INTRODUCTION:

Dapagliflozin, chemically known as (2S)-propane-1,2-diol(2S,3R,4R,5S,6R)-2-[4-chloro-3- [(4-ethoxyphenyl) methyl) phenyl]-6- (hydroxymethyl) oxane-3,4,5-triol hydrate, is shown in Fig. 1. Dapagliflozin is a sodium-glucose cotransporate inhibitor used in type-2 diabetes mellitus. Dapagliflozin enhance glycaemic control in patients with type-2 diabetes mellitus by decreasing renal glucose reabsorption leading to urinary glucose excretion^1,2

Purposeful force degradation studies can be a useful tool to predict the stability of a drug or dosage form with effects on purity, potency, and safety under various environmental conditions^3,4.

Figure 1 Chemical structure of dapagliflozin propanediol monohydrate

Stress studies should be done in different pH solutions, in the presence of oxygen and light, and at elevated temperatures and humidity levels to determine the stability of the drug substance⁵. It involves the exposure of representative samples of drug or dosage form to the relevant stress conditions of, acid/base hydrolysis, photolytic, oxidation and thermal^5,6. Stability indicating assay method can be developed by generating degraded samples for testing selectivity of method, determining limit of quantification threshold for degraded product according to ICH Q3B^6,7.

The extensive literature survey revealed that the drug is not official in pharmacopoeia, there were few analytical methods reported such as LC-MS/MS in rat plasma⁸, UV spectroscopy^9-11, RP-HPLC^12-18 in single and RP-UPLC in combination with saxagliptin¹⁹, stability indicating RP-HPLC method for estimation of single drug Dapagliflozin²⁰. This method required greater run time compared to UPLC method, degradation was carried out for dapagliflozin but not for dapagliflozin propanediol monohydrate (DPM) which is available in market and the linearity data are mismatched in abstract and in paper. Also DPM in combination with other drugs^21,22 reported. In these articles degradation was carried out for all conditions exceptphotolytic. And thermal degradation shows no degradation and alkali degradation shows only 0.5% which is inadequate stress study. Degradation chromatograms show no generated degradant peak. The percentage degradation carried out is not sufficient as it should be between 5-20%²³. In this HPLC with stress degradation of Dapagliflozin % degradation is excessive for Acidic, Basic, Oxidation, Thermal, and Photolytic and doesn’t give the complete data of peak purity test of drug. Photodegradation was carried out in sun light but it is batter to have study in expose to UV chamber with specified Lux of UV light²⁴.

From 3^21,22,23 research articles ambiguity is created because of different degradation behaviour shown by all authors for same molecule. Thus it is worthwhile to develop simple, rapid, and green chemistry based stability indicating assay method for dapagliflozin propanediol monohydrate by RP-UPLC.

According to current good manufacturing practice, the drug must be release after tested with stability indicating assay method. (International Conference of Harmonisation, 2005). Stability study was carried out in various force degradation conditions like acid hydrolysis,” base hydrolysis, oxidative, thermolytic and photolytic by applying stress. The developed RP-UPLC PDA method was validated as per ICH Q2 (R1) guideline²⁵.

MATERIAL AND METHOD:

Chemicals and reagents:

Reference standard of Dapagliflozin Propanediol Monohydrate, was given by Piramal Healthcare Ltd, Ahmedabad. Forxiga tablet (containing 5mg of dapagliflozin) was used for method application. The HPLC grade solvents used were Water, Acetonitrile, methanol and Hydrochloric acid, sodium hydroxide and Hydrogen peroxide were of analytical grade.

Equipment and chromatographic conditions:

The UPLC system consisting of Acquity UPLC-H Class (Waters, Milford, USA) equipped with quaternary pumps, online degasser module, Photo diode array detector (set at 223nm), column temperature control compartment and auto sampler integrated with Empower 2 Software. Chromatographic separation was optimized on Bridge Ethylene Hybrid (BEH) C18 analytical column (50 × 2.1mm, 1.7µm) at 30ºC. The ratio of mobile phase was 60:40 v/v of water and acetonitrile, with a flow rate of 0.5ml/minand injection volume was 3µl.

Preparation of Stock and Standard solution:

A standard stock solution of DPM was freshly prepared to obtained the concentration of 200µg/ml. The Solution was filtered through a 0.22µm Polyvinylidene fluoride (PVDF) filter before it analysed.

