Reverse Phase High Performance Liquid Chromatographic Method for Separation and Estimation of impurities present in Pharmaceutical Formulation of Empaglifozin

 

Patel N*, Patel S

Department of Quality Assurance, Shree S.K. Patel College of Pharmaceutical Education and Research,

Ganpat University, Ganpat Vidhyanagar - 384012, Gujarat, India.

*Corresponding Author E-mail: nilesh33.emcure@gmail.com

 

ABSTRACT:

Empaglifozin is a Sodium-glucose co-transporter-2 inhibitors work by inhibiting SGLT2, to prevent reabsorption of glucose and facilitate its excretion in urine. Impurities in pharmaceuticals which are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during stability testing, or develop during formulation or upon aging of both API and formulation. A simple and very sensitive method developed for estimation of impurities present in Empaglifozin formulation by Reverse Phase High Performance Liquid Chromatographic method. Method is capable to detect impurities in very low level (1g/mL). Chromatographic separation of six different impurities was achieved on Inertsil ODS-2 (250 x 4.6) mm, 3m column using gradient elution method at 30C column temperature and the detection was carried at 230nm at a flow rate of 1.0 mL/min. The method was validated as per ICH Q2(R1) guideline along with stress studies.

 

KEYWORDS: Empaglifozin, SGLT-2 Inhibitor, method development, method validation, Stress condition, Impurities, ICH Q2(R1).

 

 


1. INTRODUCTION:

Empaglifozin is a Sodium-glucose co-transporter-2 inhibitors work by inhibiting SGLT2 in the PCT, to prevent reabsorption of glucose and facilitate its excretion in urine. As glucose is excreted, its plasma levels fall leading to an improvement in all glycemic parameters. This mechanism of action is dependent on blood glucose levels and, unlike the actions of thiazolidinediones (mediated through GLUTs), is independent of the actions of insulin. Thus, there is minimal potential for hypoglycemia, and no risk of overstimulation or fatigue of the beta cells. Because their mode of action relies upon normal renal glomerular-tubular function, SGLT2 efficacy is reduced in persons with renal impairment. 1-5

 

Chemical Structure of Empaglifozin:

Due to lots of advantages of Empaglifozin, it is necessary to estimate related impurities present in these drug. So present investigation involve the development of RP-HPLC related substances method for pharmaceutical dosage form for Empaglifozin. Possibly six impurities identified base on API source so separation was done on this impurities and validate developed method. Other Assay methods are available but very few methods are available for quantification of impurities in Empaglifozin Tablet.6-22

 


1.1 Impurities Details

Table 1: Details of Impurity-I to VI

Impurity Name

Impurity-I

Impurity-II

Impurity-III

 

Chemical Structure

 

 

 

Chemical Formula

C21H23ClO7

C11H14O4 S

C23H27ClO7

Chemical Name

((2R,3R,4R,5S,6R)-6-(4-chloro-3-(4-hydroxybenzyl)phenyl)-3,4,5-trihydroxy tetrahydro-2H-pyran-2-yl)methyl acetate

(R)-Tetrahydrofuran-3-yl-4-methylbenzenesulphonate

(2S)-2-(4-chloro-3-(4-(((R)-tetrahydrofuran-3-yl)oxy)benzyl) phenyl)-5-(1,2-dihydroxyethyl)tetrahydrofuran-3,4-diol

Molecular Weight

422.86 g/moL

242.29 g/moL

450.91 g/moL

 

Impurity Name

Impurity-IV

Impurity-V

Impurity-VI

Chemical Structure

 

 

 

 

Chemical Formula

C23H27ClO7

C28H31ClO10

C18H15ClO5

Chemical Name

(2R,3R,4R,5R)-2-(4-chloro-3-(4-(((S)-tetrahydrofuran-3-yl)oxy) benzyl)phenyl)-5-((R)-1,2-dihydroxyethyl) tetrahy drofuran-3,4-diol

(2R,3R,4R,5S,6S)-2-(acetoxymethyl)-6-(4-chloro-3-(4-methox ybenzyl)phenyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate

(S)-4-chloro-3-(4-((tetrahydrofuran-3-yl)oxy)benzoyl)benzoic acid

Molecular Weight

450.91 g/moL

563.00 g/moL

346.76 g/moL

 


2. MATERIAL AND METHODS:

2.1 Reagents and Chemicals:

HPLC grade Acetonitrile and Methanol was procured from Merck. Potassium hydrogen Phosphate and Ortho-phosphoric acid (H3PO4) were purchased from Merck. Water used in the HPLC analysis was prepared by the water purifier (Merck Millipore Milli-Q). The mobile phase and all the solutions were filtered through a 0.45 m Merck HV membrane filter. Sample was filtered through 0.45m Millipore PVDF syringe filter.

 

2.2 Instruments:

HPLC system (Waters system, USA, e2695 and Agilent 1200 series) with a PDA detector, equipped with a quaternary pump, auto sampler, column compartment and empower and Chromeleon software was employed during this study.

 

2.3 Chromatographic condition:

Chromatographic separation was achieved at 30C column temperature and the detection was carried at 230 nm at a flow rate of 1.0mL/min. Run time was kept at 80 min. Prior to the injection of drug solution, column was equilibrated for 60 min with the mobile phase flowing through the system. The injection volume was 20μL. The analysis has been performed by using Inertsil ODS-2 (250 4.6mm, 3u). Use Acetonitrile and Water in ratio of 50:50%v/v as a Diluent. The mobile phase A containing 6.8 g Potassium Hydrogen Phosphate into 1000mL of Milli-Q water. Adjust pH 2.2 with OPA solution and mobile phase B contains Acetonitrile and Methanol in ratio of 50:50%v/v using following gradient.

 

Time (Min)

% Mobile Phase-A

% Mobile Phase-B

0

90

10

5

90

10

15

70

30

30

60

40

50

60

40

60

30

70

70

30

70

75

90

10

80

90

10

2.4 Standard Preparation:

The standard stock solutions 200ug/ml of Empaglifozin was prepared by dissolving working standards in Diluent and diluting with the same solvent to obtain final concentration 4g/mL.

 

 

2.5 Sample Preparation:

Twenty tablets were weighed and finely powdered. Powder equivalent to 50mg Empaglifozin was accurately weighed into a 25ml volumetric flask, 20ml of diluent was added and sonicated for 15 min with intermittent shaking, made up to the volume with diluent and mixed well. Filter the solution through 0.45m Millipore PVDF syringe filter.

 

3. Method Validation:

After method development, validation of the current test method for Empaglifozin tablets was performed in accordance with United States Pharmacopeia requirements/ICH guidelines for related substance method the parameter includes precision, accuracy, linearity, LOD and LOQ, precision and accuracy at LOQ level, selectivity, specificity includes blank, placebo, known impurity interference and interference of degradents by degradation study. Robustness was also performed.23-24

 

3.1 Specificity:

To assess the method specificity, tablet powder without Empaglifozin was prepared with the same excipients as those in the commercial formulation. For RP-HPLC, the solution was prepared using the same procedure as for the analytical sample. Placebo solution was injected into the HPLC system following test conditions, the chromatogram was recorded and the responses of the peaks if any measured. Chromatogram of the placebo has not shown any interference at the retention time of both Empaglifozin and its impurities . Blank, placebo, and impurity spiked sample preparation chromatogram shown in figures.


 

Figure 1: Chromatogram of Diluent, Placebo and Spiked sample


 

3.2 System Suitability:

20μL of standard solution six times injected into HPLC and recorded the chromatogram, %RSD of Empglifozin, area was with in the limit of 5.0%. The results summarized in Table and standard solution chromatogram shown in figure.

 

Table 2: System suitability Result

Injection No

Peak Area of Empaglifozin.

