Development and Validation of RP- HPLC Method for Estimation of Empagliflozin and its Stability Studies In vitro Simulated Gastric and Intestinal Fluids

 

Sushil D Patil*, Swapnapurti V Bharambe*, Sanjay Kshirsagar

 

ABSTRACT:

A simple and precise high performance liquid chromatography method for determination of Empagliflozin and its stability studies invitro simulated gastric and intestinal fluids. The separation was carried out on Cosmosil C18 (250mm X 4.6, 5µ) RP-HPLC column using a 0.8ml/min as flow rate with methanol: water (80:20 v/v) mobile phase and RT was 5.017min. Quantification was performed with a UV detector at 224nm, solubility and stability was determined individually in simulated biological fluids at various pH was investigated. The calibration curves of EMPA were linear in the range of 10-50 µg/ml(R²=0.999). The developed method was applied to pharmaceutical formulation successfully with no interfering peaks. The percentage recovery was 99.71-100.29%. It was observed that solubility of EMPA was increased in all in vitro interaction medium, freely soluble in pH 6.8 and slightly in 1.2 and 7.4 pH and the stability of EMPA was stable or less degraded in pH 6.8 for 24 hours.

 

 

 

INTRODUCTION:

Empagliflozin chemically 1-chloro-4-[b-D-glucopyranos-1-yl]-2-[4-([S]-tetrahydrofuran -3- yl -oxy) benzyl]-benzene (fig.1) which belongs to gliflozin class used in the treatment of type-2 diabetes. Empagliflozin is an inhibitor of the sodium glucose co transporter -2 (SGLT-2) thus SGLT-2reduces blood glucose by blocking glucose reabsorption in the kidney and thereby excreting glucose (i.e., blood sugar) via the urine which is the most commonly prescribed drug to the patients with type-2 diabetes.

 

It bring down the blood glucose levels by decreasing the hepatic glucose production, declining intestinal absorption of glucose and enhancing insulin sensitivity by elevating peripheral glucose utilization and uptake. The literature reveal that various analytical technique viz; UV spectrophotometry, high performance liquid chromatography (HPLC)[7], ultra performance liquid chromatography (UPLC). Our aim was to develop proper method which estimates both the analytes in a shorter time and to develop low cost method.and its stability in simulated fluids.

 

Fig no1: Empagliflozin

MATERIALS AND METHODS:

Drug sample:

Empagliflozin was obtained as gift samples from Macleods Laboratories, Pvt. Ltd, Gujarat. The procured drug a sample was standardized by measurement of physical properties like melting point, IR spectrum and UV absorption spectrum and comparing with the data reported in literature.

 

Chemical and Reagents:

Solvents methanol and water used for chromatographic analysis were of HPLC grade purchased from Thomas Baker (chemicals), Mumbai.

 

Instrumentation and chromatographic conditions:

Chromatographic analysis were carried out using an HPLC system consisting of pump P-3000-M Reciprocating (40 MPa) and detection was carried out using detector UV- 3000M. Column used is cosmosil C18 (250mm˟ 4.6 ID, Particle size: 5µ) was used.

 

Standard solution and chromatographic graphs:

An accurately weighed quantity of 10 mg empagliflozin was transferred to 10 ml volumetric flask, dissolved with sufficient quantity of methanol and volume was then made up to the mark with solvent and sonicated for 15 min, from the resulting solution 0.1ml was transferred to 10 ml volumetric flask and the volume was made up to the mark with same solvent. The resulting 10µg/ml of solution was subjected to chromatographic analysis using mobile phase of different strengths with chromatographic conditions. [1,5]

 

Solution for interaction studies:

Preparation of simulated gastric juice pH 1.2:

Take a 2 gm of NaCl and dissolve 3.2gm of pepsin in distilled water, add 80ml of 1 molar HCl make up the volume, diluted with water 1000ml. [13]

 

Preparation of simulated intestinal fluid pH 6.8:

Take a 2.38 gm of potassium dihydrogen phosphate and 3.55 gm of disodium hydrogen phosphate in 1000 ml volumetric flasks diluted with the water and make up to the volume upto 1000ml. adjust the pH up to 6.8 [13]

