INTRODUCTION:

Canagliflozin approved by the FDA on 29 march 2013 and become the frist sodium glucose co- transportor 2 (SGLT2) inhibitor in the united states. Canagliflozin indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Canagliflozin is not recommended in patients with type 1 diabetes mellitus or for the treatment of diabetic ketoacidosis. Metformin was discovered in 1922 French physician Jean Sterne began study in humans in the 1950. Metformin is primarily use for type-2 diabetes, but is increasingly being used in polycystic ovary syndrome. Metformin is generally well tolerated common side effect include Diarrhea, Nausea and Abdominal pain. Metformin marketed under the tradename Glucophage is the first line medication for the treatment of type-2 diabetes. Canagliflozin increases urinary glucose excretion by selectively inhibiting renal sodium- glucose transporter 2 (SGLT2), an insulin independent mechanism of action that may be complementary to that of other oral antidiabetes drugs.

Fig 1: Structure of Canagliflozin.

Canagliflozin acts by inhibiting the SGLT2 which accounts for more than 90% of renal glucose reabsorption.^[1]Hence the efficacy of this drug also is dependent upon the amount of glucose which is filtered through the glomeruli and enters the tubular lumen and therefore shows maximal effect in patients with uncontrolled T2DM.^[2]Apart from bringing down the blood glucose levels, it has many other beneficial actions like reduction of the glycosylated hemoglobin levels due to the better control of blood glucose levels. It additionally improved the sensitivity of liver to insulin by reducing the blood glucose levels thereby reducing the glucose production from liver. This reduces the general gluco-toxic state of the body in patients with T2DM and helps in bringing down the serum insulin levels. Since the calories are lost from the body in the form of glucose in urine of the patients taking this drug, it causes a negative energy balance and loss of weight, which is again beneficial in patients of T2DM. This drug also helps in reduction of blood pressure owing to the mild weight loss and diuretic action caused by it. It has a positive effect on blood lipids as well due to mild weight loss caused by it.^[3,4,5]

It is given orally and reaches the peak concentration in plasma in about 1-2 hours. The steady state of plasma concentration is achieved in around 4-5 days. Oral bioavailability of Canagliflozin is approximately 65%. It has a high plasma protein binding (99%) and it binds mainly to albumin.^[14] Food does not interfere with its absorption. It has a half-life of 11 hours with a 100 mg dose and of 13 hrs with a dose of 300 mg and is mainly metabolized via glucuronidation. A small part (~7%) of absorbed drug also undergoes oxidation through CYP3A4 enzyme. Hence drugs which induce this enzyme e.g. rifampicin, phenytoin, ritonavir etc. will decrease the plasma level of Canagliflozin. Therefore, its dose has to be increased to 300 mg/day when used in combination with these drugs. It increases the plasma concentration of digoxin, so the drug levels need to be monitored to avoid the development of digoxin toxicity. Renal function needs to be assessed whenever this drug has to be started and is contraindicated when the glomerular filtration rate is less than 45 ml/min/1.73m².^[6]

In a study done to assess the pharmacokinetics and pharmacodynamics of Canagliflozin, it was established that the profile of the drug is meant for once daily regimen.^[10]The recommended starting dose of Canagliflozin is 100 mg once daily, taken before the first meal of the day. In patients tolerating Canagliflozin 100 mg once daily who have an eGFR of 60 mL/min/1.73 m² or greater and require additional glycemic control, the dose can be increased to 300 mg once daily.^[3]

Diabetes mellitus, describes a group of metabolic diseases in which the person has high blood glucose. Metformin hydrochloride is an anti-diabetic drug from the bigunaide class of oral antihyperglycemic agents. Half-life 6.2 hours. Duration of action is 8-12 hours. Metformin decreases blood glucose levels by decreasing hepatic glucose production, decreasing intestinal absorption of glucose. Metformin hydrochloride and gliclazide were prepared by using different concentration of polymers of various grades of HPMC, Carboxy methyl cellulose, Calcium phosphate dibasic anhydrous, Micro crystalline cellulose, PVP, Lactose monohydrate. Different types of pellets i.e. Metformin pellets, coated Metformin pellets and disintegrate pellets were shown independent influence on the formulation. In order to evaluate the pHs effect on the transdermal drug delivery, HPMC/PVA based TDS-patch was prepared. Floating tablet Metformin HCl have been shown sustained release there by proper duration of action at a particular site and are designed to prolong the gastric residence time after oral administration. The Chemical of metformin HCl is -1-carbamimidamido-N, N- dimethymethanimidamide. The Molecular formula –C4H11N5.HCL and Molecular weight 165.63. their melting point is 165.63. the Solubility –Freely soluble in water, soluble in methanol is practically insoluble in acetone, ether, and chloroform.

Fig 2: Structure of Metformin HCl

Metformin's mechanisms of action differ from other classes of oral antihyper-glycemic agents. 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 sign along, whole body energy balance, and the metabolism of glucose and fats. Increased peripheral utilization of glucose may be due to improved insulin binding to insulin receptors. Metformin administration also increases AMPK activity in skeletal muscle. AMPK is known to cause GLUT4 deployment to the plasma membrane, resulting in insulin-independent glucose uptake.

