www.wjpps.com │ Vol 10, Issue 9, 2021. │ ISO 9001:2015 Certified Journal │ 2251
STABILITY INDICATING RP-HPLC METHOD DEVELOPMENT AND VALIDATION OF DAPAGLIFLOZIN IN BULK AND TABLET
DOSAGE FORM
Suraj Nagwanshi*1, Smita Aher2 and Rishikesh Bachhav3
1Department of Pharmaceutical Quality Assurance, R. G, Sapkal College of Pharmacy, Anjaneri, Nashik-422212.
2Department of Pharmaceutical Chemistry, R. G, Sapkal College of Pharmacy, Anjaneri, Nashik-422212.
3Department of Pharmacology, R. G, Sapkal College of Pharmacy, Anjaneri, Nashik-422212.
ABSTRACT
A simple, sensitive and accurate Stability Indicating RP-HPLC method has been developed for the determination of Dapagliflozin in bulk and formulation. The λmax of the Dapagliflozin was found to be 285nm in Methanol : Phosphate buffer (50:50) pH 4.0. The method shows high sensitivity with linearity 2-10 µg/ml (regression equation: y = 791514x - 428583; r2 = 0.9992). The various parameters according to ICH guidelines and USP are followed for validating and testing of this method. The Detection limit and quantitation limit were found to be 0.0437 µg ml –1 and 0.1326 µg ml–1 respectively. The % assay of Forxiga 10 (Macleod pharmaceutical) was found to be 99.71%. The force degradation study were employed on five conditions i.e. Acidic, Alkaline, Oxidative, Thermal and Photolytic degradation. The
degradation products produced during the stability study were well separated from the pure drug signifying the specificity of developed procedure. The major degradation of drug was found to be in alkali and peroxide stress condition. The results demonstrated that the procedure is accurate, specific and reproducible (RSD <2%), and also being simple, cheap and less time consuming and appropriate for the determination of Dapagliflozin in bulk and tablet dosage form.
KEYWORDS: Dapagliflozin, HPLC method, Force Degradation Study.
Volume 10, Issue 9, 2251-2262 Research Article ISSN 2278 – 4357
*Corresponding Author Suraj Nagwanshi Department of
Pharmaceutical Quality Assurance, R. G, Sapkal College of Pharmacy, Anjaneri, Nashik-422213.
Article Received on 24 July 2021,
Revised on 13 Aug. 2021, Accepted on 02 Sept. 2021, DOI: 10.20959/wjpps20219-20067
INTRODUCTION
Dapagliflozin is a SGLT2 (sodium-glucose transporter 2) inhibitor. Hypoglycemia in diabetics is achieved by removing excess sugar from the body through urine.. By chemically Dapagliflozin propanediol monohydrate is (2S)-propane-1,2-diol(2S,3R,4R,5S,6R)-2-{4- chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6(hydroxymethyl) oxane-3,4,5-triol hydrate and having atomic equation is C24H35ClO9. The construction of Dapagliflozin is appearing in fig 1.
Fig 1: Structure of Dapagliflozin propanediol monohydrate.
The atomic mass of dapagliflozin is 502.99 g/mol. Dapagliflozin is used with diet and exercise to improve blood sugar control in adults with type 2 diabetes and reduce the risk of hospitalization for heart failure in adults with type 2 diabetes and known cardiovascular disease or other risk factors. It seems to be more useful than empagliflozin. In addition, dapagliflozin is suitable for the treatment of adult heart failure with reduced ejection fraction to reduce the risk of cardiovascular death and heart failure hospitalization. It has also been shown to reduce the risk of impaired renal function, renal failure, cardiovascular death, and hospitalization for heart failure in adults with chronic kidney disease who are at risk of disease progression.
MATERIALS AND METHOD Instruments
Chromatographic sections were created using an HPLC-3000 analytical technique based on a trivial liquid chromatography procedure by adjusting Ryodine injection for 20 programmable UV-ID variable frequency circuits. liter. Reversed phase C18 [Cosmosil C18 (250 mm x 4.6 ID, particle size: 5 μm)] was used. Model: Bright two-row UV Spectrophotometer and Accurate Venser Scale from 2012 Model: PGB100 Electronic scale was used for spectrophotometer and custom measurements.
Reagents and chemicals
Drug grade unadulterated Dapagliflozin sample was secured from Invochem Lab PVT LTD.
HPLC grade Methanol and water were acquired from Merck specialities private restricted, Mumbai.
Chromatographic conditions
C18 [Cosmosil C18 (250mm x 4.6ID, Particle size: 5 micron)] was utilized for the chromatographic separation at a discovery frequency of 285 nm. Methanol: Phosphate buffer (50:50) pH 4.0 was chosen as mobile phase for elution and same blend was utilized in the arrangement of standard and sample solutions. The elution was checked by infusing the 20µl and the stream rate was changed in accordance with 1.0 ml/min.
