RP-HPLC Method for Quantification of Empagliflozin in Pharmaceutical Formulation

 

Mounika P Siridevi, Hemant T Kumar*, Srinivasa Y Rao, Vara Prasad K Rao

Department of Pharmaceutical Analysis and Quality Assurance, Vignan Institute of Pharmaceutical Technology, Beside VSEZ, Duvvada, Visakhapatnam-530049

*Corresponding Author E-mail: hemkar_pharma@yahoo.co.in

 

ABSTRACT:

A simple, sensitive and specific RP-HPLC method was developed and validated for determination of Empagliflozin in bulk and tablet dosage form. Chromatography was carried out on a Enable C18G (250 x 4.6 mm i.d.,5µ) column using filtered and degassed mixture of methanol and water in the ratio of 70:30 % v/v as mobile phase at a flow rate of 1 ml/min and effluent was monitored at 233 nm. The method was linear over the concentration range of   10 – 90 µg/ml with a correlation coefficient of 0.999. The retention time of the drug was 6.2 mins. The proposed method was validated by determining sensitivity, accuracy, precision, robustness studies. The developed method was effectively applied to tablets of empagliflozin and the % recovery of empagliflozin from tablet formulation was found to be 99.65 -99.89 %. The method is simple, accurate, precise and reproducible and hence can be applied for routine quality control analysis of empagliflozin in pure and tablet dosage form.

 

KEYWORDS: Empagliflozin, RP-HPLC, Validation, Tablet dosage form.

 

 

 

INTRODUCTION:

Empagliflozin1 is a sodium glucose co-transporter-2 (SGLT-2) inhibitor indicated as an adjunct to diet and exercise to improve glycemic control in adult patients with type 2 diabetes2-6. Inhibition of SGLT2-mediated glucose transport in the kidney lowers the threshold at which urinary glucose excretion (UGE) occurs, which leads to loss of glucose in the urine and a reduction in hyperglycemia7-9. Chemically it is known as (2S, 3R, 4R, 5S, 6R)-2-[4- chloro-3-({4-[(3S)-oxolan-3-yloxy] phenyl}methyl) phenyl]-6-(hydroxymethyl) oxane-3,4,5-triol. The chemical structure of Empagliflozin was given in Fig.1. Empagliflozin is a white to yellowish, non-hygroscopic crystalline solid. It is commercially marketed under the name Jardiance and marketed by Boehringer Ingelheim.

 

 

 

Fig.1: Structure of Empagliflozin

 

Empagliflozin is not official in any pharmacopoeia. A literature survey on empagliflozin revealed that, until now only few analytical methods were reported for its estimation of empagliflozin such as UV-visible spectroscopy10, HPLC method11-15 in bulk and API form. However the reported RP-HPLC method utilizes complex mobile composition, so there is a need to develop an RP-HPLC method having simple composition of  mobile phase. Hence an attempt has been made to develop and validate a novel, simple and sensitive RP-HPLC method in accordance with ICH guidelines7 for the estimation of  Empagliflozin in its Tablet formulation.

MATERIALS AND METHODS:

Instrumentation:

Chromatographic separation was performed on a Shimadzu LC-20AD HPLC system equipped with a C18 G column (250 x 4.6 mm i.d, 5 µm particle), binary pumps, degasser, Variable wave length detector and Rheodyne injector with 20 μl loop volume. ‘LC solution’ software was used to collect and process the data.

 

Chemicals and Reagents:

Empagliflozin pure sample was provided by pharma industry and its pharmaceutical form tablets  were purchased from online. Methanol and water of HPLC grade were used. All other chemicals and reagents are of HPLC grade.

 

Optimized chromatographic conditions:

Mobile phase

:

Methanol: Water 70:30 (v/v).

Elution type

:

Isocratic.

Column

:

Enable C18 G 250 x4.6 mm .

U.V. detection

:

233 nm.

Flow rate

:

1.0 ml/min.

Injection volume

:

20 μl.

Temperature

:

Ambient.

Runtime

:

20 min

 

 

Preparation of mobile phase:

The mobile phase Methanol and water were mixed in the ratio of 70: 30 v/v and filtered through membrane filter (Millipore Nylon disc filter of 0.45 µ). This filtered mobile phase was sonicated for 15 min in ultrasonic bath before use.