MATERIAL AND METHOD:

Table 1: List of apparatus/ instruments used.

Sr. No.	Name	Model
1.	Weighing balance	PGB 100
2.	Ultra-Sonicator	WUC-4L
4.	UV- Spectrophotometer and Software	UV2450 UV probe v 2.3.3
5	HPLC	HPLC 3000 series P- 3000-M reciprocating (binary pump) UV-3000-M (UV-Visible Detector)

Table 2: List of chemical used.

Sr. No.	Reagents and Chemicals	Details
1.	Water	HPLC grade
2.	Methanol	HPLC grade
3.	KH₂PO₄ Buffer	HPLC grade

Table 3: List of API used

Sr. No.

Name

Specification

Manufacturer/

Supplier

1. 1

Metformine hydrochloride

Working standard

APL Research center, A Division of Aurobindo Pharma Limited, Hyderabad.

2. 2

Canagliflozin

Working stand

APL Research center, A Division of Aurobindo Pharma Limited, Hyderabad.

Preparation of mobile phase

Mixed a HPLC grade Methanol: KH₂PO₄ Buffer 10mM (90:10) in volumetric flask. Filter through under vaccum filtration.

Preparation Stock Solution:

Accurately weighed quantity of Metformine hydrochloride and Canagliflozin 10mg individually dissolved in 10ml volumetric flask using mobile phase and solution was sonicated for 20 minutes and volume is make up to the mark to get 1000μg/ml and filtered through 0.45μm membrane filter.

Preparation of Sample solution:

20 tablets were weighed and powdered, tablets powder equivalent to 10mg of Metformin hydrochloride and canagliflozin was transferred 100ml volumetric flask, sufficient amount of mobile phase was added and dissolved by 20minutes ultrasonication. then made the volume up to the mark with the mobile phase and filtered with 0.45µ filter paper. Pipette out from above solution and diluted to 10 ml mobile phase and use for sample injection.

Selection of analytical wavelength:

Accurately weighed quantity of Metformin hydrochloride and Canagliflozin 10 mg dissolved in 100 ml volumetric flask and volume is make up to the mark to get 100μg/ml. Solution was scanned using UV-Visible Spectrophotometer in the spectrum mode between the wavelength ranges of 400 nm to 200 nm. The wavelength selected was 222 nm.

Fig. 3. Wavelength of Metformin hydrochloride and Canagliflozin.

Optimized chromatographic condition

In the present study the separation of Metformine hydrochloride and Canagliflozin was achieved by using column Grace C18, (250×4.6mm,5µ) with mobile phase consisting of mixture of methanol and KH₂PO₄ Buffer in the ratio of 90:10 at a flow rate 0.8 ml/min with uv detection wavelength of 222nm at ambient temperature. The run time for Metformine hydrochloride and Canagliflozin were found to be 7.50min respectively.

Figure No. 4. Chromatogram for sample solution.

Name of drug

Time

Area

Reso

lution

Th. Plate

Asym metry

Metformine HCL

3.772

4722041

6.36

9302

1.17

Canagliflozin

5.254

1082749

0.00

9964

1.07

Figure No. 5. Chromatogram for standard solution.

Name	RT (min)	Area	Res olution	Theoretical plate	Asym metry factor
Metformine HCL	3.777	4719614	6.92	9076	1.16
Canagliflozin	5.257	1083915	0.00	9616	1.07

RESULT AND DISCUSSION:

Validation of the Developed Method:

A. Linearity:

Linearity of an analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in samples within a given range.

The linearity of the analytical method is determined by mathematical treatment of test results obtained by analysis of samples with analyte concentrations across the claimed range. Area is plotted graphically as a function of analyte concentration. Percentage curve fittings are calculated.

Table 4: Result of linearity.

Conc. of Metformin HCL (µg/ml)	Area	Conc. Of Canagliflozin (µg/ml)	Area
10	250123	3	79040
20	1256085	6	316505
30	2372558	9	559160
40	3526104	12	815863
50	4719614	15	1083915

Fig. No. 6. Linearity graph for Canagliflozin.

Fig.No.7. Linearity graph for Metformin HCl.

B. % Recovery:

The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. Accuracy may often the expressed as percent recovery by the assay of known added amounts of analyte.

The accuracy of an analytical method is determined by applying the method to analyzed samples, to which known amounts of analyte have been added. The accuracy is calculated from the test results as the percentage of analyte recovered by the assay.

Table 5: % Recovery data for metformin and canagliflozin.

Drug	% Composition	% Recovery	% RSD
Metformin	50	100.32	1.10
	100	100.23	0.21
	150	100.05	0.83
Canagliflozin	50	100.18	1.01
	100	99.73	0.46
	150	99.89	0.43

C. Robustness:

The robustness of an analytical method is determined by analysis of aliquots from homogenous lots by differing physical parameters that may differ but are still within the specified parameters of the assay.

