A Validated Stability Indicating High Performance Liquid Chromatographic Method for determination of impurities in Empagliflozin Film Coated Tablets

Amruta Sainath Patil¹, Amrutkar Sunil V², Nalwade Santaji³, Abhijit Bhausaheb Patil⁴

¹M. Pharm (Pharmaceutical Chemistry), Shrimaan Sureshdada Jain College of Pharmacy,

Chandwad, Nashik, India.

²M. Pharm, PhD (Pharmaceutical Chemistry), GES’s Sir Dr. M.S. Gosavi College of Pharmaceutical Education and Research, Nashik, India.

³MSc, PhD, Callidus Research Laboratories Pvt. Ltd., 23 PAP- A-29/1, Chakan Industrial Area Phase-IV, Nighoje, Tal-Khed, India.

⁴M. Pharm, (Pharmaceutical Chemistry); Callidus Research Laboratories Pvt. Ltd., 23 PAP- A-29/1, Chakan Industrial Area Phase-IV, Nighoje, Tal-Khed, India.

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

ABSTRACT:

An entirely new gradient reversed-phase high performance liquid chromatographic technology is used to quantitatively identify empagliflozin and its six process associated contaminants in pharmaceutical dosage forms. With a buffered mobile phase made up of solvent A (orthophosphoric acid 1.0ml diluted with water up to1000mL) and solvent B (a combination of 800:200 v/v ratio of acetonitrile to methanol) delivered with a detection wavelength of 225nm and 1mL/min flow rate, chromatographic separation has been achieved on an Avantor, ACE Excel, 5µm, C18-PFP, 250mm x 4.6 mm. Resolution for every pair of empagliflozin and its six impurities is more than 2.0. The drug was exposed to oxidative, alkaline, hydrolytic, thermal, and photolytic stress conditions. Empagliflozin was observed to deteriorate significantly in the presence of oxidative, acid-base hydrolysis, thermal, and photolytic stress conditions. The method's stability was demonstrated by the distinct separation of the primary peak, its process-related contaminants, and breakdown products. The mass balance for each degradation sample was found to range from 95% to 105% when the samples kept under stress condition were analysed with respect to a standard reference. Therefore, the detection of empagliflozin by assay in pharmaceutical dose forms is suitable with this method of detection. In accordance with ICH requirements, the developed method's linearity, specificity, accuracy, precision, limit of detection, and limit of quantification, were all validated.

KEYWORDS: Empagliflozin, HPLC, Validation, Impurities, ICH guidelines.

1. INTRODUCTION:

Type 2 Diabetes mellitus is controlled with a class of pharmacologicals reffered to as sodium glucose co-transporter 2 (SGLT2) inhibitors including Empagliflozin (EMPZ), reduced absorption of glucose in kidney and increases urine glucose excretion. It facilitates blood glucose regulation in those with type 2 diabetes. For those with type 1 diabetes, it is not advised.D-Glucitol,1,5-anhydro-1-C-[4-chloro-3-[[4-[[(3S)- tetrahydro-3-furanyl] oxy] phenyl] methyl] phenyl] is its chemical name.-, (1S) Phenyl derivatives replaced with glucopyranosyl (Fig. 1). The six possible related compounds to empagliflozin are impurity One to six impurities of (2S,3R,4S,5S, and 6R)6-hydroxymethyl-2-(4-chloro-3-(4-((S)-tetrahydrofuran-3-yl)oxy)benzyl)phenyl) (EMPZ-ZBA), (S)-(5-bromo-2-chlorophenyl) (4-((tetrahydrofuran-3-yl)oxy) phenyl) (2-methoxytetrahydro-2H-pyran-3,4,5-triol) Methanone: 5-bromo-2-chlorophenyl (EMPZ-ETHF-2)(S)-3-(4-(5-bromo-2-chlorobenzyl)phenoxy) tetrahydrofuran: (EMPZ-ETHF), (2R,3R,4R,5S,6R) (4-fluorophenyl) methanone: (EMPZ-ETHF-1),-2-(4-chloro3-(4-(((S)-tetrahydro furan-3-yl)oxy)benzyl)pheny)-6-(hydroxymethyl)Tetrahydro-2H-pyran-3,4,5-triol: (S) and (EMPZ-α-DYT)4-(2-chlorobenzyl)phenoxy-3-(4-) Tetrahydrofuran: impurity (EMPZ-NBr), correspondingly (Fig. 1). It is taken orally^1-4.

