A Novel UPLC Method for Simultaneous Estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet Dosage Form

Deepthi R¹*, Gowri Sankar D²

¹Research Scholar, Department of Pharmaceutical Analysis and QA, Andhra University,

Visakhapatnam - 530003, Andhra Pradesh, India.

²Professor, Department of Pharmaceutical Analysis and QA, Andhra University,

Visakhapatnam - 530003, Andhra Pradesh, India.

*Corresponding Author E-mail: deepthi.pharma7@gmail.com

ABSTRACT:

Aim: The proposed study aimed to develop a novel ultra-performance liquid chromatography (UPLC) method for the estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet dosage form and validate the method in accordance with the International Conference on Harmonization guidelines. Methods: The optimized conditions for the developed UPLC method are Acquity UPLC HIBRA C18 (100mm × 2.1mm, 1.8µ) column maintained at 30°C with a mobile phase consisting of Buffer (0.01N sodium hydrogen phosphate) pH adjusted to 4.0 with dil. orthophosphoric acid: Acetonitrile in the ratio of 60:40%v/v on isocratic mode at a flow rate of 0.3mL/min. Results and conclusion: The sample was detected at 220nm. The retention time for Ertugliflozin and Sitagliptin was deemed at 1.873min and 1.260min. The developed method was validated for accuracy, precision, specificity, ruggedness, robustness, and solution stability. The method obeyed Beer’s law in the concentration range of 3.75µg/mL to 22.5µg/mL for Ertugliflozin and 25µg/mL to 150µg/mL for Sitagliptin with a correlation coefficient of 0.999 for Ertugliflozin and Sitagliptin respectively. Forced degradation studies were conducted by exposing the drug solution to numerous stress conditions such as acidic, basic, peroxide, neutral, photolytic, and thermal conditions. The net degradation was considered within the limits, indicating that the drug is stable in stressed conditions. The developed method for the estimation of Ertugliflozin and Sitagliptin can be utilized for the routine analysis of Pharmaceutical dosage form.

KEYWORDS: Ertugliflozin, Sitagliptin, Stability-indicating, Method development, Validation, Ultra-performance liquid chromatography.

INTRODUCTION:

Type-II Diabetes mellitus¹ is a metabolic condition which is chareterized by hypoglycemia condition. Ertugliflozin and sitagliptin together as Steglujan tablets used to treat type-II diabetes mellitus.

Ertugliflozin is chemically known as (1S, 2S,3S, 4R,5S)-5-(4-chloro-3-(4ethoxybenzyl)phenyl)-1-(hydroxymethyl)-6,8-dioxymethyl)-6,8-dioxabicyclo{3,2,1} octane-2,3,4-triol.

The molecular formula is C₂₇H₃₂ClNO₁₀ with a molecular weight of 566.0. Ertugliflozin belongs to class SGLT inhibitor which selectively inhibits sodium-dependent glucose cotransporters, thereby ertugliflozin increases urinary glucose excretion which leads to osmotic diuresis and helps in treating diabetes. Sitagliptin is chemically known as 3-amino-1-[3-(trifluoromethyl)6,8dihydro5H[1,2,4]triazolo[4,3]pyrazin-7-yl]-4-(2,4,5-trifluorophenyl)butan 1-one. The molecular formula is C₁₆H₁₅F₆N₅O.H₃PO₄.H₂O with a molecular weight of 523.324. Sitagliptin is a potent inhibitor of DPP-4 (dipeptidyl peptidase-4 used as a hypoglycemic agent in treating Diabetes by reducing blood glucose concentration.

Along with diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus when treatment with both ertugliflozin and sitagliptin has shown promised results. Though several methods^2-14were reported in literature for the estimation of Ertugliflozin and sitagliptin individually and in combination with other drugs, no methods were reported for the estimation of Ertugliflozin and Sitagliptin by UPLC. The main objective of the present study is to develop a novel, accurate, precise, stability-indicating UPLC method for the simultaneous estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet dosage form and validate the method according to ICH guidelines.

