1. INTRODUCTION:

A Stability indicating assay method can be defined as “Validated quantitave analytical method that can detect the change with time in the chemical, physical or microbiological properties of the drug substance and drug products are specific so that the content of active ingredients and degradation products can be accurately measured without interference”

Generally force degradation/stress testing is used to generate the samples for stability-indicating assay methods. Force degradation/stress testing is defined as “the stability testing of drug substance and drug product under conditions exceeding those used for accelerated stability testing”.

Degradation can be achieved by exposing the drug, for extended period of time, to extremes of pH (HCl or NaOH solutions of different strengths), at elevated temperature, to hydrogen peroxide at room temperature, and to UV light, to achieve degradation to an extent of 5-20%. Generally, trial and error experimentation is used during these experiments. This trial and error approach is generally cost, labor, and time intensive and should be substituted with some systematic approach.

Canagliflozin is a sodium glucose co-transporter-2 (SGLT-2) inhibitor and to improve the glycemic control in adult patient with type 2 diabetes. Chemically known as(2S,3R,4R,5S,6R)-2-(3-{[5-(4-fluorophenyl)thiophen-2-yl}methyl)-6-(hydroxymethyl)oxane-3,4,5-triol.The chemical structure of Canagliflozin shown in Fig.1

Fig 1: Structure of Canagliflozin

As per literature review, several methods were there for the determination of its pharmacological action. Canagliflozin was estimated by only few method UV spectroscopy, HPLC, HPTLC and UPLC. Two Stability indicating RP-HPLC method. The aim of present work was to develop and validate a accurate, cost effective and precise stability indicating RP-HPLC method for determination of Canagliflozin.

2. MATERIALS AND METHODS:

2.1. Apparatus and equipment:

A HPLC method for estimation of Canagliflozin using Analytical technologies Ltd. HPLC system on Grace C18 (250mm x 4.6ID, Particle size: 5 micron) column was used. The instrument is equipped with manual sampler and UV-3000-m detector. A 20µL injector port was used for injecting the samples. Data was analyzed by using HPLC Workstation. A Wenser ultra Sonicator was used for sonication, a Wenser High Precision balance also used for weighing.

2.2. Reagent and Chemicals:

Pharmaceutical grade Canagliflozin was supplied as a gift sample from Macleod Pharmaceutical Pvt. Ltd. Gujrat, India. Methanol used in analysis were of HPLC grade and all other chemicals and reagents were of analytical grade and were purchased from S.D. FINE chemicals, Mumbai, India. The 0.45µ Nylon filter papers were purchased from Millipore (India) Pvt. Ltd., Bengaluru, India.

2.3. Chromatographic Conditions:

All chromatographic separation were carried out on Grace C18 (250mm x 4.6ID, Particle size: 5 micron) column, using mobile phase comprising methanol: water 90:10% v/v. The flow rate was kept constant throughout analysis at 0.9mL/min and eluent was detected at 290nm by UV-detector.

2.4. Standard Preparation: (Canagliflozin 1000µg/ml)

Accurately weighed and transferred 10mg Canagliflozin working standard into a 10mL clean dry volumetric flask, add few mL of methanol, sonicated for 15minutes and make up to 10mLwith methanol. From above stock solution, 0.1ml was pipeted out into a 10mL volumetric flask and then make up to the final volume with mobile phase. The chromatogram of standard Canagliflozin solution was shown in Fig.2. And the average retention time was found to be 4.413 min.

2.5. METHOD VALIDATION:

2.5.1. Accuracy and Precision:

It is the closeness of test results obtained by the method to the true value. It was determined by percent recovery of the standard API to the blank and it is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day andinter-day precision of method. The average recovery of the analyte of 80%, 100% and 120% solution. The amount found (mg) and %RSD was calculated and were shown in Table 1.

2.5.2. Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It is calculated to be 0.7212µg/mL by using formula,

LOD= 3. 3σ/S

Where,

σ = Standard deviation of the response,

S= Slope of calibration curve.

2.5.3. Limit of Quantitaion (LOQ):

LOQ is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. It was calculated to be 2.1854 µg/mL by using formula,

LOQ = 10 σ /S

Where,

σ = Standard deviation of the response,

S= Slope of the calibration curve.

