Stability indicating assay of Dapagliflozin and Saxagliptin
Chinmaya Panda1*, Minaketan Sahoo2
1PhD Research Scholar (Pharmacy), Biju Patnaik University of Technology,
Rourkela, Odisha, Institute of Pharmacy and Technology, Salipur, Cuttack, Odisha.
2Department of Pharmaceutical Analysis, Institute of Pharmacy and Technology,
Salipur, Cuttack, Odisha, India.
*Corresponding Author E-mail: chinmayapn14@gmail.com
ABSTRACT:
The concentration of Saxagliptin(Sx) and Dapagliflozin(Dp) in the tablet was estimated simultaneously as per ICH guideline. For this purpose, a RP-HPLC method was newly established which is simple and robust showing Retention times were of 1.87 and 2.59 min for Sx and Dp respectively in the final developed condition. This method helped in separation of stable form of Saxagliptin and Dapagliflozin from their products. For the separation the conditions applied were, analytical column used was Phenomenex (Kinetex C8, 150 x 4.6mm, 5µm), wavelength- 212nm, mobile phase in isocratic elution mode was 0.1% Perchloric acid: ACN (50:40) , rate of flow-1.2ml/min, Injection volume-10µl and run time-8mins. During the study for robustness, the % RSD was <1.5%. Different mediums such as 0.1N HCl (acidic), 0.1 N NaOH (basic) and 3% H2O2(oxidative) were used to study the forced degradation and the result showed 0.93%, 3.17% and 2.24% degradation for Saxagliptin and 1.18%, 28.20% and 1.67% for Dapagliflozin respectively. During chromatographic studies distinct peaks were observed for analyte and degradant. In case of Dapagliflozin, the LOD was obtained as 0.31µg/ml and LOQ as 0.93µg/ml. Similarly, in case of Saxagliptin, the LOD was obtained as 0.37µg/ml and LOQ as 1.1193µg/ml. This developed method(RP-HPLC) was proved to be fast, new, robust, highly sensitive and efficient for estimation of Sx and Dp present together. This developed method can help in quality control tests of many combined dosage forms in general and Saxagliptin and Dapagliflozin in particular.
KEYWORDS: Saxagliptin, Dapagliflozin, Stress study, System Suitability, Dissolution study.
INTRODUCTION:
Dapaglifozin (C21H25ClO6) is white solid(crystalline) of BSC class-III antidiabetic drug soluble in organic solvents like DMSO, DMF and ethanol. It is sodium-glucose-cotransporter-2 inhibitor. pH has no effect on its solubility and partition coefficient as it is non-ionizable. It is used either alone or as a combination product with metformin in type 2 diabetes.1 Saxagliptin(C18H25N3O2) is an antidiabetic drug. It is Dipeptidylpeptidase-4 inhibitor.
It was approved by FDA in the month of July 31, 2009. The molecular weight of Saxagliptin is 315.4 g/mol. Structural formula of Dp and Sx are shown in Fig-1(a)and(b).2-4
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(a) Dapagliflozin |
(b) Saxagliptin |
Fig. 1. Structure of (a) Dapagliflozin and (b) Saxagliptin.
The reported literature on study of estimstion of Dapagliflozin (Dp)/Saxagliptin (Sx) alone or in combination as well as with other drugs like Metformin (Mt) by High-Performance Liquid Chromatographic method is shown in the Table-1.
