Method Development and Validation of Canagliflozin by using RP-HPLC in Pure and Tablet Dosage Form

 

Swapna Singh*, Prem Kumar Bichala, Abhishek Agrawal

School of Pharmaceutical Sciences, Pratap University, Sunderpura – 303104, Jaipur, Rajasthan, India.

 

ABSTRACT:

A new method was established for simultaneous estimation of Canagliflozin by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Canagliflozin by using INERTSIL column, C₁₈ (150x4.6)5µm column, flow rate was 1ml/min, mobile phase ratio was Water: Acetonitrile (70:30), detection wavelength was 264nm. The instrument used was Hitachi HPLC Auto Sampler, Separation module 1575. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study for Canagliflozin was found in concentration range of 1μg-5μg and 100μg-500μg and correlation coefficient (r2) was found to be 0.999 and 0.999, %mean recovery was found to be 100% and 100.5%, %RSD for repeatability was 0.2 and 0.4, %RSD for intermediate precision was 0.5 and 0.1 respectively.

 

 

 

INTRODUCTION:

DRUG NAME: Canagliflozin

It belongs to a new class of anti-diabetic drugs that works by inhibiting the sodium-glucose transport protein (SGLT2).^4,5

 

Category:

Hypoglycemic Agents

Drugs Used in Diabetes

 

Figure 1: Structure of Canagliflozin

 

IUPAC Name:

(2S,3R,4R,5S,6R)-2-(3-{[5-(4-fluorophenyl) thiophen-2-yl] methyl}-4-methylphenyl)-6-(hydroxymethyl) oxane-3,4,5-triol ^,4,5

 

Chemical formula: C₂₄H₂₅FO₅S

 

Molecular weight: 444.516

 

MATERIALS AND METHODS:

 

Table 1: Instruments used

UV-Visible Spectrophotometer	Lab india
HPLC	Shimadzu (LC 20 AT VP)
HPLC	Agilent 1200 series
Sonicator	PCi
pH meter	Srini electronics systems
Digital balance	Matter Toledo
Syringe	Hamilton
HPLC Column	C18 (4.6 ID x 150) 5µm

 

Table 2: Reagents used

Water	HPLC Grade
Methanol	HPLC Grade
Potassium Dihydrogen ortho Phosphate	AR Grade
CAN	HPLC Grade
Ammonium acetate buffer	AR Grade
Sodium dihydrogen phosphate	AR Grade

 

Table 3: Drug used

Canagliflozin	Chandra labs, Prashnathi nagar, kukatpally, Hyd
Invokana--100mg	From local pharmacy

 

Mobile Phase:

A mixture of ACN 60 volumes and Sodium dihydrogen ortho phosphate 40 volumes pH 3.5 were prepared. For the degassing purpose mobile phase was sonicated for 10min.^2,3

 

Preparation of Buffer:

Weigh 1.36gms of sodium dihydrogen ortho phosphate in 1000ml of water by adjusting the solution with sodium hydroxide pellets to pH 3.5.^2,3

 

RESULTS AND DISCUSSION:

Solubility Studies: Canagliflozin:

Soluble in organic solvents such as methanol, acetonitrile and water.

 

Estimation of Wavelength (λmax):

In estimation of drug wavelength maxima is used.

 

Results:

The absorption curve shows characteristic absorption maxima at 246 nm for Canagliflozin.

 

Figure 2; UV-VIS spectrum of Canagliflozin

 

Observations:

λ_max was found to be 246 nm for Canagliflozin

 

Method Development of Canagliflozin:

 

Trial - 1

Chromatographic conditions

M.P: Phosphate buffer: CAN

Ratio                      : 60:40

Column                 : BDS (250×4.6× 5µ)

Lambda max        : 246nm

Rate of Flow         : 1ml/min

pH: 3.0

 

Preparation of standard solution:

10mg of Canagliflozin in 10ml of volumetric flask and dissolve with 10ml mobile phase and make up the volume with mobile phase. 10µg/ml of Canagliflozin is prepared by reducing concentration 1ml of canagliflozin to 10ml with mobile phase.^2,3

 

Trial- 2

Chromatographic conditions:

M.P                        : Phosphate buffer: Methanol

Ratio                      : 55:45

Column                 : Zodiac C18 Column (250×4.6 ×5µ)

Wavelength          : 246nm

Rate of Flow         : 1.0ml/min

pH: 2.5

 

Standard solution Preparation:

10mg of Canagliflozin in 10ml of volumetric flask and dissolve with 10ml mobile phase and make up the volume with mobile phase. 10µg/ml of canagliflozin is prepared by reducing concentration 1ml of canagliflozin to 10ml with mobile phase.^2,3

 

Observation:

Fronting and tailing of the peak was occurred.

