INTRODUCTION:

CANAGLIFLOZIN HEMIHYDRATE^[1-6]

Canagliflozin hemihydrate belongs to the class of sodium glucosetansport protein (SGLT2) inhibitors. It is used in the treatment of type-2 diabetes.

Canagliflozin inhibits the reabsorption of glucose from kidneys and lowers the renal glucose threshold by inhibiting sodiumglucose transport protein (SGLT2). Canagliflozin can be used as monothreapy or multi-therapy in the treatment of type-2 diabetes.

Molecular Formula: 2(C24H25FO5S).H2O

Molecular weight: 904.0466 g/mol

Chemical Name: (2S, 3R, 4R, 5S, 6R)-2-[3[[S-(4-Florophenyl) thiophen-2-yl] methyl]-4-methylphenyl]-6-(hdroxymethyl) oxane- 3, 4, 5, triol; hydrate

Structure:

Description: White crystalline powder

Solubility: Soluble in methanol, acetonitrile, ethanol, DMSO, insoluble in water

Category: Antidiabetic

Goal of analytical method developed basically deals with detailed description of the analytical procedure sufficiently. An analytical method includes all important operational parametres and specific instructions such as preparation of reagents, perform system suitability tests, description of blanks used, precautions and explicit formulas for calculation of test results.

The planned research work concerns with development of analytical procedures by instrumental techniques with development of analytical procedure by instrumental technique, deals with use of spectrophotometric and chromatographic techniques i.e. absorbance spectroscopy by utilizing UV spectrophotometer and separation technique relevant with HPLC. Now a day the use of these two is very common for determination of pharmaceuticals.

The ultimate goal of stability stored program is to propose a scientifically sound shelf life. Stability testing is a primary tool used to access expiration dating and storage conditions for pharmaceutical products. Many protocols have been used for stability testing, but most of the industries are now standardizing on the recommendation of the international conference on harmonization (ICH). These guidelines are developed as co-operative efforts between regulatory agencies and industry officials in Europe, Japan, United States.

Stability test is the only way to demonstrate that the pharmaceutical product would meet the laid down specification within acceptance criteria throughout its lifetime.

It is an interesting aspect whether formulation instead of drug substance can be directly subjected to stress condition for the development of a SIAM. The ICH guidelines Q1AR suggest stress testing only of the drug substance. For drug product there is no suggestion on conduct of stress studies directly on formulation other than photostability testing. For new drugs the information on intrinsic stability behaviour of the drug substance and the stability assay methods are usually kept secret by the inventors to protect these elements from exploitation.

Literature survey reveals that only one UV spectroscopy method and stability indicating liquid chromatography analytical method has been reported for determination of Canagliflozin hemihydrate in bulk or pharmaceutical dosage form, this research work especially recounts the use of spectrophotometric technique and chromatographic technique to estimate a drug from pharmaceutical dosage form.

Instrument and Chemicals:

Instruments:

UV-visible spectrophotometers: Jasco V-630 and Shimadzu-UV-1700

High performance liquid chromatography system: Shimadzu HPLC 1100 series chrmatograph equipped with isocratic pump LC-10ADVP, UV Visible detector.

pH Meter: EI, model No. 111E

Weighing balance: Shimadzu AUX220 and Analytical CAS-44

Stability chamber: THERMOLAB, Sr. No. 398/10/09-10

Photostability chamber: TERMOLAB, Sr. No. 399/10/09-10

Chemicals:

Canagliflozin Hemihydrate reference standard, Methanol (HPLC grade), Acetonitrile (HPLC grade), Ortho phosphoric acid (HPLC grade) Hydrochloric acid (AR grade) Sodium hydroxide (AR grade), Hydrogen peroxide (AR grade), Milli Q water.

Development of UV-Spectrophotometric Methods for the Estimation of Canagliflozin Hemihydratefrom its Tablets:

Preparation of Standard Solution: An accurately weighed (~20 mg) quantity of canagliflozin hemihydrate (CANA) was transferred in a 100.0mL volumetric flask, dissolved in sufficient quantity of methanol to prepare a standard stock solution having concentration 100.0μg/mL of CANA.

Working Standard Solution: A 1.0mL of the above standard solution was diluted up to 10mL with Methanol. (Concentration: 10μg/mL of CANA)

Selection of Wavelength^[7-11]: The working standard solution of CANA (10μg/mL) was scanned in the range of 400-200nm in 1.0cm cell against solvent blank (Methanol) and spectrum was recorded.

