A Validated RP-HPLC Method for Simultaneous Estimation of Dapagliflozin and Saxagliptin in Bulk Drug and Pharmaceutical Formulations

Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor used as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Common side effects include yeast infections of the vagina or penis, urinary tract infections, and changes in urination, including urgent need to urinate more often, in larger amounts, or at night.

Fig.no: 01 Chemical structure of Dapagliflozin

It is chemically (2S,3R,4R,5S,6R)-2-[4-chloro-3-[(4-ethoxy phenyl)methyl] phenyl]-6-(hydroxymethyl) oxane-3,4,5-triol. The molecular formula is C21H25ClO6. The molecular weight is 408.873 g/mol. Dapagliflozin drug work by targeting and helping to stop sodium-glucose transport proteins from allowing glucose that has been filtered out of the blood by the kidneys to be reabsorbed back into the blood. The SGLT2 proteins are responsible for 90% of the glucose that is reabsorbed into the blood. Dapagliflozin is also used alone or in combination in adults for the treatment of type II diabetes. Dapagliflozin is a relatively new medication, the long term effects of the drug, over several years, are as yet unknown. Literature review shows minimal methods has been developed and reported for Dapagliflozin estimation in biological fluids and there are some methods reported by spectroscopy, HPTLC HPLC, UPLC and capillary electrophoresis. Two methods were reported for estimation of Dapagliflozin first is UV spectroscopy and the other is HPTLC method. Method development of HPLC estimation for Dapagliflozin is new method will fulfil all requirements of validation according to ICH guidelines.

Fig.no: 02 Chemical structure of Dapagliflozin

Saxagliptin , is a new oral hypoglycemic (anti-diabetic drug) of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. Early development was solely by Bristol-Myers Squibb; in 2007 AstraZeneca joined with Bristol-Myers Squibb to co-develop the final compound and collaborate on the marketing of the drug. In June 2008, it was announced that Onglyza would be the trade name under which Saxagliptin will be marketed. Molecular formula of Saxagliptin is C18H25N3O2. The IUPAC name is 1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl) acetyl]-2- azabicyclo [3.1.0]hexane-3- carbonitrile and Average Molar mass is 315.41 g/mol (Cole et al., 2008). Saxagliptin is mostly used for antidiabetic, it is selective and competitive, cyanopyrrolidine-based, orally administration and is metabolized into a less potent, active mono-hydroxy metabolite (5-hydroxy saxagliptin) Saxagliptin is available as a 2.5mg and 5mg tablet and orally administered once a day.

2. MATERIALS AND METHODS

Dapagliflozin and Saxagliptin pure drugs (API), Combination Dapagliflozin and Saxagliptin tablets (QTERN), Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dehydrogenate ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.

1. 1. HPLC Instrumentation and chromatographic conditions

HPLC system of WATERS HPLC 2695 equipped with quaternary pumps, Photo Diode Array detector and Auto sampler integrated with Empower 2 Software was used.For data recording the LC-solution software used. UV-VIS spectrophotometer PG Instruments T60 with special bandwidth of 2 mm and 10mm and matched quartz cells integrated with UV win 6 Software was used for measuring absorbances of Dapagliflozin and Saxagliptin solutions.

1. 2. Preparation of mobile phase

 Based up on the solubility of the drugs, diluent was selected, Acetonitrile and Water taken in the ratio of 50:50

2.2.1 Preparation of Standard stock solutions: Accurately weighed 10 mg of Dapagliflozin, 5 mg of Saxagliptin and transferred to individual 10 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labeled as Standard stock solution 1and 2. (1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin)

2.2.2 Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (100µg/ml Dapagliflozin of and 50µg/ml of Saxagliptin)

2.2.3 Preparation of Sample stock solutions: 5 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 10 ml volumetric flask, 5ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin)

2.2.4 Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (100µg/ml of Dapagliflozin and 50µg/ml of Saxagliptin )

2.3 Method Validation

The present method was preceded to obtain new, sensitive and easy method for simultaneous estimation by HPLC from capsule formulation. According to the ICH guidelines recommendations the experimental was validated and USP-30 for parameters such as, system suitability, accuracy, precision, linearity and specificity.

