AN OVERVIEW ON ANALYTICAL METHODS FOR DAPAGLIFLOZIN – AN ANTIDIABETIC DRUG

AN OVERVIEW ON ANALYTICAL METHODS FOR DAPAGLIFLOZIN - AN ANTIDIABETIC DRUG

B. V. Suma ^{* 1} and R. Deveswaran ²

Department of Pharmaceutical Chemistry ¹, Department of Pharmaceutics & Drug Design and Development Centre ², Faculty of Pharmacy, M. S. Ramaiah University of Applied Sciences, Bangalore - 560054, Karnataka, India.

ABSTRACT: Dapagliflozin (DAPA) is a drug of gliflozin class. It is a white crystalline solid. Dapagliflozin is a selective sodium-glucose co-transporter subtype 2 (SGLT2) inhibitor with antihyperglycemic activity. Dapagliflozin blocks glucose reabsorption into the kidney, resulting in the elimination of blood glucose through the urine. DAPA is not official in any pharmacopeia. It comes under EU Pharmaceutical Product Classes, as per literature survey. Till date officially no analytical method has been developed or reported for this drug. This review focuses on collective literature on the analytical methods developed for DAPA.

Dapagliflozin, Titrimetry, UV, HPLC, LC-MS

INTRODUCTION: Dapagliflozin (DAPA) is a drug of gliflozin class. Chemically it is (2S, 3R, 4R, 5S, 6R)- 2- (4- chloro- 3- (4-methoxybenzyl) phenyl)-6- (hydroxymethyl)tetrahydro-2H-pyran-3, 4,5-triol with molecular formula of C₂₁H₂₅ClO₆ and 408.875g/ mol as its molecular weight. It is a white crystalline solid, having solubility in organic solvents like DMSO dimethylformamide and ethanol. Its melting point is 55-60 ºC ¹. Dapagliflozin is a selective sodium-glucose co-transporter subtype 2 (SGLT2) inhibitor with antihyperglycaemic activity. Dapagliflozin selectively and potently inhibits SGLT2 compared to SGLT1, which is the co-transporter of glucose in the gut.

Dapagliflozin blocks glucose reabsorption into the kidney, resulting in the elimination of blood glucose through the urine ¹. DAPA is not official in any Pharmacopoeia as of now. Very few analytical methods have been reported so far. The purpose of this review is to collate all those published literature on analytical methods developed for DAPA by various researchers.

FIG. 1: CHEMICAL STRUCTURE OF DAPAGLIFLOZIN

Method of Pharmaceuticals:

Pharmacopoeial Methods: DAPA is not official in any pharmacopeia. It comes under EU Pharmaceutical Product Classes, as per literature survey. So officially no analytical method has been developed for this drug as of now.

Titrimetric Methods: Titrimetric methods have not been reported for this drug.

UV- Spectrophotometric Methods: UV spectrophotometric methods were reported ^{4, 7}. Four different methods based on a spectrophotometric method for the determination of DAPA in tablet formulation are reported. The methods included are calibration curve, Area under the curve, First and second order derivative method. All the developed methods obeyed Beer-Lambert’s Law in the concentration of 5-40 μg/mL, with correlation coefficient value less than 1, when measured at a selected wavelength using UV-Visible spectro-photometer with 1cm matched quartz cell using methanol-water mixture as solvent blank. Nearly 100% of the drug was estimated from these methods when applied to the dosage form.

In the second paper, a simple economic UV spectrophotometric method has been developed using ethanol and water as working solvent. The developed method describes that DAPA in API obeyed Beer’s-Lambert’s Law in the concentration of 0.5-0.9 μg/mL, with correlation coefficient value of 0.994, and LOD and LOQ were found to be 0.0925 μg/mL and 0.00129 μg/mL respectively. Intra-and inter-day precisions were approximately equivalent to 0.5% (relative standard deviation, RSD). The details of UV-spectrophotometric analysis are summarized in Table 1.

