INTRODUCTION:

Dapagliflozin propanediol monohydrate (DAPA), chemically (2S,3R,4R,5S,6R)-2-[4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl]-6-(hydroxymethyl) oxane-3,4,5-triol; propane-1,2 diol; monohydrate¹ is an inhibitor of Sodium-glucose co-transporter 2 (SGLT2). It induces round-the-clock glucosuria and reduces blood glucose levels after a single daily dosage² Metformin hydrochloride (MET), chemically 3-(diamino methylidene)-1, dimethyl guanidine; hydrochloride³whose effect is dependent on the presence of insulin.

Metformin does not stimulate insulin release, and it also inhibits hepatic gluconeogenesis and glucose production from the liver. This is the primary activity responsible for reducing blood glucose levels in diabetics. Improves glucose absorption and disposal in skeletal muscle and fat⁴DAPA and MET are combination dose forms intended to enhance blood sugar management in individuals with type 2 diabetes mellitus in conjunction with exercise and diet⁵ Figures 1 and 2 illustrate the chemical structures of DAPA and MET respectively. The purpose of stability and related substance study is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors^6-7, Presence of impurities critically effect the stability and pharmacological action of pharmaceutical API and drug product^8-12, Analytical quality by design (AQbD) and centralcomposite design (CCD) aid in regulatory compliance for RP-HPLC method development, stress testing or stability indicating methods Stability testing^13-16

Figure 1: Molecule Structure of Dapagliflozin Propanediol Monohydrate

Figure 2 Molecule structure of metformin hydrochloride

The literature review reveals that this DAPA and MET combination is not officially recognized by any pharmacopeia, but it has been determined by various methods including UV-Visible spectrophotometry^17-18 RP-HPLC (assay)^19-23, RP-HPLC (stability indicating) ^24-25, Ion-pair RP HPLC²⁶, LC-MS-MS^27-30, RP-UPLC³¹and UHPLC-QTOF-MS³². Few analytical methods are a simultaneous estimation of DAPA and MET in combined dosage form by UV- Visible spectrophotometry³, HPLC (assay)^], and HPLC (stability indicating)³⁴. This method required greater run time and more solvent consumption compared to the RP-UPLC method. Therefore, felt essential to develop a new rapid method for the quantification and separation of the studied drugs in their combination using an isocratic mobile phase with an RP-UPLC system. RP-UPLC method is superior as compared to other methods as it improves the selectivity, sensitivity, and resolution lowers cost, and accelerates the process of their detection. The proposed method is validated as per ICH Q2(R1) guideline³⁵, and this pharmaceutical analytical method applies to both the pharmaceutical industry and research.

MATERIALS AND METHODS:

Materials:

The working standards DAPA and MET, which were gifts from Piramal Pharma Ltd., Ahmedabad, India, have purity levels of 99.38% and 99.92%, respectively. The orthophosphoric acid, water, acetonitrile, methanol, and other chemicals were HPLC-grade. Commercial Xigduo® XR (Astra Zeneca) tablets containing 10mg of DAPA and 500mg of MET were purchased from a local pharmacy.

Instrumentation and chromatographic conditions:

The Waters Acquity UPLC H-class system (Waters, Milford, USA) includes an autosampler, quaternary gradient pump, photodiode array detector (DAD), column oven connected to a multi-instrument, along with analytical balance (Mettler Toledo, XP 504), pH meter, and data acquisition and processing system with Empower 3 version (Mettler Toledo). The optimized Chromatographic. condition as shown in Table 1.