Fig. No. 1 The study of spectra shows peak maxima for CANA at 290 nm

Study of Beer– LambertLaw^[12-16]:

Aliquots of standard stock solution were diluted with methanol to get a concentration of standard drug in the sequential series of 5, 10, 15, 20, 25, 30µg/mL. Absorbance of each solution was measured at λmax i.e., 290nm.

Table No. 1: Observation for Study of Beer-Lambert Law of CANA

Sr. No.	Concentration (µg/mL)	Absorbance
1	5	0.3956
2	10	0.7810
3	15	1.1003
4	20	1.3685
5	25	1.6388
6	30	1.9317
Coefficient of Correlation		0.995

Methodsdevelopment:

Method I: Standard absorptivity value [A(1%, 1cm)]:

Standard solutions used for Beer-Lamberts law study and absorbance of CANA were used to calculate A (1%, 1cm) value, using formula as given below. The results are shown in Table No. 2.

Absorbance

A(1%,1cm) = ------------------------------------------ (6.1.1)

Concentration (g/100 mL)

Table No. 2: Absorptivity Value [A (1%, 1cm) Method]:

Sr. No.	Concentration (g/100mL)	Absorbance	(A 1%, 1cm)
1	0.001022	0.3956	387.66
2	0.002044	0.7810	382.09
3	0.003066	1.1003	358.87
4	0.004088	1.3685	334.76
5	0.005110	1.6388	320.70
6	0.006132	1.9317	315.01
Mean			349.848

Method II: First order derivative method:

The working standard solution of CANA (10µg/mL) was scanned in the range of 200-400nm and first order derivative spectra was recorded shown in Fig. No. 2 from the spectra 271.2nm was selected for furtherstudy.

Fig. No. 2: First order derivative Spectra of CANA

The results are shown in Table No. 3 and calibration curve plotted as absorbance Vs concentration which is shown in Fig. No. 2

Table No. 3: Observation of CANA for Method II

Sr. No.	Concentration (µg/mL)	Absorbance First order derivative method
1	5	0.014
2	10	0.027
3	15	0.040
4	20	0.053
5	25	0.067
6	30	0.081
Coefficient of Correlation		0.999

Method III: (Second order derivativemethod):

The working standard solution of CANA (10µg/mL) was scanned in range of 200-400nm and second order derivative spectra was recorded shown in Fig. No. 3. From the spectra 266.8nm was selected for further study.

Fig. No. 3: Second order derivative Spectra for CANA

The results are shown in the Table No.4 and calibration curve plotted as absorbance Vs concentration which is shown in Fig. No. 3.

Table No. 4: Observation of CANA Method-ΙΙΙ

Sr. No.	Concentration (µg/mL)	Absorbance Second order derivative method
1	5	0.0012
2	10	0.0024
3	15	0.0033
4	20	0.0045
5	25	0.0053
6	30	0.0067
Coefficient of Correlation		0.996

Method IV. (Area under curve):

^{For this method wavelength selected were
295 (λ}1^{) and
285 nm (λ}2^).The area under curve for solution concentration range 5-30µg/mL of ^{CANA was noted in the range 295 (λ}1^{)
and 285 nm (λ}2^{). The AUC (Area}under curve) involves the calculation of integrated valve of absorbance ^{with respect to the
wavelength λ}1 ^{and λ}2^{. Area calculation
processing}item calculation the area bound by the curve and the horizontal axis. The horizontal axis was selected by entering the wavelength range over which the area has to be calculated. The results are shown in Table No.5. The spectra displaying AUC was show in Fig.No.4.

Fig. No. 4: Spectrum of CANA for area under curve

Table No. 5: Observation of CANA for Method-ΙV

Sr. No.	Concentration(µg/mL)	Area Under Curve
1	5	4.8063
2	10	9.3919
3	15	13.8070
4	20	18.8440
5	25	22.2873
6	30	26.1562
Coefficient of correlation		0.996

Estimation of Canagliflozin Hemihydrate in tablet was carried out by UV-Spectrophotometric methods like absorptivity value (Method-I), first order derivative (Method-II), second order derivative (Method-III) and Area under curve method (Method-IV) by using Shimadzu UV 1700 double beam spectrophotometer. The solubility of CANA was in methanol and hence it was selected as a solvent for the estimation of CANA.

