A Comprehensive Approach to Method Development and Validation for Simultaneous Quantification of Dapagliflozin, Vildagliptin, and Metformin in Tablet Formulation using HPLC

Bhuvnesh Kumar Singh¹*, Ashok Kumar Rajpoot¹, Neelanchal Trivedi²,

Harishchandra Verma³, Anil Kumar¹, Nikhil Singh¹, Neha Tamta⁴, Nishi Sharma¹

¹Moradabad Educational Trust, Group of Institutions, Faculty of Pharmacy, Moradabad, Uttar Pradesh, India.

²Invertis Institute of Pharmacy, Invertis University, Bareilly, Uttar Pradesh, India.

³Teerthanker Mahaveer College of Pharmacy,

Teerthanker Mahaveer University, Moradabad, Uttar Pradesh, India.

⁴Faculty of Pharmacy, IFTM University, Lodhipur Rajput, Moradabad, Uttar Pradesh, India.

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

ABSTRACT:

In this work, we present a novel method for determining the prescribed dosages of Dapagliflozin (DAPA), Vildagliptin (VIL), and Metformin (MET) all at once. The goal of this research is to create and test a new RP-HPLC technique that can simultaneously measure DAPA, VIL, and MET in formulation and bulk materials.The goal of this research is to create and test a new RP-HPLC technique that can simultaneously measure DAPA, VIL, and MET in formulation and bulk materials. An isocratic elution method was used with a flow rate of 1.0 ml min-1 and a diode array detector operating at 261nm to perform the chromatographic separation on a kromasil-C18 column(4.5 x 250mm; 5µm). Using orthophosphoric acid to get the pH down to 3.5, the mobile phase consisted of a combination of 0.05 mmol potassium dihydrogen phosphate buffer and acetonitrile in an 80:20 v/v ratio. With concentrations ranging from 0.1-1.0µg/ml and 2-25µg/ml, as well as DAPA, VIL, and MET values from 10 to 120µg/ml, the calibration curve displayed linearity. The research found that DAPA had a limit of detection and quantification of 0.0122µg/ml while VIL had a limit of 0.0323µg/ml. The upper limits for MET were 0.232µg/ml and 0.635µg/ml, while the lower limits were 1.124µg/ml and 3.124µg/ml, respectively. We have developed and validated a new reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantitative determination of vildagliptin and metformin. This method is very sensitive, easy to use, and stable. The suggested technique could be used to routinely measure DAPA, VIL, and MET.

KEYWORDS: Dapagliflozin, Vildagliptin, Metformin, RP-HPLC.

INTRODUCTION:

The chemical characterisation of dapagliflozin (DAPA) is (1s)-1,5-anhydro-1-C-[4-chloro-3-[(4-ethoxy phenyl) methyl]phenyl]. The novel class of oral antidiabetic drugs known as sodium-glucose cotransporter 2 (SGLT2) inhibitors includes -D-glucitol. These inhibitors specifically target sodium-glucose cotransporters, which are in charge of the kidneys' ability to reabsorb glucose^1,2. DAPA is a first-generation selective SGLT inhibitor that inhibits glucose transport with a 100-fold preference for SGLT2 over SGLT1, demonstrating remarkable specificity³. With the chemical formula [S]-1-[N-[3-hydroxy-1-adamantyl] glycyl] pyrrolidine-2-carbonitrile, vildagliptin (VIL) is a noteworthy material. It is a vital drug for the treatment of diabetes because it is a potent inhibitor of di-peptidyl peptidase IV (DPP-IV)⁴. Vildagliptin is a novel oral medicine intended to treat high blood sugar in people with type 2 diabetes. It is a kind of DPP-IV inhibitor^5,6. Metformin hydrochloride, or 3-[diaminomethylidene]-1,1-dimethylguanidine hydrochloride, is a widely used biguanide chemical that is mainly used to treat type 2 diabetes mellitus⁷. The molecular structures of MET, VIL, and DAPA are shown in Figure 1^7,8. A thorough review of the literature has demonstrated that metformin, vildagliptin, and dapagliflozin are estimated using RP-HPLC. However, for these chemicals there is a clear deficiency in stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) methods^9-11. This work aims to address the present gap in analytical techniques by developing a novel RP-HPLC method that is direct, sensitive, and stability-determining^12-14. This procedure will be verified in accordance with ICH recommendations Q2 (R1) and adhere to the stringent requirements established by the International Council for Harmonisation (ICH) Q1A (R2) and Q1B. Our study overcomes the drawbacks of traditional methods, which need lengthy retention times for vildagliptin and rely on costly solvents. Instead, our technique offers an innovative and economical solution^15-19. Our research employs an economical 80:20 ratio of acetonitrile to buffer, utilizing a straightforward yet precise methodology. This strategic combination ensures accuracy and consistency in testing the targeted chemicals while also enhancing the ability to detect subtle changes. A photodiode array detector, known for its exceptional sensitivity even at lower concentrations, further facilitates detection. The RP-HPLC method used in this study enables accurate detection of dapagliflozin, vildagliptin, and metformin in pharmaceutical dosage forms, representing a significant advancement in pharmaceutical analysis. Our technology provides a stable and efficient analytical tool that enhances the quality assessment procedures in the pharmaceutical industry. Ultimately, this ensures that patients worldwide have access to essential drugs that are both safe and effective^21-23.

Figure 1: Structure of Dapagliflozin (A) Vildagliptin (B)and Metformin (C)

MATERIALS AND METHODS:

Analytes and Reagents:

Aurobindo Pharmaceuticals Limited, headquartered in Hyderabad, India, generously provided VIL as a gift sample. Additionally, AKUMS Drugs and Pharmaceuticals Ltd, based in Haridwar, Uttarakhand, supplied DAPA and MET as gift samples. To ensure the highest quality standards in our analytical techniques, we procured HPLC-grade acetonitrile from E-Merck Specialties Private Limited, Mumbai, India, and potassium dihydrogen phosphate (KH₂PO₄) from Qualigens Fine Chemicals Limited, also located in Mumbai, India. Moreover, we employed the Milli-Q water purification system in our laboratory to obtain HPLC-grade water, thereby ensuring the integrity and reliability of our analytical procedures^24-25.

Chromatographic conditions:

The development of the analytical method for vildagliptin and metformin HCl entailed conducting experiments with different solvents. Eventually, a successful separation was accomplished using a mobile phase consisting of a 0.05mmol potassium dihydrogen phosphate buffer and acetonitrile in a ratio of 80:20 (v/v). Orthophosphoric acid was added to the mobile phase to bring its pH down to 3.5, and it was then pumped through the system at a flow rate of 0.9ml/min. A PDA detector with a 261nm wavelength was used to find the eluent. The mobile phase was degassed in an ultrasonic bath to eliminate any possible gas molecules, and it was filtered using a 0.22µm nylon membrane filter under vacuum before being employed²⁶.

Preparation of buffer solution:

The buffer solution was prepared by dissolving 0.68g of potassium dihydrogen orthophosphate in 1000 ml of water, and then adding orthophosphoric acid slowly until the necessary pH of 3.5 was reached. Afterwards, the solution was filtered using a Millipore membrane filter with a pore size of 0.45µm to guarantee transparency and eliminate any solid particles.

Wavelength Selection:

After being dissolved in the mobile phase, the medications were further diluted until they had a concentration of 1μg/ml. After filtering, a UV-Visible spectrophotometer was used to evaluate each drug solution independently, scanning a wavelength range of 200 to 400nm. The isobestic point was found by superimposing the acquired spectra, and 261nm, the wavelength connected to this point, was selected for more research.