2.3.1 System suitability

The system suitability parameters were determined by preparing standard solutions of Dapagliflozin (100ppm) and Saxagliptin (50ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The % RSD for the area of six standard injections results should not be more than 2%.

2.3.2 Precision

Preparation of Standard stock solutions: Accurately weighed 10 mg of Dapagliflozin, 5 mg of Saxagliptin and transferred to individual 10 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labelled as Standard stock solution 1and 2. (1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin)

Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (100µg/ml Dapagliflozin of and 50µg/ml of Saxagliptin)

Preparation of Sample stock solutions: 5 tablets were weighed and the average weight of  each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 10 ml volumetric flask, 5ml of diluents was  added and sonicated for 25 min, further the volume was  made up with diluent and filtered by HPLC filters(1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin )

Preparation of Sample working solutions (100% solution): 1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (100µg/ml of Dapagliflozin and 50µg/ml of Saxagliptin )

2.3.3 Linearity:

Preparation of Standard stock solutions: Accurately weighed 10 mg of Dapagliflozin, 5 mg of Saxagliptin and transferred to individual 10 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labelled as Standard stock solution 1and 2. (1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin )

2.3.4 Accuracy:

Preparation of Standard stock solutions: Accurately weighed 10 mg of Dapagliflozin, 5 mg of Saxagliptin and transferred to individual 10 ml volumetric flasks separately. 3/4 th of diluents was added to both of these flasks and sonicated for 10 minutes. Flasks were made up with diluents and labelled as Standard stock solution 1and 2. (1000µg/ml of Dapagliflozin and 500µg/ml of Saxagliptin)

Robustness: Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there was no recognized change in the result and are within range as per ICH Guide lines.

2.3.5 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 0.1ml each of Dapagliflozin and Saxagliptin solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents

2.3.6 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 0.3ml each of Dapagliflozin and Saxagliptin solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent.

2.3.7 Degradation studies:

2.3.7.a Oxidation:

To 1 ml of stock solution of Dapagliflozin and Saxagliptin, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 60⁰c. For HPLC study, the resultant solution was diluted to obtain 100µg/ml&50µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.3.7.b Acid Degradation Studies:

To 1 ml of stock s solution Dapagliflozin and Saxagliptin, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 60⁰c .The resultant solution was diluted to obtain 100µg/ml&50µg/ml solution and 10 µl solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.3.7.c Alkali Degradation Studies:

To 1 ml of stock solution Dapagliflozin and Saxagliptin, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60⁰c. The resultant solution was diluted to obtain 100µg/ml&50µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.3.7.d Dry Heat Degradation Studies:

The standard drug solution was placed in oven at 105°C for 1 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100µg/ml&50µg/ml solution and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

2.3.7.e Photo Stability studies:

The photochemical stability of the drug was also studied by exposing the 1000µg/ml&500µg/ml solution to UV Light by keeping the beaker in UV Chamber for 1days or 200Watt hours/m² in photo stability chamber^. For HPLC study, the resultant solution was diluted to obtain 100µg/ml&50µg/ml solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

2.3.7.f Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 1hrs at a temperature of 60º. For HPLC study, the resultant solution was diluted to 100µg/ml&50µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

3. RESULTS AND DISCUSSIONS

As per ICH Guidelines In this trail all system suitability parameters were passed so this method was optimized. Saxagliptin and Dapagliflozin were eluted at 2.218 min and 2.788 min respectively with good resolution. Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated.

Fig.no: 03 Chromatographic conditions of Dapagliflozin and Saxagliptin

Fig.no: 04 System suitability Chromatogram

Fig.no: 05 Calibration curve of Dapagliflozin

System 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 0.4% and 0.2% respectively for Saxagliptin and Dapagliflozin. As the limit of Precision was less than “2” the system precision was passed in this method.

Table no: 03 System precision table of Saxagliptin and Dapagliflozin

Fig.no: 07 System precision chromatogram Saxagliptin and Dapagliflozin

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 99.87% and 99.97% for Saxagliptin and Dapagliflozin respectively.

Table no: 04 Accuracy table of Saxagliptin

Table no: 05 Accuracy table of Dapagliflozin

Table no: 06 Sensitivity table of Sitagliptin and Dapagliflozin

Fig.no: 09 LOD Chromatogram of Standard              Fig.no: 10 LOQ Chromatogram of Standard