STABILITY INDICATING RP-UPLC-DAD METHOD FOR DETERMINATION OF METFORMIN AND CANAGLIFLOZIN IN BULK AND TABLET DOSAGE FORM
HTML Full TextSTABILITY INDICATING RP-UPLC-DAD METHOD FOR DETERMINATION OF METFORMIN AND CANAGLIFLOZIN IN BULK AND TABLET DOSAGE FORM
N. Padmaja 1 and G. Veerabhadram * 2
Faculty of Pharmacy 1, University College of Technology, Department of Chemistry 2, University College of Science, Osmania University, Hyderabad - 500007, Telangana, India.
ABSTRACT: The objective of the present work is to develop and validate a novel stability-indicating RP-UPLC-DAD method for the simultaneous analysis of Metformin and Canagliflozin in bulk and tablet dosage form. The chromatographic separation was accomplished on a Waters UPLC system equipped with autosampler and PDA detector. A volume of 5 µL of sample or standard was injected into the column and the analytes were separated by using the mobile phase containing mixture of 0.1% orthophosphoric acid buffer (pH adjusted to 3.0 with 0.1 N NaOH) and acetonitrile in the ratio 30:70% v/v at a flow rate of 0.25ml/min through C18 BEH (Ethylene Bridged Hybrid) UPLC (100 mm × 2.1 mm 1.7 µm) at 55 °C column temperature and the detector wavelength was set at 260 nm. Peak area and retention time of Metformin and Canagliflozin were found to be 651341 & 332544, 0.994 & 1.508 respectively. Regression analysis shows r value greater than 0.999 for Metformin and Canagliflozin. Percent recovery of Metformin and Canagliflozin was found to be 99.94 %-100.16% & 100.17%-100.68%, respectively. The developed RP-UPLC method was validated with regard to system suitability, linearity, robustness, accuracy, precision, LOD, LOQ and forced degradation studies.
Keywords: |
Forced degradation, Metformin, Canagliflozin, RP-UPLC, Stability – indicating and Validation
INTRODUCTION: Worldwide most of the patients with type 2 diabetes mellitus are commonly accomplished with singular-agent therapy, generally metformin. It is good and most commonly used antihyperglycemic agent which enhances the glucose margin in type 2 diabetes patients, lowering both basal plasma glucose and postprandial 1. Metformin lowers abdominal absorption of glucose and reduces hepatic glucose output and enhances the insulin productivity by increasing peripheral glucose more.
Metformin is a white crystalline compound with a Molecular formula C4H11N5.HCl and molecular weight 165.62 Fig. 1.
FIG. 1: CHEMICAL STRUCTURE OF METFORMIN
Canagliflozin is an inhibitor of SGLT2. SGLT2 is present in the proximal renal tubule especially in the kidney cortex, exactly in the S1 segment of the proximal tubule where it reuptakes the majority of glucose filtered by the glomerulus. Canagliflozin primarily transports glucose and sodium (Na) in a 1:1 ratio and it works as a low-attraction high-capability transporter. Quelling of SGLT2 results in enhanced urinary glucose excretion (UGE) due to reduced glucose reabsorption. Canagliflozin is a white to off-white powder, normally insoluble in water and freely soluble in acetonitrile, methanol and it is non-hygroscopic. The chemical name of canagliflozin is, (1S)-1, 5-anhydro-1-[3-[[5-(4-fluorophenyl)-2-thienyl]-methyl]-4-methylphenyl]-D-glucitol hemihydrates 2-5 Fig. 2.
FIG. 2: CHEMICAL STRUCTURE OF CANAGLIFLOZIN
Literature survey reveals that canagliflozin is estimated by UV spectrophotometric method 6, human plasma by RP-HPLC 7, HPLC method 8, rat plasma by LC-MS/MS 9, HPTLC 10 and few chromatographic methods are available for the simultaneous determination of Metformin and Canagliflozin in tablet dosage form by using RP-HPLC 11-19.
The analysis of two drugs using GC/MS, LCMS and LC-MS/MS were very expensive and very delicate methods as compared to UPLC for general quality control analysis. Because of this purpose, we need to use this advanced technique (UPLC) for estimation of stability studies of these drugs by using the RP-UPLC method.