 

Procedure for interaction:

Taken 10 mg pure empagliflozin in 10 ml volumetric flask diluted with the pH 1.2 and pH 6.8 to make a 1000ppm and kept it at room température for 24 hours. Withdrawn the sample 0.3ml at différent time interval that 0 hr, 6 hr, 12 hr and 24 hours. and inject the sample to take chromatograms on HPLC with the appropriâtes mobile phase. [8,9,10]

 

Optimization of chromatoghraphic conditions [6,12]:

Mobile phase Méthanol: ACN (70:30%) v/v, the peak shapes were improved but dilution with Méthanol: Water (80:20%) v/v it gives in good Shape for Empagliflozin and flow rate 0.8ml/min, column température of 30ºC and UV detector adjusted to 224nm.

 

Fig No. 2: Optimize chromatogram of Empagliflozin in Methanol: Water (80:20%v/v) pH 3 at λmax 224nm, RT 5.017min.

 

Method validation [11]:

Linearity:

Calibration curve representing the relation between the concentrations of drug versus the peak area were constructed by taking appropriate aliquots of the standard stock solutions of the drug in different 10 ml volumetric flasks and the solutions were made up to volume with mobile phase and subjected to regression analysis using the least square method. These standard solutions were analyzed in triplicates. [2,3] Results show linear relationship in the range of 10-50 μg/mL for Empa. The regression equations were computed and found to be: y = 50206x – 168802 R² = 0.9995 (EMPA) Where y is the integrated peak area at 224 nm and x is the concentration of the drug in μg mL-1 and r is the correlation coefficient. The results are collected in table 1.

 

Table No.1 Peak area of EMPA

 

 

Fig No.2 Linearity Graph of EMPA

 

Detection and quantification limits:

Sensitivity was determined by establishing the limit of detection (LOD) and limit of quantification (LOQ). The limit of detection (LOD) represents the concentration of analyte that would yield a signal-to-noise ratio of 3:1 and the limit of quantification (LOQ) represents the concentration of analyte that would yield a signal-to-noise ratio of 10:1. LOD and LOQ were calculated according to ICH guidelines.[2,3]  using following formula:

 

LOD= 3.3(SD)/S and LOQ= 10(SD)/S,

 

where

SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve. Results are tabulated in table 1 and conclude that the method is sensitive for the simultaneous determination of EMPA.

 

Table No 2: Analytical data of the calibration graphs of EMPA

Sr. No	Parameters	Observation
1	Linearity and range (µg/ml)	10-50µg/ml
2	Regression equation (Y)	y = 50206x - 168802
3	Slope (M)	50206
4	Intercept (c)	168802
5	correlation coefficient (r)	0.9995
6	LOD (µg/ml)	0.2675
7	LOQ (µg/ml)	0.8108

 

Précision:

The result of intra and interday variation of Empagliflozin for intraday precision inject 30ppm of EMPA at morning and at evening of the same day and for the interday precision was calculated form assaying freshly prepared solution of drug over a period of two days then, inject 30ppm of EMPA at day 1 and day 2 are depicted in table 3 and4.The relative standard deviation (RSD) was less than 2% indicating high degree of precision of the proposed method.[2,3]

 

Table No.3: Day 1Precision study of EMPA

Sr. No	Morning	Evening	Mean	%RSD
1	1351377	1346664
2	1346954	1354782	1352142	0.37%
3	1353107	1359967

 

Table No.4: Day 2Precision study of EMPA

Sr. No	Morning	Evening	Mean	%RSD
1	1351377	1355866
2	1346954	1353897	1344452	0.32%
3	1353107	1344452

 

Accuracy:

The accuracy of the method was determined by calculating recoveries of the drug by the standard addition method. For this reason, a known amount of the drug standard solution within the range of linearity was added to the mixture of sample solution previously analysed 30 μg ml-1 Empa. The solutions prepared in six replicates were analyzed and concentration of added standards were calculated each time from the corresponding regression equation, the mean recovery percentages (Table 5) were found to be 100.02 % for Empa, respectively with mean % RSD less than 1%. [2,3]

 

Table No 5: Accuracy study of EMPA

 