Wavelength selected for method-I was 290.0nm, for method-II was 271.2nm, for method-III was 266.8nm and wavelength range selected for method-IV was 285.0 nm -295.0nm. Beer-Lambert’s law was obeyed in the concentration range of 5-30μg/mL for method I and method IV. % Labelled claim of CANA for method-I, method-II, method-III and method-IV were estimated^[17-20]. The results of estimation of CANA in marketed formulation and recovery studies are summarized in Table No. 6.

Validation of the proposed methods^[21-27]:

Validation of proposed method was carried out as per ICH guideline.

Accuracy:

Accuracy of proposed methods was ascertained on the basis of recovery studies performed by standard addition method.

Precision:

Precision of any analytical method was expressed as SD and RSD of series of measurement. Precision of estimation of CANA by proposed methods was ascertained by replicated analysis of homogeneous sample of tablet content.

Linearity and Range:

Accurately measured quantity of Canagliflozin hemihydrate equivalent to 80, 90, 100, 110 and 120% of label claim were taken and dilutions were made as described under marketed formulation. The absorbance of each resulting solution was measured at 290.0nm, 271.2nm, 266.8nm and, 285.0-295.0nm in 1.0 cm cell using solvent blank. The absorbances for linearity study are shown in Table No. 6.5. The plots were constructed as concentration Vs absorbance and found to be linear as depicted in Fig. No. 7

Table No. 7: Observation for linearity and range study

Sr. No.	% labelled Claim	Absorbance
Sr. No.	% labelled Claim	290.0 nm	271.2 nm	266.8 nm	285.0-295.0 nm
1	80	0.6248	0.021	0.0019	7.5135
2	90	0.7029	0.024	0.0021	8.4527
3	100	0.7912	0.027	0.0024	9.4991
4	110	0.8591	0.029	0.0026	10.3310
5	120	0.9572	0.032	0.0028	11.2702
Coefficient of correlation		0.997	0.996	0.994	0.998

Ruggedness:

The content of CANA in tablet was analyzed by proposed methods by two different analysts. Results are shown in Table No. 8.

Different Instrument:

The content of CANA in tablet was analyzed by proposed methods by two different instrument. Results are shown in Table No. 9.

Interday and intraday variation:

An accurately measured quantity of tablet powder equivalent toabout43.5 mg transferred to 100.0 mL of volumetric flask, sufficient quantity of methanol added and sonicated for 15 min. And diluted up to the mark with methanol. The content in the flask were filtered through whattman filter paper. Form these filtrate 5.0 mL of the filtrate was diluted to 100.0 mL in a volumetric flask using methanol. The absorbance of and AUC of the final solution was recorded after 0^thhr, 3^rdhr, and 5^thhr in 1.0 cm cell at selected wavelengths. similarly, the absorbance of the same solution were measured onthe 1^st, 3^rd, 5^thand the percent label claim were calculated using formulae as described under marketed formulation. The results are shown in Table No. 10.

Table No. 10: Observation of intraday study

Sr. No.	Intraday	Wt. taken (mg)	% Labelled claim
Sr. No.	Intraday	Wt. taken (mg)	Method-𝚰	Method-𝚰𝚰	Method-𝚰𝚰𝚰	Method-𝚰V
1	1^st hr	43.68	100.19	99.76	100.02	102.02
2	2^nd hr		99.70	99.90	99.76	101.21
3	3^rd hr		100.13	100.76	99.76	101.70
Mean			100.00	100.14	99.84	101.64
± SD			0.26	0.54	0.15	0.40
%RSD			0.27	0.54	0.15	0.40

Table No. 11: Observation of interday study

Sr. No.	Day Interval	Wt. taken (mg)	% Labelled claim
Sr. No.	Day Interval	Wt. taken (mg)	Method-𝚰	Method-𝚰𝚰	Method-𝚰𝚰𝚰	Method-𝚰V
1	1^st day	43.68	99.41	99.76	100.02	102.02
2	2^nd day		99.96	99.90	99.76	101.38
3	3^rd day		100.51	99.76	99.76	101.17
Mean			99.96	99.80	99.48	101.52
± SD			0.55	0.08	0.15	0.44
%RSD			0.55	0.08	0.15	0.44

Development of Validated RP-HPLC Method for Estimation of Canagliflozin Hemihydrate from ItsTablets:

Preparation of StandardSolution:

Standard stocksolution: An accurately weighed quantity of CANA (10.0 mg) was transferred in a 100.0 mL volumetric flask, dissolved in sufficient quantity of acetonitrile to prepare a standard stock 100µg/mL.

