1. INTRODUCTION:

Canagliflozin (Cana) is a selective Sodium-Glucose Co-transporter 2 (SGLT2) inhibitor used for the management of type 2 Diabetes Mellitus. Chemically it is (2S,3R,4R,5S,6R)- 2-{3-[5-(4-fluoro-phenyl)-thiophen-2-ylmethyl]-4-methyl-phenyl}-6 hydroxymethyltetrahydro-pyran-3,4,5-triol (Figure 1)¹. It is not official in IP-2014, BP-2008 and USP-2011.

Metformin HCl (Met) is chemically N, N-dimethyldiguanide used in the treatment of type 2 diabetes. It suppresses hepatic gluconeogenesis and glucose output from liver, enhances binding of insulin to its receptors and stimulates insulin mediated glucose disposal². It is official in IP-2014, BP-2008 and USP-2011^3-5.

Fig. 1 Structure of Canagliflozin

Fig. 2 Structure of Metformin HCl

Combination of Cana and Met was approved by FDA on Aug 08, 2014 as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Tablets INVOKAMET containing Cana and Met (50:500, 50:1000, 150:500, 150:1000) by Janseen Pharmaceuticals are available in market ⁶.

Thorough literature survey revealed that there are UV-Visible spectroscopic methods reported for Cana and Met as single drugs ^7-13. Similarly, few chromatographic methods are also reported for simultaneous determination of Cana and Met^14-21. But there is no scientific reporting of UV-Visible spectroscopic methods for simultaneous analysis of Cana and Met in combined dosage form.

Therefore, the objective of current study is to develop and validate UV-Visible spectrophotometric methods for simultaneous estimation of Cana and Met in pharmaceutical formulation.

2. MATERIAL AND METHODS:

2.1 Instrumentation:

UV-Visible Spectrophotometer (Shimadzu-1800, Japan) with 10 mm matched quartz cells and electronic balance (Shimadzu model AUX 220) were used.

2.2 Reagents and Chemicals:

Pure samples of Cana and Met were kindly supplied as a gift sample by Sun Pharma and Nulife Pharmaceuticals respectively. All reagents used were of analytical grade.

Tablets Glycomet (Metformin HCl 500 mg) and Sulisent (Canagliflozin 100 mg) were purchased from local market.

2.3 Preparation of standard solutions:

Standard stock solutions containing 1000 μg/ml of Met and 300 μg/ml of Cana were prepared by dissolving standard drugs separately in methanol. Appropriate aliquots were diluted by methanol to get 2.5-15 μg/ml of Met and 0.75-4.5 μg/ml of Cana separately.

2.4 Preparation of laboratory mixtures:

Standard stock mixture solution containing 1000 μg/ml of Met and 300 μg/ml of Cana was prepared by dissolving both the drugs in methanol. Appropriate dilutions were done using methanol to get 2.5-15 μg/ml of Met and 0.75-4.5 μg/ml of Cana in combination.

2.5 Method development:

2.5.1 Selection of wavelengths for analysis:

Wavelengths for measurement of absorbances were selected by scanning solutions containing 10μg/ml of Met and 3 μg/ml of Cana separately in spectrum mode. A zero order overlain spectrum (Fig. 3) was obtained and wavelengths 233 nm (λ max of Met) and 291 nm (λ max of Cana and Zero absorbance for Met) were selected for analysis by method A. From the zero order overlain spectrum Absorbance correction method was chosen for analysis.

The zero order spectra were converted to first order derivative spectra and a first order overlain spectrum (Fig. 4) was obtained. From the first order overlain spectrum wavelengths 243 nm (Zero absorbance of Cana) and 318 nm (Zero absorbance of Met) were selected for analysis.

Fig. 3 Zero order overlain spectrum

Fig. 4 First order overlain spectrum

2.5.2 Analysis of marketed formulation:

Twenty tablets were weighed and triturated to fine powder. Powder equivalent to 10 μg of Met and 3 μg of Cana was transferred to 100 ml volumetric flask containing 50 ml Methanol. Mixture was sonicated for 20 min and volume was made up to the mark with methanol. Then it was filtered through Whatman filter paper 21 and diluted appropriately to get concentration of 5μg/ml of Met and 1.5 μg/ml of Cana.