Preparation of Standard solutions
10mg Dapagliflozin was precisely gauged and moved into 10 ml volumetric cups, broken up utilizing portable stage and the volume was made up with a similar dissolvable to acquire essential stock arrangement of focus 1000µg/ml of the medication. (Working stock arrangement).
Optimisation of RP-HPLC method
HPLC technology has simplified the pending evaluation of dapagliflozin. Several stimulation steps were performed to improve the method, but in satisfactory time virtual PAGE and methanol C18 column: excellent phosphate buffer (50:50) accuracy at pH 4.0 [Cosmosil C18 (250 mm x 4.6 ID, particle size:): 5 µm)] Table: 1 The performance of the dapagliflozin chromatograph is shown in Figure 3.
Table 1: Optimized parameter.
Parameter Condition
Column Cosmosil C18 (250mm x 4.6ID, Particle size: 5 micron)
Mobile Phase Methanol : Phosphate buffer (50:50) pH 4.0
Flow Rate 1.0 ml/min
Wavelength 285 nm
Injection Volume 20 µl
Detector UV-3000-M
Run Time 9 min
Retention Time Approx. 4.5 min
Validation of RP-HPLC method
Validation of the optimized HPLC method was performed in accordance to the ICH Q2 (R) guidelines.
1. Linearity
For linearity; prepared an appropriate sample solution of 1000 g/ml solution that can be used for linear estimation. 20-100 liters were sequentially poured into 5 volumetric flasks of 10 ml, and the volume of the mobile phase was adjusted to a dapagliflozin concentration of 2-10 g/ml. Each solution was injected into the system three times. A calibration curve was plotted as solution concentration versus observed peak area and regression and correlation coefficients were calculated. The calibration curve for the dapagliflozin sample is shown in Figure 2 and the corresponding linear parameters are shown in Table 2..
2. Accuracy
To make sure the reliability and accuracy of the recovery study data were carried out by % recovery method which is also called as standard addition method. A known quantity of pure drug of Dapagliflozin was mixed to pre-analysed sample and contents again undergoes analysis by the optimised method and the % recovery was reported in table 4.
3. Precision
The repeatability study of the proposed method was verified by calculating the percentage RSD of three replica injections of 100% concentration i.e. 6µg/ml of Dapagliflozin on the same day and for intraday precision % RSD was calculated from repeatition. The results were shown in table 5.
4. Limit of Quantitation (LOQ) & Limit of Detection (LOD)
The LOD and LOQ were analysed from the slope(s) of the calibration curve and the standard deviation (SD) of the peak areas using the formula LOD = 3.3 s/s and LOQ = 10 s/s.
5. Robustness
Robustness was calculated by changing the chromatographic conditions such as the composition of the mobile phase, the detection of the wavelength, the speed of the current, etc. It must be specified as a percentage of the RSD position. Minor modifications were allowed to determine the robustness of the method under optimal conditions. A deviation of 2 nm was detected separately for each wavelength at a flow rate of 0.1 ml/min. 100% of the test
solution was injected into system 3 for n changes under optimal conditions. Table 6 shows the percentages of RSD.
6. System suitability
It was make sure that from the system suitability parameters, the method can gives results of accuracy and precision. System suitability was performed with six replicate injections of solution of 6 µl/ml of Dapagliflozin in to the chromatographic system. Tailing factor (T) Number of theoretical plates (N) obtained was reported in table 8.
RESULT AND DISCUSSION Linearity
It was clarify from the analytical method linearity as the capability of the method to obtain test results that are directly proportional to the analyte concentration, within a specified range.
The peak area obtained from the chromatograph was plotted verses corresponding concentrations to obtain the calibration graph. The results of linearity study (Figure 1) gave linear relationship over the concentration range of 2-10 µg/ml for Dapagliflozin. From the regression analysis, a linear equation was obtained y = 791514x - 427583, and the goodness- of-fit (r2) was found to be 0.9992, indicating a linear relationship between the concentration of analyte and area under the peak.
Table 2: Summary of results of Linearity.
Conc. (µg/ml) Peak Area
2 1194511
4 2765674
6 4226519
8 5856613
10 7564179
Figure 2: Linearity.
Figure 3: Typical chromatograph of Dapagliflozin.
Accuracy
The accuracy of the method determines the closeness of results obtained by that method to the true value. From the results of accuracy testing it was showed that the method is accurate within the acceptable limits. The % RSD is calculated for the Dapagliflozin and all the results are within limits. Acceptable accuracy was within the range and not more than 2.0% RSD, as demonstrated in Table -3.
Table 3: Summary of Results of Accuracy.