MATERIALS AND METHODS:
Chemicals and Reagents: Qualified Canagliflozin standard was kindly obtained from Manus akkteva (Ahmadabad, Gujarat, India) and Metformin was obtained from Aurobindo pharmaceuticals Ltd. (Hyderabad, India). Invokamet (Canagliflozin -50 mg and Metformin-500 mg film-coated tablets) (USA) were purchased from local market. Orthophosphoric acid (AR grade) purchased from Merck (Darmstadit, Germany). Acetonitrile (HPLC grade) was obtained from JVR fine chemicals Ltd (Hyderabad, India). Milli-Q ® type 1 ultrapure water system (Sartorius, Germany), vacuum pump, purchased from PCI Analytics Pvt., Ltd (Mumbai, India) were also used.
Instrumentation and Chromatographic Conditions: The UPLC waters system was used for the method development and method validation. It consists of a binary solvent manager with connected with photodiode array detector (MA, USA) controlled with Empower software and auto sampler and auto-injector. C18 BEH UPLC (100 mm × 2.1 mm 1.7 µm) column from waters was used. Photostability studies were performed in photo stability chamber (Osworld Scientific Equipment Pvt. Ltd, Mumbai, India). Thermal stability studies were carried out in a dry air oven (Newtronic lifecare, Mumbai, India). The mobile phase consists of mixture of 0.1% orthophosphoric acid buffer and acetonitrile in the ratio of 30:70% v/v at a flow rate of 0.25 ml/min. The column temperature was maintained at 55 °C with detection wavelength of 260 nm and it maintained at 35 °C with an injection volume of 5 µl.
Preparation of Solutions:
Preparation of Mobile Phase: 0.1% orthophosphoric acid buffer solution was prepared by taking 1 ml of orthophosphoric acid in a 1000 ml volumetric flask, dissolved in water, made up to the mark after adjusting the pH of solution equal to pH = 3.0 with 0.1 N NaOH solution. Then, the resulting solution was filtered through 0.45 µ filter under vacuum filtration. A mixture of buffer and acetonitrile in the ratio 30:70% v/v was degassed in an ultrasonic water bath for 5 min allowed to cool at room temperature and then filtered through 0.45 µ filter under vacuum filtration. This prepared mobile phase was used as diluent.
Preparation of Standard Solution: Accurately weighed 100 mg of Metformin and 10 mg of Canagliflozin working standard was taken into a 10 ml clean dry volumetric flask, added about 7 ml of diluent and sonicated to dissolve it totally and made volume up to the mark with the same solvent (Stock solution). After that, pipetted 0.3 ml of the above stock solutions into a 10 ml volumetric flask and dilute up to the mark with diluent.
Preparation of Sample Solution: Accurately weighed 100 mg of Metformin and 10 mg of Canagliflozin working standard was transferred into a 10 ml clean dry volumetric flask, and about 7 ml of diluent was added and sonicated to dissolve completely and make volume up to the mark with the same solvent. After that 0.3 ml of the above stock solution was pipetted into a 10 ml volumetric flask and diluted up to the mark with diluent.
Method Validation: The developed method was validated as per ICH guidelines for its specificity, system suitability, linearity, accuracy, precision, LOQ, LOD, and robustness by using the following procedures 20-21.
System Suitability: A system suitability test was defined based on the analytical results obtained in various representative chromatograms. The concentration of the standard solution was found to be 300 µg/mL for Metformin and 30 µg/mL for Canagliflozin respectively. The column efficiency resolute from the sample peak >20,000, the tailing factor <5.0% and resolution between peaks of two drugs should be >1.5.
Precision: Precision was evaluated by studying the repeatability and intermediate precision. The precision of each level was determined by three replicate of concentrations (25, 50, and 75µg/mL) of Metformin and (5, 10, and 15 µg/mL) of Canagliflozin respectively.
Accuracy: The accuracy of the assay method was evaluated in triplicate by a recovery experiment using three different levels (50, 100, and 150% of the normal assay concentration). The standard solutions of Metformin (120, 240 and 360 μg/mL) and Canagliflozin (4.0, 8.0 and 12.0 μg/mL) were injected in triplicate (n=3) into UPLC system.
Linearity: Calibration curve of Metformin and Canagliflozin solutions was constructed by plotting peak area vs. concentration. The least-square regression equation was applied, and the calibration curve was plotted over the concentration range of 100-500 µg/mL for Metformin and 10-50 µg/mL for Canagliflozin.