2.5.2.1 Method A Absorbance Correction Method:

The above solution was scanned in UV spectrum mode and Absorbances at selected wavelengths were recorded. The % drug contents were calculated by given formulae.

Concentration of Cana was calculated by formula,

A _{(291 nm)}= a.b.c₁ --------------------------------------------(1)

Where,

A _{(291 nm)}= absorbance at 291 nm

a = absorptivity of Cana at 291 nm

b = path length (1 cm)

c₁ = concentration of Cana

Concentration of Met was calculated by formula,

A _{(233 nm) =}a₁. b. c₁ + a₂.b.c₂--------------------------------(2)

Where,

A _{(233 nm)}= absorbance at 233 nm

a_{1 =}absorptivity of Cana at 233 nm

c_{1 =}concentration of Cana calculated from formula 1

a_{2 =} absorptivity of Met at 233 nm

c_{2 =}concentration of Met

b = path length (1 cm)

2.5.2.2 Method B First Order Derivative Method:

The zero order spectra were manipulated to first order derivative spectra and amplitude at selected wavelengths were recorded. Linearity graphs were plotted to get linear regression equations and % drug contents were calculated.

Concentration of Cana was calculated by formula,

Y₁ = - 0.0013 x₁ – 0.001

Where

Y₁ = amplitude at 318 nm

x₁ = concentration of Cana in μg/ml

Concentration of Met was calculated by formula,

Y₂ = - 0.0045 x₂ – 0.0053

Where

Y₂ = amplitude at 243 nm

x₂ = concentration of Met in μg/ml

2.6 Method validation [20]:

2.6.1 Linearity and Range:

Linearity of the method was determined by preparing a series of dilutions of mixture containing 2.5 - 15 μg/ml of Met and 0.75 - 4.5 μg/ml of Cana from standard stock mixture solution. Graphs were plotted as concentration Vs response and values of regression equation, Correlation coefficients were obtained (Fig. 5).

Fig. 5 Zero Order Linearity graph of Method A

Fig. 6 First order derivative linearity graph of Cana

Fig. 7 First order derivative linearity graph of Met

2.6.2 Accuracy:

Accuracy of the method was determined by recovery studies at three different levels. Known amount of pure drugs were spiked to sample at 50, 100 and 150 % level and analyzed by the developed method in triplicate.

2.6.3 Precision:

Precision of the method was determined by repeatability and intermediate precision studies. The developed method was repeated six times on the same day for repeatability study. Intermediate precision was estimated by inter-day analysis where developed procedure was repeated on three consecutive days in triplicate. Results were expressed as the standard deviation and % RSD.

3 RESULT AND DISCUSSION:

3.1 Analysis of marketed formulation:

Marketed formulation was analyzed by proposed methods and % drug content were found to be 98.48 ± 0.83 and 100.76 ± 1.29 for Cana and Met respectively by method A, whereas by method B the percentage drug content were found to be 97.94 ± 0.96 and 97.22 ± 1.15 for Cana and Met respectively (Table 1).

Table 1- Results of analysis of marketed formulation

Method	Drug	% Drug content ± SD	% RSD
Method A (Absorbance Correction Method)	Metformin HCl	100.76 ± 1.29	1.28
Method A (Absorbance Correction Method)	Canagliflozin	98.48 ± 0.83	0.84
Method B (First Order Derivative Method)	Metformin HCl	97.22 ± 1.15	1.18
Method B (First Order Derivative Method)	Canagliflozin	97.94 ± 0.96	0.98

*Average of three determinations

3.2 Method validation:

3.2.1 Linearity and range:

The Beer’s law was obeyed in the concentration range of2.5-15 μg/ml of Met and 0.75-4.5μg/ml of Cana at selected wavelengths. The values of correlation coefficients were found near to one, indicates linearity of proposed methods. Results of linearity parameters are given in (Table 2).