Level of addition
Standard
added conc. Total
conc. Area obtained*
Std Area
Drug
recovered %Recovery
(μg/ml) (μg/ml) (μg/ml) (μg/ml)
2 4 6 4215659 5.984583 99.743051
50% 2 4 6 4203659 4226519 5.967547 99.4591294
2 4 6 4236598 6.014308 100.23847
4 4 8 5863179 8.008969 100.112113
100% 4 4 8 5852136 5856613 7.993884 99.9235565
4 4 8 5846312 7.985929 99.8241134
6 4 10 7552169 9.984122 99.8412253
150% 6 4 10 7574812 7564179 10.01405 100.14057
6 4 10 7594612 10.04023 100.402331
Table 4: % recovery study.
Level of
addition % Mean recovery* SD % RSD
50% 99.81 0.3944 0.395161
100% 99.95 0.1463 0.146348
150% 100.1 0.2808 0.280403
Precision
Precision is “the closeness of results obtained from multiple sampling of the same homogeneous sample under the prescribed conditions,” and it is expressed in the form of relative standard deviation. The repeatability, intra-day and inter-day precision results are shown in the table 5 and table 6. The RSD were calculated for all the results are within limits.
Precision was not more than 2.0% RSD, as demonstrated in Table 5 and Table 6.
Table-5: Summary of Intraday Precision.
Sr.
No.
Conc.
(μg/mL) Area Mean SD %RSD
1 2 1208945
1196841.67 11849.71 0.990082
2 2 1196317
3 2 1185263
4 6 4236518
4236087.67 19495.25 0.460218
5 6 4226132
6 6 4245613
7 10 7551638
7551958.67 10499.67 0.139032
8 10 7541623
9 10 7562615
Table-6: Summary of Interday Precision.
Sr. No. Conc.
(μg/mL) Area Mean SD %RSD
1 2 1216354
1214802 11541.5292 0.95007493
2 2 1202563
3 2 1225489
4 6 4225618
4224458 11862.6135 0.28080794
5 6 4235698
6 6 4212058
7 10 7558963
7563286 24472.5619 0.32357049
8 10 7589632
9 10 7541263
LOD and LOQ
The LOD and LOQ were calculated by the equations LOD and LOQ = where, std. Deviation taken from accuracy and slope is from linearity. Based on these equations, the calculated LOD and LOQ values for Dapagliflozin were 0.0437 and 0.1326 µg/ml, respectively.
Robustness
Robustness of the method reflects that the results are unaffected or reliable even if the minute changes in the method parameters. Here, the flow rate and wavelength were slightly changed to lower and higher sides of the actual values to find if the change in the peak area and retention time were within limits. The results obtained with changes in the parameters on a 6µg/mL solution are as shown in Table No. 7.
Table 7: Data of robustness Study.
Sr.No Parameter Condition Area Mean SD %RSD 1 Change in
Flow rate (ml/min)
0.9 4236598
4221938 12736.3 0.30167
2 1 4213592
3 1.1 4215625
1 Change in Wavelength (nm)
283 4216523
4235580 20105.9 0.47469
2 285 4256592
3 287 4233626
Ruggedness
Ruggedness is the study to determine effect of external parameters on the method. To evaluate ruggedness of the developed method, parameter was deliberately varied. These parameter included variation of system, different analyst, Atmospheric changes. Ruggedness was studied by different analyst. The %RSD were calculated for all the results are within limits.
Table 8: Data of robustness Study.
Sr.No Analyst Conc.
(μg/ml) Area Mean
area* SD % RSD
1 Analyst-Ι 6
4216542
4217834.67 3279.95874 0.07776404 4215398
4221564 2 Analyst-ΙΙ 6
4203651
4219948.33 22508.7419 0.53338904 4210563
4245631 Specificity
Excipients and impurities were not interacting with the standard drug, hence method is specific. Results of specificity are shown in Table no. 9.
Table No.9: Data for specificity study.
Drug
conc. Excipients Total conc.
(μg/ml)
Area Mean SD %RSD
(μg/ml) (μg/ml)
2 4 6 1195612
2 4 6 1194452 1196461.67 2543.27355 0.21256624
2 4 6 1199321
4 4 8 2765780
4 4 8 2764890 2767119.33 3122.42876 0.11284041
4 4 8 2770688
6 4 10 4236543
6 4 10 4230894 4231675.67 4527.39487 0.10698823
6 4 10 4227590
% Assay of Marketed formulation
The % Assay of Forxiga 10 marketed formulation of Macleod Pharmaceutical was calculated and given in table No. 10.
Table No. 10. Data of % Assay of marketed formulation.
Marketed Formulation Area of Standard Area of Sample % Assay
Forxiga 10 4226519 4214301 99.71
System Suitability Parameters
System suitability was performed by injecting six replicate injections of 100% test concentration, number of theoretical plate, asymmetry factor are satisfactory. The chromatographs confirm the presence of Dapagliflozin at 4.5 min without any interference.