2.5.4. Linearity:

Linearity is the ability of the method to elicit test results that are proportional to concentration of the analyte in the sample.

It was found to be in the range of 1-5µg/ml. The calibration graph was plotted, equation was obtained and the drug was found to be linear with a correlation coefficient (r2) of 0.999 were shown in Fig 1.

2.5.5. Robustness:

It is the capacity of the method to remain unaffected by small but deliberate variations in method parameters. The analysis was performed by slightly changing the wavelength (288nm and 292nm), and flow rate (0.80 and 1.0 mL/min).The variables are shown in Table 2.

2.6. Degradation studies:

Force degradation experiment were carried out on Canagliflozin under various conditions explained in ICH guidelines Q1A (R2), namely acidic, alkali, oxidative and photolytic conditions

2.6.1. Acid Degradation:

To 0.1ml of stock solution of canagliflozin, 10ml of 0.1N Hydrochloric acid was added and refluxed for 30 min at 60 °C. The resultant solution was diluted up to 50mL with methanol to obtain 200µg/mL solution and then 0.5mL pipette out and dilute upto 10mL with mobile phase and10µL solutions were injected into the system and the chromatograms were recorded to assess the stability of sample. The chromatogram of acid degradation studies were shown in figure.

2.6.2. Alkali degradation studies:

To 0.1ml of stock solution of canagliflozin, 10ml of 0.1N Sodium hydroxide was added and refluxed for 30mins at 60°C. The resultant solution was diluted upto 50mL with methanol to obtain 200µg/ml solution and then 0.5mL pipette out and diluted upto 10mL with mobile phase and10µL solution were injected into the system and the chromatograms were recorded to assess the stability of sample. The chromatogram of alkali degradation studies were shown in figure

2.6.3. Oxidation studies:

To 0.1ml of stock solution of canagliflozin, 1ml of 3% hydrogen peroxide (H₂O₂) was added separately. The solutions were kept for 24 hours at room temperature (RT). From the above stress solution, 0.1ml was pipette out into a 10 mL volumetric flask and then make up to the final volume with mobile phase 10 µg/mL solution and 10µL were injected into the system and the chromatograms were recorded to assess the stability of the sample.

2.6.4 Photo Stability studies:

In a petri plate drug powder form was directly expose to sunlight for 48 and 72 hours for photo stability study. For HPLC study, the resultant drug was weigh 10mg accurately and transfer into 10ml clean and dry volumetric flask, add few mL of methanol, sonicated for 15minutes and make up to 10mL with methanol. From the stress solution, 0.1ml was pippeted out in to a 10mL volumetric flask add then make up to the final volume with mobile phase 10µg/ml solution and 10µL were injected into the system and the chromatograms were recorded to assess the stability of the sample. The chromatogram of photo stability degradation studies were shown in figure.

2.7. Chromatographic Analysis of Forced Degradation samples:

After degradation, each sample obtained under each forced degradation condition was diluted appropriately with mobile phase to get a final concentration of 10µg/mL; the resulting solution was injected in the column under described chromatographic condition. The chromatogram obtained was diluted for area of drug peak and appearance of secondary peaks. The decrease in the area of drug peak and the occurrence of secondary peaks was considered as indication of degradation. The % degradation was calculated by using formula and was result shown in Table- 3.

Peak area of stressed sample

% Degradation = ------------------------------ X 100

Peak area of unstressed sample

3. RESULTS AND DISCUSSION:

Parameter	Condition
Mobile Phase	Methanol: water (90:10 v/v)
Column	Grace C18 (250mm x 4.6ID, Particle size: 5 micron)
Wavelength	290nm
Flow Rate	0.9 mL/min
Run Time	6.88 min

Figure 1: Representative Chromatogram of Canagliflozin in Methanol: Water (90:10% v/v)

