INTRODUCTION:

Empagliflozin chemically is (2S,3R,4R,5S,6R)-2-[4-chloro-3-[[4-[(3S)-oxolan-3-yl] oxyphenyl] methyl]phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol.]

Structure of Empagliflozin

Diabetes mellitus is a condition which requires continuous medical care. It is a progressive disease which at first causes insulin secretory defect which is characterized by insulin resistance and some degree of insulin deficiency¹.

The prevalence of type 2 diabetes mellitus has a tremendous impact on obesity, which is a growing problem. Because of the rise in obesity, there is an increased demand for the researchers to develop drug therapies that not only effectively treat hyperglycemia but also promotes weight loss. Metformin, a biguanide², is the preferred oral hypoglycaemic agent for initial therapy in patients with type-2 diabetes and later combination therapy in which metformin along with other medication is given to the patient and in other scenarios where the condition worsens the patient, they are advised to take insulin with metformin. But long-term use of Metformin is associated with lactic acidosis which is serious side effect³.

There are many other options for further oral therapy. Agents that can be used along with metformin include: sulfonylureas, thiazolidinediones, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 receptor agonists, sodium-glucose cotransporter 2 (SGLT2) inhibitors and insulin^4,5.

Empagliflozin (Jardiance, Boehringer Ingelheim), a sodium-glucose cotransporter 2 (SGLT2) inhibitor, belongs to a new class of oral hypoglycemic agents, which includes canagliflozin and dapagliflozin. In August 2014, empagliflozin became the most recent medication in its class to be approved by the Food and Drug Administration⁶. Empagliflozin has less side-effects when used in combination with other anti-diabetic medications. Empagliflozin had the highest selectivity for SGLT2 over SGLT1 compared to dapagliflozin and canagliflozin. It is a sodium glucose co-transporter-2 inhibitor (SGLT2). SGLT2 co-transporters reabsorbs glucose from the glomerular filtrate in the kidney. By inhibiting the SGLT2 co-transporter, it reduces renal absorption of glucose and it increases glucose excretion through urine⁷.

There is minimum or no risk of hypoglycemia with empagliflozin because the mechanism of action is not dependent on beta-cell function and insulin pathway. Empagliflozin is preferred over metformin for early diabetes and in combination it can prevent lactic acidosis caused by Metformin. Empagliflozin is suggested for the improvement of glycemic control along with diet and exercise in adults with type-2 diabetes mellitus⁸. It has additional cardioprotective effects.

Long-term complications of type 1 diabetes due to exposure to chronic hyperglycaemia, blindness, cardiovascular disease and kidney failure, remain a fear for many patients living with type1 diabetes⁹. Intensive insulin therapy reduces the risk of complications, but the majority of people with type 1 diabetes fail to achieve the acceptable glycaemic targets. It is also seen that long-term intensive insulin therapy is associated with weight gain which is a relevant side-effect as a significant proportion of patients with type 1 diabetes are now overweight. Empagliflozin can be used even for type-1 diabetes patients for greater glycemic control.

UV spectroscopy is an absorption spectroscopy in which ultra violet light which having a range of 200-400 nm is absorbed by the molecule. Every molecule has either n, π or σ or electrons. Either bonding(σ and π) and non-bonding (n) electrons absorb the radiation and undergo transition from ground state to excited state¹⁰. By the absorption peaks obtained for a particular molecule and the nature of the electron present in that particular molecule, the molecular structure of a molecule can be elucidated.

A literature search has shown that there are very few quantitative analytical methods for estimation of empagliflozin. Thus, it was clear that there was a necessity to develop a rapid, simple and economical method.

MATERIALS AND METHOD:

Instrumentation:

Double beam UV spectrophotometer; Model: SL 210; Make: ELICO. The data was obtained using Spectra Treats 3.11.01Rel 2b software.

Materials:

Empagliflozin was obtained as a gift sample from a pharmaceutical company. Empagliflozin tablets of 25 mg was obtained from market. Analytical grade ethanol was obtained from SD fine chemicals, Mumbai, India.

Method:

Selection of wavelength:

10mg of empagliflozin drug was accurately weighed and transferred into 10ml of volumetric flask and the volume was made up to the mark with ethanol as diluent. Then from this 0.1ml was pipetted out and transferred into another 10ml volumetric flask and the volume was made up to the mark with water to give 10ppm solution and this was scanned between 200 to 400nm and its absorption maxima was identified as to 223nm (Figure-1)

Figure 1: UV Spectrum of Empagliflozin

Assay:

Standard preparation:

10mg of empagliflozin drug was accurately weighed and transferred into 10ml of volumetric flask and the volume was made up to the mark with ethanol to give 1000ppm. From this 0.1ml was pipetted out and transferred into 10ml of volumetric flask and the volume was made up to the mark with water to give 10ppm solution and its absorbance was measured at 223nm.

Test preparation :

5 tablets were weighed and powdered. Powdered tablet equivalent to 10mg of empagliflozin was weighed and taken into 10ml volumetric flask then volume was made up to the mark with ethanol. From that 0.1ml of solution was pipetted out and taken in 10ml volumetric flask. The volume was adjusted with water up to 10ml to get 10ppm solution and its absorbance was measured at 223nm.

The % assay is calculated by using the regression equation obtained by linearity studies.