TABLE 1: PERFORMANCE CHARACTERISTIC OF UV SPECTROPHOTOMETRIC METHOD

LOD: Limit of detection; LOQ: Limit of Quantification

Chromatographic Methods: In an effort to optimize a quantitative analysis of DAPA in formulation a liquid chromatographic method was developed ⁶, the separation was achieved on Waters C18 column, 5 μm particle size, 25 cm × 4.6 mm, with phosphate buffer and acetonitrile in the ratio of 60:40 v/v as a mobile phase and a flow rate of 1 mL/min with UV detection of 237 nm, showing a retention time of 3.461 min with linearity range concentration of 10-60 μg/mL with correlation coefficient value of 0.995 and LOD and LOQ values of 0.02 μg/mL and 0.06 μg/mL respectively. The method was applied to determine DAPA in tablet with the recovery of 99.4%-100.90%. The method was also shown to be accurate, precise, specific and robust.

A Reverse Phase-HPLC method for estimation of DAPA from its tablet dosage form was developed. Aristocratic mode chromatographic separation was achieved with a mixture of Acetonitrile: 0.1% Triethylamine (pH-5.0) in the ratio of 50:50 v/v as mobile phase using Princeton  C18 column at flow rate of 1mL/min and a detection wavelength of 224 nm, the retention time of drug was found to be 5.163 min. The proposed method obeyed Beer’s-Lambert’s law in the concentration range of 10-70 μg/mL, with correlation coefficient value 0.999 and LOD and LOQ values of 2.1 μg/mL and 6.39 μg/mL respectively. The method was applied to the marketed formulation and shown 100.57% mean value concerning label claim. Stress testing under various conditions such as pH (acid/base), oxidation, temperature, light, humidity, etc. was also carried out ⁸.

DAPA in API was chromatographed using Waters HPLC system, BDS column, a mixture of acetonitrile and orthophosphoric acid (55:45 v/v) was used as mobile phase at a detection wavelength of 245 nm ¹⁰. The proposed method obeyed Beer’s-Lambert’s law in the concentration range of 25-150 μg/mL, with correlation coefficient value 0.999 and LOD and LOQ values of 0.60 μg/mL and 1.81 μg/mL respectively. The authors have also developed a UV spectroscopic method using methanol as solvent at a detection wavelength of 203 nm. The developed UV spectroscopic method showed a linearity range of 1-5 μg/mL with correlation coefficient value 0.999; LOD and LOQ values of 0.01 μg/mL and 0.05 μg/mL respectively. Both the methods were validated as per ICH guidelines.

A reverse phase high performance liquid chromatographic method was developed for the estimation of Dapagliflozin in tablet formulation and bulk drug using Zorbax Eclipse Plus C8 column, (150 × 4.6 mm) using buffer (pH 7.6) Tris: Methanol (60:40 v/v) as a mobile phase at a flow rate of 1 ml/min by employing UV detection at 224 nm, retention time was found to be 1.467 min, with linearity concentration range of 1-32 μg/mL. LOD and LOQ were found to be 0.207 and 0.693 μg/ml respectively. As per ICH guidelines, the method was found to be simple, precise and accurate when validated ².

An RP-HPLC method was developed and validated for the analysis of DAPA in bulk and tablet formulation. The separation has been achieved using C18 column reversed-phase using mobile phase composition of methanol: water (75:25 v/v), utilizing Shimadzu SPD-20A Prominence UV-Visible detector at a flow rate of 1 mL/min and the absorption maxima 230 nm, retention time was found to be 3.107 min for DAPA. The proposed method obeyed Beer’s-Lambert’s law in the concentration range of 5-25 μg/mL, with correlation coefficient value 0.9252 and LOD and LOQ values of 2.5 μg/mL and 10 μg/mL respectively. The method was validated as per the ICH guidelines ⁵.

