RP-HPLC Method Validation for Estimation of related Substances of Empagliflozin

 

Chandni Chandarana, Aarti Panchal, Vishal Modi*

Department of Pharmaceutical Quality Assurance, SSR College of Pharmacy, Silvassa - 396230,

UT of Dadra and Nagar Haveli, India.

*Corresponding Author E-mail:  vishalmodi1111@gmail.com

 

ABSTRACT:

In this study, researchers aimed to develop and validate a reproducible and precise Reverse Phase High-Performance Liquid Chromatography technique for accurately estimating a chemical related to empagliflozin. The primary objective was to establish a method suitable for quality control of empagliflozin batches and its impurities. To achieve this, effective chromatographic separation has done by using two mobile phases: Mobile phase A, which comprised water adjusted to pH 3.9±0.5 using glacial acetic acid, and Mobile phase B, consisting of pure acetonitrile. The separation was conducted on a Phenomenex (Kinetex) C18 stationary phase (250mm × 4.6mm, particle size 5μm) at a flow rate of 1.5mL/min. Detection occurred at 240nm at 50°C. An injection volume of 50μL was used for analysis. Empagliflozin was quantified at a concentration of 0.03g/mL, while impurities were measured between 0.052-0.090g/mL. The results indicated successful chromatographic separation and accurate quantification. The proposed method demonstrated its efficacy for quality monitoring of bulk samples containing Empagliflozin, ensuring the reliability and consistency necessary for routine quality control purposes in the pharmaceutical industry.

 

KEYWORDS: Empagliflozin, RP-HPLC, Validation, Type 2 Diabetes Mellitus, Impurity profile.

 

 

INTRODUCTION: 

Empagliflozin (Figure 1) belongs to a relatively newer class of agents, called as gliflozin class, Sodium glucose co- transporter 2 inhibitors, which has emerged as a novel treatment to help manage type 2 diabetes. It is a novel hypoglycaemic drug that stimulates glucose-dependent insulin release. Empagliflozin's chemical name is (2S,3R,4S,5R,6S)-2-[4-[(3S)-oxolan-3- yl]. oxyphenyl] methyl] phenyl-6-(hydroxymethyl) oxane-3,4,5-triol. In the proximal tubules of the kidney's nephronic components1. A human protein called SGLT2 aids in the kidney's reabsorption of glucose. SGLT2 inhibitors enhance glucose excretion, decrease blood glucose levels, and prevent the kidneys from reabsorbing glucose. There may be a small risk of hypoglycemia with SGLT 2 inhibitors.

 

Additional effects of SGLT 2 inhibition may include weight loss from calories lost from increased glucose excretion as well as a possible modest diuretic effect-related drop in blood pressure. It is mandatory to find the purity of the pharmaceutical drug substances and drug products before their release into the market because of its potential toxicity and may be its interference with the quality. Related substances are similar in structure to the drug substances, which are also called as impurities. Impurities can be either Process impurities, Degradation product, By products. Intermediate product, Related products or Transformation products. Exhaustive Literature survey reveals that few analytical methods like UV, HPLC, and Stability indicating method for determination of Empagliflozin as individual and as combined dosage form with other drugs have been reported2-5. There is no method reported to detect impurities in the drug substance of Empagliflozin.6-12 However newer approach has been made to develop specific, precise, accurate and reproducible method for estimation.

 

Figure 1. Structure of Empagliflozin

 

MATERIAL AND METHODS:

Materials:

Empagliflozin were supplied by Macleod’s Pharmaceutical Ltd. The RS (Reference Substances) for system suitability and accuracy, consisting of a mixture of all the impurities i.e. Impurity-A Empagliflozin (s)-3-(4-(5-bromo-2- chlorobenzyl) phenoxy)-tetrahydron impurity], Impurity-E [Empagliflozin Des Bromo Product], Impurity-L [Empagliflozin Dimer product], Impurity-G [Empagliflozin Ring open product] was procured from Macleod’s Pharmaceutical Ltd. The Ethanol, KBr, Potassium Hydroxide, Methanol, ACN, Perchloric acid, Na2Co3, Isopropyl ether, Glacial acetic acid was obtained by Fischer Scientific and Dimethyl Formamide,1,4-Dioxane, Methylene Dichloride, n-Butyraldehyde were obtained by Sisco Research Laboratory.