Table 11: System suitability parameter.
Sr. No. conc. (μg/ml) Retention Time (min)
Theoretical plates
Asymmetry Factor
1 6 4.503 8487 1.25
2 6 4.515 8552 1.24
3 6 4.505 8462 1.25
4 6 4.589 8359 1.23
5 6 4.531 8252 1.24
6 6 4.509 8539 1.25
Mean 4.525333333 8441.83333 1.2433333
SD 0.032776007 115.691688 0.008165
%RSD 0.775702291 1.37045691 0.6566997
DEGRADATION STUDIES
Stress testing of the drug substance can help to identify the likely degradation products, the stability and specificity of the analytical procedure.
Table No. 12: Results of Forced Degradation Studies.
Sr.
No. Condition Area of sample
Area of Standard
% Drug recovered
% Degradation
1 Acid stress 3903190 4226519 92.35 7.65
2 Alkali Stress 3426438 4226519 81.07 18.93 3 Oxidative Stress 3091698 4226519 73.15 26.85 4 Thermal Stress 3881635 4226519 91.84 8.16 5 Photolytic Stress 4155090 4226519 98.31 1.69
a) Acidic stressed b) Alkaline stressed
c) Oxidative stressed d) Thermal stressed
e) Photolytic stressed
Fig no.4: Force degradation studies.
CONCLUSION
The proposed method was found to be simple, precise, accurate, rapid and specific for determination of Dapagliflozin from pure and its dosage forms. The mobile phase used for method development is very simple to prepare and economical also. The sample recoveries in the formulation were showing good results. And hence, this method can be easily and conveniently adopted for routine analysis of Dapagliflozin in pure form. The degradation products produced during the stability study were well separated from the pure drug signifying the specificity of developed procedure. The major degradation of drug was found to be in alkali and peroxide stress condition.
ACKNOWLEDGEMENT No conflict of interest REFERNCES
1. https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q2_R 1/Step4/Q2_R1 Guideline.pdf
2. G. V. Mante, A. T. Hemke, RP-HPLC Method for Estimation of Dapagliflozin from its Tablet, International Journal of ChemTech Research, 2018; 11(01): 242-248.
3. R. Aswini, M. Mukkanti Eswarudu, a novel rp-hplc method for simultaneous estimation of dapagliflozin and saxagliptin in bulk and pharmaceutical dosage form, JPSR, 2018;
9(12): 5161-5167
4. Jitendra Debata, Sundeep Kumar, A New RP-HPLC Method Development and Validation of Dapagliflozin in Bulk and Tablet Dosage Form, , Int J Drug Dev & Res, 2017; 9: 2.
5. Mitali v. verma, chirag j patel, development and stability indicating hplc method for dapagliflozin in api and pharmaceutical dosage form, int j app pharm, 2017; 9(5): 33-41.
6. Md. Rageeb Md. Usman, Sufiyan Ahmad, Stability Indicating RP-HPLC Method for Determination of Saxagliptin and Dapagliflozin in Bulk and Tablet Dosage Forms, J.
Pharm. Sci. & Res, 2020; 12(4): 499-506.
7. Merugu Manasa, Vijey Aanandhi M. Stability Indicating Simultaneous Method development and Validation of Dapagliflozin and Saxagliptin by RP-HPLC. Research J.
Pharm. and Tech, 2021; 14(2): 1045-1049.
8. Manasa, S., D. K., N. R. G, and S. S. “METHOD DEVELOPMENT AND VALIDATION OF DAPAGLIFLOZIN IN API BY RP-HPLC AND UV-
SPECTROSCOPY”. International Journal of Pharmaceutical Sciences and Drug Research, July 2014; 6(3): 250-252.
9. GRACE A, C., P. T, and S. T. “DEVELOPMENT AND VALIDATION OF HIGH-
PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR
DETERMINATION OF DAPAGLIFLOZIN AND ITS IMPURITIES IN TABLET DOSAGE FORM”. Asian Journal of Pharmaceutical and Clinical Research, Mar. 2019;
12(3): 447-453.
10. Goday S, Rahaman ASK, Prameelarani A. Development and Validation of a LC-ESI- MS/MS Based Bioanalytical Method for Dapagliflozin and Saxagliptin in Human Plasma.
Indian J of Pharmaceutical Education and Research, 2018; 52(4 Suppl 2): S277-S286.
11. https://go.drugbank.com/salts/DBSALT001101
12. https://www.webmd.com/drugs/2/drug-165640/dapagliflozin-oral/details
13. https://pubchem.ncbi.nlm.nih.gov/compound/Dapagliflozin-propanediol-monohydrate
