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REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR SEPARATION AND ESTIMATION OF IMPURITIES PRESENT IN PHARMACEUTICAL FORMULATION OF CANAGLIFOZIN

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REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR SEPARATION AND ESTIMATION OF IMPURITIES PRESENT IN PHARMACEUTICAL FORMULATION OF CANAGLIFOZIN

N. Patel * and S. Patel

Department of Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidhyanagar - 384012,  Gujarat, India.

ABSTRACT: Canagliflozin is sodium-glucose co-transporter-2 inhibitors work by inhibiting SGLT2 to prevent reabsorption of glucose and facilitate its excretion in urine. Impurities in pharmaceuticals which are unwanted chemicals that remain with the active pharmaceutical ingredients (APIs), or develop during stability testing, or develop during formulation or upon aging of both API and formulation. The presence of these unwanted chemicals, even in small amounts, may influence the efficacy and safety of the pharmaceutical products. A simple and very sensitive method developed for estimation of impurities present in Canaglifozin formulation by Reverse Phase High Performance Liquid Chromatographic method. A method is capable to detect impurities at very low level (0.1 µm/mL). Chromatographic separation of six different impurities was achieved on inertsil C8-3 (250 × 4.6) mm, 3 µm column using gradient elution method.

Keywords:

Canagliflozin, SGLT-2 inhibitor, Method development, Method validation, Stress condition, Impurities, ICH Q2(R1)

INTRODUCTION: Canagliflozin is a sodium-glucose co-transporter-2 inhibitor work by inhibiting SGLT2 in the PCT, to prevent reabsorption of glucose and facilitate its excretion in urine. As glucose is excreted, its plasma levels fall, leading to an improvement in all glycemic parameters. This mechanism of action is dependent on blood glucose levels and, unlike the actions of thiazolidinediones (mediated through GLUTs), is independent of the actions of insulin. Thus, there is minimal potential for hypoglycemia and no risk of overstimulation or fatigue of the beta cells.

Because their mode of action relies upon normal renal glomerular-tubular function, SGLT2i efficacy is reduced in persons with renal impairment. Due to lots of advantages of canagliflozin, it is necessary to estimate related impurities present in this drug. So present investigation involves the development of RP-HPLC related substances method for pharmaceutical dosage form for canagliflozin. Possibly six impurities identified base on API source, so the separation was done on this impurities and validated developed method.

FIG. 1: CHEMICAL STRUCTURE OF CANAGLIFOZIN

TABLE 1: DETAILS OF IMPURITY-I TO III:

Impurity Name Impurity-I Impurity-II Impurity-III
Chemical structure
Chemical formula C24H25FO5S C18H14FIS C32H33FO9S
Chemical name (3R, 4R)-2-((S)-1, 2 dihyd roxyethyl)-5-(3-((5-(4-flurophenyl)thiopen-2-yl)methyl)-4-methyl phenyl) tetrahydrofuran-3, 4-diol 2-(4-flurophenyl)-5-(5-iodo-2-methyl benzyl) thiophene (2R, 3R, 4R, 5S, 6S)-2-(acetoxymethyl)-6-(3-((5-(4-flurophenyl) thiopen-2-yl)methyl)-4-methyl phenyl) tetrahydro-2H-pyran-3, 4, 5-triyl  triacetate
Molecular Weight 444.53 g/mol 408.27  g/mol 612.64 g/mol

TABLE 2: DETAILS OF IMPURITY-IV TO VI

Impurity Name Impurity-IV Impurity-V Impurity-VI
 

Chemical structure
Chemical formula C18H14FS C32H33FO9S C23H35FO6S
Chemical name 2-(4-flurophenyl)-5-(2-methyl benzyl) thiophene (2R, 3R, 4R, 5S, 6S)-2-(acetoxymethyl)-6-(3-((5-(4-flurophenyl) thiopen-2-yl)methyl)-2-methy lphenyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate {{5-{4-Flurophenyl) thiopen-2-yl) (5-(tetrahydro-3, 4, 5-trihy droxy-6-{hydroxymethyl } -2H-pyran-2-yl}-2-methylphenyl) methanone
Molecular Weight 355.27 g/mol 612.64 g/mol 458.0  g/mol