Method validation parameters:

Linearity:

A series of dilutions were prepared and their absorbance was measured at 223nm. Six aliquots ranging from 1-30µg/ml were found to be linear after plotting concentration on X-axis and absorbance on Y-axis.

Figure 2: Calibration Plot

Table 1: Linearity Study Data

Concentration (µg/ml)	Absorbance at 223nm (n=3)
1	0.0899
5	0.3330
10	0.6758
15	0.9231
20	1.2346
25	1.5678
30	1.9000

Precision:

Intra-day precision:

Intra-day precision was evaluated by analyzing six replicates of the standard solution in a day.

Inter-day precision:

Inter-day precision was evaluated by analyzing single aliquot of the standard solution on 5 different days.

The % Relative standard deviation (% RSD) for intra-day and inter-day precisions were calculated by using the formula

Standard deviation of the measurement

% RSD = –––––––––––––––––––––––––––––––– × 100

Mean value of measurement

Table 2: Intra-Day Precision and Inter-Day Precision Study Data

Intra-day precision		Inter-day precision
Sample No.	Absorbance at 223nm	Day	Absorbance at 223nm
1	0.6835	Day 1	0.6835
2	0.6803	Day 2	0.6821
3	0.6793	Day 3	0.6801
4	0.6810	Day 4	0.6781
5	0.6779	Day 5	0.6770
6	0.6824
Mean	0.6807	Mean	0.68016
SD	0.002038	SD	0.002699
%RSD	0.2993	%RSD	0.3968

Accuracy:

Accuracy of the method was ascertained by standard addition method at three levels which is at 50% (20µg/ml), 100% (25µg/ml) and 150% (30µg/ml).

In standard addition method known quantities of standard was spiked to sample at three different levels.

% Recovery was calculated using the below formula.

Spiked absorvance

% Recovery formula = –––––––––––––––––– × 100

Original absorbance

Table 3 : Accuracy Study Data

Level	Concentration in µg/ml		Mean % Recovery	%RSD
Level	Spiked	Sample	Mean % Recovery	%RSD
50%	5	15	99.49	0.84
100%	10	15	99.40	0.46
150%	15	15	99.20	0.12

Limit of detection:

The LOD of the standard drug in the developed methods were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines. LOD value was calculated using the relation,

LOD = 3.3 × σ / S

where

σ = the standard deviation ;

S = the slope of the calibration curve

Limit of quantification:

The LOQ of the standard drug in the developed methods were determined by using standard deviation of the response and slope approach as defined in International Conference on Harmonization (ICH) guidelines. LOQ value was calculated using the relation,

LOD = 3.3 × σ / S

where

σ = the standard deviation;

S = the slope of the calibration curve

Robustness:

Robustness is assessed by measuring the absorbance at ±1nm of the absorption maxima and also at absorption maxima.

% Relative Standard Deviation (% RSD) was calculated by using the formula

Standard deviation of the measurement

% RSD = –––––––––––––––––––––––––––––– × 100

Mean value of measurement

Table 4 : Robustness Study Data

Sample No.	Absorbance at 222m	Absorbance at 223nm	Absorbance at 224nm
1	0.6820	0.6837	0.6809
2	0.6813	0.6826	0.6800
3	0.6821	0.6835	0.6803
Mean	0.6818	0.6832	0.6804
Mean of mean	0.6818
SD	0.0014
%RSD	0.2053

Ruggedness:

Ruggedness study is carried out using different analyst and the absorbance of the solution was measured at 223nm.

% Relative Standard Deviation (% RSD) was calculated by using the formula

Standard Deviation of the measurement

% RSD = –––––––––––––––––––––––––––––––– × 100

Mean value of measurement

Table 5 : Ruggedness Study Data

Analyst	Absorbance at 223nm	Analyst	Absorbance at 223nm
Analyst 1	0.6836	Analyst 2	0.6827
	0.6821		0.6816
	0.6816		0.6822
Mean	0.6824	Mean	0.6821
Mean of mean	0.68225
SD	0.0002121
%RSD	0.031

RESULTS AND DISCUSSION:

The calibration curve which is obtained was evaluated using its correlation coefficient. The absorbance of the samples in the range of 1.0–30.0μg/mL was linear with a correlation coefficient (r²) of 0.9988. The intra-day and inter-day precision study (Table 2) of the developed method confirmed that the developed method is reliable, where all the RSD’s were <2%. Accuracy results were within the range of 99.20–99.49% which ensures that the developed method is accurate (Table 3 ) as well as it that indicates that there is no interference of excipient with the drug. The LOD and LOQ were calculated as 0.10 μg/mL and 0.33μg/mL respectively. Robustness and ruggedness studies were performed and the method was found to be robust and rugged. The amount of drug present in the tablet is calculated from regression equation obtained from the calibration curve and it was found to be 99.87%.

CONCLUSION:

The obtained results demonstrate that the proposed UV spectrophotometric method is simple, rapid, accurate and precise. Therefore, this method can be used for routine quality control of empagliflozin in bulk and in dosage formulation without interference.

ACKNOWLEDGMENT:

We are thankful to management of RBVRR Women’s College of Pharmacy for supporting and encouraging us to carry out this research.

CONFLICT OF INTEREST:

The authors declare that they don’t have any conflict of interest.

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Received on 28.11.2020 Revised on 14.01.2021

Asian Journal of Pharmaceutical Analysis. 2021; 11(2):123-126.

DOI: 10.52711/2231-5675.2021.00021