A Validated RP-HPLC Method for Simultanious Determination of Metformin and Canagliflozin in Pharmaceutical Formulation

 

Dr. Gandla. Kumara Swamy1*, R. Lalitha2, Ch. Mounika, B. Soumya2, D. Sudheer Kumar2

1Associate Professor and H.O.D Department of Pharmaceutical Analysis, Care College of Pharmacy, Oglapur (Vill), Damera, (Mdl), Warangal (Dist)-506001. Telangana, India.

2Care College of Pharmacy, Department of Pharmaceutical Analysis, Oglapur (Vill), Damera, (Mdl), Warangal-Rural,(Dist)-506001.Telangana,India.

*Corresponding Author E-mail: kumaraswamy.gandla@gmail.com, drkumaraswamygandla@gmail.com

 

ABSTRACT:

The proposed study a new validated RP-HPLC method has been developed for estimation of metformin and canagliflozin in bulk and pharmaceutical dosage form. The present method was a sensitive, precise and RP-HPLC method for analysis of metformin and canagliflozin. To optimize the mobile phase various combinations of organic solvents were used on Primesil C18 250 column, Then the mobile phase containing a mixture of Methanol and phosphate buffer in the ratio of 70:30% v/v was selected at a flow rate of 1.0ml/min for developing the method and the peak with a good shape and resolution were found resulting in short retention time baseline stability and minimum noise. The retention time metformin and canagliflozin found to be 2.413 and 3.47 min respectively, quantitative linearity was obeyed in the concentration range of 50-250 μg/mL for metformin and 5-25 μg/mL for canagliflozin. The limit of detection and limit of quantitation were found to be 2.1μg/ml and 0.0372μg/ml (metformin), 4.13μg/ml and 0.112μg/ml (canagliflozin) respectively, which indicates the sensitivity of the method. The high percentage recovery indicates that the proposed method is highly accurate no interfering peaks were found in the chromatogram indicating that excipients used in tablet formulation didn’t interfere with the estimation of the drug by the proposed HPLC method.

 

KEYWORDS: RP-HPLC, Method development, metformin, canagliflozin; ICH Validation.

 

 


 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION:

Metformin (MET)1-3 is chemically named as N, N-dimethylimidodicarbonimidic diamide.[1,7] (Fig.1) It is of oral antihyperglycemic agent .metformin decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose and improving insulin sensitivity by increasing peripheral glucose uptake and utilization.

 

These effects are mediated by the initial activation by metformin of AMP-activated protein kinase (AMPK), a liver enzyme that plays an important role in insulin signaling, whole body energy balance and the metabolism of glucose and fats. Activation of AMPK is required for metformin’s inhibitory effect on the production of glucose by liver cells. [1]

 

 

Fig 1: chemical structure of metromin.

 

Canagliflozin (CANA)1-3 is chemically named as (2S,3R,4R,5S,6R)-2-[3-{[5-(4-fluorophenyl) thiophen-2-yl]methyl}-4-methylphenyl)-6-(hydroxymethyl) oxane-3,4,5-triol.[4,7] (Fig. 2). Sodium-glucose co-transporter 2 (SGLT2), expressed in the proximal renal tubule, is responsible for the majority of the reabsorption of filtered glucose from the tubular lumen. Canagliflozin is an inhibitor of SGLT2. By inhibiting SGLT2, canagliflozin reduces reabsorption of filtered glucose and lowers the renal threshold for glucose (RTG) and thereby increases urinary glucose excretion.

 

 

Fig 2: chemical structcture of canagliflozin.

 

The review of literature4-6 revealed that several analytical methods have been reported for metformin and canagliflozin in spectrophotometry.7, 8 HPLC, HPTLC, LC/MS9-12 individually and in international journal of sciences. To date, there have been no published reports about the stability indicating studies and simultaneous estimation of metformin and canagliflozin by RP-HPLC in bulk drug and in pharmaceutical dosage forms. This present study reports for the first time stability indicating simultaneous estimation of metformin and canagliflozin by RP-HPLC in bulk drug and in pharmaceutical dosage form.

 

MATERIALS AND METHODS:

Chemicals and reagent:

Metformin and Canagliflozin were obtained as gift sample from spectrum pharma research laboratory in Hyderabad and marketed formulation purchased from local market. Methanol, water, methanol, were obtained from Merck, Mumbai. and phosphate buffer, obtained from MERCK Mumbai. All solvents used in this work are HPLC grade.

