1. INTRODUCTION:

The quality of a drug plays an important role in ensuring the safety and efficacy of the drugs. Diabetes is a condition that hampers the body ability to regulate blood sugar levels. It can result in sugar levels, in the bloodstream, which can lead to health complications such, as liver damage, heart problems and kidney dysfunction.¹

Diabetes raises the risk of atherosclerotic cardiovascular events, such as peripheral arterial disease, cerebrovascular disease, and coronary artery disease, by two to four times.² A reduction in the secretion and activity of insulin causes type 2 diabetes mellitus (T2DM), which is generally encountered in adults. Although many anti-diabetic medications have been created to lower high blood sugar levels, T2DM still causes significant morbidity and mortality, with more than 6 million deaths from diabetes in 2021.³

Most diabetics need a combination of medications to keep their blood sugar levels under control. The more recent pairing of DPZ and VGT was introduced to the Indian market in late 2022. Fixed-dose combination of VGT (100mg) and DPZ (10mg) in tablet form (sustained release that helps people with type 2 diabetes keep blood sugar levels under control) Only a few of the analytical techniques described for determining DPZ and VGT concentrations in single and mixed dosage forms included ultraviolet (UV) spectrophotometry, high-performance liquid chromatography, and high-performance thin layer chromatography.⁴

Vildagliptin is a pharmaceutical compound that belongs to a class of medications known as dipeptidyl peptidase-4 (DPP-4) inhibitors. Vildagliptin is an orally active DPP-4i that enhances islet function and stops the inactivation of glucagon-like peptide-1. Vildagliptin works by inhibiting DPP 4, which enhances the levels of these hormones. As a result, insulin release is stimulated while the production of glucagon is reduced. This ultimately leads to control of blood sugar levels. When taken as a stand-alone medication or as an adjunct to existing treatment, vildagliptin effectively decreases HbA1c in patients.⁵ The European Medicines Agency authorised oral vildagliptin in 2008 for the treatment of people with type II diabetes mellitus as a monotherapy or in combination with metformin.⁶ Glucagon-like peptide-1 (GLP-1) is an incretin hormone that the L-cells of the stomach release into the gut wall following food consumption. This hormone is quickly removed by DPP-4, reduces glucagon secretion, and promotes insulin secretion.⁷

Dapagliflozin is a pharmaceutical compound belonging to a class of medications known as sodium-glucose co-transporter 2 (SGLT2) inhibitors. Dapagliflozin is mainly prescribed to manage type 2 diabetes mellitus and regulate blood sugar levels. It works by preventing the kidneys from reabsorbing glucose. This medication offers advantages such, as control over blood sugar levels, weight loss and a lowered risk of cardiovascular incidents, in certain patients. At least 90% of the kidney's glucose reabsorption is caused by the sodium-glucose transport protein subtype 2 (SGLT2), which dapagliflozin inhibits.⁸ Adults with type II diabetes may also use dapagliflozin alone or in combination with other medications. Since dapagliflozin is a relatively new drug, its long-term effects over several years are not yet known.⁹ With an increase in disease incidence, population exposure levels to many environmental toxins have increased.¹⁰ Numerous anti-diabetic drugs have been developed, but issues with sustained glycemic control, complications, and compliance continue to exist. Therefore, there is always a need for new medications to address these problems. As we continue to explore the outcomes and safety aspects linked to this combination our goal is to offer insights, into how it can optimize diabetes care.¹¹

After conducting research, it was found that various analytical techniques have been documented in literature. There is a lack of reported methods, for stability studies While more cost effective and simple methods have been identified for the estimation of Dapagliflozin, Vildagliptin by RP HPLC^{12,13,14,15,16} n Therefore there is a need to develop and validate an affordable approach in pharmaceutical dosage form using following the guidelines set by ICH (Q2 specification).^17,18,19

Fig. 1: Structure of Dapagliflozin

Fig. 2: Structure of Vildagliptin

2. MATERIALS AND METHODS:

Instrument used: WATERS HPLC 2695 SYSTEM equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2 Software.

UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Dapagliflozin and Vildagliptin acid solutions.

Materials used: Dapagliflozin and Vildagliptin pure drugs (API) received from vendor. Combination Dapagliflozin and Vildagliptin tablets (Vildaily-DZ) received from local market.

Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.

Chromatographic Conditions:

Mobile phase	:	Acetonitrile: Na2hpo4 (70:30)
Flow rate	:	1 ml/min
Column	:	BDS C18 (4.6 x 150mm, 5µm)
Detector wave length	:	228nm
Column temperature	:	25°C
Injection volume	:	10L
Run time	:	5 min
Diluent	:	Acetonitrile and Buffer taken in the ratio of 60:40

Methods:

Preparation of buffer:

0.01N Na2HPO4 Buffer: Accurately weighed 1.41gm of Potassium dihyrogen Ortho phosphate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then PH adjusted to 5.2 with dil. Formic acid.