MATERIALS AND METHODS:

Reagents and Chemicals: Canagliflozin and its impurities were the generous gifts from MSN Laboratories PVT Ltd, India. HPLC grade Acetonitrile was procured from Merck. Orthophosphoric acid (H3PO4) was purchased from Merck. All other chemicals and solvents used were of analytical grade. Water used in the HPLC analysis was prepared by the water purifier (Merck Millipore Milli-Q). The mobile phase and all the solutions were filtered through a 0.45 µm Merck HV membrane filter. The sample was filtered through a 0.45 µm millipore PVDF syringe filter.

Instruments: HPLC system (waters system, USA, e 2695 and Agilint 1200 series) with a PDA detector equipped with a quaternary pump, autosampler, column compartment, and empower and chrome eon software was employed during this study.

Chromatographic Condition: Chromatographic separation was achieved at  30 °C column temperature, and the detection was carried at 290 nm at a flow rate of 1.0 mL/min. Run time was kept at 80 min. Prior to the injection of drug solution, the column was equilibrated for 60 min with the mobile phase flowing through the system. The injection volume was 15 μL. The analysis has been performed by using Inertsil C8-3 (250 × 4.6 mm, 3 u). The mobile phase a containing 1 mL orthophosphoric acid in 1000 mL of purified water, and mobile phase B contains acetonitrile using following gradient.

TABLE 3:  GRADIENT PROGRAMME

Time (min) % Mobile phase-A % Mobile phase-B
0 55 45
5 55 45
15 45 55
45 10 90
70 10 90
75 55 45
80 55 45

Standard Preparation: The standard stock solutions 200 ug/ml of canagliflozin were prepared by dissolving working standards in diluents and diluting with the same solvent to obtain a final concentration of 4 µg/mL.

Sample Preparation: Twenty tablets were weighed and finely powdered. Powder equivalent to 50 mg Canaglifozin was accurately weighed into a 25 ml volumetric flask, 20 ml of diluent was added and sonicated for 15 min with intermittent shaking, made up to the volume with diluent and mixed. Filter the solution through a 0.45 µm Millipore PVDF syringe filter.

Method Validation: After method development, validation of the current test method for canagliflozin tablets was performed in accordance with united states pharmacopeia requirements / ICH guidelines for related substance method the parameter includes precision, accuracy, linearity, LOD and LOQ, precision and accuracy at LOQ level, selectivity, specificity includes blank, placebo, known impurity interference and interference of degradants by degradation study. Robustness was also performed.

Specificity: To assess the method specificity, tablet powder without canagliflozin was prepared with the same excipients as those in the commercial formulation. For RP-HPLC, the solution was prepared using the same procedure as for the analytical sample. Placebo solution was injected into the HPLC system following test conditions; the chromatogram was recorded, and the responses of the peaks, if any, measured. The chromatogram of the placebo has not shown any interference at the retention time of both canagliflozin and its impurities. Blank, placebo, and impurity spiked sample preparation and impurities mixture chromatogram shown in Fig. 2-6.

FIG. 6: CHROMATOGRAM OF IMPURITY MIXTURE SOLUTION

TABLE 4: SYSTEM SUITABILITY RESULT

Injection no Peak area of canagliflozin Theoretical plates Tailing factor
1 66.511 29312 1.0
2 66.897 29874 1.0
3 65.234 29354 1.0
4 67.842 29541 1.0
5 66.325 29456 1.0
6 66.251 29328 1.0
Mean 66.510
SD 0.9
%RSD 1.3

System Suitability: 15 μL of standard solution six times injected into HPLC and recorded the chromatogram; % RSD of canagliflozin, area was within the limit of 5.0%. The results summarized in Table 4 and the standard solution chromatogram shown in Fig. 7.