Preparation of stock and standard solutions:

A 100mL solution of methanol was used to dissolve precisely measured 10mg of each medication, resulting in separate stock solutions of 100µg/ml for DAPA, VIL, and MET. For DAPA, VIL, and MET, working standard solutions with concentrations ranging from 0.5 to 50 µg/ml were created by diluting the stock solutions using the same mobile phase. Other analytical procedures and linearity testing were conducted using these solutions. Prior to usage, the resulting solutions were filtered using a 0.45µm-pore-diameter Millipore membrane filter.

Solution Stability:

The solutions containing DAPA, VIL, and MET were stable for 24hours at 25°C and for one week when stored at 2 to 8°C in the refrigerator. After computing the percentage difference between the stability results, it was discovered that there was no degradation observed in the peak areas of DAPA, VIL, or MET under the stipulated conditions.

Method optimization:

This study's primary objective was to develop and validate a better method for simultaneously detecting DAPA, VIL, and MET by methodically altering several parameters. The parameters included changes in the pH, wavelength, and flow rate of the sodium acetate buffer system, as well as changes in the organic solvent composition (methanol and acetonitrile). Achieving clearly defined peaks, the maximum number of theoretical plates, a minimal tailing factor, and shorter separation times were the goals. The best outcome was achieved by using a mobile phase in which orthophosphoric acid was added to adjust the pH to 3.5. A phosphate buffer and acetonitrile combination in a ratio of 80:20 (v/v) made up the mobile phase. A 1 ml/min flow rate was used when pumping it. Using a diode array detector set to run at a particular wavelength of 261 nm, the effluents were investigated. Figure 2 illustrates example chromatograms from the research that showed multiple profiles, including a placebo, individual components (DAPA, VIL, and MET), and a combination combining DAPA, VIL, and MET.

Figure 2: System suitability Chromatogram of Dapagliflozin, Vildagliptin and Metformin

RESULTS AND DISCUSSION:

Method Development:

The primary objective of this study is to develop and validate a high-performance liquid chromatography (HPLC) technique capable of simultaneously analysing DAPA, VIL, and MET in active pharmaceutical ingredients (API) and various formulations. The goal is to ensure compliance with ICH standards while achieving an approach that is straightforward, accurate, reliable, efficient, and successful. In order to do this, a number of factors were changed, including the pH level, elution procedure, flow rate, composition of the mobile phase, and selection of appropriate columns, as part of an updated and verified technique. In order to increase the overall efficacy and efficiency of the analytical process, the optimising approach aims to generate identifiable peaks, maximise the number of theoretical plates, decrease tailing variables, and shorten analysis times.

System suitability:

The system suitability test is essential for validating analytical procedures and ensuring adequate discrimination between the various peaks of interest. Throughout the experiment, crucial parameters like theoretical plates, retention duration, and tailing factor constantly met the criteria for full acceptance. The percent relative standard deviation (RSD) for DAPA, VIL, and MET in the peak region was calculated to be 1.81%, 1.43%, and 0.45%, respectively, based on six replicates. Furthermore, the percentage relative standard deviation (RSD) for retention time was determined to be 0.112%, 0.342%, and 0.465% for DAPA, VIL, and MET, respectively. The system's suitability measures, such as a tailing factor below 2 and a theoretical plate count above 2000, indicate the effectiveness and dependability of the technique.

Specificity:

This method is particularly suitable for the simultaneous evaluation of both medications due to its outstanding resolution and lack of interference from blanks or excipients. The chromatogram, which presented precise and comprehensive data, exhibited no additional peaks. This validates that the technique is both precise and reliable. Figure 3's chromatogram showcases the selectivity of DAPA, VIL, and MET.