0.1% Formic acid Buffer: 1ml of Conc Formic acid was diluted to 1000ml with water.

Preparation of Standard stock solutions:

Accurately weighed 5mg of Dapagliflozin, 50mg of Vildagliptin and transferred to 100ml volumetric flask. 3/4 Th of diluents was added to the flasks and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution 1. (50µg/ml of Dapagliflozin and 500µg/ml of Vildagliptin)

Preparation of Standard working solutions:

(100% solution): 1ml from stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (5µg/ml of Dapagliflozin and 50µg/ml of Vildagliptin)

Preparation of Sample stock solutions:

10 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters. (100µg/ml of Dapagliflozin and 1000µg/ml of Vildagliptin)

Preparation of Sample working solutions:

(100% solution): 0.5ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (5µg/ml of Dapagliflozin and 50µg/ml of Vildagliptin).

3. RESULTS AND DISCUSSION:

a. System Suitability:

The system suitability parameters were determined by preparing standard solutions of Dapagliflozin (5ppm) and Vildagliptin (50ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. (Table-1).

The % RSD for the area of six standard injections results should not be more than 2%.

b. Specificity:

Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

c. Linearity:

Six linear concentrations of Dapagliflozin (1.25-7.5µg/ml) and Vildagliptin (12.5-75µg/ml) were injected in a duplicate manner. Average areas were mentioned above and linearity equations obtained for Dapagliflozin was y = 42540x + 1488.4. and of Vildagliptin was y = 38485x + 2186.5. Correlation coefficient obtained was 0.999 for the two drugs. (Table-2).

Table 2: Linearity of Dapagliflozin and Vildagliptin

Dapagliflozin		Vildagliptin
Conc (μg/mL)	Peak area	Conc (μg/mL)	Peak area
0	0	0	0
1.25	53879	12.5	465341
2.5	110689	25	976032
3.75	162543	37.5	1453473
5	214053	50	1926125
6.25	264482	62.5	2439526
7.5	321439	75	2857076

Fig. 5: Calibration curve of Dapagliflozin

Fig. 6: Calibration curve of Vildagliptin

d. Accuracy:

Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 100.41% and 99.94% for Dapagliflozin and Vildagliptin respectively. (Table-3).

Table 3: Accuracy table of Dapagliflozin

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	2.5	2.52	100.78	100.41%
	2.5	2.62	104.97
	2.5	2.52	100.62
100%	5	5.01	100.29
	5	4.98	99.58
	5	4.97	99.43
150%	7.5	7.45	99.39
	7.5	7.43	99.08
	7.5	7.47	99.54

Table 4: Accuracy table of Vildagliptin

% Level	Amount Spiked (μg/mL)	Amount recovered (μg/mL)	% Recovery	Mean % Recovery
50%	25	25.11	100.46	99.94%
	25	24.76	99.06
	25	24.83	99.31
100%	50	50.10	100.20
	50	49.74	99.48
	50	50.00	99.99
150%	75	75.53	100.71
	75	75.53	100.71
	75	74.63	99.51

e. Precision:

From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated for two drugs. % RSD obtained as 0.9% and 0.7% respectively for Dapagliflozin and Vildagliptin. As the limit of Precision was less than “2” the system precision was passed in this method. (Table-5).

Table 5: Precision of Dapagliflozin and Vildagliptin

S. No	Area of Dapagliflozin	Area of Vildagliptin
1.	213975	1934284
2.	214477	1963915
3.	216471	1959490
4.	215235	1960000
5.	214711	1960937
6.	213620	1949851
Mean	214748	1954746
S.D	1015.0	11092.0
%RSD	0.5	0.6

f. Detection and quantification limits:

LOD sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.3ml each of Dapagliflozin, Vildagliptin, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents LOQ sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.9ml each of Dapagliflozin, Vildagliptin, and solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent. (Table-6).

Table 6: LOD and LOQ

Molecule	LOD	LOQ
Dapagliflozin	0.02	0.07
Vildagliptin	0.24	0.72

g. Robustness:

Robustness conditions like Flow minus (0.7ml/min), Flow plus (0.9ml/min), mobile phase minus (65B:35A), mobile phase plus (75B:25A), temperature minus (27°C) and temperature plus(33°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit. (Table-7).