Theoretical Plates

Tailing Factor

1

94.528

21521

1.00

2

95.574

21589

1.00

3

94.362

21547

1.00

4

95.874

21543

1.00

5

94.587

21547

1.00

6

94.300

21544

1.00

Mean

94.871

SD

0.7

%RSD

0.7

 

Figure 2 : Chromatogram of Standard Solution

 

3.3 Precision:

Precision was measured in terms of repeatability of application and measurement. Repeatability of standard application (System precision). Precision study of Empaglifozin and its impurities were carried out by spiking known concentration in sample and calculating % recovery of impurities in sample. Intermediate precision carried out using same manner but on other day using different column and HPLC. The results summarized in Table 5 and 6.

 


Table 3: Method Precision

Sr. No.

Impurity-I

Impurity-II

Impurity-III

Impurity-IV

Impurity-V

Impurity-VI

Single Unk

Total Imp

1

0.61

0.51

0.46

0.56

0.52

0.50

0.03

3.19

2

0.61

0.51

0.48

0.56

0.53

0.50

0.03

3.22

3

0.63

0.51

0.45

0.58

0.52

0.52

0.03

3.24

4

0.62

0.50

0.46

0.54

0.52

0.51

0.03

3.18

5

0.61

0.51

0.48

0.56

0.52

0.52

0.03

3.23

6

0.61

0.51

0.45

0.55

0.51

0.50

0.03

3.16

Mean

0.62

0.51

0.46

0.56

0.52

0.51

0.03

3.20

SD

0.008

0.004

0.014

0.013

0.006

0.010

0.00

0.03

%RSD

1.4

0.8

2.9

2.4

1.2

1.9

0.0

1.0

 


3.4 Linearity:

To evaluate linearity of the method, six levels calibration curve made includes LOQ level. Signal to noise ratio was observed. The linearity of method is obtained by preparation of the calibration curve. The calibration curve for Empaglifozin and its impurities were obtained by plotting the peak area of Empaglifozin versus concentration of Empaglifozin over the range of 115 μg/ml. The results are summarized and overall linearity graph for Empaglifozin and its impurities was shown in figure 3 .

 

3.5 Accuracy:

Accuracy of the method was studied for three levels from 50% to 150% by spiking known concentration of impurities in sample. 0.05% for LOQ, 0.25% for 50%, 0.50% for 100%level and 0.75% for 150% level from the target concentration of Empaglifozin impurities spiked in sample preparation and analyzed with unspiked sample preparation, recorded the chromatogram. Triplicate preparation for LOQ, 50%, 100%, 150% level prepared as per method. Results are summarized in Table 4.

 

3.6 Robustness:

Robustness of the current method was investigated by analyzing the standard solution and established system suitability with the deliberate variation of flow rate and column temperature at 10 percentage level from the original value. RSD of six replicate injections of standard solution was found below 5.0% for all the chromatographic condition and all peaks in standard solutions. The conditions with the variation and the results are presented in Table 5.

3.7 LOD and LOQ:

LOD and LOQ were calculated by using the formula 3.3S.D/S and 10S.D/S where D is the standard deviation of Y-intercept and S is the slope of the calibration curve.

 

3.9 Solution Stability:

Solution stability optimized by injected standard solution and sample at different time interval and calculated % deviation against initial area of standard solution. It was found that standard and sample were stable upto 69 hrs.

 

Table 4: Recovery / Accuracy Results

Name

Level

% Recovery

% RSD

Impurity-I

LOQ

98.1

1.2

50%

98.7

0.5

100%

99.3

1.9

150%

95.8

3.0

Impurity-II

LOQ

99.3

0.3

50%

97.9

1.0

100%

99.2

1.1

150%

96.7

1.4

Impurity-III

LOQ

98.0

1.2

50%

98.9

0.8

100%

100.9

0.8

150%

99.9

0.6

Impurity-IV

LOQ

98.0

1.3

50%

101.1

1.5

100%

101.8

0.5

150%

100.7

0.5

Impurity-V

LOQ

100.4

1.1

50%

100.9

1.6

100%

100.3

1.0

150%

100.5

0.3

Impurity-VI

LOQ

98.2

1.6

50%

100.1

0.8

100%

100.4

1.7

150%

99.3

0.8

 


