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

A novel approach was used to develop and validate a rapid isocratic Reversed Phase-High Performance Liquid Chromatographic method for the simultaneous estimation of Metformin Hydrochloride and Canagliflozin in bulk and pharmaceutical tablet dosage form with forced degradation studies. The separation was performed by using Kromasil C18 column (250mm×4.6 mm, 5mm particle size), Waters Alliance e2695 HPLC system with 2998 PDA detector and mobile phase contained a mixture of 0.01M Ammonium acetate (pH adjusted to 3.5 with orthophosphoric acid) and Acetonitrile (65:35, v/v). The flow rate was set to 1ml/min with responses measured at 254nm. The retention time of Metformin Hydrochloride and Canagliflozin was 2.440min and 3.713min respectively with resolution of 8.95.Linearity was established in the range of 50-300μg/ml for Metformin Hydrochloride and 5-30μg/ml for Canagliflozin with correlation coefficients (r2=0.999). The percentage recoveries were between (99.45%-100.65%) and (99.95%-100.74%) for Metformin Hydrochloride and Canagliflozin respectively. Validation parameters were evaluated according to the International Conference on Harmonization (ICH) Q2 R1 guidelines. The forced degradation studies were performed by using HCl, NaOH, H2O2, thermal, UV radiation and water. Metformin Hydrochloride and Canagliflozin are more sensitive towards oxidative degradation condition. The developed method was successfully applied for the quantification and hyphenated instrumental analysis.


Chemicals and reagents
Metformin Hydrochloride (API) was obtained from Rantus Pharma Pvt. Ltd., Hyderabad, India and Canagliflozin (API) was obtained from Manus Aktteva Biopharma Ltd., Ahmedabad, Gujarat.HPLC grade of Ammonium Acetate was obtained from Rankem Ltd., India and HPLC grade of Acetonitrile was obtained from Merck Specialities Private Limited, India.HPLC grade of Water and Ortho phosphoric acid was obtained from Rankem Ltd., India.Invokamet tablet contains Metformin Hydrochloride 500mg and Canagliflozin 50mg were kindly supplied by Janssen Pharmaceuticals, Inc.

Instrumentation
The analysis was performed by using a chromatographic system from Waters Alliance e2695 HPLC system with 2998 PDA detector.The HPLC system was equipped with Empower 2 software.Semi-micro analytical balance (India), Ultrasonic bath sonicator (Frontline FS 4, Mumbai, India), Digital pH meter (Systronics model 802) and Whatmann filter paper No. 41 (Whatmann International Ltd., England) were used in the study.

Chromatographic conditions
Metformin Hydrochloride and Canagliflozin was analysed in Kromasil C 18 column (250mm×4.6mm, 5mm particle size) column for the chromatographic separation.The mobile phase was composed of 0.01M Ammonium acetate (pH adjusted to 3.5 with orthophosphoric acid) and Acetonitrile (65:35, v/v).Filtered through 0.45µm nylon membrane filter under vacuum filtration and pumped at ambient temperature, at a flow rate of 1 ml/min with UV detection wavelength at 254nm.Injection volume was 20ìl.The run time was 8 min and the retention time of Metformin Hydrochloride and Canagliflozin was found to be 2.440min and 3.713min respectively with resolution of 8.95.The resulting HPLC chromatogram was shown in (Figure 6).

Solutions and sample preparation Preparation of Phosphate buffer
A 0.01M Ammonium acetate buffer was prepared by dissolving 0.77gm of Ammonium acetate in 1000ml of HPLC grade water and pH was adjusted to 3.5 with orthophosphoric acid.The buffer was filtered through 0.45ìm nylon membrane filter to remove all fine particles and gases.

Preparation of mobile phase
The above prepared Ammonium acetate buffer and Acetonitrile HPLC grade were mixed in the proportion of 65:35, v/v and was filtered through 0.45µm nylon membrane filter and degassed by sonication.

Preparation of diluent
Mobile phase was used as diluent.

Preparation of standard stock solutions of Metformin Hydrochloride and Canagliflozin
Standard stock solutions of Metformin Hydrochloride and Canagliflozin were prepared by dissolving 500mg of Metformin Hydrochloride and 50mg of Canagliflozin in100ml of diluent into a 100ml clean dry volumetric flask and the standard solutions was filtered through 0.45 ìm nylon membrane filter and degassed by sonicator to get the concentration of 5000µg/ml of Metformin Hydrochloride and 500µg/ml of Canagliflozin.