Tablet Sample Solution:

Weighed the 20 tablets of Forxiga (dapagliflozin 5mg) and the average weight were calculated. The tablets were finely powdered and quantities of the powder equivalent to two tablets were transferred into a 50ml volumetric flask. 35ml of the mobile phase was added to it and sonicated for 10 minutes, then the volume was made upto the mark with the mobile phase and mixed well to prepare the sample stock solution. 5ml of the filtrate were diluted to 20ml to obtain concentration equal to 50µg/mlof DPM. This solution was passing it through a 0.22µm PVDF filter. Triplicate samples were analysed.

Degradation Study:

The hydrolysis degradation study was performed at room temperature using 0.1 N HCl (75 hours), 0.1 N NaOH (24 hours) then the samples were neutralized with appropriate solvent and the solution prepared to get concentration of 50µg/ml. For oxidative degradation study 3% H₂O₂(6 hours) used. For thermal and photolytic degradation, samples were kept at 60⁰C (1 hour) and 1 ICH cycle in photolytic chamber respectively.

Method Validation:

Linearity:

Linear calibration plot of the present method was obtained over concentration range of 25-75µg/ml(25, 40, 50, 60 and 75µg/ml) for DPM.

Accuracy:

The accuracy of developed method was determined by spiking API in placebo. Sample (100% concentration) of DPM (50µg/ml) was prepared. Three levels of samples were prepared corresponding to 50%, 100%, and 150% of nominal concentration and analyzed by the developed method.

Precision:

Repeatability of the proposed method was evaluated by performing six independent test sample preparation. Intra-day precision was evaluated by injecting 3 different concentrations (25, 50 and 75µg/ml) of DPM on the same day. Inter-day precision was determined by injecting the same solutions for three consecutive days. The results were presented in the form of RSD.

Specificity:

Interference with the active ingredient was evaluated by injecting the diluent, placebo and sample solution individually into UPLC as per proposed method.

Limit of detection (LOD) and Limit of quantification (LOQ):

It was determined on the basis of standard deviation of the response and slope.

Robustness:

The robustness of method was established by introducing small change in experimental condition like mobile phase composition, flow rate and temperature. The robustness of method was evaluated by calculating RSD of peak area.

Estimation in tablet dosage form:

Tablet was analysed by the developed analytical method.

RESULTS AND DISCUSSION:

Optimization of chromatographic condition:

In RP-UPLC analysis the stationary phase selection depends on the properties and chemical structure of the target drug. Due to high carbon content and nature ^{of DPM can be separated through BEH
C}18 ^{column. The}process of ^methoddevelopment was begin with different ratios of water and acetonitrile. Symmetrical and sharp peaks of DPM were obtained with water: acetonitrile (60: 40, v/v) ^{on C}18 ^{columns at}retention time of 0.77 min^.The chromatographic data under the mentioned condition were presented in Fig. 2.

Fig. 2 Sample chromatogram of DPM

Force degradation:

All the stress conditions were applied which was enough to degrade the drug. As compared to the acid, base and oxidative condition DPM is more stable in thermal and photolytic stress. Under acidic condition DPM was degraded up to 8.4%. Under basic stress DPM was degraded up to 12.31%. Under oxidative stress DPM was degraded up to 10.91%. Under thermal and photolytic stress DPM was degraded up to 5.94% and 5.25%, respectively. Information regarding degradation has been given in Fig. 3. Results of stress testing have been given in table 1.

Fig. 3 Chromatograms of (A) DPM under acid degradation, (B) DPM under base degradation, (C) DPM under oxidative degradation, (D) DPM under thermal degradation, (E) DPM under photolytic degradation

Method Validation:

The developed chromatographic method was validated according to ICH Q2(R1) guideline.

Linearity:

Linear calibration plot for the proposed method was obtained in concentration range of 25-75 µg/ml(25, 40, 50, 60 and 75 µg/ml) (fig.5). The linearity equation for DPM was found to be y = 21457x + 36465 with correlation coefficient greater than 0.9977. Overlain chromatogram of DPM is demonstrated in Fig. 4.

Fig. 4 Overlain Chromatogram of linearity

Fig. 5 Linearity curve of Dapagliflozin

Table 2: Regression and Optical characteristics of DPM

Parameters	Value For DPM
Beer’s law limit (µg/ml)	25-75
Correlation Coefficient (r)	0.9977
Slope	21457
Intercept	36465

Specificity:

No interference was found at Rt of analyte.