Figure 3 : Linearity curve of Empaglifozin, Impurity I/II/III/IV/V/VI

 


Table 5 : Robustness results

Condition

% RSD

Tailing Factor

Theoretical Plates

RT (Min)

Normal

0.7

1.0

21521

18.31

Column temperature -5C (25C)

0.7

1.1

21644

18.94

Column temperature +5C (30C)

0.6

1.0

20584

17.83

Flow rate -10% (0.9 mL/min)

0.7

1.0

21584

20.04

Flow rate +10% (1.1 mL/min)

0.7

1.0

20615

16.72

Organic Ratio -2% Absolute

0.5

1.1

21478

21.25

Organic Ratio +2% Absolute

0.4

1.0

21321

16.11

pH of Buffer -0.2 unit (2.0)

0.9

1.0

19589

17.98

pH of Buffer +0.2 unit (2.4)

1.1

1.0

20851

18.25


3.8 Force Degradation:


Table 6: Result of Force Degradation study

Sample

Imp-I

Unk-1

Unk-2

Unk-3

Total Imps

Assay

Mass Balance

Peak Purity

Acid

0.18

4.80

2.30

3.80

11.08

87.3

98.9

998

Base

0.13

0.11

0.05

0.04

0.33

98.3

99.1

995

Peroxide

0.15

0.10

0.07

0.03

0.35

99.6

100.5

996

Thermal

0.12

0.06

0.04

0.03

0.25

100.3

101.1

998

Photo

0.11

0.08

0.02

0.01

0.22

101.3

102.0

999

Humidity

0.12

0.05

0.04

0.02

0.23

98.8

99.5

1000

Sample As such

0.12

0.02

ND

ND

0.14

99.5

-

1000

API

0.11

0.02

ND

ND

0.13

-

-

1000

 

Figure 4: Chromatogram of force degradation study

 


4. RESULTS AND DISCUSSION:

The main objective of the chromatographic method development was to separate Empaglifozin from the impurities were carried out for accurate and precise method development and impurities were coeluted. After using several columns and buffers, suitable column chemistry and good peak shape were obtained with Inertsil ODS-2 (2504.6) mm 3 particle size, column temperature was adjusted at 30C, with gradient mobile phase system consisting the mobile phase A containing 6.8g Potassium Hydrogen Phosphate into 1000mL of Milli-Q water. Adjust pH 2.2 with OPA solution and mobile phase B contains Acetonitrile and Methanol in ratio of 50:50%v/v using above mentioned gradient.

HPLC method has been development and validated for determination of related substances of Empaglifozin in tablets with gradient evaluation. The method is selective, because we have very good separation between impurities. The method described in this study is suitable to determine impurities at very low level. These parameters showed a good linearity with correlation coefficients. We have shown that the method is robust with little change critical chromatographic parameters. Validation parameters have proved that our method can used as stability indicating method for determination of related substances of Empaglifozin in tablet.

 

5. CONCLUSION:

A novel, reverse phase liquid chromatographic method has been developed and validated for the estimation of Empaglifozin and its impurities with a very recent and advanced HPLC method. The proposed method is found to be simple, accurate, precise, sensitive, specific and robust. Hence, it can be successfully used for the routine analysis of Empaglifozin in pharmaceutical dosage forms.

 

6. ACKNOWLEDGEMENT:

The author would like to thank guide Mrs. Sejal Patel and all family members for their valuable support throughout the work. The Authors acknowledge Ganpat University and Shree S. K. Patel College of Pharmaceutical Education and Research for providing the infrastructure facility for carrying out this work..

 

7. CONFLICT OF INTEREST :

The authors declare no conflicts of interest.

 

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Received on 14.06.2020 Modified on 18.08.2020

Accepted on 24.09.2020 RJPT All right reserved

Research J. Pharm. and Tech. 2021; 14(9):4595-4601.

DOI: 10.52711/0974-360X.2021.00799