Preparation of standard solutions of Metformin Hydrochloride and Canagliflozin for assay
From the above standard stock solution of 5000µg/ml of Metformin Hydrochloride and 500µg/ ml of Canagliflozin further pipette 0.4ml and transferred into a 10ml volumetric flask and dilute up to the mark with diluent to get the concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ ml of Canagliflozin.

Preparation of sample solutions of Metformin Hydrochloride and Canagliflozin
Twenty tablets were accurately weighed and powdered and tablet powder equivalent to 500mg of Metformin Hydrochloride and 50mg of Canagliflozin were taken into 100ml clean dry volumetric flask, diluent was added and sonicated to dissolve it completely and volume was made up to the mark with the same diluent.Further pipette out 0.4ml from the above Metformin Hydrochloride and Canagliflozin sample stock solution into a 10ml volumetric flask and diluted up to the mark with diluent to get the concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin.20ml from standard and sample solution were injected into the chromatographic system and the peak areas were measured for Metformin Hydrochloride and Canagliflozin which was shown in (Figure 6 and 7) and the % assay was calculated by comparing the peak area of standard and sample chromatogram by using the formula given below and the assay results was shown in (Table 1).

Selection of wavelength
In simultaneous estimation of Metformin Hydrochloride and Canagliflozin isosbestic wavelength is used.Standard stock solutions of Metformin Hydrochloride and Canagliflozin were prepared by dissolving 500mg of Metformin Hydrochloride and 50mg of Canagliflozin in 100ml of diluent into a 100ml clean dry volumetric flask and the standard solutions was filtered through 0.45ìm nylon membrane filter and degassed by sonicator to get the concentration of 5000µg/ml of Metformin Hydrochloride and 500µg/ml of Canagliflozin.From the above standard stock solution of 5000µg/ml of Metformin Hydrochloride and 500µg/ml of Canagliflozin further pipette 0.4ml and transferred into a 10ml volumetric flask and dilute up to the mark with diluent to get the concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin.The wavelength of maximum absorption (ëmax) of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin were scanned using UV-Visible spectrophotometer within the wavelength region of 200-400 nm against mobile phase as blank.The isosbestic wavelength (ëmax) was found to be 254nm for the combination shown in (Figure 3).

Method validation
The developed method for the simultaneous estimation of Metformin Hydrochloride and Canagliflozin was validated as per the ICH guidelines for the parameters like system suitability, specificity, linearity, accuracy, precision, ruggedness, robustness, limit of detection (LOD) and limit of quantitation (LOQ) 17 .

System suitability
At first the HPLC system was optimized as per the chromatographic conditions.One blank followed by six replicates of a single calibration standard solution of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin was injected to check the system suitability.To ascertain the system suitability for the proposed method, the parameters such as retention time, theoretical plates, peak asymmetry and resolution were taken and results were presented in (Table 2).

Specificity
The effect of excipients and other additives usually present in the combined tablet dosage form of Metformin Hydrochloride and Canagliflozin in the determination under optimum conditions was investigated.The specificity of the RP-HPLC method was established by injecting the blank and placebo solution into the HPLC system.The representative chromatogram of blank and placebo was shown in (Figure 4 and 5).

Linearity and range for Metformin Hydrochloride and Canagliflozin
Aliquots of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6ml of mixed standard working solutions of Metformin Hydrochloride and Canagliflozin was pipette out from the standard stock solution of 5000µg/ml of Metformin Hydrochloride and 500µg/ml of Canagliflozin and transferred into a series of 10ml clean dry volumetric flask and make volume up to the mark with the same diluent to get the concentration of 50, 100, 150, 200, 250 and 300µg/ ml of Metformin Hydrochloride and 5, 10, 15, 20, 25 and 30µg/ml of Canagliflozin.The calibration standard solutions of Metformin Hydrochloride and Canagliflozin were injected using a 20ìl Hamilton Rheodyne injector and the chromatograms were recorded at 254nm and a calibration graph was obtained by plotting peak area versus concentration of Metformin Hydrochloride and Canagliflozin respectively.The linearity data is presented in (Figure 26) and (Table 3).

Acceptance Criteria: Correlation coefficient should be not less than 0.999 Accuracy studies for Metformin Hydrochloride and Canagliflozin
The accuracy of the method was determined by calculating recovery of Metformin Hydrochloride and Canagliflozin by the method of standard addition.Known amount of standard solution of Metformin Hydrochloride and Canagliflozin at 50%, 100% and 150% was added to a pre quantified sample solution and injected into the HPLC system.The mean percentage recovery of Metformin Hydrochloride and Canagliflozin at each level was calculated and the results were presented in (Table 4 and 5).