Fig.1. Chemical structure of Ertugliflozin

Fig.2. Chemical structure of Sitagliptin

MATERIALS AND METHODS:

Reagents and chemicals:

Ertugliflozin and Sitagliptin working standards were procured from Spectrum pharma research solutions, Hyderabad as a gift sample.

The Steglujan tablets were procured from a local pharmacy. All the chemicals used were of AR grade purchased from Merck, Mumbai.

Chromatographic conditions and instruments:

The Acquity UPLC system equipped with a binary solvent manager, sample manager, Ultraviolet (UV) detector, and Hibra C18 (100mm × 2.1mm, 1.8µm) column was used for the determination of Ertugliflozin and Sitagliptin. The optimized conditions included Buffer: Acetonitrile (60:40%v/v) as mobile phase run on an isocratic mode at a flow rate of 0.3mL/min. The column was held at 30⁰C and detection was done at 220nm. Further, a piece of equipment is used are ultrasonic bath sonicator and weighing balance (Denver).

Preparation of the mobile phase:

A Mixture of Acetonitrile and Water in the ratio of 50:50%v/v was used as the mobile phase.

Preparation of 0.01N Na₂HPO₄Buffer:

1.41g of sodium hydrogen phosphate was dissolved in a 900mL of milli-Q water in a 1000mL volumetric flask, sonicated and finally the volume was made up with water and pH was adjusted to 4.0 with dil. ortho phosphoric acid.

Preparation of standard and sample solutions:

About 3.75mg of Ertugliflozin and 25mg of Sitagliptin working standards was dissolved in 25ml of diluent. Then, 1mL of the above stock solution was diluted to 10mL using diluent to get a concentration of 15µg/mL of Ertugliflozin and 100µg/mL of Sitagliptin respectively. 5 Tablets (Steglujan) were weighed accurately and the average weight was calculated. An amount equivalent to 3.75mg of Ertugliflozin and 25mg of Sitagliptin was dissolved in 25ml of diluent, filtered the solution, and diluted 1mL of the above solution to 10mL with diluent.

Method Validation:

The developed method was validated in accordance with the International Conference on Harmonization (ICH) guidelines^15,16

Specificity:

The specificity of the method was determined by comparing the drug solution to the placebo solution and observed for the interference of placebo peak with drug peak.

Accuracy:

Accuracy of the method was determined in accordance with percentage of recovery. The drug solution along with sample was prepared in three concentration levels, i.e., 50%, 100%, and 150%. Then, the percentage recovery was calculated.

Precision:

The Precision of the method was established by injecting the standard solution 6 times into the UPLC system and percentage relative standard deviation (%RSD) was calculated.

Linearity:

The Linearity of the method was determined by preparing a series of dilutions ranging from 3.75 µg/mL to 150 µg/mL and injecting them into a UPLC system.

Ruggedness:

Ruggedness was determined by injecting the standard solution into UPLC 6 times for different days. The percentage of RSD was calculated.

Robustness:

Robustness of the method was determined by varying the optimized analytical conditions, such as mobile composition by ±5%, flow rate by ±10% and column oven temperature by ±5°C.

Forced degradation studies:

Forced degradation studies^17,18were carried out for drug by exposing the drug solution to the various stress conditions such as acidic (2N hydrochloric acid for 30 min for 60°C), basic(2N sodium hydroxide for 30 min for 60°C), peroxide(20% hydrogen peroxide {H₂O₂}for 30 min for 60°C), neutral (refluxing the drug in water for 6h at 60°C), photolytic (exposing the drug solution to UV light by keeping the beaker in UV chamber for 7 days or 200 t h/m² in photo stability chamber, and thermal (exposing the drug solution in hot air oven at 105°C for 6 h) conditions.

RESULTS AND DISCUSSION:

From the UV spectrum, detection wavelength for Ertugliflozin and Sitagliptin was found to be 220nm.