 

Preparation of standard stock solution:

A stock solution was prepared by taking accurately weighed 10 mg of Empagliflozin  in 100 ml volumetric flask and initially dissolved in 50 ml mobile phase. Then the solution was made upto mark with mobile phase to obtain a concentration of 100 µg/ml and the resulting solution was sonicated for 15 min and filtered through 0.45 µ nylon membrane filter. The chromatogram for standard Empgliflozin was shown in Fig. 2.

 

Preparation of sample solution:

Weighed accurately 10 tablets individually, average weight was  calculated  and crush in to fine powder in a mortor by using pestle. Weigh accurately and transfer tablet powder(equivalent to 50 mg of empagliflozin) in to 100 ml dry volumetric flask, added about 60 ml dry volumetric flask, sonicated for about 5 minutes, cooled to room temperature, diluted to volume with mobile phase and mixed.

 

 

Fig 1: Chromatogram of standard Empagliflozin

 

 

Transferred 5 ml of above solution to 50 ml volumetric flask and volume made with mobile phase and mixed. Pipetted 2.0 ml from the above solution in to a 20 ml volumetric flask, diluted to volume with mobile phase and mixed. Filtered a portion of the clear solution through 0.45 μm Nylon syringe filter.

 

RESULTS AND DISCUSSION:

Method Validation:

System suitability:

A standard solution was prepared by using empagliflozin working standard as per test method and was injected five times into the HPLC system. The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from replicate injections for Empagliflozin monohydrate peak areas. the system suitability parameters met the requirement of method validation. The % RSD for the retention times and peak areas were found to be within the limits. The data for system suitability parameters is shown in Table 1.

 

Table 1: System Suitability Parameters Data For Empagliflozin

Parameter

 

%RSD for peak areas

0.47

No. Of Theoretical plates

15900 

Tailing factor

1.21

Retention time

6.2 min

 

Specificity:

A study to evaluate the interference of placebo was conducted. Test solutions were prepared in duplicate with placebo equivalent to the amount present in test preparation and analyzed as per the test method. The placebo should not show any peak at the retention time of empagliflozin indicates the specificity of   the method.

 

Linearity:

The calibration curve was prepared in a concentration range of 10-90 µg/ml by taking 1-9 ml of standard stock in 10 ml volumetric flask and the volume was made upto the mark with mobile phase. The resulting solutions were filtered through 0.45 µ membrane filter paper and the filtrate was used for analysis. Calibration curve was plotted for different concentrations of working standards prepared from standard drug solution of pure drug and showed linearity over a concentration range of 10-90 µg/ml. The method was found to be linear in the concentration range of 10-70 µg/ml. Correlation coefficient was found to be 0.9994. The calibration curve was shown in Fig.4 and linearity data was shown in Table 2.

 

Table 2: Calibration Table Of Empagliflozin

S.No.

Concentration(µg/ml)

Peak area

1

10                                 

500256

2

20

999028

3

30

1503793

4

40

2003900

5

50

2498118

6

60

2997181

7

70

3412377

8

80

4002048

9

90

4512321

 

 

Fig.4 Calibration curve of Empagliflozin

 

Table 3 : Precision 

Precision

Intra-day

Inter-day

Day 1

Day 2

Day 3

Mean

2491218

2405612

2435990

240222

Standard deviation

18520.4

11889.4

13965

12147.3

% RSD

0.44

0.35

0.43

0.36

 

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. Six solutions of same concentrations were prepared and peak area was noted. The results were shown in terms of % RSD. The inter and intra day precision results of Empagliflozin showed % RSD less than 1, which showed that the method was precise.  The table for precision was shown in Table 3.

 

Accuracy:

Accuracy (recovery) of the method was obtained by spiking 80, 100 and 120 % of Empagliflozin working standard concentrations, in which the amount of marketed formulation was kept constant and the amount of pure drug was varied. Solutions were prepared in triplicates and accuracy was indicated by % recovery.  The recovery was well within the limit. Hence, the method was accurate. The accuracy results are shown in Table 4.