FIG. 7: CHROMATOGRAM OF STANDARD SOLUTION

Precision: Precision was measured in terms of repeatability of application and measurement. Repeatability of standard application (system precision).

Precision study of canagliflozin and its impurities were carried out by spiking known concentration in the sample and calculating % recovery of impurities in the sample. Intermediate precision carried out using the same manner but on another day using different columns and HPLC. The results summarized in Tables 5 and 6.

TABLE 5: METHOD PRECISION

Method Precision
S. no.  Impurity-1 Impurity-2 Impurity-3 Impurity-4 Impurity-5 Impurity-6 Single unk Total Imp
1 0.64 0.48 0.63 0.50 0.58 0.46 0.00 3.29
2 0.62 0.49 0.64 0.52 0.59 0.48 0.00 3.34
3 0.62 0.48 0.63 0.51 0.56 0.46 0.00 3.26
4 0.62 0.47 0.63 0.51 0.60 0.47 0.00 3.30
5 0.62 0.48 0.63 0.50 0.61 0.45 0.00 3.29
6 0.63 0.47 0.65 0.51 0.61 0.46 0.00 3.33
Mean 0.63 0.48 0.64 0.51 0.59 0.46 0.00 3.30
SD 0.008 0.008 0.008 0.008 0.019 0.010 0.00 0.03
%RSD 1.3 1.6 1.3 1.5 3.3 2.2 0.0 0.9

TABLE 6: INTERMEDIATE PRECISION

 S. no. Impurity-1 Impurity-2 Impurity-3 Impurity-4 Impurity-5 Impurity-6 Single unk Total Imp
1 0.50 0.52 0.61 0.55 0.49 0.60 0.00 3.27
2 0.50 0.52 0.61 0.54 0.50 0.62 0.00 3.29
3 0.51 0.51 0.61 0.55 0.49 0.61 0.00 3.28
4 0.49 0.53 0.62 0.52 0.50 0.60 0.00 3.26
5 0.50 0.52 0.61 0.55 0.49 0.60 0.00 3.27
6 0.50 0.51 0.61 0.55 0.49 0.60 0.00 3.26
Mean 0.50 0.52 0.61 0.54 0.47 0.61 0.00 3.27
SD 0.006 0.008 0.004 0.012 0.005 0.008 0.00 0.01
%RSD 1.3 1.5 0.7 2.2 1.1 1.4 0.0 0.4

Linearity: To evaluate the linearity of the method, six levels calibration curve made includes LOQ level. Signal to noise ratio was observed. The linearity of the method is obtained by the preparation of the calibration curve. The calibration curve for canagliflozin and its impurities were obtained by plotting the peak area of canagliflozin versus the concentration of canagliflozin over the range of 1-15 μg/ml. The results are summarized, and the overall linearity graph for canagliflozin and its impurities was shown in Fig.

Accuracy: Accuracy of the method was studied for three levels from 50% to 150% by spiking 0.05% for LOQ, and 0.25% for 50% level from the target concentration of canagliflozin impurities and 0.50%, 0.75% for 100%, 150% level from the target concentration of canagliflozin impurities spiked in sample preparation and analyzed with unspiked sample preparation, recorded the chromatogram. Six preparation for 50%, 150% level, and triplicate preparation of median level concentration was done. The results are summarized in Table 7.

Robustness: Robustness of the current method was investigated by analyzing the standard solution and established system suitability with the deliberate variation of flow rate and column temperature at 10 percentage levels from the original value. RSD of six replicate injections of the standard solution was found below 5.0% for all the chromatographic conditions and all peaks in standard solutions. The conditions with the variation and the results are presented in Table 8-9.

