Development of Validated RP-HPLC Method for Estimation of Empagliflozin and Metformin in Combined Formulation

Aim: The aim of the present study include development of validated RP-HPLC method for estimation of Empagliflozin and Metformin in combined dosage form by using LC-MS compatible volatile mobile phase. Methodology: Appropriate separation of drugs was achieved using C18 column as a stationary phase and Acetonitrile: Water (50: 50, v/v) at a flow rate 1mL/min as mobile phase. Detection was done at 230 nm. Results: The Rt of Metformin and Empagliflozin was found to be 2.20 ± 0.02 min and 3.64 ± 0.02 min respectively. When the marketed formulation was analyzed by the developed method, the % drug contents were found to be 98.57 ± 1.28 and 99.86 ± 1.02 %w /w for Empagliflozin and Metformin, respectively. The method was found to be linear in a range of 11.25 – 56.25 μg/mL for Empagliflozin and 85 – 425 μg/mL for Metformin. Detection limit and quantitation limit were found to be 0.30 and 0.92 μg/mL for Empagliflozin and 1.12 and 3.36 μg/mL for Metformin, respectively. The accuracy and precision results were found to be near 100 % w/w for both the drugs. The method was also found to be robust and specific. Original Research Article Vijaya et al.; JPRI, 33(60A): 1-7, 2021; Article no.JPRI.80162 2 Conclusion: The developed RP-HPLC method was found to be linear, sensitive, accurate, precise, specific and robust for the analysis of Empagliflozin and Metformin in combined dosage form.

Metformin decreases blood glucose levels by increasing insulin sensitivity, increasing glucose uptake and utilization in the peripheral tissue. It also decreases glucose production in liver as well as absorption of glucose by intestine [1]. Its chemical name is 1, 1-dimethylbiguanide hydrochloride (Fig. 2). It is official in IP, BP and USP [2][3][4].

Fig. 2. Metformin
Combination of EM and MH is approved by FDA on 26 Aug 2015 and CDSCO on 17 Oct 2017 as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus [5-6].

MATERIALS AND METHODS
Pure standard sample of Empagliflozin (99.33 % w/w) was procured from Manus Aktteva Biopharmaceutical, Gujarat. Pure sample of Metformin (99.7 %, w/w) was obtained as a gratis sample from Nulife Pharmaceuticals, Pune. Tablet Synjardy containing EM and MH was purchased from local market. AR grade chemicals and solvents were procured from Research Lab, Mumbai.

Preparation of Standard Solutions
Accurately weighed 11.25 mg of EM and 85 mg of MH powder was dissolved in 10 mL of methanol to get stock solution. This stock solution was further diluted by taking appropriate alliquotes to get working standard solution of 11.25 μg/mL of EM and 85 μg/mL of MH.

Selection of Wavelength for Analysis
The standard drug solutions were scanned in 200-400 nm range in UV spectroscopy. From the UV spectroscopy overlay spectrum of EM and MH it was observed that both the drugs absorb radiations of 230 nm. Therefore analysis was done at 230 nm.

Optimization of Chromatographic Conditions
Combination of water and methanol in various ratios was tried as a mobile phase. Tailing of MH was observed. Therefore methanol was replaced by acetonitrile. Combinations of water and acetonitrile in various proportions were tried.

Method Validation
The developed method was validated as per ICH guidelines Q2(R1) [21].

Linearity and range, limit of detection and quantitation
Linearity was checked by preparing a series of dilutions in a range from standard stock solution and verifying the linear relationship between concentration and response.
Detection limit and quanitation limits were determined by formulae, LOD = 3.3 σ/ S and LOQ = 10 σ / S Where, σ is SD of smallest response and S is slope of the calibration curve.

Accuracy
Tablet powder was spiked with pure drugs at 80, 100 and 120 % levels and analysis of samples were carried out by developed method to determine % drug recovery.

Precision
Repeatability and intermediate precision studies were performed to establish precision. In case of repeatability studies, sample containing 100 % of the test concentration was analyzed six times within a short interval of time in a day. Interday analysis was done to confirm the intermediate precision.
Values of % RSD were found to be less than 2 indicated method is precise.

Robustness
Robustness was estimated by checking the stability of analytical solutions at RT. The effect of variations like change in detection wavelength (± 2 nm) and the composition of mobile phase (± 2 mL) was also determined.

Specificity
The specificity of the method was determined by peak purity analysis. Purity angle and purity threshold were determined.

Optimization of Chromatographic Conditions
An excellent resolution with good peak shapes was obtained by using Acetonitrile: Water (50: 50, v/v) at a flow rate 1mL/min as mobile phase. MH is highly hydrophilic and polar drug with negative log P value eluted out faster at 2.20 ± 0.02 min. Whereas, EM a lipophilic drug eluted out later with R t 3.64 ± 0.02 min (Fig. 3). The resolution between the two drugs was 2.78. The number of theoretical plates for EM and MH were 2900 ± 100 and 2100 ± 100 respectively. The Tailing factor for EM was 1.08 ± 0.02 and for MH was 1.12 ± 0.02.

Analysis of Marketed Formulation
Analysis of this formulation solution was done by the developed method (Fig. 4) and % drug contents were determined. The % drug contents were found to be 98.57 ± 1.28 and 99.86 ± 1.02 %w /w for EM and MH, respectively.

Linearity and range
The method was found to be linear in a range of 11.25 -56.25 μg/mL for EM and 85 -425 μg/mL for MH with correlation coefficients 0.9999 and 0.9991 for EM and MH respectively. Values close to 1 indicated good linearity (Fig. 5 and 6). Detection limit and quantitation limit were found to be 0.30 and 0.92 μg/mL for EM and 1.12 and 3.36 μg/mL for MH, respectively. From such small values of limits, sensitivity of the method was proposed.

Accuracy
The % drug recovery was found to be in a range of 98.86 to 99.08 and 100.59 to 101.27 % w/w for EM and MH, respectively. Values close to 100 confirmed the accuracy of the method.

Precision
The % drug contents were found to be in a range of 99.12-100.34 % w/w for both the drugs.Values of % RSD were found to be less than 2 indicated method is precise.

Robustness
Robustness was estimated by checking the stability of analytical solutions at RT and solutions were found to be stable for 8 h at RT. The % RSD values of the deliberate variations like change in detection wavelength (± 2 nm) and the composition of mobile phase (± 2 mL) were found to be less than two proved robust nature of the method.

Specificity
The purity angle was found to be less than purity threshold, concluded peak purity.

CONCLUSION
The HPLC methods reported for EM and MH have used buffers like phosphate as a part of mobile phase. Use of buffers decrease column life as well as it requires thorough washing after analysis which increases analysis time. Therefore, a RP-HPLC method using Acetonitrile: Water (50:50, v/v) as a mobile phase was developed and validated for the determination of EM and MH simultaneously in tablet formulation. The assay values and Recovery values were found to be nearer to 100% w/w. Whereas %RSD for precision and robustness studies was found to be les than two. The developed HPLC method was validated and found to be linear, sensitive, accurate, precise, specific and robust.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.