Jardiance is the brand name of the film-coated 10 mg and 25 mg tablets of Empagliflozin that are used orally. Empagliflozin drug substance and its film coated tablets are not official in any of the pharmacopeia. Numerous techniques for estimating the amount of empagliflozin alone or in combination with other medications have been documented in the literature^5-14. Ultra high-performance liquid chromatography technique is used to identify two process-related contaminants in a fixed dose combination of linagliptin and empagliflozin¹⁵. Research that was published in the literature detailed the use of UPLC/DAD technology to assess empagliflozin and three related chemicals simultaneously in spiking human plasma¹⁶. At present, there is no well-established technique for determining the presence of empagliflozin and its six impurities. In order to separate and identify empagliflozin and its six impurities—EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-ETHF-1, EMPZ-ETHF, EMPZ-α-DYT, and EMPZ-NBr, we have created a stability-indicating RP-HPLC method (Fig. 1).

2. MATERIALS AND METHODS:

2.1. Reagents and chemicals:

Callidus Research Laboratories, located in Pune, India, supplied the empagliflozin standards and tablets. Jiangxi Synergy Pharmaceuticals Co., LTD supplied all the impurity standards. We purchased acetonitrile and methanol of HPLC-grade from Merck (Mumbai, India). The ELGA Purelabs purifier was used to produce purified water.

2.2. Equipment:

Throughout the analysis, a Thermo Fischer Scientific Ultimade 8000 HPLC system equipped with a UV detector and photodiode array detector was used. Software called Chromeleon was used to process and monitor the output stream. For the hydrolysis studies, a digital water bath from Bio-Technics India was utilized. In a photostability chamber made by Mack PharmaTech, photo stability experiments were conducted. In a dry air oven (Bio-Technics India), thermal stability tests were conducted.

2.3. Chromatographic conditions:

A chromatographic column of 250mm x 4.6mm, 5µm, C18-PFP, made by Avantor, was utilized. The separation was established using a gradient method. The mobile phase was buffer-buffered and supplied at a flow rate of 1 mL/min with a identification wavelength of 225 nm. Solvent A was 1.0ml orthophosphoric acid diluted to 1000mL water, and solvent B was a mixture of acetonitrile and methanol in the 800:200 v/v ratio. The HPLC gradient method was configured as follows: Time (min)/% solution B: 0/20, 35/50, 65/80, 75/20. The temperature within the column was maintained at 35^◦C. The injection volume was 10µL. A 700:300 v/v mixture of acetonitrile and water was used as a diluent or solvent.

2.4.1 Preparation of Diluted Standard Solution: (About 2.0µg/mL of Empagliflozin):

Weigh about 20mg of the working standard empagliflozin and put into volumetric flask of 100mL. Once the diluent has been added, sonicate to dissolve it. Let it come to room temperature. Dilute well and use diluent to adjust the final volume. 2.0mL of this solution should be pipetted into volumetric flask of 200mL. Use diluent to adjust the volume and mixthoroughly.

2.4.2 Preparation of Sample Solution: (About 1000 µg/mL of Empagliflozin):

Weigh about 20 tablets and crush them into a fine powder using a clean, oven-dried mortar and pestle. Volumetric flask of 100ml should be filled with weighed and ground sample powder equal to 100mg of empagliflozin. Add 70 milliliters of diluent, sonicate for half an hour while shaking intermittently, then dilute to the appropriate level using diluent. For ten minutes, centrifuge the above-mentioned quantity of solution at 5,000 rpm. Place the filtrate (about 3–4milliliters) into an HPLC vial and proceed with the HPLC analysis by filtering the mixture with a 0.45μ Nylon syringe filter.