 

Instrument and chromatographic condition:

RP-HPLC waters 2965 separation module equipped with photodiode array detector was employed in this method. The empower 2 software was used for LC peak integration along with data acquisition and data processing. The column used for separation of analytes is Primesil C18 250 (250×4.6 mm, 5μ). Mobile phase consisting of phosphate buffer Methanol in the ratio of 65: 35 % v/v at flow rate of 1.0ml/min. it was filtered through 0.45 μm nylon filter and sonicated for 5 min in ultrasonic bath. Sample were analyzed at 248nm at an injection volume 10μL.

 

OPTIMIZED                     CHROMATOGRAPHIC                                             CONDITIONS:

Instrument used  :              Waters HPLC with auto                                                 sampler and PDA Detector                                              996 model.

Temperature        :              25ºC

Column                :              Primesil C18 (4.6×250mm) 5µ

Mobile phase      :              Methanol: Buffer (70:30v/v)

Flow rate              :              1.0 mL/min

Wavelength         :              220nm

Injection volume               :              10 ml

Run time              :              6min

 

 

Fig.3.Typical Chromtograph of Metformin and Canagliflozin

 

Preparation of buffer pH 3.2:

Accurately weighed 1.36gm of sodium dihydrogen phosphate in a 1000ml of volumetric flask add about 900ml water added and degas to sonicate and finally make up the volume with water and pH adjusted to 4.5 with dil. OPA.

 

Preparation of solutions:

Standard preparation: (20μg/ml of metformin, 20μg/ml canagliflozin):

Accurately weighed and transferred 50mg and 5mg of metformin and canagliflozin working standards in to two 25ml clean and dry volumetric flask separately, add ¾ volume of diluent, sonicated for 30 minutes and make up to the final volume with diluents. From the above stock solution 1ml was pippeted out in to a 10ml volumetric flask and then make up to the final volume with diluent.

 

Sample preparation: (10μg/ml of metformin, 10μg/ml canagliflozin):

The 20 tablet were weighed and calculate the average weight of each tablet then the tablet powder weight equivalent to 500mg of metformin and 50mg of canagliflozin was transferred into a 250ml volumetric flask, 200ml of diluent added and sonicated for 30 min, further withdraw volume make up with the diluent and filtered. From the filtered solution 1ml pipetted out into a 10ml volumetric flask and make up to 10ml with diluent.

 

METHOD VALIDATION:

The validation of the method was carried out as per ICH guidelines the parameter assessed were specificity, linearity, precision, accuracy, stability, LOD and LOQ.

 

Specificity:

Specificity is the ability of analytical method to measure the analyte response in the presence of interferences including degradation product and related substances.

 

Accuracy:

The accuracy was determined by calculating % recoveries of metformin and canagliflozin. It was carried out by adding known amount of each analytes corresponding to three concentration levels (505, 100%, 150%) of the labelled claim to the excipients. At each level, six determinations were performed and the accuracy result was expressed as percent analytes recovered by the proposed method. The accuracy results are shown in below tables.

 


 

 

Table 1.The accuracy results for Metformin

%Concentration

(at specification Level)

Peak Area

Amount Added (µg/ml)

Amount Found (µg/ml)

% Recovery

Mean Recovery

50%

1184204.3

5

4.96

99.91%

99.56%

100%

2121872.4

10

9.98

99.18%

150%

3525766.1

15

15.02

99.60%

 

 

Table 2.The accuracy results for Canagliflozin

%Concentration

(at specification Level)

Peak Area

Amount Added (mg/ml)

Amount Found (mg/ml)

% Recovery

Mean Recovery

50%

52228.2

0.5

0.99

99.53%

99.47%

100%

979319

1.0

1.05

99.38%

150%

1576651

1.5

1.495

99.52%

 

 

Table 3. Results of method precession for Flupentixol

S.NO

Retention times

Peak Area

USP plate count

USP Tailing

USP Resolution

Metformin

Canagliflozin

Metformin

Canagliflozin

1

3.0

4.7

2285660

1005320

5761

1.3

9.7

2

3.0

4.7

2286224

1006642

5489

1.3

9.6

3

3.0

4.8

2292264

1007252

5367

1.3

10.6

4

3.0

4.79

2301304

1001606

5845

1.3

9.6

5

3.0

4.9

2355896

1014319

5347

1.3

9.7

Mean

 

 

2304269

1009027.9

 

 

 

S.D

 

 

29539

3782.8

 

 

 

% RSD

 

 

0.3

0.4

 

 

 

 

 


Precision:

Precision of an analytical method is usually expressed as the standard deviation. The repeatability studies were carried out by estimating response of metformin and canagliflozin six times. The intra-day and interday precision studies (intermediate precision) were carried out by estimating the corresponding responses three times on the same day and on three different days for three same concentrations and the result are reported in terms of relative standard deviation.