For the development of a method for the estimation of Ertugliflozin and Sitagliptin in pharmaceutical dosage form, initially many mobile phases and many columns were tried to elude the drug peak with less tailing factor, more plate count and better resolution. Acquity UPLC Hibra C18 (100mm×2.1mm, 1.8µm) column and Buffer: Acetonitrile in the ratio 60:40% v/v were selected as mobile phase based on the peak parameters. The detection wavelength was found to be 220nm.

Prepared standard solution, sample solution, and the blank solution were injected into the UPLC system, and system suitability parameters were noted as summarized in Table 1 along with Optimized chromatogram as showed in Fig 3.

Table 1: Optimized chromatographic conditions for determination of Ertugliflozin and Sitagliptin

Parameter	Description
Stationary phase	Hibra C18 (100 mm× 2.1 mm, 1.8 µ)
Mobile phase	Buffer: Acetonitrile (60:40)
Flow rate	0.3mL/min
Detection wavelength	220nm
Detector	UV
Column temperature	30⁰C
Diluent	Water: Acetonitrile (50:50)

The developed method was identified to obey Beer’s law in the concentration range of 3.75µg/mL to 22.5µg/mL for Ertugliflozin and 25µg/mL to 150µg/mL Sitagliptin with a correlation coefficient of 0.999 each.

A linearity graph was plotted between concentration and peak area as showed in the Fig 4 and Fig 5 results as presented in Table 2.

Table 2: System suitability and validation parameter results

Parameter	Result
	Ertugliflozin	Sitagliptin
USP Plate count	3935	3266
USP Tailing factor	1.7	1.8
USP Resolution	-	5.6
Precision (%RSD)	0.6	0.5
Accuracy	99.10-100.50	98-1.02
Specificity	Specific, No interference
Linearity range (µg/mL)	3.75-22.5	25-150
Correlation coefficient, r²	0.999	0.999
LOD (µg/ml)	0.09	0.28
LOQ (µg/ml)	0.32	0.95

The method was found to be accurate as the percentage recovery was 99.03 - 100.90% for Ertugliflozin and 99.50 - 101.00% for Sitagliptin and was within the limits. The percentage of RSD was determined to be 0.6% RSD and 0.52% RSD, which indicates that the method was precise. The method was shown to be specific, as there is no interference of retention time of placebo peak with that of drug peak.

Forced degradation studies results indicate that the drug was reported to be stable in various stress conditions as net degradation was to be within the limits and the results were outlined in Table 3.

Table 3: Forced degradation studies result Stress condition

Condition	Ertugliflozin		Sitagliptin
	%Assay	%D	%Assay	%D
Acid	94.59	5.41	95.62	4.38
Base	94.24	5.76	96.05	3.95
Neutral	99.24	0.76	99.29	0.71
Peroxide	94.90	5.10	93.58	6.42
Photolytic	98.65	1.35	98.52	1.48
Thermal	97.34	2.66	97.38	2.62

%- Percentage Degradation

CONCLUSION:

A novel, accurate, and precise stability-indicating method was developed for the estimation of Ertugliflozin and Sitagliptin in bulk and tablet dosage form using UPLC. The method was validated using numerous validation parameters, and the method was found to be linear, precise, accurate, specific and robust. From the degradation studies conducted, it infers that Ertugliflozin and Sitagliptin were stable at high concentrations of acid, base, peroxide, thermal, UV and water stress conditions. The retention time for both the drugs was 2min, which enables rapid quantification of many samples in routine and quality control analysis of tablet formulation.

ACKNOWLEDGEMENT:

The authors are thankful to the Spectrum pharma research solutions, Hyderabad, for providing the Ertugliflozin and Sitagliptin gift samples and also for providing required facilities to undertake this research work.

AUTHOR’S CONTRIBUTIONS:

All the authors contributed equally to this manuscript.

CONFLICTS OF INTEREST:

The authors claim that they have no conflict of interest.

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Received on 08.08.2020 Modified on 12.10.2020

Research J. Pharm. and Tech. 2021; 14(9):4859-4862.

DOI: 10.52711/0974-360X.2021.00844