Simultaneous Estimation and Validation of Dapagliflozin and Saxagliptin in Bulk Drug and Dosage Form by RP-HPLC

 

Rahul K. Godge*, Ganesh S. Shinde, Shraddha Joshi

Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Maharashtra, India

*Corresponding Author E-mail:Rahul_godge@rediffmail.com

 

Abstract:

A simple, rapid, precise, accurate RP-HPLC method has been developed and validated to estimate Saxagliptin hydrochloride and Dapagliflozin in bulk and in dosage form. The samples were eluated Grace C18 (250mm x 4.6 ID, Particle size: 5micron with mobile phase Methanol: water (pH adjusted to 3 with potassium dihydrogen orthophosphate) (80:20) at wavelength 225nm. A good linear response was obtained in the range from 10-50μg/mL for Saxagliptin hydrochloride and Dapagliflozin. The method was quantitatively evaluated in terms of linearity, precision, accuracy (recovery), selectivity and robustness as per USP guideline.

 

KEY WORDS: Saxagliptin, Dapagliflozin, RP-HPLC.

 

 


INTRODUCTION:

Type 2 diabetes mellitus (T2DM) is a chronic progressive metabolic disorder characterized by absolute or relative insulin deficiency. Saxagliptin is chemically known as (1S, 3S, 5S)-2[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]2azabicyclohexane-3-carbonitrile) with molecular formula of C18H25N3O2 and molecular weight of 315.41g/mol. Saxagliptin is a selective and potent dipeptidylpeptidase(DPP)-4inhibitor, approved as an adjunct to diet and exercise to improve glycemic control in type2 diabetes mellitus (T2DM). In patients with T2DM, Dapagliflozin is chemically known as (1s)-1, 5-anhydro-1-C-[4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl]-D-glucitol. It has a molecular formula of C24H33ClO8 with molecular weight 408.98. Dapagliflozin is selective Sodium Glucose Co Transporter 2 inhibitor (SGLT2). It acts by reducing the reabsorption of glucose by the kidney, leading to excretion of excess glucose in the urine, there by improving glycemic control in patients with type 2 diabetes mellitus. Though several methods are reported in literature for the estimation of Saxagliptine and Dapagliflozin individually, no methods are reported for estimation of Saxagliptine and Dapagliflozin in combination. The objective of the present study is to develop a novel, simple, accurate, precise, economic method for the simultaneous estimation of Saxagliptine and Dapagliflozin and validate according to ICH guidelines.

 

Chemical Structure of Dapagliflozin Chemical Structure of Saxagliptin

EXPERIMENTAL:

Reagents and Chemicals:

Methanol (HPLC grade) and was purchased from Merck Specialties Pvt. Ltd. (Worli, Mumbai, India) and Water (HPLC grade) was purchased from Loba Chemie (Mumbai, India). All other reagents used were of HPLC grade. Working standard of Saxagliptin and Dapagliflozin were provided by Swapnroop Drug and Chemicals, Aurangabad India.

 

Pharmaceutical formulation:

Commercial tablets, each containing Saxagliptin and Dapagliflozin (Qtern); were procured from the local market.

 

Method Development:

Different mobile phases containing methanol, water, Acetonitrile, and different buffers in different proportion were tried and finally of Methanol: Water (pH 3 with potassium dihydrogen ortho phosphate) in the ratio 80: 20% V/V was selected as mobile phase which gave good resolution and acceptable peak parameters for both Saxagliptin and Dapagliflozin.

 

System Suitability Studies:

The resolution, number of theoretical plates and peak asymmetry were calculated for the standard solutions and is as shown in Table 1. The values obtained demonstrated the suitability of the system for the analysis of these drugs in combinations. The typical chromatogram of standard solution is as shown in Fig.1.

 

Apparatus and chromatographic Conditions:

Chromatographic separation was performed on a HPLC binary gradient system and 20l sample injection loop. The output signal was monitored and integrated using HPLC workstation software was used for the separation; mobile phase of a mixture of Methanol and water was delivered at a flow rate of 0.9 ml/min with detection at 225nm. The mobile phase was filtered through a 0.2 membrane filter and degassed. The injection volume was 20 l; analysis was performed at ambient temperature.

 

Table 1: System Suitability Studies

Sr. No.

Parameter

Method

1

Stationary phase (column

Grace C18(250mmx 4.6ID)

2

Mobile phase

Methanol: Water pH 3 with potassium dihydrogen ortho phosphate

3

Flow rate (ml / min)

0.9ml

4

column temperature ( 0C)

Ambient

5

Volume of injection (l)

20

6

Detection wavelength (nm)

225nm

 

Table 2: Recovery Studies of Saxagliptin and Dapagliflozin:

Level of % Recovery

% Mean Recovery

% R.S.D.

Saxagliptin

Dapagliflozin

Saxagliptin

Dapagliflozin

50

99.63

99.65

0.06993

0.3388

100

99.56

99.98

0.04553

0.4066

150

99.85

99.87

0.0766

0.28283

 

Preparation of Standard Solutions:

Accurately weighed 10mg of saxagliptin and Dapagliflozin was added in volumetric flask containing some amount of mobile phase and volume was made upto the mark using mobile phase. The resulting solution was filtered through 0.45 membrane filter and sonicated for three cycle seach of 10min. From the stock solution 1.0ml of stock was pipette out in triplicate and kept in three different volumetric flasks, cleaned previously and diluted upto10ml by using mobile phase to obtain resultant solution of 10g/ml. This solution was injected for given chromatographic system in triplicate and mean area was determined.