Table 1: Chromatographic condition

Parameters	Conditions
Column	Acquity UPLC HSS T3 C18 (50 × 2.1 mm, 1.8 μm)
Mobile phase	Ortho Phosphoric Acid (pH 3.00): Acetonitrile (55:45 % V/V)
Flow Rate	0.2 mL/min
Wavelength	222 nm
Injection Volume	2 μL
Column-temperature	35 °C
Run time	3 min
Diluent	Water: Acetonitrile (50:50 % V/V)

Preparation of standard solution and sample solution:

Standards:

Individual standard stock solutions of DAPA 100μg/ml and MET 1000μg/ml in water: acetonitrile (50:50% v/v) at appropriate dilution were finally used to prepare DAPA 10μg/ml and MET 500μg/ml from stock solutions.

Sample preparation procedure: Accurately weigh and transfer 20 intact tablets into a 200ml volumetric flask. Add 70% of diluents and sonicate. Dilute up to. the mark. with diluents. and mix well. Filter the solution through a 0.45µm Nylon filter, discarding the first few mL of the filtrate. Dilute 10ml of the filtrate to 100ml with diluents. Further, dilute 1ml of the above solution to 10ml and mix well and sample concentration of 10 μg/ml Dapagliflozin Propanediol Monohydrate and 500 μg/ml Metformin Hydrochloride analyzed for assay.

METHOD VALIDATION:

System Suitability:

To ensure the validity of the analytical procedure, a system suitability test was established. The following parameters like asymmetry factor, resolution (Rs) No. of theoretical plate number (N), and retention time (tR) were analyzed DAPA (10μg/mL) and MET (500μg/mL) injecting five times into the UPLC system.

Linearity:

The calibration curve solution was prepared by using dilution in the concentration range of 5-15μg/mL for DAPA and 250- 750μg/mL for MET.

Specificity:

The specificity of the method was ascertained by analyzing diluent, placebo, standard and tablet formulations to examine the % interference of excipients and their impurities with the analytes peak.

Accuracy:

The accuracy was prepared by spiking 3 concentration levels 50, 100, and 150% of the targeted concentration of the analytes. At each level, three replicates were performed and the results were recorded. The accuracy was revealed as the percent analyte recovered by the proposed method.

Precision:

The intra-day and inter-day precision of the method was carried out by analyzing the sample solution in three sets each of 50%, 100%, and 150% on the same day and 3 different days, at 5, 10, and 15μg/mL and 250, 500 and 750μg/mL for DAPA and MET respectively.

Robustness:

The robustness of the proposed method was determined by analysis of the sample, during which Change in wavelength (222nm±1), Temperature (35±5), Mobile phase composition (±1% for Buffer: Acetonitrile) and Flow rate (0.2ml/min ±0.1) were changed, and the retention times, peak areas and theoretical plates were observed.

RESULTS AND DISCUSSIONS:

Optimized UPLC conditions:

RP-UPLC method of chromatographic conditions was optimized with system suitability parameters. Acceptable separation of DAPA and MET drug with less run time was achieved on Acquity UPLC HSS T3 C18 (50 × 2.1mm,1μm) column. Acetonitrile, Water, Methanol, Heptanes sulfonic acid, and Otho Phosphoric Acid in various ratios of mobile phase at different compositions and pH and different flow rates were investigated. After trials, an appropriate result was achieved using the mobile phase ortho Phosphoric acid (pH 3.00): Acetonitrile (55:45 %V/V). The chromatogram was recorded at 222nm. The DAPA and MET observed retention times were 0.845 and 1.889 min at a flow rate of 0.2mL/min, respectively. The analyte both peaks were well separated and tailing <2 for both the analytes which are shown in Figure3.

Method validation:

The proposed method was validated concerning various parameters including linearity, accuracy, precision, specificity, and robustness according to ICH Q2(R1) guidelines.²⁹

Figure 3: Chromatogram of DAPA and MET standard

System suitability:

The drug concentration prepared for the system suitability test was DAPA (10μg/mL) and MET (500 μg/mL). The system suitability parameters of the RP-UPLC method include tailing factor (T), resolution (Rs), and RSD in terms of area and No. of theoretical plate count (N) were results obtained for DAPA and MET were acceptable shown in Table 2.