Figure 3: Chromatogram representing the specificity of Dapagliflozin, Vildagliptin and Metformin

Table 1: Accuracy study of Dapagliflozin, Vildagliptin and Metformin

Name of the drug	Spiked amount	Amount of drug (Tablet) µg	Amount of drug (Standard) µg	Total Drug (µg)	Total Found (µg) Mean ± SD	% RSD	% Recovery
DAPA	50%	0.5	0.25	0.75	0.754 ± 0.006	0.779	100.533
	100%	0.5	0.5	1.00	1.00 ± 0.0180	1.800	100.860
	150%	0.5	0.75	1.25	1.25 ± 0.0193	1.549	99.360
VIL	50%	10	5	15.00	15.00 ± 0.215	1.436	99.867
	100%	10	10	20.00	20.00 ± 0.210	1.046	100.700
	150%	10	15	25.00	25.00 ± 0.218	0.869	100.224
MET	50%	50	25	75.00	75.00 ± 0.719	0.948	101.139
	100%	50	50	100.00	100.00 ± 1.525	1.516	100.640
	150%	50	75	125.00	125.00 ± 0.646	0.513	100.609

Table 2: System and Method precision study for Dapagliflozin, Vildagliptin and Metformin

Injection Number	System Precision*			Method Precision
Injection Number	Peak areas			% Assay
	DAPA	VIL	MET	DAPA	VIL	MET
1	14992	329493	1530303	100.24	99.89	98.49
2	14929	330203	1549242	99.88	98.48	98.99
3	15003	320403	1559393	101.48	100.98	101.49
4	15402	334939	1549293	99.58	99.87	101.49
5	14892	340202	1549292	100.48	100.49	100.74
6	15023	330492	1563728	99.48	100.59	99.4
Mean	15040.168	330955.333	1550208.5	100.19	100.05	100.1
SD (±)	183.973	6567.211	11540.997	0.738	0.880	1.310
RSD (%)	1.223	1.984	0.744	0.736	0.880	1.309
Acceptance criteria	The maximum allowable RSD should not exceed 2.

*System precision was evaluated by measuring the peak response of standard drug solutions containing Dapagliflozin at 0.5 µg/ml, Vildagliptin at 10 µg/ml, and Metformin at 50 µg/ml in six replicates.

Table 3 The study evaluated the intraday and interday precision for Dapagliflozin, Vildagliptin, and Metformin.

Concentration	Intraday Precision			Interday Precision (Day 1,2,3)
Concentration	Area of DAPA	Area of VIL	Area of MET	Area of DAPA	Area of VIL	Area of MET
Dapagliflozin 0.4 (µg/ml), Vildagliptin 8 (µg/ml) and Metformin 40 (µg/ml)	13183	284636	1256732	13294	293832	1302832
	12837	279540	1257327	13182	293483	1304842
	12988	275844	1285968	13028	283934	1304822
Mean	13002.672	280006.666	1266675.667	13168	290416.333	1304165.333
SD (±)	173.463	4414.538	16710.299	133.551	5616.577	1154.744
%RSD	1.333	1.576	1.319	1.014	1.934	0.088
Dapagliflozin 0.5 (µg/ml), Vildagliptin 10 (µg/ml) and Metformin 50 (µg/ml)	15183	353474	1582828	15284	352636	1583932
	15032	352738	1572742	15199	349292	1583922
	15193	352762	1572842	15294	349992	1593832
Mean	15136	352991.333	1576137.333	15259	350640	1587228.667
SD (±)	90.212	418.174	5794.503	52.201	1763.664	5718.657
%RSD	0.604	0.118	0.368	0.342	0.503	0.360
Dapagliflozin 0.6 (µg/ml), Vildagliptin 12 (µg/ml) and Metformin 60 (µg/ml)	20429	437272	2003924	21039	440293	2028382
	20483	429483	2038238	20987	443721	2030392
	21039	429984	2038212	21442	443838	2039392
Mean	20650.333	432246.333	2026791.333	21156	442617.333	2032722
SD (±)	337.676	4359.558	19803.696	249.044	2013.782	5863.165
%RSD	1.635	1.008	0.977	1.177	0.455	0.288

Linearity and range:

The high correlation coefficient R² values, approaching 1 (R² = 0.9999), show that the analytical calibration curves for DAPA, VIL, and MET were linear within the specified ranges. Y = 30763x + 412.42 (R² = 0.9995), Y = 35528x + 1957 (R² = 0.9994), and Y = 31285x + 446.3 (R² = 0.9997) are the linear regression equations for MONT and EBAS. Additional proof of the linearity may be found in these equations. The linearity of the chromatograms is shown in Figure 4.

