Estimation Of Metformin Hydrochloride And Ertugliflozin Combination

Type 2 diabetes is a disease in which the body does not make enough insulin to control the level of glucose in the blood or when the body is unable to use insulin effectively. The result is a high level of glucose in the blood. The two active substances such as Metformin hydrochloride and Ertugliflozin, work in different ways to lower glucose levels. Metformin hydrochloride is an AHA (anti hyperglycaemic agent) that improves glucose tolerance in patients with T2DM by lowering both basal and post-prandial plasma glucose (PPG). It is not chemically or pharmacologically related to any other class of oral AHA. Metformin decreases hepatic glucose production, decreases intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization Ertugliflozin helps to lower blood glucose by making the patient pass out glucose in the urine. It does this by blocking a protein in the kidneys (called SGLT2) that normally takes glucose back into the blood from the kidneys. Metformin hydrochloride chemically called as N,N- dimethylimido dicarbonimidic diamide hydrochloride and does not related to any other classes of oral antihyperglycemic agents. It is a white to off-white crystalline compound with a molecular formula of C4H11N5*HCl.

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Ertugliflozin is the fourth sodium-glucose co-transoporter-2 (SGLT2) inhibitor approved by the FDA. The chemical name of ertugliflozin L-pyroglutamic acid is (1S,2S,3S,4R,5S)-5-(4-chloro-3-(4-ethoxy benzyl)phenyl)-1-(hydroxymethyl)-6,8-dioxabicyclo octane-2,3,4-triol, compound with (2S)-5oxopyrrolidine- 2-carboxylic acid and its molecular formula isC8H15NO2. Even though numerous methods are available for the estimation of Metformin hydrochloride individually and in combination with other drugs, no method has been reported for the estimation of Metformin hydrochloride and Ertugliflozin simultaneously.

Materials and methods

The reference sample of Metformin hydrochlorideand Ertugliflozin was obtained as a gift samples and the tablet containing Metforrmin hydrochloride 500mg and Ertugliflozin 7.5 mg was procured from local market. Water (HPLC grade) from Rankem and acetonitrile (HPLC grade), ortho phosphoric acid (AR grade), sodium hydroxide (pure), hydrogen peroxide (pure) from Merck Limited, 0.45 µm Nylon filter was from Zodiac life sciences were used.

Instrumentation: Waters HPLC 2695 system equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2 Software. UV-VIS spectrophotometer, PG Instruments T60 with special bandwidth of 2 mm and 10 mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Metformin hydrochlorideand Ertugliflozin solutions, Electronics Balance-Denver pH meter -BVK enterprises, India Ultrasonicator-BVK enterprises.

Chromatographic conditions: Optimized chromatographic conditions for the separation were used are Standard BDS C8 (150 x 4.6 mm, 5 mm particle size) column. Temperature was maintained ambient, mobile phase used was Buffer: Acetonitrile (55:45 v/v) and flow rate was maintained at 1 ml/min. Diluent used throughout the method was Water: Acetonitrile (50:50 v/v) and the run time was 6 mins. All the samples and mobile phase were degassed for 30 mins and filtered by ultrasonic filtration by using 0.45 µm Nylon (N66) 47 mm membrane filter. Detection was carried out at 224 nm using PDA detector with an injection volume of 10 μl. By using the above optimized conditions method was developed.

Preparation of Buffer:

Buffer: (0.01%KH2PO4)

About 1.36 gm of potassium dihydrogen phosphate was weighed and transferred into a 1000 ml volumetric flask added about 100 ml of milli-Q water and finally made the volume up to 1000 ml with milli-Q water.

Ortho Phosphoric Acid

Buffer1 ml of ortho phosphoric acid was diluted to 1000 ml with HPLC grade water Preparation of mobile phase:Buffer (55%) and Acetonitrile (45%) were mixed and degassed in an ultrasonicate water bath for 10 mins and then filtered through 0.45 µ filter under vacuum filtration.

Diluent: Diluent used throughout the method was Water: Acetonitrile (50:50 v/v) chosen purely based on the solubility of the drugs.

Preparation of Standard Solutions

Accurately weighed and transferred 500 mg of Metformin hydrochloride and 7.5 mg of Ertugliflozin working Standards into a 100 ml clean dry volumetric flasks, add 10 ml of diluent was added and sonicated for 10 mins and made up to the final volume with diluent.

