Stability Indicating Method Development and Validation of Empagliflozin in Bulk and Pharmaceutical Dosage form by using RPHPLC

High Performance Liquid Chromatography

HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.

Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at differentiates due to differences in their partition behavior between the mobile phase and the stationary phase.

High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster.

All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation [1, 2, 3]. Types of HPLC In general there are five techniques of HPLC. They are as follows:

Principal involved in HPLC

HPLC principle is based on both Adsorption as well as Partition, depending upon the nature of stationary phase. If stationary phase is solid then it is adsorption and if stationary phase is liquid then it is partition. When a mixture of components are introduced into a HPLC Column, they travel according to their relative affinities towards the stationary phase. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster. Since no two components have the same affinity towards the stationary phase, the components are separated [4, 5].

Determination of λmax by UV spectrophotometer

Preparation of Primary stock solution: Stock solution is prepared by transferring accurately weighed 10 mg of Empagliflozin in 10 ml volumetric flask and dissolved in 5 ml acetonitrile and sonicate it for 15 min by using ultrasonicator. Then volume was make up to the 10 ml with acetonitrile to get the concentration of 1000 μg/ml. Preparation of Secondary stock solution: Stock solution is prepared by transferring accurately 1ml of primary stock solution of Empagliflozin into 10 ml volumetric flask and dissolved in 5 ml acetonitrile and sonicate it for 15 min by using ultrasonicator. Then volume was make up to the 10 ml to get the concentration of 100 μg/ml. Preparation of working standard: The standard solution of 10 μg/ ml was prepared by taking 1ml of solution from 100 μg/ ml stock solution and diluted up to the 10 ml. This solution was scanned between the range 200-400 nm in uv spectrophotometer against the acetonitrile as blank after base line correction. The optimum wavelength for Empagliflozin was found to be 301nm. Preparation of calibration curve: Working solution was prepared from stock solution by further dilution with acetonitrile to obtained a concentration range 10, 12, 14, 16, 18, 20 μg/ml, respectively. These solutions were scanned from the range 301nm and calibration curve was obtained between concentrations of 10-20 μg/ml.

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Preparation of Mobile Phase

Accurately measured 700 ml (70%) of acetonitrile and 300 ml of potassium dihydrogen ortho phosphate (30%) were mixed and degassed in a digital ultrasonicater for 10 minutes and then filtered through 0.45 µm filter under vacuum filtration. Diluent Preparation: The Mobile phase was used as the diluent. Mobile Phase Optimization: Initially the mobile phase tried was Methanol: Water and Acetonitrile and water with varying proportions. Finally, the mobile phase was optimized to Acetonitrile and potassium dihydrogen ortho phosphate in proportion of 70:30 v/v respectively.

Optimization of Column

The method was performed with various C18 columns like ODS column, Xterra and kromasil column.) Kromasil 100-C18 (250 X 4.6 mm, loaded with 5µ) column was found to be ideal as it gave good peak shape and resolution at 1.0ml/min flow.

System Suitability

Selecting a proper system suitability testing mixture is essential to check the specifications of a chromatographic method. System suitability testing methods are the acceptance criteria that must be met prior to sample analysis. Some parameters that can be checked using system suitability are resolution, retention time , plate number etc.

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Assay (Standard)

The % purity of Empagliflozin pharmaceutical dosage form was found to be 99.57%

Linearity

Linearity Plot

The plot of Concentration (x) versus the Average Peak Area (y) data of DRUG is a straight Line. Y = mx + c (Slope (m) = 1358k, Intercept (c) = 2288, Correlation Coefficient (r) = 0.99)

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Precision

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

Repeatability

Obtained Five (5) replicates of 100% accuracy solution as per experimental conditions. Recorded the peak areas and calculated % RSD

Limit of Detection for Empagliflozin

The detection limit of an individual analytical procedure is the lowest amount of analyte In a sample which can be detected but not necessarily quantitated as an exact value. LOD= 3.3 × σ / s Where σ = Standard deviation of the response S = Slope of the calibration curve

Result

LOD =1.5µg/ml

Quantitation limit The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined. LOQ=10×σ/S Where σ = Standard deviation of the response S = Slope of the calibration curve

Result

LOQ = 4.5µg/ml

Acid Degradation Studies

To 1 ml of Empagliflozin stock, 1 ml of 2N HCl was added and refluxed for 30 min at 60 ºC. The resultant solution was neutralized with 1 ml 2N NaOH and makeup to final volume to obtain (100μg/mL) solution. Cool the solution to room temperature and filtered with 0.45μm membrane filter. A sample of 10μl was injected into the HPLC system, and the chromatograms were recorded to assess the stability of the sample.

Alkali Degradation Studies

To 1 ml of stock solution of Empagliflozin 1 ml of 2N sodium hydroxide was added and refluxed for 30 min at 60 ºC. The resultant solution was neutralized with 1 ml 2N HCl and makeup to final volume to obtain (100μg/mL) solution. Cool the solution to room temperature and filtered with 0.45μm membrane filter. The sample of 10μl was injected into the system, and the chromatograms were recorded to an assessment of sample stability.

Oxidation Degradation Studies

To 1 ml of stock solution of Empagliflozin 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solution was kept for 30 min at 60ºC. For HPLC study, the resultant solution was diluted to obtain (100μg/mL) solution. Cool the solution to room temperature and filtered with 0.45μm membrane filter. A sample of 10μl solution was injected into the system, and the chromatograms were recorded to assess the stability of the sample [6, 7, 8, 9, 10]. Photo Degradation Studies The photo stability of the empagliflozin was studied by exposing the stock solution to UV light for 1day or 200Watt- hours/m2 in photo stability chamber [11, 12, 13, 14, 15, 16, 17, 18]. For HPLC study, the resultant solution was diluted to obtain (100μg/ mL) solution and filtered with 0.45μm membrane filter. A sample of 10μl solution was injected into the system, and the chromatograms were recorded for the assessment of sample stability.

% 100 Sample Area Degradation Standard Area = ×