 

Table 4: Recovery Results Of Empagliflozin

% Spike level

Sample

(µg/ml)

Amount added (Std.)

Amount found (µg/ml)

% Recovery

Statistical parameters

 

80

50

40

40.74

101.85

Mean=99.94

SD=1.77

%RSD=1.77

50

40

39.34

98.35

50

40

39.85

99.62

100

50

50

49.67

99.34

Mean=99.11

SD=0.68

%RSD=0.69

50

50

49.17

98.34

50

50

49.83

99.66

120

50

60

59.74

99.56

Mean=99.68

SD=0.11

%RSD=0.11

50

60

59.87

99.78

50

60

59.82

99.70

 

Robustness:

To evaluate the robustness of the developed method, small deliberate variations in optimized method parameters were made. The effect of change in flow rate, change in composition of mobile phase and detection wavelength on retention time, tailing factor and theoretical plates were studied. The method was found to be unaffected by small changes in flow rate, change in composition of mobile phase and detection wavelength as shown in Table 5.

 

 Table 5: Robustness Results

Conditions

% Assay

System Suitability parameters

Theoretical Plates

Tailing Factor

Flow Rate  0.8 ml/min

99.68

16112

1.22

Flow Rate  1.2 ml/min

99.49

15734

1.17

Mobile Phase-Methanol (75): Water(25)

99.67

15921

1.21

Mobile Phase- Methanol(65): Water(35)

99.84

15023

1.21

Wavelength  231 nm

99.83

15891

1.19

Wavelength  235 nm

99.61

15887

1.17

 

Ruggedness:

The ruggedness was studied by evaluating by different analysts but in the same chromatographic conditions. The results of ruggedness of developed method are started in the (Table 6). The results are shown during by different analysts but in the same chromatographic condition of the test solution wasn’t affected & in the accordance with the actual. The suitability parameters are also been found good; hence this method was concluded as rugged.

 

Table 6: Evaluation Data Of Ruggedness Study Of Empagliflozin

ID Precisions

No. of Injections

Empagliflozin

Peak Area

RT

ID Precision - 1

1

249537

6.143

2

242874

6.189

3

248593

6.205

ID Precision - 2

1

245487

6.118

2

240784

6.197

3

245292

6.183

MEAN

245427.8

 

STDEV

3317.0

% RSD

1.0

 

Sensitivity:

The limit of detection (LOD) is defined as the lowest concentration of an analyte that an analytical process can reliably differentiate from background levels. The limit of quantification (LOQ) is defined as the lowest concentration of the standard curve that can be measured with acceptable accuracy, precision and variability. The LOD and LOQ were calculated from linear curve using formulae

 

LOD= 3.3 * σ / S

LOQ= 10 * σ / S

 

(Where σ = the standard deviation of the response and S = Slope of calibration curve).

The results were shown in Table 7.

 

Table 7: LOD AND LOQ 

Drug

LOD (µg/ml)

LOQ (µg/ml)

Empagliflozin

0.42

1.16

 

Application of Proposed method

The assay of marketed sample (Tablet formulation) for empaliflozin is summarized in Table 8.

 

Table 8: Data Derived From Assay Of Empagliflozin

Drug

Label Claim

Mean amount found*

%Purity* ±SD

Empagliflozin

50 mg

49.80 mg

99.6 ± 0.32

*mean of three determinations

 

CONCLUSION:

A high performance liquid chromatography method for the quantitative estimation of Empagliflozin in bulk and tablet dosage form has been developed. The method was validated and found to be applicable for the routine analysis of Empagliflozin in capsule dosage form without interference from the excipients. Statistical results and low % RSD values indicate that the method is precise, accurate, robust, specific, and can be used across a wide range of concentrations. Considering already proposed methods in literature, advantages of this new proposed method simple, economic mobile phase, user friendly and convenient approach. All these key features proposed that this method can be considered as advantageous over other methods.

 

ACKNOWLEDGEMENT:

The authors are grateful to Dr.L.Rathaiah, Chairman of Lavu educational society for providing necessary facilities to carry out the above research work.

 

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Received on 24.05.2019            Accepted on 21.06.2019           

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Tech.  2019; 9(3):208-211.

DOI: 10.5958/2231-5713.2019.00035.7