 

 

 

Linearity:

The purpose of the test for linearity is to demonstrate that the entire analytical system (including detector and data acquisition) exhibit a linear response and is directly proportional over the relevant concentration range for the target concentration of the analytes. The linear regression data for the calibration plot is indicative of a good linear relationship between peak and concentration over a wide range. The correlation coefficient was indicative of high significance.

 

 

Fig.4 Calibration curve of Metformin

 

 

Fig .5 Calibration curve of Canagliflozin

 

Table .4.Linearity data of Metformin and Canagliflozin

S.NO

Metformin

Canagliflozin

Metformin

Canagliflozin

 

Concentration

(µg/mL)

Peak Area

1.                  

50

5

800199

339009

2.                  

100

10

1589391

689527

3.                  

150

15

2264300

994963

4.                  

200

20

3071625

1385006

5.                  

250

25

3894075

1766425

LIMIT OF DETECTION:

The    detection  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

 

Metformin: LOD =3.3× 371827/563365963 =2.17µg/ml

 

Canagliflozin:LOD=3.3×5401.60/479884400=0.0372µg/ml

 

LIMIT OF QUANTITATION:

The  quantitation  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte  in  a  sample  which  can  be  quantitatively  determined. 

 

Metformin: LOQ=3.3×371827/563365963 =6.60 µg/ml

 

Canagliflozin:LOQ=3.3×5401.60/479884400=0.112µg/ml

 

ROBUSTNESS:

The robustness was performed for the flow rate variations from 0.9 ml/min to 1.1ml/min and mobile phase ratio variation from more organic phase to less organic phase ratio for Metformin and Canagliflozin. The method is robust only in less flow condition and the method is robust even by change in the Mobile phase ±5%. The standard and samples of Metformin and Canagliflozin were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count.

 

 

 

 

 


 

 

Table 5. Robustness data of Metformin

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

247392

2.061

7243

1.2

Less Flow rate of 0.9 mL/min

69214

2.267

4713

1.3

More Flow rate of 1.1 mL/min

388838

1.864

4740

1.2

Less organic phase

445628

2.165

4709

1.2

More organic phase

69404

1.967

5590

1.4

 

Table 6. Robustness data of Canagliflozin

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

3530866

2.462

3389

1.1

Less Flow rate of 0.9 mL/min

527373

2.690

5275

1.0

More Flow rate of 1.1 mL/min

4363129

2.284

5611

1.0

Less organic phase

3965572

2.590

5550

1.0

More organic phase

527708

2.390

6273

1.0


Assay of metformin and canagliflozin:

Assay of marketed product was carried out by using the developed method. Sample solution were prepared and injected in to RP-HPLC system. The sample solution was scanned at 240nm

 

RESULT AND DISCUSSION:

A simple, accurate, precise method was developed for the simultaneous estimation of the metformin and canagliflozin in tablet form, the retention time (Rt’s) of metformin and canagliflozin were found to be 3.419 min and 4.548 min respectively. Percentage RSD of the metformin and canagliflozin were found to be 0.50 and 1.55 respectively, the percent recoveries were gained as 100.2% for metformin and 99.6% for canagliflozin. LOD, LOQ values are from regression equation of metformin and canagliflozin were 2.1, 0.0310 μg/ml and 4.161, 0.112μg/ml respectively. Regression equation of metformin is y =21314 x + 6745, and canagliflozin is y = 12032 x + 578.4. In this proposed method retention time were decreased so the method developed was simple and economical that can be adopted in regular quality control test in industries.

 

CONCLUSION:

A new stability indicating RP-HPLC method has been developed for simultaneous estimation of metformin and canagliflozin in bulk and pharmaceutical dosage form. The developed method was validated and it was found to be simple, sensitive, precise and robust it can be used for the routine analysis of metformin and canagliflozin in both bulk and pharmaceutical dosage form. The forced degradation studies were carried out in accordance with ICH guidelines and the result reveled suitability of the method to study stability of metformin and canagliflozin under various degradation condition like acid, base, oxidative, thermal and water degradations. Finally it was concluded that the method is simple, sensitive and has the ability to separate the drug from degradation product and excipients found in the dosage form.

 

ACKNOWLEDGEMENT:

The authors are very thankful to CARE College of Pharmacy, Warangal,for providing necessary facilities and supporting to his research work and also thankful to Sura Pharma Lab,Dilshuknagar,Hyderabad,Telangana.

 

REFERENCES:

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Received on 22.02.2018       Accepted on 25.03.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(2): 73-77.

DOI: 10.5958/2231-5675.2018.00014.5