 

Assay:

Preparation of Sample Solutions:

Twenty Tablets were weighed and finely powdered. A quantity of powder equivalent to 10mg of Saxagliptin was weighed and transferred to 50 ml volumetric flask. To this, methanol was added and sonicated for 10min; the volume was made up to 50 ml with methanol to get solution of 100 g/ml. The solution was filtered using Whatman filter paper. From the filtrate appropriate dilutions were made to obtain concentration in the range of 10 to 50g/ml for both saxagliptin and Dapagliflozin respectively.

 

With the optimized chromatographic conditions, a steady baseline was recorded, the mixed standard solution was injected and the chromatogram was recorded. The retention time of saxagliptin and dapagliflozin were 3.999 and 5.387 min respectively. This procedure was repeated for the sample solution obtained from the formulation. The proposed method was found to be specific and no interference from common tablet excipents like lactose, starch etc was observed. The assay was calculated from the equation of regression line for each drug. The percentage of individual drugs found in formulations was calculated and presented in table 2. The results of analysis shows that the amounts of drugs were in good agreement with the label claim of the formulations.

 

Method Validation:

As per the USP guidelines, the method validation parameters checked were linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.

 

Linearity and Range:

The linearity of the method was determined for the formulation at five concentration levels ranging from 10 to 50g/ml for both saxagliptin and Dapagliflozin respectively. The equation for regression line for Saxagliptin was y =18121x + 96224 (R2 = 0.9955) and For Dapagliflozin y =44679x -17965 (R2 = 0.998). The results show that an excellent correlation exists between Peak area and concentration of drugs within the concentration range indicated above.

 

 

Figure-1: Chromatogram of Saxagliptin (3.999min), Dapagliflozin 5.387min respectively.

 

Table3:Intraday and Interday Precision Studies System precision)

Conc. (g/ml)

 

Saxagliptin

Conc. (g/ml)

Dapagliflozin

% RSD

% RSD

Intra- day

Inter- day

Intra- day

Inter- day

10

0.16

0.25

10

0.53

0.62

20

0.29

0.30

20

0.69

0.72

30

0.24

0.27

30

0.31

0.39

40

0.31

0.25

40

0.92

0.7

50

0.30

0.37

50

0.13

0.2

RSD=Relative Standard Deviation(n=3).

 

Table4: Intraday and Interday Precision Studies (Method precision)

Conc. (g/ml)

 

Saxagliptin

Conc. (g/ml)

 

Dapagliflozin

% RSD

% RSD

Intra- day

Inter- day

Intra- day

Inter- day

20

0.36

0.52

25

0.45

0.63

40

0.42

0.48

50

0.61

0.76

60

0.56

0.43

75

0.35

0.39

80

0.36

0.21

100

1.2

1.33

100

0.21

0.34

125

0.9

0.37

RSD=Relative Standard Deviation(n=3).

Calibration Curve:

Linearity of the system was investigated by serially diluting the stock solutions to give concentrations in the range of 10 g/ml to 50 g/ml for Saxagliptin and 10 g/ml to 50g/ml for Dapagliflozin. An aliquot (20 l) was injected using mixture of Methanol: Water (80:20) v/v, as mobile phase. Calibration curves were obtained by plotting the peak area vs. concentration. The calibration curves are as shown in

 

 

Fig.2 Calibration Curve of Saxagliptin

 

 

Fig 3Calibration Curve of Dapagliflozin

 

Accuracy and Precision:

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 50, 100 and 150% and the percentage recovery was calculated and presented in Table 2. Recovery was within the range of 100 2% which indicates accuracy of the method. The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, 3 repeated injections of standard and sample solutions were made in a day and percentage RSD were calculated. In the inter day variation studies, 3 repeated injections of standard and sample solutions were made on 3 consecutive days and percentage RSD were calculated and presented in Table 3 and 4. The data obtained, %RSD not more than 1.5%, indicates that the developed RP-HPLC method is precise.

 

Limit of Detection and Limit of Quantification:

The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula

LOD = (3.3 x standard deviation)/ Slope of calibration curve

The LOD for saxagliptin and Dapagliflozin were found to be 0.47106 g/ml and 0.23757g/ml, respectively.

The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula

LOQ = (10 x standard deviation) / Slope of calibration curve.

The LOQ was 1.4273 g/ml and 0.71991g/ml for saxagliptin and Dapagliflozin respectively.

Robustness:

Robustness is checked by making slight deliberate change in the experimental procedures. In the present method a deliberate change of Wavelength, pH, and flow rate was made and the effects were noted. The method was found to be robust with respect to change in wavelength, pH, and flow rate.

 

RESULT AND DISCUSSION:

The proposed method was found to be simple and linear in the concentration range of 10 to 50 g/ml for both saxagliptin and Dapagliflozin respectively. The method was found to be accurate and precise as indicated by recovery studies and % RSD not more than 1.5. Moreover LOD and LOQ for saxagliptin were found to be 0.47106 g/ml and 1.4273 g/ml, respectively and for Dapagliflozin were 0.23757g/ml and 0.71991g/ml, respectively. Thus the method is specific and sensitive.

 

CONCLUSION:

The Proposed RP-HPLC method for the simultaneous estimation of saxagliptin and Dapagliflozin in combined dosage form was found to be sensitive, accurate, precise, simple and rapid. Hence the present RP-HPLC method may be used for routine analysis of the raw materials and formulations.

 

ACKNOWLEDGEMENT:

The author wish to thanks to Swapnroop drug and chemicals, Aurangabad Ltd. Aurangabad, India for providing Working standard of saxagliptin and Dapagliflozin and also thankful to Dr. Priya Rao for providing guidance for carrying research.

 

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Received on 18.07.2018 Modified on 31.07.2018

Accepted on 16.08.2018 A&V Publications All right reserved

Research J. Science and Tech. 2019; 11(1):59-63.

DOI:10.5958/2349-2988.2019.00008.1