TABLE 7:  RECOVERY/ACCURACY

Name Level % Recovery % RSD
Impurity-1 LOQ 93.2 1.8
50% 92.5 2.7
100% 99.7 2.4
150% 96.8 2.9
Impurity-2 LOQ 97.7 2.3
50% 99.4 0.7
100% 99.7 2.4
150% 95.9 3.5
Impurity-3 LOQ 100.8 1.5
50% 99.6 0.5
100% 102.2 2.7
150% 99.0 1.2
Impurity-4 LOQ 96.9 1.3
50% 94.9 3.5
100% 96.0 2.7
150% 96.8 1.4
Impurity-5 LOQ 100.7 0.6
50% 99.8 0.5
100% 102.9 0.4
150% 99.8 0.5
Impurity-6 LOQ 96.6 2.0
50% 98.7 1.3
100% 99.3 2.6
150% 98.4 1.1

TABLE 8: ROBUSTNESS (FLOW RATE)

Normal Condition Flow + Flow -
RT RRT RT RRT RT RRT
Impurity - VI 13.35 0.92 12.06 0.92 14.23 0.93
Canaglifozin 14.49 1.00 13.15 1.00 15.32 1.00
Impurity - I 16.03 1.11 14.45 1.10 17.89 1.17
Impurity - V 18.62 1.29 16.81 1.28 21.52 1.40
Impurity - III 31.79 2.19 28.65 2.18 35.83 2.34
Impurity - VI 41.47 2.86 37.81 2.88 44.87 2.93
Impurity -II 46.47 3.21 42.32 3.22 52.36 3.42

TABLE 9: ROBUSTNESS (COLUMN TEMPERATURE)

Normal condition Column temp + Column temp -
RT RRT RT RRT RT RRT
Impurity - VI 13.35 0.92 13.25 0.93 13.68 0.89
Canaglifozin 14.49 1.00 14.31 1.00 15.36 1.00
Impurity - I 16.03 1.11 15.48 1.08 17.02 1.11
Impurity - V 18.62 1.29 17.36 1.21 19.25 1.25
Impurity - III 31.79 2.19 30.17 2.11 33.1 2.15
Impurity - VI 41.47 2.86 39.51 2.76 44.05 2.87
Impurity -II 46.47 3.21 44.03 3.08 48.36 3.15

TABLE 10: LOD AND LOQ OF CANAGLIFOZIN AND ITS IMPURITIES

Name LOD LOQ
Canaglifozin 0.02% 0.05%
Impurity-I 0.02% 0.05%
Impurity-II 0.02% 0.05%
Impurity-III 0.02% 0.05%
Impurity-IV 0.02% 0.05%
Impurity-V 0.02% 0.05%
Impurity-VI 0.02% 0.05%

TABLE 11: FORCE DEGRADATION SAMPLE CONDITION

Sample Condition
Acid 5N HCL (5 mL) 80 °C , 3H
Base 5N NaOH (5 mL) 80 °C , 1H
Peroxide 0.1M KmnO4(5 mL) RT,1H
Thermal 40 °C, 10 Days
Photo Sun Tester, 1.2 Lux/H, 24H
Humidity 75% RH, 10Days

LOD and LOQ: LOD and LOQ were calculated by using the formula 3.3 S.D / S and 10 S.D / S where S.D is the standard deviation of Y-intercept, and S is the slope of the calibration curve.

TABLE 12: RESULT OF FORCED DEGRADATION STUDY

Sample Furanose Oxidative Methyl phenyl Unk-1 Unk-2 Unk-3 Total im. Assay Mass balan. Peak  purity
Acid 0.02 ND 0.01 0.04 0.07 0.02 0.44 100.2 98.9 995
Base ND ND 0.01 0.11 0.05 0.04 0.27 99.8 99.4 996
Peroxide ND 9.80 0.01 1.30 0.07 0.25 11.53 86.3 102.3        996
Thermal ND ND 0.01 0.04 0.04 0.04 0.22 98.7 100.6 997
Photo ND 0.09 0.01 0.13 0.09 0.06 0.45 99.0 100.1 998
Humidity ND ND BQL 0.05 0.04 0.07 0.16 98.8 100.5 998

TABLE 13: RESULT OF STABILITY OF STANDARD SOLUTION

Standard Solution
Time (h) Area % Deviation
Initial 66.511 -
18 66.200 -0.47
24 66.897 0.58
36 67.847 2.01
48 68.500 2.99
54 69.874 5.06

Solution Stability: Solution stability optimized by injected standard solution and sample at a different time interval and calculated % deviation against the initial area of the standard solution.