Table 2 - Linearity parameters of proposed method

Parameters	Method A		Method B
Parameters	At 233 nm	At 291 nm	243 nm	318 nm
Linearity range (μg/ml)	2.5 - 15	0.75 - 4.5	2.5 - 15	0.75 - 4.5
Regression equation	y = 0.1946x + 0.0831	y = 0.0326x + 0.0077	y = -0.0045x - 0.0053	y = -0.0013x - 0.001
Correlation Coefficient	0.9977	0.995	0.9952	1

*Average of three determinations

3.2.2Accuracy:

Accuracy of the developed method was determined by recovery studies and results were expressed in terms of % recovery. The % recovery of was found to be in a range of 96.86 - 100.14 and 97.77 - 101.19 % w/w for Met and Cana respectively by both the methods. The % RSD values were found to be less than 2 confirming accuracy of the method (Table 3).

3.2.3Precision:

Results of repeatability and intermediate precision studies were estimated in terms of % drug content, SD and % RSD. Values of % RSD were found to be less than 2 proving the proposed method is precise in nature. Results of repeatability study are shown in (Table 4 and 5).

4. CONCLUSION:

Combination of Canagliflozin and Metformin HCl was approved by FDA on Aug 08, 2014 as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. Till date there is no scientific reporting of UV-Visible spectroscopic methods for simultaneous analysis of Cana and Met in combined dosage form.

Therefore, two simple, economic and fast UV-Visible spectroscopic methods were developed for simultaneous estimation of Canagliflozin and Metformin HCl in combined pharmaceutical formulation. The developed methods were Absorbance correction method and First order derivative method. The developed methods were validated as per ICH guidelines and found to be accurate and precise. Hence, they could be successfully used for routine quality control analysis in industry.

5. ACKNOWLEDGEMENT:

Authors are thankful to management, PES’s Modern College of Pharmacy (for ladies), Moshi, Pune for providing necessary facilities for research.

6. DECLARATIONS OF INTEREST:

None

7. REFERENCES:

1. www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=BasicSearch.process, visited on (accessed on Dec 7, 2017).

2. Essentials of Medical Pharmacology, K D Tripathi, Jaypee Brothers Medical Publishers Ltd., 4^th Edition, 2001, 279.

3. Indian Pharmacopoeia, Government of India, Ministry of Health and family welfare. Published by Indian Pharmacopoeia Commission, Ghaziabad, Vol-II, 2186-2187 (2014).

4. British Pharmacopoeia, The Stationary Office on behalf of Medicines and Health Care Products Regulatory Agency (MHRA), London, United Kingdom, Vol-II, 1410-1411 (2008).

5. United States Pharmacopoeia-34 National Formulary-29, United States Pharmacopoeial Convection, Rockville, Vol-III, 3442 (2011).

6. www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=204353 (accessed on Dec 7, 2017).

7. Krupa Karuna Vani B. Extractive Visible Spectrophotometric Method development and validation for the estimation of Canagliflozin in pharmaceutical formulations. The Experiment. 2017; 40(3): 2376-2381.

8. Krupa Karuna Vani B. UV and Derivative Spectrophotometry Method for the estimation of Canagliflozin in pharmaceutical formulations. International Journal of Applied Pharmaceutical Sciences and Biological Sciences. 2017; 6(1): 022-029.

9. Kaur I et al. Development and Validation of Stability indicating UV Spectroscopic Method for Determination of Canagliflozin in Bulk and Pharmaceutical Dosage Form. Pharm Methods. 2016; 7(1): 63-69.

10. Kaur I et al. Development and Validation of UV Spectroscopic Method for Determination of Canagliflozin in Bulk and Pharmaceutical Dosage Form. Pharm Methods, 2015; 6(2): 82-86.

11. Mubeen G, Khalikha N. Spectrophotometric Method for Analysis of Metformin Hydrochloride. Indian Journal of Pharmaceutical Sciences. 2009; 71(1): 100-102.

12. Arayne MS et al. Spectrophotometric Quantitation of Metformin in Bulk Drug and Pharmaceutical Formulations using Multivariate Technique. Indian Journal of Pharmaceutical Sciences, 2009; 71(3): 331–335.

13. Umapathi P et al. Quantitative Determination of Metformin Hydrochloride in Tablet Formulation Containing Croscarmellose Sodium as Disintegrant by HPLC and UV Spectrophotometry. Tropical Journal of Pharmaceutical Research. 2012; 11(1): 107-116.