Limit of Quantitation (LOQ) and Limit of Detection (LOD): The quantitation limit of an analytical procedure is the lowest amount of analyte in a sample, which can be quantitatively determined with suitable precision and accuracy. The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample, which can be detected but not necessarily quantitated as an exact value.
Robustness: To determine the robustness of the method, system suitability parameters were verified by making deliberate changes in the chromate-graphic conditions, viz., changing flow rate by ± 0.04 mL/min, column temperature (± 5 °C) and changing the organic phase in the mobile phase from 100% to 90% and 110% during the development stage itself using 4 µg/mL of Metformin and 24 µg/mL of Canagliflozin solutions respectively.
Degradation Study: The forced degradation studies were performed to establish the stability-indicating nature of the assay method and to observe any degraded compounds 21. In this study, Metformin and Canagliflozin were exposed to different chemical and physical conditions such as 0.1 N HCl (acid hydrolysis), 0.1 N NaOH (base hydrolysis), 30% H2O2 (oxidation), heat (thermal decay) and UV-light (radiation decay) for specified time then diluted similar as standard stock solution and chromatograms were obtained. The percentage of degradation of compounds were calculated from the peak area of the chromatograms. In the study of base or acid hydrolysis, an amount of fine powdered sample equivalent to 100 mg Metformin and 10 mg of Canagliflozin was transferred into 10 ml of volumetric flask and added 3 ml of freshly prepared 0.1 N NaOH or 0.1N HCl shaken well and allowed for 12 h at a temperature of 60 °C. Then the solution was filtered through 0.45µ filter into 10 ml standard flasks and neutralized the unreacted acid or base with 0.1 N HCl or 0.1N NaOH and was made up to the mark. In case of peroxide degradation same amount of sample was transferred into a 10 ml volumetric flask, added 3 ml of freshly prepared 30% H2O2 and kept at 75 °C for 24 h and filtered the solution through 0.45µ filter and made up to the mark. In the study of thermal degradation same amount of sample was transferred into clean and dry watch glass, placed in an oven over a period of 24 h at 110 °C. In case of photodegradation same amount of sample was exposed to UV light of 254 nm for 10 days.
RESULTS AND DISCUSSION:
Method Development and Optimization of Chromatographic Conditions:
Wavelength Selection: The standard Metformin and Canagliflozin solutions were scanned in UV spectrophotometer, and isoabsorptive wavelength was found at 260 nm. Hence, a wavelength at 260 nm is selected for the method development purpose and it is shown in Fig. 3.
FIG. 3: UV SPECTRA OF CANAGLIFLOZIN AND METFORMIN
Optimized Method: After trying different columns like, cyano, PFP, C8 the final chance of the stationary phase that gave a good resolution and eluting all peaks with better separation was reverse phase C18 BEH UPLC (100mm × 2.1mm, 1.7µm particle size) column, this column was only showing elution time less than 2 min. Compare to methanol; acetonitrile was showing good separations and less column backpressure. In acidic conditions, drugs were showing good peak shape. Finally, the best results were obtained by use of mixture of 0.1% orthophosphoric acid buffer (pH adjusted to 3.0 with 0.1 N NaOH) and acetonitrile in the ratio 30:70% v/v at a flow rate of 0.25ml/min through C18 BEH UPLC (100mm × 2.1mm, 1.7µm) at 55 °C column temperature and the detector wavelength was set at 260 nm. Optimized UPLC chromatogram of Metformin and Canagliflozin was shown in Fig. 4 and optimized chromatographic conditions are finalized in Table 1.
TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS
Optimized chromatographic conditions | |
Column | BEH UPLC C18 (100 × 2.1mm × 1.7µm) |
Mobile phase | 0.1% orthophosphoric acid buffer and acetonitrile in the ratio 30:70% (v/v) |
UPLC program | Isocratic |
Flow rate | 0.25mL/min |
Wavelength of detection | 260 nm |
Column temperature | 35oC |
Injection volume | 5 µL |
Diluent | Mobile phase |
Retention time | 1.017 min (Metformin)
1.556 min (Canagliflozin) |
FIG. 4: OPTIMIZED UPLC CHROMATOGRAM OF METFORMIN AND CANAGLIFLOZIN
Method Validation:
System Suitability: The concentration of the standard solution was found to be 300 µg/mL for Metformin and 30 µg/mL for Canagliflozin, respectively. The column efficiency resolute from the sample peak >20,000, the tailing factor <5.0% and resolution between peaks of two drugs should be >1.5. The results of system suitability were expressed in Table 2.