Table No.6: Change in flow rate

 

Table No.7: Change in wavelength

 

Robustness and ruggedness:

The robustness of proposed method was tested by changing parameters such as wavelength range, slit width, temperature, pH and shaking time. None of the variables significantly affected the peak area of the drugs indicating that the method could be considered as robust. [2,3,1]

 

 

System suitability:

A typical chromatographic parameter of empagliflozin. [4,5]

 

Table No 8: System suitability Parameter of EMPA

Sr. No	Parameter	Empagliflozin
1	Theoretical plates	7160
2	Retention time (min)	5.017
3	Asymmetry	1.28

 

Invitro stability studies in simulated fluid:(8,9)

 

Fig No.3:  Indicate the invitro studies in pH 1.2 with 0.8 flow rate at 224 nm

 

 

Fig No.4: indicate that invitro studies in pH 6.8 with 0.8 flow rate at 224 nm

 

 

 

Table No.9: Degradation studies.[9] (A, B)

A.     Calculated % for degradation in pH 1.2

Sr. No	Time in hours	Degradation %
1.	0hr	58.72
2.	6hr	70.6
3.	12hr	75.64
4.	24hr	76.27

 

B.       Calculated % for degradation in pH 6.8

Sr. No	Time in hours	Degradation%
1.	0hr	14.52%
2.	6hr	7.51%
3.	12hr	11.39%
4.	24hr	16.29%

 

DISCUSSION:

The proposed method was found to be simple, accurate and rapid for the routine determination Empagliflozin in tablet formulation. To study validity and reproducibility of proposed method, recovery studies were carried out. The method was validated in terms of linearity, accuracy, precision, specificity and reproducibility. The developed methods can be successfully used for estimation of Empagliflozin in formulation.

 

ACKNOWLEDGMENT:

The authors are thankful to the Management and Trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Macleods Pharmaceutical Pvt. Ltd. Gujarat, India, for providing pharmaceutical grade Empagliflozin and Metformin Hydrochloride as gift sample.

 

 

REFERENCES:

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2.      ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005.

3.      International Conference of Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures: Methodology, Adopted in Geneva (1996).

4.      N. Padmaja, Method development and validation of RP-HPLC method for the estimation of empagliflozin in API, International Journal of Pharmaceutical Sciences and Research 2017,724-727

5.      Sushil H. Jaiswal, Validated stability indicating HPLC method for determination of process related impurties in empagliflozin drug substances, World Journal of Pharmaceutical Research 2017, (6), 1025-1037.

6.      Beckett A, Stenlake J, Practical Pharmaceutical Chemistry. CBS Publication, Fourth edition, Part II^nd, New Delhi, 285-288.

7.      Shyamala, Validated stability indicating RP-HPLC method for determination of empagliflozin. Scholar Library, 2016,8(2),457-464

8.      Amit singh A, Pramod K. Sharaba B and Dipak K. Majumdar development and validation of new HPLC method for estimation of fluconazole in different simulated biological fluids: A comparative study Journal of Liquid Chromatography and Related Technologies 2014, 597-607.

9.      Magda M. Ibrahim, Development and validation of a RP-HPLC Method for the simultaneous determination of carvedilol, glimepride or glibenclamide in binary combination; and its application for in vitro-interaction studies.2015, 5(8), 2231-6876.

10.   N. Sultana, M.S. Arayne, S Naveed, H. Shamshad An RP-HPLC Method for Simultaneous Analysis and Interaction Studies on, Enalapril Maleate and H2-Receptor Antagonists Acta Chromatographic 2009,21, 547–558.

11.   Chan, C.C., Analytical Method Validation: Principles and Practices, 2008, 727-742

12.   Sethi, p.D., High performance Liquid Chromatography quantitative Analysis of Pharmaceutical Formulation, First Edition, CBS, New Delhi, 2001,161-196

13.   Indian Pharmacopoeia, Government of India, Indian Pharmacopoeia Comission Ministry of Health and Welfare Ghaziabad India, 2014.

 

 

 

Received on 15.06.2019                 Accepted on 11.07.2019

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Asian J. Pharm. Ana. 2019; 9(3):156-160.