 

Trial-3:

Mobile Phase: Water: Acetonitrile

Ratio                      : 70:30

Column                 : Zodiac, C18 (250×4.6× 5µ)

Wavelength          : 246nm

Rate of flow          : 1ml/min

 

Standard solution Preparation:

Canagliflozin 10mg in 10ml of volumetric flask and dissolve with 10ml mobile phase and make up the volume with mobile phase. 10µg/ml of canagliflozin is prepared by reducing conc. 1ml of canagliflozin to 10ml with mobile phase.^2,3

 

Table 4: Optimized Chromatographic Conditions

M.P	Water: ACN
Column	INERTSIL column, C18 (150x4.6 ID) 5µm
Rate of Flow	1.0 ml/min
Column Temperature	Room Temperature (20-25֯C)
Sample Temperature	Room Temperature (20-25֯C)
λmax	256 nm
Volume of injection	20µl
Run Time	6 min
Rt	Around 20187 min for Canagliflozin

 

 

Table 5: Assay results of Canagliflozin

Canagliflozin
	Standard Area	Sample Area
Injection-1	3522.517	3521.076
Injection-2	3544.340	3542.298
Injection-3	3612.255	3514.071
Injection-4	3541.000	3537.099
Injection-5	3535.187	3544.651
Average Area	3551.06	3531.839
Tablet average weight	0.0478gm
Standard weight	10mg
Sample weight	10mg
Label amount	90mg
std. purity	98.5%
Assay(%purity)	95.96%

 

Validation:

Specificity by Direct comparison method:

There is no interference of mobile phase, solvent and placebo with the analyte peak and also the peak purity of analyte peak which indicate that the method is specific for the analysis of analytes.

 

Linearity and range:

Standard solution Preparation:

10mg of Canagliflozin in 10ml of volumetric flask and from this, 1ml dissolve in 10ml of M.P and make up the volume.¹

 

Table 5: Linearity Preparations

Preparations	Volume from standard stock transferred in ml	Volume made up in ml (with mobile phase)	Concentration of solution (µg /ml)
Preparation 1	6	10	60
Preparation 2	8	10	80
Preparation 3	10	10	100
Preparation 4	12	10	120
Preparation 5	14	10	140

Table 6: Linearity of Canagliflozin

S. No.	Conc. (µg/ml)	Area
1	60	2259.834
2	80	2863.357
3	100	3518.693
4	120	4180.489
5	140	4890.409

 

Figure 3: Calibration Curve of Canagliflozin

 

Acceptance criteria:

The relationship between the conc. and area of Canagliflozin must be linear in the particular vary and the correlation should NLT 0.999.¹

 

Observation:

The regreration coefficient for linearity obtained between Area Vs Conc. for standard preparations of Canagliflozin is 0.999.

 

Accuracy:

By the Recovery profile Accuracy was estimated. The referral STD (100µg/ml, 120µg/ml and 140µg/ml) were added at the level of 100%, 120%, 140%

 

 

Table 7: Canagliflozin Recovery results

 

Observation:

The % recovery of Canagliflozin is 98.40%.

 

Precision:

Method precision:

The prepared Canagliflozin sample preparations was injected 6 times in to the column as per test method.¹

 

Acceptance criteria:

The Assay % RSD of Canagliflozin must be NMT 2.0%.

 

Table 8: Canagliflozin Method precision Results

Canagliflozin
S. No.	Rt	Area
1	2.850	3506.392
2	2.770	3524.283
3	2.830	3499.880
4	2.827	3540.192
5	2.843	3536.313
6	2.840	3544.651
avg	2.826667	3525.285
stdev	0.0290	18.55
%RSD	1.02	0.52

 

Observation:

Canagliflozin values were exhibiting that the % RSD results were within the range.

 

LIMIT OF DETECTION^2,3

                3.3 σ

LOD = -------

                 S

Observation:

The LOD was originated to be 0.003µg/ml.

 

LIMIT OF QUANTIFICATION (LOQ)^2,3

                10 σ

LOQ = -------

                 S

Observation:

The LOQ was originated to be 0.011µg/ml

 

Robustness:

Table 9: Robustness Results

Parameter	Canagliflozin
	Rt (min)	TF
Flow 0.8ml/min 1.2ml/min	4.240 2.120	1.44 1.38
Wavelength 244nm 248nm	2.817 2.770	1.42 1.42

 

Observation:

From the above obtained values the system suitability parameters were originated within the limit at all different conditions.

 

Ruggedness:

The ruggedness was by the estimating by analyst-to-analyst variant by conducting the Assay by 2 different analysts

 

Acceptance criteria:

The Assay % RSD values between 2 analysts must be NMT 2.0%.

 

Table 10: Results for Ruggedness

Canagliflozin	%Assay
Analyst _01	98.96
Analyst _02	98.12
%RSD	0.64

 

OBSERVATION:

From the observation the %RSD between 2 analysts Assay values NMT 2.0%, consequently the process was rugged.

 

CONCLUSION:

A new accurate, rapid, precise technique has been developed for the determination of Canagliflozin in pharmaceutical dosage form by RP-HPLC.

 

From the proposed one it was successfully applied for the quantification of API contented in the profitable formulations of Canagliflozin in Educational institutions and Quality Control department of various industries.

 

REFERENCE:

1.      International Conference on Harmonization, "Q2A: Text on Validation of Analytical Procedures," Federal Register. 1995, 60, 11260–11262.

2.      Ghulam, A. S. PLC Method Development and Validation for Pharmaceutical Analysis. Pharmaceutical Technology Europe. 2004, 7, 55 -63.

3.      Radhika, R.; Alfred, D. G. Guidance for Industry- Analytical Procedures and Methods Validation. Federal Register, 2000, 2396, 1-32.

4.      https://www.drugbank.ca/drugs/DB08907

5.     Sandouk P, Bouvierd'Yvoire M, Chretien P, Tillement JP, Scherrmann JM (January 1994). "Single-dose Apixaban in healthy volunteers". Biopharmaceutics and Drug Disposition 15 (1): 87–92.doi:10.1002/bdd.2510150108. PMID 8161719.

 

 

Received on 19.11.2020            Modified on 21.12.2020

Accepted on 19.01.2021       ©A&V Publications All right reserved