 

Methods:

Using a gradient elution and a Phenomenex C18 column, relevant substance determinations were carried out. A: Water (pH 3.9); B: Acetonitrile (100%) as an organic phase; pH was adjusted to 3.9 with glacial acetic acid was used at flowrate of 1.5ml/min. At 240nm, detection was carried out.

 

Mobile phase Preparation:

A 300mL (30%) mixture of acetonitrile and 700mL (70%) of water pH 3.9 (with glacial acetic acid added to modify pH to 3.9 0.5) was used to create the mobile phase. Before use, the mobile phase was degassed and filtered by using 0.45m nylon membrane filter.

 

Preparation of Standard Empagliflozin Stock Solution: (Solution A):

A precise quantity of Empagliflozin (10mg) has been accurately measured and placed into a 100mL of volumetric flask. To this flask, 50mL of the mobile phase, consisting of a mixture of water and acetonitrile in a 70:30 v/v ratio (pH 3.9), was added as the solvent. The mixture was subjected to 15 minutes of sonication to ensure proper dissolution and homogenization. Subsequently, the volume was adjusted to 100g/mL.

 

Preparation of working Empagliflozin Standard Solution: - (Solution-B):

By transferring 3ml from Solution A and adding diluent to the mixture (up to 10mL), a working standard solution of empagliflozin (30g mL-1) was created.

 

Preparation of Impurity Stock Solution: - (Solution-C):

Each impurity was precisely measured at 10mg and subsequently merged in a flask containing 50mL of the mobile phase as the diluent (a mixture of water with pH 3.9 and acetonitrile in a 70:30 v/v ratio). After 15 minutes of sonication, the volume was adjusted to 100 mL using the same mobile phase, yielding a concentration of 100g/mL. The solution underwent filtration through 0.2-micrometer membrane filter for subsequent processing.

 

Preparation of working Impurity Standard Solution: - (Solution-D):

A standard working solution of the impurity was created by transferring 1.5mL from solution-C leading to a concentration of 3µg/mL.

 

Preparation of Spiked Solution: - (100 % level):

Empagliflozin, weighing 10mg, was precisely measured and diluted to the desired concentration using the mobile phase as the diluent (Water with pH 3.9 and ACN in a 70:30 v/v ratio). Subsequently, 1.5mL of each impurity were added to the solution, and the mixture was further diluted to the appropriate concentration using same mobile phase, resulting in a final solution containing Empagliflozin at a concentration of 200g/mL along with 3g/mL of contaminants.

 

Method Development selection of wavelength:

Standard stock solution of Empagliflozin and the Impurity –A, E, L and G each of about 10µg mL-1 in Acetonitrile was prepared. (10mg of Empagliflozin and 10mg of each impurity dilute to 100ml Acetonitrile which gives 100µg mL-1 solution. (Stock-1). Pipette out 1ml from stock-1 and make up to 10ml with acetonitrile gives 10µg mL-1 solution). Standard stock solutions were scanned in the range 200-400nm using PDA detector in HPLC. Based on trial method the given standard stock solution of Empagliflozin and all other Impurity showed significant absorbance at about 240 nm. (Figure 2).

 

 

Figure 2. Overlay UV Spectrum of Empagliflozin and impurity

Optimization of Chromatographic conditions:

From many trials it was concluded that the peaks are highly influenced by the mixture of organic phase and water as the aqueous phase. Acetonitrile showed improvement in peak shapes but Pure Acetonitrile did not separate all the peaks. Hence a mixture of water and Acetonitrile was selected with pH adjusted towards acidic side i.e. pH 3.9. Peaks are well resolved; all the peaks were eluted out under minimum retention time. (Figure 3). (Table-1 and 2).