Preparation of sample solution

A total of 10 tablets were weighed, their mean weight was determined and crushed in mortar. An amount of powder weight equivalent to 500 mg of Metformin hydrochloride and 7.5 mg of Ertugliflozin was taken and transfer to 100 ml volumetric flask. The powder obtained was dissolved in mobile phase and sonicated for 20 mins for complete extraction. The solution was made up to the volume with mobile phase. The solution was filtered through membrane filter. The stock solution was further diluted with diluent to get concentration of 500 μg/ml of Metformin hydrochlorideand 7.5 μg/ml of Ertugliflozin.

Quantitative estimation

Where:AT = Average area of each main peak obtained from chromatogram of the sample solution AS= Average area of each main peak obtained from chromatograms of the standard solution WS = Weight of each Metformin hydrochloride and Ertugliflozin in standard solution (mg/ml) WT= Weight of each Metformin hydrochloride and Ertugliflozin in sample (mg/ml) DS =Dilution of standard solution DT =Dilution of test solution P= Potency of each standard.

Method validation

System suitability

System suitability tests are a fundamental part of liquid chromatographic method. It ensures that system is working correctly. System suitability parameters such as number of theoretical plates, retention time, and tailing factor were evaluated. This was performed by injecting mixture of standard in six replicates.

Linearity

The linearity of the proposed method was determined by quantitative dilution of the standard solution of Metformin hydrochloride and Ertugliflozin to obtain solution in concentration range of 125-750 µg/ml and 1.875-11.25 µg/ml for Metformin hydrochloride and Ertugliflozin respectively. A graph of peak area versus concentration in μg/ml was plotted for all three drugs in triplicate. The slope, intercept, and correlation coefficient of regression line were determined.

Limit of detection (LOD) and limit of quantitation (LOQ)

The LOD and LOQ represent the concentration of analyte that would yield to signal-to-noise ratio of 3 for LOD and 10 for LOQ. LOD and LOQ were calculated using following formula, LOD=3.3 σ/S LOQ= 10 σ /S where,σ = standard deviation of response (peak area) and S = average of slope of the calibration curve.

Method precision

The method precision of the proposed method was determined by injecting six replicates of sample and standard on the same day to ensure that the analytical method is repeatable.

System precision

The system precision is checked by injecting six replicates of standard solution to ensure that the analytical system is working properly.

Accuracy

The accuracy of this method was performed at three different levels (50%, 100%, 150%), by the addition of a known amount of standard to the sample at each level. Each level was repeated three times (n=3).

Robustness

Robustness is the measure of optimized method capacity to remain unaffected by small, but deliberate variations in method parameters such as mobile phase flow rate (±0.2 ml/min), wavelength nm (±1 nm), and column oven temperature (±1°C).

Degradation studies

Oxidative degradation

To 1 ml of stock solution of Metformin hydrochloride and Ertugliflozin 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 mins. For HPLC study, the resultant solution was diluted to obtain (500 µg/ml and 7.5 µg/ml) solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Acid degradation

As described in the oxidative degradation same method was followed by using 2N Hydrochloric acid and refluxed for 30 mins.

Alkali degradation

Same procedure was followed as the above in which 1ml of 2N sodium hydroxide was used and refluxed for 30 mins. Thermal degradation The standard drug solution was placed in oven at 105°C for 6 hrs to study dry heat degradation. For HPLC study, the resultant solution was diluted to (500 µg/ml and 7.5 µg/ml) solution and10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample. Photo Stability The photochemical stability of the drug was also studied by exposing the solution to UV light by keeping the beaker in UV Chamber for 7days in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain (500 µg/ml and 7.5 µg/ml) solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Water degradation

Stress testing under neutral conditions was studied by refluxing the drug in water for 6 hrs at a temperature of 60º C. For HPLC study, the resultant solution was diluted to (500 µg/ml & 7.5 µg/ml) solution and 10 µL were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Conclusions

No method is available for estimation of Metformin hydrochloride and Ertugliflozin combination, so a new simple, precise, accurate and repeatable RP-HPLC method for estimation of these simultaneously have been developed and validated according to ICH guidelines. All the validation parameters including system suitability, linearity, accuracy, precision, LOD, LOQ and robustness were within the recommended limits of ICH. The developed method has a total run time of 6 mins, which permits the analysis of large number of samples in a short period of time, thereby reducing solvent cost. Exposure of the drugs to different stress conditions such as acid, base, UV, thermal, photolytic and water showed the drugs are stable in all the conditions. Developed method can be used for route analysis of these compounds simultaneously.

03 December 2019

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