Table 2: Results of the System Suitability Test

Parameters	Observation		Specification
Parameters	MET	DAPA	Specification
Repeatability	0.23	0.18	RSD ≤ 1%
Resolution (RS)	-	5.9	RS > 2
Tailing Factor (T)	1.1	1.1	T ≤ 2
Theoretical Plates (N)	7158	9214	≥2000

Linearity:

The correlation coefficients and regression equations for DAPA and MET were found to be R²=0.9987, y = 5,921.2586x+1,477.0138 and R²=0.9998, y=15,253.8838x+22,529.1034 respectively. The calibration curve of DAPA and MET are shown in Figure 4.

Figure 4: Calibration curve of DAPA and MET

Specificity:

The % interference was found to be DAPA (0.088 %) and MET (0.107%), which is less than 0.5 %, thus making specificity to be acceptable. It was found by comparing chromatograms of blank, placebo, sample solution, and standard solution allow that there was no % interference of excipients with DAPA and MET peak in Figure 5.

Diluent

Placebo

DAPA and MET standard

DAPA and MET sample.

Figure 5 Chromatograms of (A) Diluent, (B) Placebo, (C) DAPA and MET standard, and (D) DAPA and MET sample.

Accuracy:

The percentage recovery for DAPA was found to be within 98.77 %-99.57 % and that for MET was found to be in the range of 99.15 %-101.21 %. Thus, the % recovery of both drugs is acceptable as shown in Table 3.

Table 3: Accuracy of DAPA and MET determination

Level (%)	Amount added μg/mL (n = 3)		% Recovery (n = 3)
Level (%)	DAPA	MET	DAPA	MET
50	5	250	99.57	101.2
100	10	500	99.46	100.16
150	15	750	98.77	99.15

Precision:

Intraday and interday precision was performed using solutions preparing DAPA and MET mixed as 5 + 250, 10 + 500, and 15 + 750 μg/mL, respectively. The samples were analyzed on 3 different days for interday precision and 3 times within the same day for intraday precision. The peak area was determined, and the intraday and interday precision of RSD was found less than 2%. Data of RSD results are shown in Table 4.

Table 4: Precision data for DAPA and MET

Precision test		Intraday precision	Interday precision
Drug	% Level (n=3)	RSD	RSD
DAPA	50	0.69	0.63
	100	0.33	0.69
	150	0.67	0.71
MET	50	1.21	1.32
MET	100	1.09	0.92
	150	0.52	0.51

Robustness:

The robustness of the suggested method was confirmed by performing the analysis with modifications to the flow rate, mobile phase composition, and detection wavelength and temperature. The relative standard deviation of the peak area was found to be <2%, indicating that the proposed method is robust. Results remained unaffected by small changes in these parameters.

Assay of Tablet Dosage form:

The purity of Xigduo® XR tablets was calculated and the result was found to be acceptable. These assay results are given in Table 5.

Table 5: Results of market formulation assay

Drug

Label claim (mg)

Amount Found

(mg/Tablet)

% Assay (n=3)

Dapagliflozin Propanediol Monohydrate

9.95

99.57

Metformin Hydrochloride

500

499.86

99.97

CONCLUSION:

A convenient, rapid, accurate, precise, and robust diode array detector-based RP-UPLC method was developed and validated for the simultaneous determination of dapagliflozin propanediol monohydrate (DAPA) and metformin hydrochloride (MET) in the tablet dosage form. The method was shown to be better than the previously described methods. In comparison to standard HPLC, RP- UPLC reduces sample run times, consumes less solvent, and considerably boosts lab productivity. The method was fully validated, and the results for all the parameters examined were acceptable. The technique is appropriate for figuring out how much of these medications are in tablets, therefore it may be used for regular quality checks on DAPA and MET in this dose form.

ACKNOWLEDGMENTS:

The authors are thankful to Piramal Pharma Ltd. For providing samples and laboratory facilities for this research work.

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