Table 1: Literature survey for estimation of Saxagliptin and Dapagliflozin:
|
Analyte |
Column and Flow rate (ml/min) |
Mobile phase |
Rt (min) |
Wavelength (nm) |
Ref. |
|
Dp |
C-18 and 1 |
ACN: tri-ethylamine (50:50) |
5.163 |
224 |
1 |
|
Dp and Sx |
Discovery C-18 and 0.98 |
ACN and O – H3PO4 (50:50) |
2.67(Sx) , 3.420(Dp) |
210 |
2 |
|
Dp and Sx |
SPOLAR C-18 and 0.96 |
ACN: KH2PO4 pH5.8 (26:74) |
3.5 (Dp) , 5.0 (Sx) |
236 |
3 |
|
Dp and Sx |
Eclipse XBD C-18 and 1 |
0.1 % O-H3PO4 (pH – 5) and ACN (50:50) |
2.75(Lina-IS) 5.173(Sx) 7.218(Dp) |
254 |
4 |
|
Dp |
BDS and 1 |
ACN and O-H3PO4 |
2.718 |
220 |
5 |
|
Dp |
Thermo BDS; C-18 and 1 |
Mobile phase –A 88% O-H3PO4 in 2000ml of H2O (pH 6.5) adjusted with tri-ethylamine Mobile phase –B ACN: H2O (90:10) (Gradient mode) |
16.95 |
245 |
6 |
|
Dp and Mt |
Cosmosil , C-18 and 1 |
CH3OH : KH2PO4 (80:20) (pH 3) |
3.6 (Mt), 5.2 (Dp) |
228 |
7 |
|
Dp and Mt |
Hypersil BDS, C-18 and 1 |
0.2% O-H3PO4 (pH 6.8 adjusted by tri-ethyl amine) (50:50 v/v) |
2.791(Mt), 3.789 (Dp) |
240 |
8 |
|
Dp and Mt |
Symmetry® C-18 (100 mm, .1 mm, 2.2 µm) C-18 and 0.4, 60 0C LC-MS/MS |
K H2 PO4 (pH 3.5), ACN (50:50) |
0.8 (Mt), 1.52(Dp) |
228 |
9 |
|
Dp and Sx |
Acquity C18 BEH column and 0.5 |
CHCl3 Ethyl acetate and CH3OH (8:1:1 v/v) (Spectroden-sitometric), UPLC : 0.02 M KH2PO4 buffer: ACN CH3OH (2:1:1) |
Rf-0.17(Sx) Rf-0.31(Dp) 8.3(Dp) 2.227(Sx) |
Developed spot can scanned at225, and 210and UPLC used at 220 |
10 |
|
Sx and Dp |
Intersil C-18 (250 mm×4.6mm) and1.5 |
NH4 H2 PO4 buffer (pH 6.8) and CH3 OH (65:35) |
4.57(Dp) , 6.74(Sx) |
280 |
11 |
MATERIALS AND METHOD:
Chemicals and Reagents: Saxagliptin, Dapagliflozin, Acetonitrile, Water, 0.1% Perchloric acid ( all of HPLC grade).
Instruments: HPLC System: Schmidazu LC-2010-CHT with quaternary constant flow system equipped with auto-sample injector, Analytical balance (Contech), pH meter (Eutech), Ultra-sonicator (Leela sonic)
Software: Schimadzu LC Solution software for monitoring and integrating the output signals.
Saxagliptin Stock Solution (SSS): 5mg of Sx was taken in a 10 ml flask(volumetric) having diluents-7-8ml. Sx was dissolved by sonication and volume was made to 10ml(500μg/ml).
Dapagliflozin Stock Solution (DSS): 10mg of Dp was taken in a 10 ml flask(vol.) having diluents-7-8ml. Dp was dissolved by sonication and volume was made to to 10ml(1000μg/ml).
Optimized Chromatographic conditions:
a. Oven Temp : 30 oC
b. Rate of Flow : 0.8ml/min.
c. Mobile Phase: 0.1% Perchloric acid:ACN(50:40 v/v)
d. Runtime : 8 minutes
e. Injection Volume: 10µl
f. Wavelength : 212 nm
g. Diluent: 50%-0.1% Perchloric acid: 50%-ACN
h. Column: Phenomenex KinetexC8(150 x 4.6 mm,5 µ)
Preparation of Calibration curve:
Linearity was performed for 6 different levels of concentration of drugs. 0.1, 0.2, 0.4, 0.8, 1.0, 1.2ml of SSS were transferred into six volumetric flask(10ml), diluents was added to produce 10 ml each. The prepared solutions were estimated and the data of Sx is noted in the Table-2 and the graph is shown in Figure-2. Same procedure was adopted for Dp using DSS and estimated data is given in the Table-3 and shown in Figure-3.
Estimation of Saxagliptin and Dapagliflozin in Marketed Tablets:
Preparation of Sample Solution using Marketed Tablets:
QTERN 5mg/10mg (AstraZeneca) tablet was procured from drug store. Twenty tablets were weighed to get average weight. Tablets were finely crushed and the average weight of powder was transferred to 10ml flask(vol.). Drugs were dissolved by sonication in a part of diluent and volume was made to 10ml. The liquid was filtered and 0.2 ml each of filtrate was transferred into five volumetric flasks (10ml) and volume was made to 10 ml. Each ml of the solution contained about 10µg of Saxagliptin and 20µg of Dapagliflozin.
Estimation of Saxagliptin and Dapagliflozin:
The above sample solution was assayed by HPLC following the Optimized Chromatographic conditions. The estimated data is given in Table-4 and the the chromatogram is shown in Figure-4.
Method validation: 12
The ICH Q2 (R1) guideline was followed for validation of HPLC method The validation included assessing system suitability(following USP), linearity, specificity, sensitivity, precision, accuracy, and robustness.
Precision: Three working standard solutions and test sample solutions were estimated by HPLC on the same day (intraday precision/repeatability). The area of each injection was measured, and the % Relative Standard Deviation (RSD) was calculated. Similarly, for interday precision study, three different working standard solutions and three different test sample solutions were estimated by HPLC on three different days. The areas of all findings were recorded, and % RSD was counted.