Figure 4: Linearity and range of Dapagliflozin (A), Vildagliptin (B) and Metformin (C)

LOD and LOQ:

In Table 4, the reported values pertain to (LOD) and (LOQ) for DAPA, VIL, and MET. For DAPA, the LOD is 0.0122 µg/mL, and the LOQ is 0.0323 µg/mL. For VIL, the LOD is 0.232 µg/mL, and the LOQ is 0.635 µg/mL. For MET, the LOD is 1.124 µg/mL, and the LOQ is 3.124 µg/mL.

Figure 5: Chromatogram illustrating the LOQ (A) and LOD (B) of Dapagliflozin, Vildagliptin and Metformin

Table 4: Result of LOD and LOQ study

Parameters	DAPA	VIL	MET
LOD* (µg/mL)	0.0122	0.232	1.124
LOQ* (µg/mL)	0.0323	0.635	3.124

Assay of Tablets

Table 5: Result of assay of tablet formulation

Drugs	Label Claim (mg)	Amount Found (mg)	Label claim (%)	SD^*
Dapagliflozin	5	5.03	100.6	0.9373
Vildagliptin	100	101.34	101.34	0.8472
Metformin	500	499.56	99.912	0.4653

* The tablet formulation was estimated based on the average of six measurements.

CONCLUSION:

We conducted a thorough investigation for improving the chromatographic settings in order to produce the best possible separation and form of peaks for the compounds DAPA, VIL, and MET. Our proposed RP-HPLC technique has been thoroughly validated and has shown outstanding accuracy and precision. It successfully meets the strict standards for limit of detection (LOD) and limit of quantification (LOQ). However, during stability testing, we discovered that the medication solutions maintained stability exclusively under laboratory benchtop conditions. The RP-HPLC method we have recently devised is notable for its straightforwardness, rapidity, exactness, consistency, and sensitivity. Consequently, it becomes a handy instrument for the regular examination of DAPA, VIL, and MET in their respective pharmaceutical formulations. The notable importance of our created method lies in its versatility, which allows it to be used in both routine and unfamiliar sample analyses in different sectors of the pharmaceutical industry. Our approach plays a crucial role in maintaining product quality standards by enabling the quick evaluation of DAPA, VIL, and MET levels in pharmaceutical formulations. The implementation of this practice in pharmaceutical enterprises guarantees comprehensive quality assurance, therefore protecting the authenticity and effectiveness of pharmaceutical products for final consumers. Looking ahead, further research could focus on adapting this RP-HPLC method for application under different environmental conditions to enhance its robustness and versatility. Additionally, exploring its potential use in combination with other analytical techniques could provide a more comprehensive quality assessment framework. Future studies may also investigate the method's applicability in real-time monitoring of pharmaceutical manufacturing processes, potentially leading to more efficient production and better quality control.

ACKNOWLEDGEMENT:

The authors express their gratitude for the samples provided by Aurobindo Pharmaceuticals Limited, Hyderabad, India, and AKUMS Pharmaceuticals Ltd., Haridwar, Uttarakhand, India.

CONFLICT OF INTEREST:

The authors affirm that there are no conflicts of interest to disclose.

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Received on 01.05.2024 Revised on 15.10.2024

Accepted on 04.01.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3473-3479.

DOI: 10.52711/0974-360X.2025.00500

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