Few stability indicating analytical method development studies using HPLC were also reported. In a method, separation was effected on a Symmetry C18 column, 25 cm × 4.6 mm i.d. 5μm, particle size column using a mobile phase mixture of Methanol: Acetonitrile: Orthophosphoric acid in a ratio of 75:25:05 v/v/v at a flow rate of 1.0 mL/min. The detection was maintained at 246 nm. DAPA showed the linearity concentration range of 0-70 μg/mL. The LOD and LOQ were found to be 0.04 and 0.12 μg/mL respectively. The retention time of DAPA was found to be 2.797 min. The developed method was found to be simple, precise, accurate, specific, robust and rapid for the estimation of DAPA in bulk and tablet dosage form. This paper also reported the effect of stress conditions on DAPA. The various degradation pathway studied were acid hydrolysis, basic hydrolysis, thermal degradation and oxidative degradation ¹².

An RP-HPLC method has been developed for the DAPA in bulk and pharmaceutical dosage form. Chromatographic separation of DAPA was achieved on Symmetry C18, 250 mm × 4.6 mm i.d.5μm particle size and the mobile phase containing phosphate buffer: methanol in the ratio of 35:65 v/v with the flow rate was 1.0 ml/min, detection was carried out by absorption at 215nm using a UV detector at ambient temperature. The linearity of the proposed method was excellent over the range 0-70 μg/mL with the correlation coefficient of 0.999 with LOD of 0.09 μg/mL and LOQ of 0.36 μg/mL respectively ¹¹.

DAPA in the presence of its degraded products was identified by RP HPLC method. DAPA was subjected to acid, alkali hydrolysis, oxidation, thermal and photolytic degradation. The degradation studies indicated that DAPA was more susceptible to thermal degradation. The separation was achieved using Agilent C18 (4.6 × 150 mm, 5 μm), mixture of acetonitrile: di-potassium hydrogen phosphate with pH-6.5 adjusted with orthophosphoric Acid  (40:60% v/v) as a mobile phase with the flow rate of 1 mL/min and the effluent was monitored at 222 nm using photodiode array detector.

The linearity of the proposed method was excellent over the range 50-150 μg/mL with the correlation coefficient of 0.99 with LOD of 5.14 μg/mL and LOQ of 15.6 μg/mL respectively. The retention time of DAPA was reported as 3.160 min for API and 3.067 for DAPA in tablets ⁹. The details of HPLC analysis were summarized in Table 2 and LC-MS analysis in Table 3. A fully automated high throughput liquid chromatography/ tandem mass spectrometric (LC-MS/MS) method was developed for DAPA using normal and Zucker diabetic fatty rat plasma. The assay was carried out using solid phase extraction and LC-MS/MS analysis in negative ion electrospray ionization mode. The mobile phase used was water and acetonitrile ³.

TABLE 2: PERFORMANCE CHARACTERISTICS OF HPLC METHODS

• LOD: Limit of Detection
• LOQ: Limit of Quantification
• SIAM: Stability Indicating Analytical Method
• API: Active Pharmaceutical Ingredient

TABLE 3: PERFORMANCE CHARACTERISTICS OF LIQUID CHROMATOGRAPHY-MASS SPECTROPHOTO-METRIC (LC-MS/MS) METHODS

CONCLUSION: The review of the analytical methods reported for dapaglifloxin showed that spectrophotometric, HPLC with UV- visible detector have been reported. So far only one paper has been published based on its assay in rat plasma. In general, for the determination of the drugs in biological samples, HPLC-MS/MS is ideal for estimation.

So, it greater option to estimate the dapagliflozin in a biological sample by HPLC MS/MS can be carried out. This review represents an overview of the current analytical methods for the determination of dapagliflozin in an active pharmaceutical ingredient, tablet, and pharmaceutical dosage forms.

ACKNOWLEDGEMENT: The authors are thankful to Dean, Faculty of Pharmacy, Pro-Vice Chancellor, M. S. Ramaiah University of Applied Sciences, Bangalore for their guidance and support.

CONFLICT OF INTEREST: The authors declare no conflict of interest

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    How to cite this article:

    Suma BV and Deveswaran R: An overview on analytical methods for dapagliflozin - an antidiabetic drug. Int J Pharm Sci & Res 2019; 10(6): 2688-92. doi: 10.13040/IJPSR.0975-8232.10(6).2688-92.

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