Accuracy: Standard addition method was performed by a spiking recovery investigation at 50, 100 and 150 % level. The findings are noted in Table-5.
Peak purity: Rt of standard mixture samples with tablet samples of Saxagliptin and Dapagliflozin was compared to optimize peak purity.
Robustness: The assay method was verified by changing the chromatographic condition. The wavelength was taken as 210nm and 214nm, Rate of flow-0.7ml/min and 0.9ml/min and organic phase (+5% to-5%).
System suitability: This test was performed to prove the optimized chromatographic condition suitable to establish the studied method for estimation of Sx and Dp in the reference and prepared tablets with validation. A blank assay was conducted following optimized chromatographic condition to get the interference of the system (Figure-5). The representative chromatogram of Saxagliptin and Dapagliflozin (50 and 100µg/ml) was shown in Figure-6. The results of the test parameter is shown in the Table-6. Every parameter of system suitability test was passed the acceptable criteria.
Forced degradation study: This study was conducted using optimal RP-HPLC settings with acidic, basic, and oxidative conditions. Investigating solution was made from tablet containing Saxagliptin as 50μg/mL and Dapagliflozin as 100μg/mL. The findings are given in Table-7. The acidic, basic and oxidative degradation chromatogram of both the drugs in three selected medium are shown in the Figure 7, 8 and 9 respectively.
Sensitivity:
The sensitivity was assessed by determining LOD and LOQ using the standard calibration curve.
RESULTS AND DISCUSSION:
The linear regression equation of Saxagliptin was “Y = 15087x + 3253 and r 2 = 0.999”. and that of Dapagliflozin was “Y = 39462x + 7571 and r 2 = 1” which were obtained from linearity data. The precision result showed that the % RSD was found to be less than 2 % in standard and test sample solutions. The recovery results were found within 98%-102%. From the peak purity determination it was found that there was a good correlation between the chromatogram. The robustness study showed that the % RSD remained within 1.2. System suitability test was conducted and found that every parameter of system suitability test was passed. Force degradation study was performed and % degradation was found to be within the limit of 0.93 to 3.17%. The LOD of Dp=0.31µg/ml and of Sx=0.37µg/ml. Similarly LOQ of Dp=0.93µg/ml and of Sx=1.1193µg/ml.
Table 2: Linearity table of Saxagliptin
|
% Level |
Conc. (ug/ml) |
Area |
|
0 |
0 |
0 |
|
5 |
5 |
80982 |
|
10 |
10 |
153745 |
|
20 |
20 |
305338 |
|
40 |
40 |
609502 |
|
50 |
50 |
759376 |
|
60 |
60 |
904858 |
Fig. 2. Standard graph of Saxagliptin.
Table 3: Linearity table of Dapagliflozin:
|
% Level |
Conc. (ug/ml) |
Area |
|
0 |
0 |
0 |
|
10 |
10 |
401688 |
|
20 |
20 |
801279 |
|
40 |
40 |
1589699 |
|
80 |
80 |
3167847 |
|
100 |
100 |
3958568 |
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120 |
120 |
4734959 |
Fig. 3. Standard graph of Dapagliflozin.
Table 4: Results of Tablet analysis:
|
Analyte |
Marketed tablet(mg/Tablet) |
Calculated Conc.(mg/Tablet) |
C.I. |
C.V. |
SE |
Analyte |
|
Sx |
5 |
5.026 |
100.52±1.35 |
1.086 |
0.489 |
Sx |
|
Dp |
10 |
10.006 |
100.06±1.19 |
0.965 |
0.433 |
Dp |
The confidential interval denoted as C. I. is the range within which the real value may be found with a 95% confidence level = R ± ts/√n, It is calculated by using this equation C.I.= R ± ts/√n where n=5 and . ‘t’ (theoretical) values at 95% confidence level for n - 1 degrees of freedom is t(0.05,4) = 2.776.
Fig. 4. Representative chromatogram of tablet sample Saxagliptin (10µg/ml) and Dapagliflozin (20µg/ml).
Table 5: The results of recovery study of the drug:
|
% Recovery study |
Analyte |
Formulation |
Pure drug added |
Drug found |
CI |
C.V. |
SE |
t |
|
50 |
Saxa |
10 |
5 |
15.020 |
100.134±1.154 |
0.928 |
0.422 |
0.324 |
|
Dapa |
20 |
10 |
30.128 |
100.473±1.54 |
1.249 |
0.570 |
0.848 |
|
|
100 |
Saxa |
10 |
10 |
20.136 |
100.680±1.44 |
1.155 |
0.528 |
1.13 |
|
Dapa |
20 |
20 |
39.98 |
99.95±0.445 |
0.358 |
0.160 |
0.311 |
|
|
150 |
Saxa |
10 |
15 |
25.004 |
100.016±0.399 |
0.321 |
0.144 |
0.111 |
|
Dapa |
20 |
30 |
49.964 |
99.928±0.707 |
0.570 |
0.255 |
0.282 |
Fig. 5. Representative blank chromatogram of the method.