The sample along with standard was injected under different chromatographic conditions as shown below.

Table 6: Robustness Data. (At different Flow Rate)

Drug sample	Flow rate (conc.)	Area	Mean	SD	% RSD
Metformin HCL	10	1256085	1268 432	191 97.4	1.513477
	20	1258661
	30	1290549
Canagliflozin	3	316505	306 905	162 61.9	5.298667
	6	316081
	9	288129

Table No. 7. (At different wavelength)

Drug sample	Area	Mean	SD	% SD
Metformin HCL	1256085	1256225	1074.86	0.0855627
	1257363
	1255227
Canagliflozin	316505	316196	296.47	0.0937615
	316168
	315914

E. Limit of Detection (LOD) and limit of quantitation (LOQ):

Limit of detection:

The lowest conc. of the analyte in the sample that the method can detect but not necessarily quantify under the stated experimental conditions simply indicates that the sample is below or above certain level. Limit test prescribed as percentage or as parts per million. The limit of detection will not only depend on the procedure of analysis but also on type of instrument.

S/N= 2/1 or 3/1

Where, S= Signal ; N=Noise

It may be calculated based on standard deviation (SD) of the response and slope of the curve(S).

3.3 SD

LOD=----------

Where,

SD= Standard deviation

S= Slope

LOD was found to be 0.4648 μg/ml for Metformin hydrochloride and 0.1065μg/ml for Canagliflozin.

Limit of quantitation:

From the linearity data calculate the limit of quantitation, using the following formula

10 SD

LOD=----------

Where,

SD = standard deviation

S = slope

LOQ was found to be 1.4085 μg/ml for Metformin hydrochloride and 0.3227μg/ml for Canagliflozin.

Table 8: Result of LOD and LOQ.

Drug	LOD (µg/ml)	LOQ (µg/ml)
Metformin	0.4648	1.4085
Canagliflozin	0.1065	0.3227

Table No. 9: Ruggedness datafor Metformin HCL and Canagliflozin

Conc. of Metformin HCL (µg/ml)	Area	Conc. of Canagliflozin (µg/ml)	Area
10	252796	3	80391
20	2369640	6	557851
30	3532761	9	817266
40	4713783	12	1077646

Fig .No.8: Ruggedness graph for Canagliflozin.

Fig. No. 9: Ruggedness graph for Metformin HCL

Table No.10: Data for assay of sample of metformin hydrochloride and Canagliflozin

Drug Name	Area of Std.	Area of Sample	% Assay
Metformin HCl	2372558	2368944	99.847%
Canagliflozin	559160	560329	100.209%

CONCLUSION:

The proposed simultaneous estimation and validation method was found to be simple, precise, accurate and rapid for the determination of Metformin HCL and Canagliflozin. The coefficient of correlation was obtained in acceptable range. The percentage recovery obtained in acceptable range .variation in flow rate, wavelength, does not have any effect on the % RSD of standard and assay value. The relative standard deviation of main peak area, tailing factor and theoretical plate is well within the acceptable range . Hence the precision of given method is confirmed. Thus from the above result of the individual method is conclude that the analytical method is validated and found to be satisfactory.

REFERENCES:

1. Nair S, Wilding JP. Sodium glucose cotransporter 2 inhibitors as a new treatment for diabetes mellitus. J Clin Endocrinol Metab, (2010); 5(1), page no:34-42.

2. Strojek K, Yoon KH, Hruba V, Elze M, Langkilde AM, Parikh S, et al. Effect ofdapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control withglimepiride: a randomized, 24-week, double- blind, placebo-controlled trial. Diabetes Obes Metab,(2011); 13(10):page no : 928-938.

3. Kim Y, Babu AR. Clinical potential of sodium- glucose cotransporter 2 inhibitors in the management of type 2 diabetes. Diabetes Metab Syndr Obes, (2012); 5(3),page no :313-327.

4. Janssen Research and Development, LLC. Canagliflozin as an adjunctive treatment to diet and exercise alone or coadministered with other antihyperglycemic agents to improve glycemic control in adults with type 2 diabetes mellitus ,(2013), page no :224-231.

5. http://www.mediclnewstoday.com

6. http://www.thediabeticvoice.com

7. Cindy Green, RAC. A Step By Step Approach to Establishing a Method Validation. Journal of Validation Technology August 2007; 13(4):p.317-323.

8. Vasant D. Khasia, Hetal V. Khasia, Dhara Desai, Dharmishtha N. Bhakhar, Ashok R. Parmar, “Development and Validation of Stability Indicating RP-HPLC Method for Immediate Release Tablet Dosage Form”, Journal of Pharmacy research, 2012, 5(8),4115-4118.

Received on 12.06.2019 Modified on 19.07.2019

Research J. Pharm. and Tech. 2019; 12(10):4953-4957.

DOI: 10.5958/0974-360X.2019.00859.X