Working standard solution:

The standard stock solution was appropriately diluted with acetonitrile to get the final concentration of 25µg/mL.

Selection of mobile phase:

Using the following chromatographic parameters, various mobile phases was tried to select a suitable one.

The following chromatographic parameters were maintained for the present study.

^Column:^{ACE C}18 ^{(150 × 4.6 ×5µ)}

Detection wavelength: 290nm

Flowrate: 1mL/min

Temperature: 25-30ºC

Injection volume: 20µL

Mobile phase: Aetronitrile: orthophosphoric acidsolution

Preparation of orthophosphoric acid solution: 1.0mL of orthophosphoric acid added in 1000.0mL of double distilled water and prepared 0.1% orthophosphoric acid solution. Each mobile phase was sonicated and filtered through o.45µm membrane filterpaper.

Diluent: Acetronitrile

selection of wavelength:

The working standard solution of CANA (25µg/mL) was scanned in the range of 400-200 nm in 1.0cm cell against solvent blank (Acetonitrile) and the spectra was recorded.

Trial I-(For - Column):

In the first trial, the different column was used for same mobile phase Acetonitrile:Orthophosphoric acid solution (60:40).

^{1. Phemomax ODS column C}18 ^(250×4.6mm)

^2.^{Column ACE C}18 ^{(100×4.6×5µ)}

Trial II (For-Mobile phase)

Table No. 12: Selected for Mobile phase trails

Sr. No.	Mobile phase	Remarks
1	Methanol: Acetonitrile (75:25)	Peak not proper
2	Methanol: Acetonitrile (80:20)	Tailing occur
3	Methanol: Acetonitrile: OPA solution (70:25:5)	Tailing, symmetry 1.9
4	Acetonitrile: Orthophosphoric acid solution (70:30)	Asymmetry 1.12, R.T is 2.3
5	Acetonitrile: OPA solution (60:40)	Sharp peak, R.T is 2.9

2.1 Methanol: Acetonitrile (75:25)

2.2 Methanol: Acetonitrile (80:20)

2.3 Methanol:Acetonitrile: Orthophosphoric acid solution (70:25:5)

2.4 Acetonitrile: Orthophosphoric acid solution (70:30)

2.5 Acetonitrile: Orthophosphoric acid solution (60:40)

Validation of Method^[28-32]:

Validation of the proposed method was carried out as per ICH guideline.

System Suitability:

As per methodology, injected blank, standard solution for five times and checks standard solution into HPLC system

Table No. 13: Observation of system suitability parameters

Sr. No.	Standard weight taken (mg)	AUC of CANA (mV)
1	20.4 mg	1517.256
2		1574.096
3		1551.501
4		1530.501
5		1585.676
6		1512.586
	Mean	1545.293
	% RSD	1.96
	Theoretical plate/column	3526
	Retention time	2.94
	Asymmetry	0.744

Accuracy:

Accuracy of the proposed method was ascertained on the basis of recovery studies performed by standard addition method.

Precision:

Precision of any analytical method was expressed as SD and %RSD of series of measurements. Precision of estimation of CANA by proposed method was ascertained by replicate analysis of homogeneous samples of tablets.

Linearity and Range:

An accurately weighed tablet powder equivalent to 80, 90, 100, 110 and 120% of label claim was taken and dilutions were made as described under marketed formulation. then each solution was injected and chromatogram were recorded.

The correlation coefficient was found to be and results are recorded in Table No.14.

Table No.14: Linearity and range study

Sr. No.	Wt. of table powder (mg)	% of level	Area (mV)
1	33.72	80	1268.20
2	39.06	90	1426.54
3	43.40	100	1585.25
4	47.74	110	1743.77
5	52.08	120	1902.30
Coefficient Correlation			0.995

Ruggedness:

Differentanalyst:

The ruggedness of the proposed method has been verified by analyzing the tablet sample used for method precision by two different analysts using same instrument. The ruggedness results were compared with method precision data. The overall mean, standard deviation (SD) and %RSD of the assay value are shown in Table No. 15

Table No. 15: Observation of ruggedness study (Different analyst)

