ICH and FDA guidelines, and the validation parameters included specificity, linearity, range, accuracy, precision, sensitivity (LOQ and LOD) robustness, and solution stability [5-7]. a) Specificity: Specificity is one of the significant features of HPLC, and it refers to the ability of the analytical method to discriminate between the analyte and the other components in the complex mixture [7]. Specificity of the method was evaluated by injecting 20 μL solutions of standard, sample, blank, and placebo separately. b) Linearity: To evaluate the linearity and range of the method, Direct standard solutions were prepared by diluting the standard stock solution with the diluent in different concentrations Metformin Hydrochloride: 350, 450, 500, 550 and 600 μg/ml, and Ertugliflozin 6.80, 8.74, 9.72, 10.69 and 11.66 μg/ml which cover 70%, 90%, 100%, 110% and 120%, of the target concentration, respectively. Three replicate injections from each concentration were analysed under the same conditions. Linear regression analysis was used to evaluate the Indo Global Journal of Pharmaceutical Sciences, 2021; 11(1): 62-69 64 linearity of the calibration curve by using the least square linear regression method. c) Sensitivity: Limit of detection (LOD)/limit of quantitation (LOQ) of Metformin Hydrochloride and Ertugliflozin were determined by measuring the signal-tonoise ratio. limit of detection (LOD) is the concentration that gives a signal-to-noise ratio of approximately 3: 1, while the limit of quantification (LOQ) is the concentration that gives a signal-to-noise ratio of approximately 10 :1 with %RSD (n = 3) of less than 10%. d) Accuracy: The accuracy of the assay method was determined by recovery studies at three concentration levels (70%, 100%, and 120%), i.e., 350, 500, and 600 μg/ml for Metformin Hydrochloride and Ertugliflozin 6.8, 9.72 and 11.66 μg/ml and three samples from each concentration were injected. percentage recovery of Metformin Hydrochloride and Ertugliflozin added and RSD were calculated for each of the three replicate samples. e) Precision: The system precision and method precision (repeatability) of the proposed methods were determined by several measurements of standard solution and sample solution, respectively [7-9]. System precision was established by six measurements of the standard solution at the 100% concentration levels on the same day. Method precision was established by six assay determinations of the sample solution at the 100% concentration levels on the same day [9-12]. The RSD of obtained results was calculated to evaluate repeatability results. f) Robustness: Robustness of the method was verified by applying minor and deliberate changes in the experimental parameters, for example: (i) Column temperature: ±2°C (ii) Flow rate: ±0.2 mL/min. Change was made to evaluate its effect on the method. Obtained data for each case was evaluated by calculating % RSD and percent of recovery. g) Stability of Analytical Solutions: The stability of analytical solutions was determined by analysing the standard and sample preparations at Initial, 12 Hr and 24 Hr at ambient room temperature 25°C. Three injections from each solution were analysed, and the average of the peak and the RSD were calculated. RESULTS AND DISCUSSION Method Development and Optimization Several physical and chemical properties of Metformin Hydrochloride and Ertugliflozin were obtained from the literature. The analytical method was developed to select preliminary reversed phase HPLC method chromatographic conditions, including detection wavelength, mobile phase, stationary phase, and sample preparation procedure. For this purpose, a series of trials were performed by varying the ratio of include trials. Table 1. Results of method optimization Column Used Mobile Phase Flow Rate Wavelength Observation Kromasil C18 (5 μm) (250 mm x 4.6 mm ) Water : Methanol (60:40) 1.0 ml/min 235 nm Improper Peak Shape observed for both drugs Kromasil C18 (5 μm) (250 mm x 4.6 mm ) Water : Methanol (50:50) 1.0 ml/min 235 nm Improper Peak Shape observed for both drugs Kromasil C18 (5 μm) (250 mm x 4.6 mm ) Water: Acet