TABLE 2: SYSTEM SUITABILITY PARAMETERS
Name | Peak area
(μV* sec) |
Theoretical plate (N) | Tailing factor
(Tf) |
Retention Time | Resolution |
Metformin | 651341 | 3871.59 | 1.26 | 0.994 | 3.67 |
Canagliflozin | 332544 | 3711.90 | 1.30 | 1.508 |
Precision: All the samples exhibited RSD values < 2% confirming that the analytical method was precise. The results of precision were expressed in Table 3.
TABLE 3: PRECISION STUDIES OF METFORMIN AND CANAGLIFLOZIN
Name of drug | Amount applied(µg/mL) | Repeatability (Mean peak area ±SD) n=3 | % RSD | Intermediate precision (Mean peak area ±SD) n=3 | % RSD |
Metformin | 25 | 659181 ± 3993.20 | 0.605 | 661892 ± 3696.75 | 0.569 |
50 | 661942 ± 3696.75 | 0.547 | 661892 ± 3626.04 | 0.547 | |
75 | 661992 ± 3484.62 | 0.526 | 662042 ± 3555.33 | 0.537 | |
Canagliflozin | 5 | 334705 ± 3837.46 | 1.146 | 334752 ± 4044.65 | 1.208 |
10 | 334741 ± 3887.67 | 1.161 | 334742 ± 3903.22 | 1.160 | |
15 | 334842 ± 3973.94 | 1.186 | 334445.3 ± 3123.3 | 0.933 |
Accuracy: The acceptable percentage recovery of Metformin and Canagliflozin in bulk and pharmaceutical dosage form ranges from 99.94%-100.16% and 100.17%-100.68%, respectively. The results are tabulated in Table 4.
TABLE 4: PERCENTAGE RECOVERY OF METFORMIN AND CANAGLIFLOZIN
Name of drug | Spiked level (%) | Amount added | Amount recovered | % Recovery | % RSD |
Metformin | 50 | 48.51 | 49.08 | 100.16 | 0.61 |
100 | 98.5 | 99.02 | 100.09 | 0.42 | |
150 | 147.1 | 148.32 | 99.94 | 0.65 | |
Canagliflozin | 80 | 4.62 | 4.95 | 100.58 | 0.62 |
100 | 9.82 | 9.94 | 100.68 | 0.45 | |
120 | 14.24 | 14.56 | 100.17 | 0.24 |
Linearity: The least square regression equation was applied, and the calibration curve was plotted over the concentration range of 100-500 µg/ml for Metformin and 10-50 µg/ml for Canagliflozin. The linear regression equations for Metformin and Canagliflozin were y = 2341.5x –13257 and y = 12416x – 9952.6 with correlation coefficient (r) being 0.999 for Metformin and Canagliflozin. The results of linearity were shown in Table 5. The linearity graphs were shown in Fig. 5 and 6.
TABLE 5: LINEARITY DATA OF METFORMIN AND CANAGLIFLOZIN
S. no. | Metformin
Conc. (µg/mL) |
Peak area
(μV* sec) |
Canagliflozin
Conc. (µg/mL) |
Peak area
(μV* sec) |
1 | 0 | 0 | 0 | 0 |
2 | 100 | 209137 | 10 | 109847 |
3 | 200 | 449696 | 20 | 229695 |
4 | 300 | 683462 | 30 | 356543 |
5 | 400 | 931615 | 40 | 492390 |
6 | 500 | 1158818 | 50 | 614238 |
Slope | 2341.5 | 12416 | ||
Y-intercept | 13257 | 9952.6 | ||
Correlation coefficient | 0.999 | 0.999 |
FIG. 5: CALIBRATION CURVE OF METFORMIN
FIG. 6: CALIBRATION CURVE OF CANAGLIFLOZIN
Limit of Quantitation (LOQ): The quantification limit of Metformin and Canagliflozin was found to be 1.536 µg/mL and 1.962 µg/mL.
Limit of Detection (LOD): The detection limit of Metformin and Canagliflozin was found to be 0.612 µg/Ml and 0.458 µg/mL. The low value of LOQ and LOD indicates the high sensitivity of the method.