It was found that standard and sample were stable upto 54 h. The results are presented in Table.

TABLE 14: RESULT OF STABILITY OF SAMPLE SOLUTION

Time (HR) Impurity-1 Impurity-2 Impurity-3 Impurity-4 Impurity-5 Impurity-6 Single unk Total im.
Initial 0.64 0.48 0.63 0.50 0.58 0.46 0.00 3.29
18 0.65 0.48 0.63 0.48 0.58 0.46 0.00 3.28
24 0.64 0.48 0.63 0.50 0.58 0.46 0.00 3.29
36 0.64 0.48 0.63 0.50 0.57 0.46 0.00 3.28
48 0.63 0.48 0.63 0.49 0.58 0.46 0.00 3.27
54 0.64 0.48 0.63 0.50 0.59 0.47 0.00 3.31
Time (HR) Impurity-1 Impurity-2 Impurity-3 Impurity-4 Impurity-5 Impurity-6 Single unk Total Im.
Initial 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
18 0.01 0.00 0.00 0.02 0.00 0.00 0.00 0.01
24 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
36 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.01
48 0.01 0.00 0.00 0.01 0.00 0.00 0.00 0.01
54 0.00 0.00 0.00 0.00 0.02 0.01 0.00 0.01

RESULTS AND DISCUSSION: The main objective of the chromatographic method development was to separate canagliflozin from the impurities that were carried out for accurate and precise method development, and impurities were coeluted.

After using several columns and buffers, suitable column chemistry and good peak shape were obtained with inertsil C8-3 (250 × 4.6) mm 3 µ particle size, column temperature was adjusted at 30 °C, with gradient mobile phase system consisting the mobile phase A containing 0.1% orthophosphoric acid solution in water and mobile phase B contains acetonitrile and water in a ratio of 80: 20% v/v using an above-mentioned gradient.

HPLC method has been the development and validated for the determination of related substances of canagliflozin in tablets with gradient elution. The method is selective because we have very good separation between impurities. The method described in this study is suitable to determine impurities at a very low level.

These parameters showed good linearity with correlation coefficients. We have shown that the method is robust, with little change in critical chromatographic parameters. Validation parameters have proved that our method can use as a stability-indicating method for the determination of related substances of canagliflozin in the tablet.

CONCLUSION: A novel, reverse phase liquid chromatographic method has been developed and validated for the estimation of canagliflozin and its impurities with a very recent and advanced HPLC method. The proposed method is found to be simple, accurate, precise, sensitive, specific, and robust. Hence, it can be successfully used for the routine analysis of canagliflozin in pharmaceutical dosage forms.

ACKNOWLEDGEMENT: The author would like to thank guide Mrs. Sejal Patel and all family members for their valuable support throughout the work. The Authors acknowledge Ganpat University and Shree S. K. Patel College of Pharmaceutical Education and Research for providing the infrastructure facility for carrying out this work.

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

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    How to cite this article:

    Patel N and Patel S: Reverse phase high performance liquid chromatographic method for separation and estimation of impurities present in pharmaceutical formulation of canaglifozin. Int J Pharm Sci & Res 2020; 11(6): 2814-22. doi: 10.13040/IJPSR.0975-8232.11(6).2814-22.

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Article Information

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English

IJPSR

N. Patel * and S. Patel

Department of Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidhyanagar, Gujarat, India.

nilesh33.emcure@gmail.com

11 July 2019

26 November 2020

17 April 2020

10.13040/IJPSR.0975-8232.11(6).2814-22

01 June 2020

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