Preparation of pre quantified sample solution for accuracy studies
Tablet powder equivalent to 200mg of Metformin Hydrochloride and 20mg of Canagliflozin were taken into 100ml clean dry volumetric flask and diluent was added and sonicated to dissolve it completely and volume was made up to the mark with the same diluent and was filtered through 0.45 ìm nylon membrane filter.Further pipette out 1ml from the above Metformin Hydrochloride and Canagliflozin sample stock solution into a 10ml volumetric flask and diluted up to the mark with diluent to get the concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin.

Preparation of standard solution of Metformin Hydrochloride and Canagliflozin for accuracy studies
Standard stock solutions of Metformin Hydrochloride and Canagliflozin were prepared by dissolving 200mg of Metformin Hydrochloride and 20mg of Canagliflozin in100ml of diluent into a 100ml clean dry volumetric flask and the standard solutions was filtered through 0.45 ìm nylon membrane filter and degassed by sonicator to get the concentration of 2000µg/ml of Metformin Hydrochloride and 200µg/ml of Canagliflozin.

Preparation of 50% standard solution
From the standard stock solution of 2000µg/ml of Metformin Hydrochloride and 200µg/ ml of Canagliflozin further pipette 0.5ml and transferred into a 10ml volumetric flask and dilute up to the mark with diluent to get the concentration of 100µg/ml of Metformin Hydrochloride and 10µg/ ml of Canagliflozin.

Preparation of 100% standard solution
From the standard stock solution of 2000µg/ml of Metformin Hydrochloride and 200µg/ml of Canagliflozin further pipette 1ml and transferred into a 10ml volumetric flask and dilute up to the mark with diluent to get the concentration of 200µg/ ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin.

Preparation of 150% standard solution
From the standard stock solution of 2000µg/ml of Metformin Hydrochloride and 200µg/ ml of Canagliflozin further pipette 1.5ml and transferred into a 10ml volumetric flask and dilute up to the mark with diluent to get the concentration of 300µg/ml of Metformin Hydrochloride and 30µg/ ml of Canagliflozin.

Acceptance Criteria
The % Recovery for each level should be between 98.0 to 102.0%.

Precision studies for Metformin Hydrochloride and Canagliflozin Method precision (Repeatability)
Tablet powder equivalent to 200mg of Metformin Hydrochloride and 20mg of Canagliflozin were taken into 100ml clean dry volumetric flask, diluent was added and sonicated to dissolve it completely and volume was made up to the mark with the same diluent and was filtered through 0.45 ìm nylon membrane filter.Further pipette out 1ml from the above Metformin Hydrochloride and Canagliflozin sample stock solution into a 10ml volumetric flask and diluted up to the mark with diluent to get the concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin.A homogenous sample of a single batch is analysed six times and was checked whether the method is giving consistent results.The %RSD for the assay of six replicate injections was calculated as mentioned in (Table 6).

Acceptance Criteria
The % RSD for the assay of six sample injections should not be more than 2%.

System precision
The system precision was carried out to ensure that the analytical system is working properly.The standard preparation concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ ml of Canagliflozin was injected six times into the HPLC system and the %RSD for the area of six replicate injections was calculated as mentioned in (Table 7).

Acceptance Criteria
The % RSD for the peak area of six standard injections should not be more than 2%.

Intermediate precision/ruggedness
The intermediate precision (also known as Ruggedness) of the method was evaluated by performing precision on different laboratories by different analysts and different days.The sample preparation concentration of 200µg/ml of Metformin Hydrochloride and 20µg/ml of Canagliflozin was injected six times into the HPLC system and the %RSD for the assay of six replicate injections was calculated as mentioned in (Table 8).

Acceptance Criteria
The % RSD for the assay of six sample injections should not be more than 2%.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limit of Detection (LOD) and Limit of Quantification (LOQ) were calculated as 3.3×SD/S and 10×SD/S respectively as per ICH guidelines, Where SD is the standard deviation of the response (Y-intercept) and S is the slope of the calibration curve.The LOD is the smallest concentration of the analyte that gives a measurable response (signal to noise ratio of 3).The LOD of Metformin Hydrochloride and Canagliflozin was calculated and shown in (Table 9).The LOQ is the smallest concentration of the analyte which gives response that can be accurately quantified (signal to noise ratio of 10).The LOQ of Metformin Hydrochloride and Canagliflozin was calculated and shown in (Table 9).

Robustness
As part of the Robustness, deliberate change in the flow rate, mobile phase proportion of ±10% and column temperature was made to evaluate the impact on the method.The results reveal that the method is robust.The results are summarized in ( Table 10, 11 and 12).