Robustness: The analysis of Metformin and Canagliflozin was carried out at different conditions of flow rate by ±0.04 mL/min, column temperature (±5 °C) and change in composition of the mobile phase. Robustness was expressed by % RSD values of Metformin and Empagliflozin peak areas as quantitative responses. All values were found to be less than 2%, indicating the robustness of the method. The results are shown in Table 6.
Degradation Studies: The drug substances were exposed to 0.1N HCl at 60 °C for 12 h. The drugs gradually underwent degradation with time and got degraded. No degradation took place over 12 h in basic conditions.
The drug substances were not degraded by oxidative conditions. The drug substances were subjected to thermal degradation at 110 °C for 24 h and no degradation was observed. The drug substances were stable on the effect of photolysis. Drugs did not undergo any degradation with time. The results of forced degradation studies are given in Table 7, and chromatograms were shown in Fig. 7-11.
TABLE 6: ROBUSTNESS OF METFORMIN AND CANAGLIFLOZIN
S. no. | Metformin
peak area ± SD |
%RSD | Theoretical plates | Canagliflozin peak area ± SD | % RSD | Theoretical plates |
Flow rate (mL/min) | ||||||
0.80 | 651974 ± 895.1 | 0.13 | 3762.62 | 331689 ± 1209.1 | 0.36 | 361.80 |
1.00 | 652474 ± 1602.3 | 0.24 | 3785.12 | 331189 ± 502.0 | 0.15 | 3618.05 |
Mobile phase composition (v/v/v) | ||||||
40:60 | 654474 ± 1226.1 | 0.18 | 3761.24 | 333389 ± 2043.5 | 0.61 | 3625.52 |
30:70 | 654874 ± 1367.5 | 0.28 | 3808.12 | 333889 ± 2750.6 | 0.82 | 3705.50 |
Column temperature | ||||||
50 °C | 654624 ± 1013.9 | 0.15 | 3901.46 | 334889 ± 1336.4 | 0.39 | 3874.84 |
55 °C | 653874 ± 2781.7 | 0.42 | 3912.74 | 335389 ± 2043.5 | 0.60 | 3625.88 |
TABLE 7: DEGRADATION STUDIES OF METFORMIN AND CANAGLIFLOZIN
Type of degradation | Metformin Peak area | %
Recovery |
%
Degraded |
Canagliflozin
Peak area |
%
Recovery |
%
Degraded |
Acid | 622195 | 91.45 | 8.55 | 305160 | 91.94 | 8.06 |
Base | 612185 | 94.04 | 5.96 | 318326 | 95.90 | 4.10 |
Peroxide | 624049 | 95.86 | 4.14 | 309199 | 93.15 | 6.85 |
Thermal | 628411 | 96.53 | 3.47 | 319758 | 96.34 | 3.66 |
Photo | 612185 | 94.04 | 5.96 | 318326 | 95.90 | 4.10 |
CONCLUSION: A new RP-UPLC method was developed and validated for the determination of Metformin and Canagliflozin in bulk and tablet dosage form. The developed method was simple, accurate, precise, and satisfactory results were obtained through the analytical method validation data.
The current method can be easily applied for routine drug analysis in the pharmaceutical industry and laboratories. Forced degradation studies were performed to assess the stability of the compound and prove the stability-indicating nature of the developed chromatographic method. The method was validated as per ICH guidelines.
ACKNOWLEDGEMENT: One of the authors (Padmaja) is thankful to RGNF for awarding financial assistance in the form of SRF and Head Department of chemistry, Osmania University, for providing necessary facilities.
CONFLICTS OF INTEREST: The authors did not report any conflict of interest
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How to cite this article:
Padmaja N and Veerabhadram G: Stability indicating RP-UPLC-DAD method for determination of metformin and canagliflozin in bulk and tablet dosage form. Int J Pharm Sci & Res 2020; 11(7): 3442-49. doi: 10.13040/IJPSR.0975-8232.11(7).3442-49.
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Article Information
43
3442-3449
766
750
English
IJPSR
N. Padmaja and G. Veerabhadram *
Department of Chemistry, University College of Science, Osmania University, Hyderabad, Telangana, India.
gvbhadram@gmail.com
14 August 2019
04 January 2020
21 January 2020
10.13040/IJPSR.0975-8232.11(7).3442-49
01 July 2020