2.4.3 Preparation of Empagliflozin Related Impurities stock solution:

Weighed about 1.155mg of EMPZ-α DYT, 1.104mg of EMPZ-ZBA, 1.331mg of EMPZ-ETHF-2, 1.060mg of EMPZ-NBr, 1.359mg of EMPZ-ETHF-1, 1.049mg of EMPZ-ETHF impurity standard individually and transfer into a volumetric flask of 10mL. To dissolve, add 5mL of acetonitrile and sonicate. Take out and let cool to room temperature. Use diluent to dilute to the appropriate amount and thoroughly mix.

2.4.4 Preparation of Spiked Sample Solution: (About 1000µg/mL of Empagliflozin, 5µg/mL of each impurity i.e 0.5% of test concentration)

To 10ml volumetric flask about 83.92mg of crushed sample powder equivalent to 10mg of empagliflozin, is transferred. Add 3ml of diluent, sonicate it for 30 minutes with sporadic shaking, impurity stock solutions (EMPZ-α DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-ETHF-1, EMPZ-NBr) are diluted to the desired level. The portion of above solution is centrifuged for 10 minutes at 5000rpm. Place the filtrate in an HPLC vial for HPLC analysis after filtering the mixture through a filter of 0.45μ Nylon syringe, discard around 3ml of the filtrate.

2.4.5 Preparation of Placebo Solution:

Transfer the 700mg of placebo powder equal to 100mg of empagliflozin into a volumetric flask of 100ml. Add 70ml of diluent, shake occasionally for 30 minutes, and then dilute with diluent until. Centrifuge the portion of above solution at 5000rpm for 10 minutes. Filter the solution through 0.45μ Nylon syringe filter by discarding about 3 ml of filtrate and then fill into HPLC vial for further analysis by HPLC.

2.4.6 Preparation of System suitability solution:

Empagliflozin working standard was weighed out and then put into a 10mL volumetric flask. Sonicate to dissolve after adding 5ml of diluent. Take out and let cool to room temperature. One milliliter each of Impurity stock solutions-1 and 2 should be added. Use diluent to dilute to the appropriate amount and thoroughly mix.

2.5 Force Degradation Study:

In order to give a sign of stability that shows certain traits and specificity of the technique, force degradation experiments of empagliflozin were conducted 15–19. ICH criteria were followed in conducting the stress studies. The stress conditions used were acid hydrolysis (2N HCl/5.0ml/80°C/6hours), base hydrolysis (2.0 N NaOH/5.0ml/ 80°C/ 6hours), heat (80ºC/24 hours), light (1.2million lux hours and UV light of 200-watt hours/ square meter), oxidation (30% H2O2/5ml/80°C/ 30min), and deterioration of humidity (40ºC/75% RH for 72 hours).

3. RESULT AND DISCUSSION:

3.1. Method optimization and development:

The chromatographic method's goal was to separate empagliflozin from the main degradation peaks and all known closely eluting contaminants. The primary objectives of the chromatographic approach, as we discovered throughout investigation, are to separate the closely eluting contaminants and the empagliflozin peaks with the proper resolution for each component. We also noticed that several columns, buffer ratios, and organic modifier compositions were used in the procedure to produce a symmetrical peak of empagliflozin and its six impurities. Multiple trials were conducted using various compositions of the mobile phase. Separating each component, though, wasn't done well. A change in the mobile phase's composition led to the observation of satisfactory separation and resolution. The gradient method with buffered mobile phase, which included solvent A (1.0ml of orthophosphoric acid diluted to 1000mL of water) and solvent B (a combination of acetonitrile and methanol in an 800:200 v/v ratio), was used because the isocratic method was not providing enough selectivity during the fine-tuning. The gradient method was done at a flow rate of 1 mL/min, with a identification wavelength of 225nm, and the HPLC gradient program as follows: Time in minutes/percentage of solution B: 0/20, 35/50, 65/80, 75/20. A constant temperature of 35^◦C was maintained in the column.10 microliters was the injection volume. Diluent of water and acetonitrile in a 700:300 v/v ratio, respectively was used. All six impurities and empagliflozin were free of excipient and solvent interference at the specified chromatographic conditions. Empagliflozin and the six impurities separated well under optimal conditions, with a resolution of more than two. With respect to empagliflozin, the relative response factors for each of the six impurities have been calculated (Table I).