Accuracy:

Three concentration levels (50%, 100% and 150%) of solution were prepared and analysed by the developed method. Recovery studies exhibit the method to be highly accurate and suitable for intended use. Percentage recoveries of analyte with relative standard deviations are tabulated in Table 3.

Table 3: Accuracy of proposed RP-UPLC PDA method

Conc. Level	Amount added (µg/ml)	Area Observed ±% Recovery	SD	% RSD
50%	25	560060 ±101.16	0.7	0.69
100%	50	1116203 ±100.81	0.1	0.1
150%	75	1639389 ±98.71	0.03	0.03

N= average of 3 analyses; SD= Standard Deviation ; RSD = Relative Standard Deviation

Precision:

Repeatability study was determined by injecting six standard solutions of same concentration. Results of repeatability are presented in Table 4.

To represent precision, relative standard deviation (RSD < 2%) of the peak area was calculated. Results of intra-day and inter-day precision presented in Table 5.

Table 4: Repeatability study of proposed RP-UPLC PDA method.

S. No.	Area	Mean	SD	%RSD
1	1104411	1101587	2518.25	0.22
2	1100221
3	1100341
4	1102088
5	1098082
6	1104376

SD= Standard Deviation; RSD = Relative Standard Deviation

Table 5: Intra- and Inter-day precision of proposed RP-UPLC PDA method.

Conc. (µg/ml)	Intra day		Inter day
Conc. (µg/ml)	Area ± SD	% RSD	Area ± SD	% RSD
25	560679±3187.65	0.56	561083±2407.59	0.42
50	1116253±795.49	0.07	1112469±4098.07	0.36
75	1626303±400.42	0.02	1626555±2221.54	0.13

n = average of 3 analyses; RSD = Relative Standard Deviation

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

LOD and LOQ of DPM were determined using average of slope and standard deviation of intercepts. LOD was found to be 0.42 μg/ml. LOQ was found to be 1.42μg/ml.

Robustness:

The results showed that by making a slight variation in chromatographic conditions, RSD was found to be < 2%. Thus the method is robust. The result was represented in Table 6.

Assay:

Application of the developed method was examined by analysing the DPM in commercially available tablet dosage form. The assay was found to be 98.5%.

CONCLUSION:

A rapid stability indicating RP-UPLC PDA method for estimation of DPM was developed and validated according to ICH guideline. Force degradation studies were conducted at five different conditions. In acid, base and oxidative condition percentage degradation were found 8.4%, 12.31% and 10.91% respectively. During thermal and photo degradation conditions the drug was stable as compare to other degradation condition. The order of stability is photo, thermal, acidic, oxidative and basic. Force degradation studies confirmed to its stability because no excipient, and impurity interfered with the active pharmaceutical ingredient. The method was found to be specific, precise, accurate, reproducible and robust. Hence the developed RP-UPLC assay method can be used for the routine analysis of Dapagliflozin propanediol monohydrate in tablet dosage form.

ACKNOWLEDGEMENT:

The authors are thankful to Piramal Healthcare Ltd., Ahmedabad, for providing necessary facilities to carry out the research work.

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Received on 25.05.2020 Modified on 17.08.2020

Research J. Pharm. and Tech. 2021; 14(9):4635-4639.

DOI: 10.52711/0974-360X.2021.00805

Types of degradation	Degradation Condition	Purity Angle	Purity Threshold	%
Types of degradation	Degradation Condition	Purity Angle	Purity Threshold	Degradation
Acid	0.1 N HCl (75 h)	0.235	0.466	8.4
Base	0.1 N NaOH (24 h)	0.144	0.428	12.31
Oxidation	3% H₂O₂(6 h)	0.234	0.472	10.91
Thermal	60 ⁰C (1 h)	0.21	0.45	5.94
Photolytic	1.2 million lux (168 h)	0.213	0.483	5.25

Conc. (50 µg/ml)	Flow rate(ml/min)		Temperature(ºC)		Mobile phase
Conc. (50 µg/ml)	0.45	0.55	25	35	55:45:00	65:35:00
Area	1163129	914985	1024955	1028562	978019	1069005
SD	7626.14	1955.7	16239.27	17373.24	9691.26	4641.04
RSD	0.65	0.21	1.58	1.68	0.99	0.43