Table 1: Accuracy and Precision studies

Amount added(mg)	Amount Found(mg)
Amount added(mg)	Day 1	Day 2	Day 3
80%(8mg)	10.678	10.469	10.514
	10.465	10.666	10.572
	10.642	10.719	10.648
Mean	10.595	10.618	10.578
Mean %Recovery	105.9562	106.1846	105.7852
SD	0.1138	0.1318	0.0669
%RSD	1.07469	1.24151	0.63304
100% (10mg)	29.331	29.237	29.5
	29.116	29.299	29.297
	29.436	29.216	29.219
Mean	29.294	29.251	29.338
Mean %Recovery	97.64995	97.50447	97.79652
SD	0.1628	0.0432	0.1455
%RSD	0.5559	0.1478	0.4959
120%(12mg)	50.257	49.548	50.420
	49.945	50.509	49.655
	50.129	49.871	49.871
Mean	50.110	49.976	49.9824
Mean %Recovery	100.2218	99.95296	99.96484
SD	0.1564	0.4892	0.3942
%RSD	0.3122	0.9789	0.7888

Table 1: Linearity for Canagliflozin

Sr. No.	Conc. (µg/mL)	Mean peak Area
1	1	448519
2	2	750967
3	3	1071920
4	4	1446027
5	5	1796872

Figure 1: Linearity graph of Canagliflozin

Table 2: Robustness Studies

Factors	Lower Limit	Upper Limit
Change in Flow rate	0.80	1.0
R.T.	5.010	3.926
Area	971978	966135
Theoretical plate	7897	7887
Asymmetry	1.21	1.20

Factors	Lower Limit	Upper Limit
Change in Wavelength (nm)	288	292
R.T.	4.351	4.266
Area	968373	975530
Theoretical plates	7792	7966
Asymmetry	1.91	1.22

Table 3: Degradation Data for Canagliflozin

Sr. No.	Degradation Conditions	% Degradation
1	Acid	7.42 %
2	Alkali	6.51 %
3	Oxidation	14.20 %
4	Photolytic	10.53 %

Figure 2: Representative Chromatogram of Acid Degradation of Canagliflozin

Figure 3: Representative Chromatogram of Alkali Degradation of Canagliflozin

Figure 4: Representative Chromatogram of Oxidative Degradation of Canagliflozin

Figure 5: Representative Chromatogram of Photolytic Degradation of Canagliflozin

4. CONCLUSION:

In conclusion, a simple, selective, sensitive and accurate stability indicating RP-HPLC method was developed and validated for the analysis of Canagliflozin. Further the method was found to be linear, precise, accurate and robust. The degradation studies reveal the stability of the drug.

Hence the proposed method can be used for the estimation of Canagliflozin in routine analysis.

5. REFERENCE:

1. Bakshi, M. and Singh S. Development of validated stability-indicating assay methods-critical review. Journal of Pharmaceutical and Biomedical Analysis. 2002; 28(6):1011-1040.

2. S. Singh and M. Bakshi, Guidance on Conduct of Stress Testing to Determine Inherent Stability of Drugs, Pharmaceutical Technology. 2000.

3. ICH Harmonised Tripartite Guideline, “Stability testing of new drug substances and products Q1A (R2),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, February 2003.

4. ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005.

5. Maddu Suma et al. (2014). RP-HPLC Method Development and Validation for the Estimation of Canagliflozin in Tablet Dosage Form. International Journal of Pharmacy, 5(4), pp.1288-1292.

6. A. Suneetha et al. (2015). A validated stability indicating RP-HPLC method for estimation canagliflozin in dosage form. Research Journal of Pharmaceutical, Biological and Chemical Science, 6(5), pp.1186-1194.

7. K. Ishpreet et al. (2016). Development and validation of a stability indicating RP-HPLC-PDA method for determination of canagliflozin in bulk and pharmaceutical dosage form. Pharm Methods, 7(1), pp.54-62.

8. Deepak Gaware et al. (2015). A validated stability indicating RP-HPLC method for simultaneous determination of metformin and canagliflozin in pharmaceutical formulation. World Journal of Pharmaceutical Sciences, 4(12), pp.631-640.

9. Beckett, A.H. and Stenlake, J.B. (Eds.) Practical Pharmaceutical Chemistry, 4th ed. 2-parts, New Delhi: CBS Publishers and Distributors.

10. British Pharmacopoeia, The Stationary Office, London, 2005.

11. The United States Pharmacopoeia- the National Formulary, United States Pharmacopoeial convention, Rockville, 2007.

Received on 01.07.2018 Modified on 05.10.2018

Asian J. Research Chem. 2019; 12(1): 11-15.

DOI: 10.5958/0974-4150.2019.00003.8