3.2. Method Validation:

3.2.1. Specificity and forced degradation studies:

The applicability of a technique for examining a material when possible contaminants are present is known as its specificity^17,18. A drug's intrinsic stability and potential degradation routes can be determined by stress testing the drug substance. It can also be used to confirm that the analytical techniques employed have the potential to indicate stability. It has been demonstrated that the HPLC method for empagliflozin is selective when six impurities and degradation products are present. Light (as described under ICH Q1B), heat (80^◦C), acid hydrolysis (2N HCl at 80^◦C for 8h), alkaline hydrolysis (2N NaOH at 80^◦C for 6 h), aqueous hydrolysis at 40^◦C for 72 h, and oxidation (30% H2O2 at 80^◦C for 30 min) were the stress conditions employed for the degradation experiment. Empagliflozin's peak purity in stress samples has been verified using a PDA detector. The mass balance (percentage assay, impurities and degradation products) computed by comparing the assay results of stressed samples to the reference standard. The blend's chromatograms showed no interference from the blank or placebo, and there was no peak during the empagliflozin retention period. When exposed to light, heat, oxidation, bases, acids, and water hydrolysis, empagliflozin degrades (Fig. 2).

Table I. Chromatographic performance data

Compound	RT (Min)	RRT^a (n=3)^d	Resolution^c (n = 3)^d	Tailing factor (n = 3)^d	RRF^b
Empagliflozin	25.435	1.00	--	0.98	1.00
EMPZ-α-DYT	26.993	1.06	5.49	1.01	0.95
EMPZ-ZBA	27.631	1.09	2.61	1.01	0.82
EMPZ-ETHF-2	51.943	2.04	81.14	0.98	1.30
EMPZ-NBr	54.835	2.16	8.09	0.98	1.00
EMPZ-ETHF-1	56.438	2.22	4.81	0.97	1.21
EMPZ-ETHF	60.798	2.39	13.12	0.97	1.40

a. Relative retention times (RRT) were calculated against the retention time (RT) of Empagliflozin.

b. Relative response factor were calculated against the response factor of Empagliflozin.

c. Resolutions were calculated between two adjacent peaks.

d. Mean ± SD.

A: Empagliflozin test spiked with its six impurities

B: HPLC Chromatogram of Acid Stress Empagliflozin sample

C: HPLC Chromatogram of Base Stress Empagliflozin sample

D: HPLC Chromatogram of Peroxide Stress Empagliflozin sample

E: HPLC Chromatogram of Thermal Stress Empagliflozin sample

F: HPLC Chromatogram of Humidity Stress Empagliflozin sample

G: HPLC Chromatogram of Photolytic Stress Empagliflozin sample

Figure 2: Typical chromatograms of Empagliflozin test spiked with its six impurities and forced degradation samples at optimized method conditions.

The homogenous and pure empagliflozin peak generated from all stress samples was validated by the PDA detector's peak-purity test results (Table II). For the samples under stress, the mass balance was nearly 99% (Table II). Contaminants or degradation products had no effect on the empagliflozin assay, indicating the method's capacity to indicate stability.

3.2.2. Limits of detection and quantification:

Empagliflozin was assessed at 0.05% Level (0.5ppm) for all impurities, whereas LOD and LOQ were determined for the six known impurities. By estimating the S/N Ratio and using (NLT 10) solutions with known concentrations, the LOQ level was projected ¹⁹. By analyzing six distinct preparations of the six contaminants and computing the RSD (%) of the peak area for each impurity, precision was also ascertained at the LOQ level (Table III).

Table II: Stress testing (forced degradation) data.