Sr. No.	% Estimation of CANA
Sr. No.	Analyst-I	Analyst-II
1	100.56	99.78
2	98.62	99.15
3	99.71	100.56
Mean	99.63	99.83
±SD	0.97	0.70
%RSD	0.98	0.71

Intraday and Interdayvariation:

The sample was prepared and analysed as per the proposed method. After equilibration of stationary phase, sample solution were injected separately at 0 Hr, 3 Hr, 5Hr, and chromatograms were recorded. Similarly the same solutions were injected on 1^st, 3^rd, 7^th, 10^th day. The chromatogram so recorded and results were calculated. The contents of CANA were calculated by comparing the peak of sample with that of standard using formula given under marketed formulation.

Robustness:

The robustness of the method was evaluated by injecting the sample at deliberately varied chromatographic conditions viz. Composition of mobile phase, wave length ± 5 nm, flow rate. The system suitability was evaluated and amount of CANA were calculated from sample solution in each varied condition. Results are tabulated in Table. No. 18

Table No. 16: Observation of intraday study

Sr.No.	Time (Hr)	Wt. of tab. Taken (mg)	AUC (mV)	% Labelled claim
1	0	43.5	1543.844	99.78
2	3		1551.446	100.27
3	5		1533.645	99.12
			Mean	99.72
			±SD	0.57
			%RSD	0.58

Table No. 17: Observation of interday study

Sr. No.	Days	Wt. of tab. taken (mg)	AUC(mV)	% Labelled claim
1	Day 1	43.5	1526.325	98.65
2	Day 2		1561.945	100.95
3	Day 5		1542.101	100.98
4	Day 7		1559.025	98.65
			Mean	99.80
			±SD	1.33
			%RSD	1.34

CONCLUSION:

The results obtained by UV and RP- HPLC methods for determination of Canagliflozin Hemihydrateare reliable, accurate and precise. The method does not have any interference of excipients while determining Canagliflozin hemihydrate from their formulation. Hence, developed methods can be employed for routine quality control analysis of Canagliflozin hemihydrate in tablet.

Hence, the present analytical method proved as stability indicating the results were within the acceptance criteria therefore can be used for regular analysis in Pharmaceutical bulk drug industry and at the formulation manufacturing industry for drug estimation,There is no interference of excepients in the injection formulation such as diluents, solubulizers etc.

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Received on 09.10.2020 Revised on 29.10.2020

Asian J. Pharm. Ana. 2021; 11(1):29-37.

DOI: 10.5958/2231-5675.2021.00006.5

Sample		% Label Claim				% Recovery
Sample		M-I	M- II	M- III	M- IV	M -I	M -II	M- III	M-IV
MF	Mean	99.88	99.78	99.80	99.94	101.64	100.19	100.46	101.61
MF	SD	0.52	0.80	0.32	0.61	0.50	0.69	1.16	0.43

Sr. No.	Analyst	Wt. of tablet powder taken (mg)	% Labelled claim
Sr. No.	Analyst	Wt. of tablet powder taken (mg)	M-I	M-II	M-III	M-IV
1	Analyst 1	43.68	99.89	99.89	100.14	102.47
2	Analyst 2	43.71	99.63	99.97	100.53	102.82
Mean			99.63	99.93	100.33	102.64

Sr. No.	Instrument	Wt. taken(mg)	% Labelled claim
Sr. No.	Instrument	Wt. taken(mg)	Method𝚰	Method𝚰𝚰	Method𝚰𝚰𝚰	Method𝚰V
1	Shimadzu-2030	43.68	100.55	99.75	100.25	100.36
2	Shimadzu-1100	43.68	99.63	99.56	100.51	100.75
Mean			100.09	99.65	100.38	100.55

Sr. No.	Deliberate condition	Wt. tablet taken (mg)	RT(min)	Theoretical plate	Asymmetry
1	Standard condition	43.5	2.970	3590	0.714
2	Mobile phase (65:35)		2.807	4087	0.706
3	Mobile phase (55:45)		2.813	3045	0.712
4	Wavelength (285 nm)		2.960	3371	0.705
5	Wavelength (295 nm)		2.953	3550	0.706
6	Flow rate (1.2 mL/min)		2.457	3131	0.738
7	Flow rate (0.8 mL/min)		3.930	3639	0.707
				Mean	0.712
				±SD	0.011
				%RSD	1.64