 

 

Figure 3. Chromatogram using Water: ACN (pH 3.9)

 

Table No. 1. Optimized Chromatographic Conditions

Column

Phenomenex (kinetex) c₁₈ (250 mm x 4.6 mm, 5 µm )

Mobile phase

Water (pH 3.9): ACN

Flow rate

1.5 ml/min

Detection wavelength

240 nm

Sample injection volume

50 µl

Run time

70 min

Column oven temperature

50°c

 

Limit of Quantitation:

LOQ level was estimated by injecting progressively lowest concentration of the standard solution and measuring the S/N ratio. Lowering of the concentration was continued till the value of S/N ratio was greater than 10.0.

 

Table No. 2. Data of optimized condition of Empagliflozin

Name

Retention Time

Resolution

Tailing Factor

Number of

Theoretical Plates

Impurity A

5.761

-

1.07

9546

Empagliflozin

10.07

3.01

1.03

150880

Impurity E

22.12

10.80

0.99

77733

Impurity L

39.09

15.70

1.02

86430

Impurity G

41.12

3.07

1.03

87764

 

Limit of Detection:

LOQ level was estimated by injecting progressively lowest concentration of the standard solution and measuring the S/N ratio. Lowering of the concentration was continued till the value of S/N ratio was greater than 3.0.

 

Robustness:

The robustness of the proposed method was checked by changing various parameters. These parameters included the flow rate, which was adjusted within ±0.2 ml/min, the column temperature varied within ±5 ˚C, pH levels were changed within ±0.2 units, and the wavelength was adjusted within ±2.0 nm.

 

System suitability:

The number of theoretical plates which is a measure of column efficiency, Resolution between drug and tailing for all the peak was found to be less than 2%. The % RSD for all these parameters was found to be less than 2 %.

 

 

Table 3. Results of system suitability parameters of drug and impurity

Parameters Number of Theoretical Plates

Impurity A

9546

9549

9582

9589

9625

9634

Empagliflozin

150880

150892

150927

150952

150968

150991

Impurity E

77733

77745

77765

77789

77625

77682

Impurity L

86430

86465

86487

86499

86523

86545

Impurity G

87764

87782

87821

87841

87893

87900

Resolution

Impurity A

-

-

-

-

-

-

Empagliflozin

3.01

3.03

3.05

3.02

3.04

3.06

Impurity E

10.80

10.84

10.88

10.90

10.97

10.99

Impurity L

15.70

15.75

15.79

15.85

15.93

15.97

Impurity G

3.01

3.17

3.14

3.04

3.12

3.01

Tailing Factor

Impurity A

1.07

1.09

1.10

1.15

1.17

1.19

Empagliflozin

1.03

1.09

1.10

1.19

1.20

1.14

Impurity E

0.99

0.87

0.92

0.98

0.96

0.93

Impurity L

1.02

1.09

1.17

1.29

1.14

1.12

Impurity G

1.03

1.00

1.29

1.24

1.18

1.12

Area

Impurity A

94064

93800

93985

94000

93975

94007

Empagliflozin

394632

398735

391347

385473

396539

396489

Impurity E

58845

58456

57985

59004

58754

58465

Impurity L

56273

56002

58154

56120

56001

57632

Impurity G

82989

82195

81987

82002

81982

81456

RESULT AND DISCUSSION:

The study confirmed the method's specificity, demonstrating no interference of impurities with Empagliflozin, and clear separation of all peaks. The proposed method exhibited excellent linearity across the concentration range of 0.9 to 6µg mL-1, both for the drug and impurity, with regression coefficients ranging from 0.9994 to 0.9999. The % RSD for Empagliflozin at 100% specification level was 1.218%, and for impurities, it was below 2%. In terms of precision, the interday % RSD for Impurity – A, Impurity – E, Impurity – L, Impurity – G, and Empagliflozin fell between 0.82% and 1.63%, while the intraday % RSD ranged from 1.00% to 1.89%. The LOQ for Impurity – A, E, L, G and Empagliflozin were 0.090, 0.090, 0.052, 0.052, and 0.030 respectively, with corresponding LOD of 0.045, 0.054, 0.045, 0.042, and 0.010 respectively. Recoveries at various concentration levels (LOQ, 50%, 100%, and 150%) were within the range of 98.018% to 101.72% for Impurity – A, E, G, L and Empagliflozin, indicating accurate quantification. Additionally, deliberate changes made to the method parameters, including flow rate, column temperature, wavelength, and pH, did not significantly affect the results for both impurities and Empagliflozin, affirming the method's robustness. The % RSD for these robustness parameters was found to be less than 2%, confirming the reliability of the developed method under varying conditions.