Fig.6. The representative chromatogram of Sx and Dp(50 and 100µg/ml).
Table- 6: The results of System suitability of Sx and Dp:
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Sample ID |
Sx |
Dp |
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|
Area |
Rt |
Asymmetry |
TP |
Res. |
Area |
Rt |
Asymmetry |
TP |
Res |
|
|
100% Rep 1 |
777787 |
1.87 |
1.27 |
4673 |
0 |
3954146 |
2.59 |
1.38 |
7030 |
6.24 |
|
100% Rep 2 |
778738 |
1.87 |
1.23 |
4729 |
0 |
3963999 |
2.59 |
1.4 |
6986 |
6.2 |
|
100% Rep 3 |
777360 |
1.87 |
1.15 |
4729 |
0 |
3957801 |
2.59 |
1.35 |
7018 |
6.19 |
|
100% Rep 4 |
779559 |
1.87 |
1.23 |
4705 |
0 |
3959385 |
2.59 |
1.36 |
7003 |
6.24 |
|
100% Rep 5 |
779698 |
1.87 |
1.24 |
4734 |
0 |
3969415 |
2.59 |
1.33 |
7041 |
6.26 |
|
100% Rep 6 |
779632 |
1.87 |
1.24 |
4710 |
0 |
3962667 |
2.59 |
1.32 |
7031 |
6.25 |
|
Average |
778795.7 |
1.87 |
|
3961236 |
2.59 |
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STDEV |
1017.66 |
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5330.51 |
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% RSD |
0.13 |
|
0.13 |
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Table 7: The results of forced degradation study of Sx and Dp:
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Stress condition |
Sx |
Dp |
||||
|
Area |
% Assay |
% Degradation |
Area |
% Assay |
% Degradation |
|
|
0.1N HCl for 45mins |
771545 |
99.07 |
0.93 |
3914589 |
98.82 |
1.18 |
|
3% H2O2 for 45 mins |
761317 |
97.76 |
2.24 |
3895254 |
98.33 |
1.67 |
|
0.1N NaOH for 45mins |
754114 |
96.83 |
3.17 |
2844152 |
71.8 |
28.2 |
Fig.7. Acidic degradation chromatogram of drug.
Fig.8. Basic degradation chromatogram of drug.
Fig. 9. Oxidative Degradation chromatogram of drug.
CONCLUSION:
In order to enhance the efficiency of the suggested approach, a series of systematic experiments were conducted on various chromatographic settings, such as the kind of column and the selection of the mobile phase. The optimization of HPLC columns included the study of solvents preferred for reverse phase, i.e. [water: organic solvent] or [buffer:organic solvent] at various proportions. The validation findings confirmed that all the parameters complied with the permitted limits set by the ICH standards. The system suitability test results accounted for well-resolved symmetric peaks with efficient separation (N>2000; R>2; t<2). Furthermore, the analytical results were consistent as the percentage of relative standard deviation was below 2% for the retention duration of system suitability evaluations. The approach was determined to be selective as the results of blank and solution with formulation excipients showed no interference. The coefficient of determination (r2) was close to 1 as resulted from the linear regression analysis, indicating a high level of linearity in the suggested technique. The approach demonstrated a recovery rate between 98% and 102%, indicating its accuracy. The %RSD results for both repeatability and intermediate precision measurements consistently remained below 1.5%, showing the proposed HPLC method’s excellent accuracy. The analytical findings obtained from an intentional modest modification of the optimal method condition showed that the adjustments had no major impact on the analytical output. The purpose is to compare the findings obtained from the newly created RP-HPLC method with those obtained from established reference methods. This comparison aims to illustrate that the new method is superior to the reference methods in terms of accuracy and precision. Therefore, it can be concluded that the technique is resilient.
STATISTICAL ANALYSIS:
All the statistical data were calculated by using Excell and SSP.exe software.
CONFLICT OF INTEREST:
No conflict of interest.
AUTHOR CONTRIBUTIONS:
All authors equally participated.
ACKNOWLEDGMENTS:
I fully acknowledge the Supervisors and Principal, Institute of Pharamcy and Technology, Salipur, Dist- Cuttack, Odisha.
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Received on 17.06.2024 Revised on 28.11.2024 Accepted on 11.02.2025 Published on 02.05.2025 Available online from May 07, 2025 Research J. Pharmacy and Technology. 2025;18(5):2393-2398. DOI: 10.52711/0974-360X.2025.00342 © RJPT All right reserved
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