Table 7: Robustness of Dapagliflozin and Vildagliptin

S. No.	Condition	%RSD of Dapagliflozin	%RSD of Vildagliptin
1	Flow rate (-) 0.7ml/min	1.3	0.7
2	Flow rate (+) 0.9ml/min	1.4	0.9
3	Mobile phase (-) 65B:35A	0.9	0.9
4	Mobile phase (+) 75B:25A	1.7	0.9
5	Temperature (-) 27°C	0.2	0.5
6	Temperature (+) 33°C	0.3	0.4

h. Degradation Studies:

Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation. (Table-8 and 9).

Table 8: Degradation Data of Dapagliflozin

S. No.	Degradation Condition	% Drug Recovered	% Drug Degraded
1	Acid	97.68	2.32
2	Alkali	97.89	2.11
3	Oxidation	93.80	6.20
4	Thermal	97.87	2.13
5	UV	98.85	1.15
6	Water	99.66	0.34

Fig. 9: Degradation Data of Vildagliptin

S. No.	Degradation Condition	% Drug Recovered	% Drug Degraded
1	Acid	97.61	2.39
2	Alkali	98.32	1.68
3	Oxidation	93.51	6.49
4	Thermal	97.71	2.29
5	UV	98.01	1.99
6	Water	99.01	0.99

4. CONCLUSION:

A simple and precise method was developed for simultaneously estimating Dapagliflozin and Vildagliptin in tablet form. The retention times were 2.307 min for Dapagliflozin and 2.865 min for Vildagliptin, with %RSD values of 0.9 and 0.7, respectively. High recovery rates were achieved, with 100.41% for Dapagliflozin and 99.94% for Vildagliptin. The method exhibited low LOD and LOQ values (0.02, 0.07 for Dapagliflozin; 0.24, 0.72 for Vildagliptin) and had regression equations for Dapagliflozin (y = 42540x + 1488.4) and Vildagliptin (y = 38485x + 2186.5). The method's efficiency was further highlighted by reduced retention times and overall run time, making it a cost-effective choice for quality control testing in industries.

5. CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

6. REFERENCES:

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2. Arintaya Phrommintikul et.al. Effects of dapagliflozin vs vildagliptin on cardiometabolic parameters in diabetic patients with coronary artery disease: a randomised study. Br J Clin Pharmacol. 2019 Jun; 85(6): 1337–1347.

3. Mahesh Attimarad et.al. Smart eco-friendly mathematically manipulated UV spectroscopic methods to resolve severely overlapped spectra of a binary mixture of dapagliflozin with sitagliptin and vildagliptin. Microchemical Journal. 2023; 190: 108700

4. Sen AK et.al. Various innovative UV spectroscopic methodologies for concurrent estimation of dapagliflozin and vildagliptin in combined tablet. J Appl Pharm Sci. 2023; 13(09): 213–223

5. Joshi A et.al. Bioequivalence study of a fixed-dose combination of Dapagliflozin/ Vildagliptin Sustained Release tablets in healthy adult male subjects: A Randomized, Open-Label, Crossover Study. Journal of Drug Delivery and Therapeutics. 2022; 12(6): 105-109

6. Summary of Product Characteristics: Galvus (vildagliptin) oral tablets.

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9. Gunasekar Manoharan et.al. Stability-Indicating RP-HPLC Method Development for Simultaneous Determination and Estimation of Dapagliflozin in Raw and Tablet Formulation. Chemistry Research Journal. 2018; 3(2): 159-164

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11. Gautam K, et al. Dapagliflozin Versus Vildagliptin as an Adjuvant to Metformin in Patients with Type 2 Diabetes Mellitus: A Randomized, Open-label Study. Cureus. 2023; 15(4): e38200. DOI 10.7759/cureus.38200

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17. IUPAC. Compendium of Chemical Terminology, 2nd edn. (The Gold Book). PAC69, 1137 (1997). Glossary of terms used in Computational Drug Design (IUPAC Recommendations.

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Received on 17.10.2023 Revised on 06.02.2024

Accepted on 23.05.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):229-233.

DOI: 10.52711/2231-5675.2024.00041

S. No.	Vildagliptin			Dapagliflozin
Inj	RT(min)	USP Plate Count	Tailing	RT(min)	USP Plate Count	Tailing	Resolution
1	2.291	5447	1.55	2.854	8567	1.27	4.4
2	2.292	5641	1.55	2.854	8898	1.25	4.5
3	2.301	5363	1.52	2.864	8501	1.21	4.4
4	2.304	5643	1.55	2.864	8761	1.20	4.4
5	2.305	5392	1.55	2.865	8923	1.20	4.4
6	2.308	5721	1.51	2.869	9402	1.23	4.5


Fig. 3: _{Chromatogram of Blank}	Fig. 4: _C_h_r_o_m_{atogram of placebo}