 

Table 4. Results of Validation Parameters of Impurity- A, E, L and G

Sr. No.

Parameters

Acceptance Criteria

Result of Impurity A

Result of Impurity E

Result of Impurity L

Result of Impurity G

1

Range

 

 

 

 

 

 

 

Intra-

 

Inter- day

-

0.76-5.82

0.75-5.87

0.83-5.84

0.87-5.89

2

Linearity

R2 >0.99

0.9995

0.9994

0.999

0.9998

3

Precision

% RSD <2

0.067-1.86

0.56-1.69

0.98-1.44

0.98-1.44

 

 

Accuracy

% RSD <2

0.73-1.22

0.87-1.88

0.97-1.51

0.97-1.51

4

98-102%

99.58-

99.16-101.48

99.16-100.18

99.12-99.81

 

 

LOD

 

101.66%

 

 

 

5

S/N>3

0.045

0.054

0.045

0.042

 

LOQ

 

 

 

 

 

6

 

S/N>10

0.090

0.092

0.052

0.052

7

 

pH(+0.2Units)

 

1.01

0.68

0.58

0.58

 

 

pH(-0.2Units)

 

1.73

0.56

0.72

0.72

 

 

Flow rate

 

0.10

0.127

0.143

0.143

 

 

(+0.2 units)

 

 

 

 

 

 

 

(-0.2 units)

 

1.16

1.125

0.65

0.65

 

 

 

%

 

 

 

 

 

 

Wavelength (+2% units)

RSD

1.29

1.12

0.56

0.56

 

 

<2

 

 

 

 

 

 

(-2% units)

 

0.97

0.98

0.63

0.63

 

 

Column Temp (+2 ˚C)

 

 

 

 

 

 

 

 

0.76

1.55

0.68

0.68

 

 

(-2˚C)

 

0.80

0.35

0.44

0.44

 

Table 5: Results of Validation Parameters of Empagliflozin

Sr. No.

Parameters

Acceptance Criteria

Result

Remarks

1

Range

 

 

0.85-5.99 (µg mL-1)

 

 

 

 

 

 

 

 

 

 

 

Complies

2

Linearity

 

R2 >0.99

0.9998

3

Precision

Intra-day

 

1.06-1.15

 

 

Inter-day

% RSD <2

0.58-1.63

4

Accuracy

 

98-102%

99.06-99.71

5

LOD

 

S/N>3

0.01

6

LOQ

 

S/N>10

0.03

7

Robustness

pH(+0.2Units)

 

0.58

 

 

(-0.2Units)

 

0.74

 

 

Flow rate

 

0.78

 

 

(-0.2 units)

 

0.97

 

 

Wavelength

 

0.96

 

 

(+2% units)

 

 

 

 

(-2% units)

% RSD <2

0.90

 

 

Column Temp

 

0.85

 

 

(+2 ˚C)

 

 

 

 

(-2 ˚C)

 

0.94

 

CONCLUSION:

The optimized method, developed through method development trials, was determined to be accurate, specific, precise, and reproducible using reverse phase HPLC for estimating the Related Substances of Empagliflozin in the Active Pharmaceutical Ingredient (API). The method's performance was validated in accordance with the current ICH guidelines Q2 (R1) for parameters such as system suitability, specificity, limit of detection, limit of quantification, linearity, accuracy, precision, and robustness. The results obtained were satisfactory, indicating that the proposed method is suitable for monitoring the quality of bulk samples containing Empagliflozin.

 

ACKNOWLEDGMENT:

I express my gratitude to Macleod’s Pharmaceutical Ltd, Sari gam, for their generous provision of empagliflozin samples for this study. Their valuable technical support greatly contributed to the success of our research work.

 

CONFLICT OF INTEREST:

There is no conflict of interest, according to the authors.

 

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Received on 29.11.2023            Modified on 13.02.2024

Accepted on 11.04.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(10):5041-5045.

DOI: 10.52711/0974-360X.2024.00775