Stability of solution
The %RSD of the assay of Metformin Hydrochloride and Canagliflozin from the solution stability and mobile phase stability experiments was within 2%.The results of the solution and mobile phase stability experiments confirm that the sample solutions and mobile phase used during the assays were stable upto 48hours at room temperature was calculated and shown in (Table 13 and 14).

Forced degradation studies Acid Degradation Studies
To 1ml of stock solution of Metformin Hydrochloride and Canagliflozin, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 60 0 C. The resultant solution was diluted to obtain 200µg/ml and 20µg/ml solution and 20µl solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 8) and purity plot of acid degradation for Metformin Hydrochloride and Canagliflozin was shown in (Fig. 14-15).

Alkali Degradation Studies
To 1ml of stock solution of Metformin Hydrochloride and Canagliflozin, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60 0 C.The resultant solution was diluted to obtain 200µg/ml and 20µg/ml solution and 20µl solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 9) and purity plot of alkali degradation for Metformin Hydrochloride and Canagliflozin was shown in (Fig. 16-17).

Oxidative degradation Studies
To 1ml of stock solution of Metformin Hydrochloride and Canagliflozin, 1 ml of 3% Hydrogen peroxide (H 2 O 2 ) was added and the solution was kept for 30 mins at 60 0 C.For HPLC study, the resultant solution was diluted to obtain 200µg/ml and 20µg/ml solution and 20µl solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 10) and purity plot

Thermal Degradation Studies
The standard drug solution was placed in oven at 105 0 C for 6hrs to study dry heat degradation.For HPLC study, the resultant solution was diluted     11) and purity plot of thermal degradation for Metformin Hydrochloride and Canagliflozin was shown in (Figure 20 and 21).

Photolytic degradation studies
The photochemical stability of the drug was also studied by exposing the drug solution to UV light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m 2 in photo stability chamber.For HPLC study, the resultant solution was diluted to obtain 200µg/ml and 20µg/ml solution and 20µl solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 12) and purity plot of photolytic degradation for Metformin Hydrochloride and Canagliflozin was shown in (Figure 22 and 23).

Water Degradation Studies
Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60ºC.For HPLC study, the resultant solution was diluted to 200µg/ml and 20µg/ml solution and 20µl solutions were injected into the HPLC system and the chromatogram were recorded to assess the stability of sample was shown in (Figure 13) and purity plot of water degradation for Metformin Hydrochloride and Canagliflozin was shown in (Figure 24 and 25).

RESULTS AND DISCUSSION
To optimize the RP-HPLC parameters, several mobile phase compositions were tried.A satisfactory separation and good peak symmetry for Metformin Hydrochloride and Canagliflozin were obtained with a mobile phase containing a mixture of 0.01M Ammonium acetate (pH adjusted to 3.5 with orthophosphoric acid) and Acetonitrile (65:35, v/v) was delivered at a flow rate of 1ml/min to get better reproducibility and repeatability.
Quantification was achieved with PDA detection at 254nm based on peak area.The retention time of Metformin Hydrochloride and Canagliflozin was found to be 2.440min and 3.713min respectively with resolution of 8.95was shown in (Figure 6).Linearity was established for Metformin Hydrochloride and Canagliflozin in the range of 50-300µg/ml for Metformin Hydrochloride and 5-30µg/ml for Canagliflozin with correlation coefficients (r 2 =0.999) and the percentage recoveries were between 99.45 % to 100.65% and 99.95% to 100.74% for Metformin Hydrochloride and Canagliflozin respectively, which indicate accuracy of the proposed method.The % RSD values of accuracy for Metformin Hydrochloride and found to be 0.27µg/ml and 0.01µg/ml respectively and LOQ values for Metformin Hydrochloride and Canagliflozin were found to be 0.83µg/ml and 0.04µg/ml respectively was shown in (Table 9).The % RSD values of robustness studies were found to be < 2% reveal that the method is robust enough was shown in ( Table 10, 11 and 12).These data show that the proposed method is specific and sensitive for the determination of Metformin Hydrochloride and Canagliflozin.The results of system suitability testing are given in (Table 2).
peak area of sample preparation AS= Average peak area of standard preparation WS = Weight of standard taken in mg WT=Weight of sample taken in mg P = Percentage purity of working standard DS= Dilution factor for standard preparation DT=Dilution factor for sample preparation

Table 10 : Summary of robustness (change in flow rate) for Metformin Hydrochloride and Canagliflozin Drug Change in Retention Change in flow Rate (0.9 ml/min to 1.1 ml/min) Flow rate Time Average peak SD % RSD USP Asymmetry (ml/min) (Mins) area (n=6) Plate Count
Fig. 5: Chromatogram of placebo