Stress Condition	% Net Degradation	Purity Match	Mass balance^a
Stress Condition	Empaglifozin	Empaglifozin	Empaglifozin
Control	0.15	963	NA
Acid hydrolysis	0.33	975	100.19
Base hydrolysis	0.19	957	100.24
Peroxide Oxidation	5.15	973	95.48
Humidity stress	0.17	971	100.96
Photolytic-UV light.	0.15	957	101.45
Heat stress	0.17	958	98.98

^aMass balance = % assay + % impurities + % degradation products

Table III: Regression and precision data

Parameter	EMPZ-α-DYT	EMPZ-ZBA	EMPZ-ETHF-2	EMPZ-NBr	EMPZ-ETHF-1	EMPZ-ETHF	EMPZ
LOD (µg/ml)	0.1470	0.1382	0.1462	0.1418	0.1441	0.1505	0.1450
LOQ (µg/ml)	0.4900	0.4606	0.04872	0.4726	0.4802	0.5015	0.4833
Slope (b)	23439	22041	34460	21495	30689	36340	26582
Intercept (a)	602	1313	-3914	10136	-129	-1818	-477
Correlation coefficient	0.999	0.999	0.999	0.996	0.999	0.999	0.999
Precision (% RSD)#	1.59	1.22	1.88	2.34	2.37	0.99	2.17
Ruggedness (% RSD)#	1.01	1.12	0.90	0.99	1.09	1.61	---
LOQ Precision (% RSD)#	0.94	1.50	0.53	3.37	2.61	1.17	0.66

Linearity range is LOQ - 200 % with respect to 0.50 % of EMPZ-α-DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-NBr, EMPZ-ETHF-1 and EMPZ-ETHF for 1000 µg/ml of Empagliflozin.

# six determinations

3.2.3. Linearity:

For an analyte concentration of 1000µg/ml, the stock solution of impurity was diluted to five different dilutions. This resulted in the LOQ being 200% of the maximum amount of the impurity that was allowed (i.e., 0.5% for EMPZ-α-DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-NBr, EMPZ-ETHF-1, and EMPZ-ETHF, and 0.2% for empagliflozin). This made it possible to find solutions for the associated compounds' linearity tests. The y-intercepts, slopes, and correlation coefficients of the calibration plots are provided. The calibration plots for the six related chemicals were linear over the tested ranges. The correlation coefficients were more than 0.998 for each component (Table III). These results reveal a high correlation between the concentration and peak area for each of the six impurities.

3.2.4. Precision:

Six distinct preparations of Empagliflozin (10mg film coated tablets) were spiked with 0.5% of its six impurities EMPZ-α-DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-NBr, EMPZ-ETHF-1, and EMPZ-ETHF—in order to assess the test method's precision. The percentage RSD of the impurity results was discovered to be within the acceptable limits for both the total and individual impurities. These outcomes validated the method's accuracy and robustness (Table III).

3.2.5. Accuracy:

Recovery was estimated for Sample of Empagliflozin film coated 10mg and 25mg Tablets were spiked with known impurities (EMPZ-α-DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-NBr, EMPZ-ETHF-1 and EMPZ-ETHF) at LOQ, 100%, and 200% of specification limit i.e., 0.50% for EMPZ-α-DYT, EMPZ-ZBA, EMPZ-ETHF-2, EMPZ-NBr, EMPZ-ETHF-1 and EMPZ-ETHF in triplicate (total nine determinations). There were computations made for the impurity recovery (Table IV). The chromatogram obtained from an empagliflozin sample spiked at the 0.5% level with all six contaminants is shown in Fig. 2.

3.2.6. Stability in solution and mobile phase:

Investigations into Mobile Phase and Solution Stability revealed no appreciable shifts in the concentrations of the six contaminants.For up to 24 hours during the identification of related chemicals, both the sample and standard solutions remained stable. The mobile phase was stable for up to 48 hours.

4. CONCLUSION:

A fast, accurate, linear, and highly specific gradient HPLC method has been devised for the quantitative detection of the contaminants in pills containing empagliflozin. The method's validation outcomes met expectations. This stability-indicating method can be applied to routine manufacturing sample analysis as well as evaluating the stability of dosage forms of empagliflozin. In addition to identifying unknown contaminants, the new HPLC technique can be used to confirm existing impurities or degradants generated during stability testing.

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Received on 26.03.2024 Revised on 20.06.2024

Accepted on 06.08.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):639-646.

DOI: 10.52711/0974-360X.2025.00095

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