14. Kumara Swamy G et al. A Validated RP-HPLC Method for Simultaneous Determination of Metformin and Canagliflozin in Pharmaceutical Formulation. Asian Journal of Pharmaceutical Analysis. 2018; 8(2): 73-77.

15. Wafaa A et al. Comparative Liquid Chromatographic Study for Concurrent Determination of Canagliflozin and Metformin in Combined Tablets. Hindawi Journal of Analytical Methods in Chemistry. 2017:1-9.

16. Vinutha K et al. Development and Validation of a New RP-HPLC Method for Simultaneous Estimation of Metformin Hydrochloride and Canagliflozin and its Comparison with the Reported Methods. World Journal of Pharmacy and Pharmaceutical Sciences. 2017; 6(03): 696-713.

17. Uppalapati J, Parimi U. Analytical Method Development and Validation for the Simultaneous Estimation of Canagliflozin and Metformin in Drug Product by RP-HPLC. European Journal of Biomedical and Pharmaceutical Sciences. 2017; 4(11): 298-303.

18. Panigrahy UP, Sunil Kumar Reddy A. A Novel Validated RP HPLC DAD Method for the Simultaneous Estimation of Metformin Hydrochloride and Canagliflozin in Bulk and Pharmaceutical Tablet Dosage Form with Forced Degradation Studies. Oriental Journal of Chemistry. 2015; 31(3): 1489-1507.

19. D souza S et al. Stability Indicating Assay Method Development and Validation to Simultaneously Estimate Metformin Hydrochloride and Canagliflozin by RP-HPLC. Current Trends in Biotechnology and Pharmacy. 2016; 10(4): 334-342.

20. Bhole RP et al. Development and Validation of HPTLC Method for Simultaneous Estimation of Canagliflozin and Metformin Hydrochloride in Bulk and Tablet Dosage Form. South African Pharmaceutical Journal. 2017; 84(6): 65-71.

21. Ali SI et al. Stability Indicating Simultaneous Estimation of Metformin and Empagliflozin in Pharmaceutical Tablet Dosage Form by RP-HPLC. Asian Journal of Research in Chemistry. 2017; 10(6):783-788.

22. Validation of Analytical Procedures: Text and Methodology Q2 (R1), ICH Harmonised Tripartite Guideline, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005).

Received on 31.10.2018 Modified on 12.12.2018

Asian J. Research Chem. 2019; 12(1): 16-20.

DOI: 10.5958/0974-4150.2019.00004.X

Drug	% Level of addition	Amount of standard added (μg/ml)	Method A		Method B
Drug	% Level of addition	Amount of standard added (μg/ml)	% Recovery ± SD	% RSD	% Recovery ± SD	% RSD
Met	50	2.5	99.09 ± 0.65	0.65	96.86 ± 0.82	0.85
	100	5	99.57 ± 0.68	0.68	99.33 ± 0.87	0.87
	150	7.5	100.14 ± 0.87	0.87	100.12 ± 0.86	0.86
Cana	50	0.75	98.71 ± 1.30	1.32	97.77 ± 0.58	0.59
	100	1.5	99.70 ± 1.53	1.53	100.46 ± 0.71	0.71
	150	2.25	100.80 ± 1.15	1.14	101.19 ± 1.25	1.24

Drug	Amount of drug (μg/ml)	Method A		Method B
Drug	Amount of drug (μg/ml)	% Drug Content ± SD	%RSD	% Drug Content ± SD	%RSD
Met	5	98.79 ± 0.86	0.87	99.22 ± 0.89	0.90
Cana	1.5	101.27 ± 0.57	0.56	100.18 ± 0.93	0.93

Drug	Amount of drug (μg/ml)	Method A		Method B
Drug	Amount of drug (μg/ml)	% Drug Content ± SD	%RSD	% Drug Content ± SD	%RSD
Met	5	98.82 ± 0.86	0.87	100.55 ± 0.98	0.97
Cana	1.5	101.37±0.77	0.78	101.34 ± 1.07	1.05