Stability Indicating Simultaneous Method development and Validation of Dapagliflozin and Saxagliptin by RP-HPLC

 

Merugu Manasa¹, Vijey Aanandhi M²*

¹Research Scholar, Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Pallavaram, Chennai,

Tamil Nadu, India.

²Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences,

Vels Institute of Science Technology and Advanced Studies (VISTAS), Chennai, India.

 

ABSTRACT:

The developed method was validated according to ICH guidelines with respect to specificity, linearity, limits of detection, quantification, accuracy, precision, and robustness. The stability indicating simultaneous method development and validation of Dapagliflozin and Saxagliptin by RP-HPLC in drug substance and product. The chromatographic separation was done in an isocratic mode using the BDS C18 (150 x 4.6mm, 5.0m) column with mobile phase containing a Ammonium acetate buffer: ACN (40:60 %v/v) used for efficient chromatographic separation. The flow rate of the mobile phase was adjusted to 1.0mL/min with 30^oC column temperature and detection of wavelength at 220nm, injection volume 10µl was fixed for achieving good elution of eluents. The retention time for DAPA and SAXA were found to be 2.207 min and 2.889 min respectively. DAPA and SAXA were linear in the concentration ranges from 0-15µg/ml and 0 -8µg/ml respectively. Regression analysis showed that R² value (correlation coefficient) for DAPA and SAXA were found to be 0.999. LOD and LOQ of DAPA was found to be 21.6µg/ml and 9.1µg/ml, SAXA was found to be 11.5µg/ml 9.1µg/ml. The developed method was validated and found to be accurate, specific and robust. Both the drugs were subjected to the stress conditions like acidic, basic, oxidative, photolytic and thermal conditions. The degradation results were found to be satisfactory. In acidic, alkaline and peroxide stress conditions. The forced degradation test results confirmed that the DAPA and SAXA peaks were homogenous in all stress conditions, thus proving the stability-indicating nature of the method. This method could be applied for the simultaneous estimation of DAPA and SAXA in drug substance.

 

 

 

INTRODUCTION:^(1-3)

Dapagliflozin (DAPA), 2S,3R,4R,5S,6R)-2-(4-Chloro-3-(4-ethoxybenzyl) phenyl)-6- (hydroxymethyl) tetrahydro-2H- pyran-3,4,5-trio is widely used for the treatment of type 2 diabetes mellitus, It shows its action by reducing glucose levels by excreting through kidney. Dapagliflozin molecular formula is C₂₁H₂₅ClO₆, and molecular weight is 408.873gram/mole.

 

Saxagliptin (SAXA) is competitive dipeptidyl peptidase-4 inhibitor used to treat diabetes mellitus type-2. Its empirical formula is C₁₈H₂₅N₃O₂,and corresponding molecular weight of the compound 315.41gram/mole.

 

The combination of DAPA and SAXA is used to treat diabetes mellitus type-2, The chemical structures of DAPA and SAXA and are shown in Image 1.

 

Image 1 Structure of Dapagliflozin and Saxagliptin

 

DAPA and SAXA drug substances are as combined dosage form is official in the United States pharmacopeia. In the literature survey, there were several LC assay approaches that have been reported for the determination of DAPA and SAXA in pharmaceutical preparation either individually or in combination with other drugs and LC-MS in human plasma. Few procedures were available for the determination of DAPA and SAXA^(4–20). It has shown aspiration to develop a stability-indicating method for simultaneous determination of DAPA and SAXA in pharmaceutical formulation.

 

MATERIALS AND METHOD:

Chemicals and Reagents:

DAPA, SAXA Reference Standards and tablets were gifted from the formulation research and development laboratory of Aizant Pharma Ltd., Hyderabad, India. India. HPLC grade Acetonitrile, Methanol, Millipore MilliQ water and Glacial acetic acid were purchased from Merck, Germany, Regis Technologies Inc, USA.

 

Equipment:

The LC system used for method development and method validation. Detection was carried by Waters with a diode array detector (model: 2996 detector 2487 separation module). The output signal was supervised and processed using Waters Empower 2 Software. LC GC Ragward Dual Range balance was used to perform weighing. Photostability studies were carried out in a photo stability chamber. Thermal stability studies were performed in at thermostat dry air.

 

Chromatographic Conditions:

RP-HPLC measurements were carried out using a reversed phase BDS C18 (150 x 4.6mm, 5.0m) column with mobile phase containing a Ammonium acetate buffer: ACN (40:60 %v/v) used isocratic chromatographic separation. The flow rate of the mobile phase was 1.0mL/min with ambient column temperature and wavelength detection at 220nm, injection volume 10µl was fixed for achieving good elution of eluents.

 

Preparation of Standard Solution and System Suitability Solution:

Accurately Weighed and transferred 2.5mg of Dapagliflozin, 1.25mg and of Saxagliptin working Standards into a 25ml clean dry volumetric flasks, add 10ml of diluent, sonicated for 10 minutes and make up to the final volume with diluents individually. 1ml from the above two stock solutions was taken into a 10ml volumetric flask and made up to 10ml. (10µg/ml Dapagliflozin, and 5µg/ml Saxagliptin).

 

Preparation of test Solution:

Accurately weighed equivalent weight of the combination (qtern) powder sample transfer into a 50ml volumetric flask, 25ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters (200µg/ml Dapagliflozin and 100µg/ml Saxagliptin) 0.5ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (10µg/ml Dapagliflozin, and 5µg/ml Saxagliptin).

 

METHOD VALIDATION:

The proposed method was validated as per ICH guidelines.

 

System Suitability:

System suitability parameters were evaluated to verify the system performance. System precision was determined by six replicate injections of standard preparations. All the important characteristics, including the relative standard deviation, peak tailing, and theoretical plate number were measured.

 

Specificity/Stability:

Stress studies were performed at concentration of 5 µg/mL of DAPA and 2.5µg/mL of SAXA in active pharmaceutical ingredients (API) and formulated sample to provide the stability-indicating property and specificity of the proposed method. Intentional degradation was attempted by the stress conditions of exposure to photolytic stress (1.2million lux hours followed by 200Watt hours ), heat (exposed at 105°C for 15h), acid (1N HCl for 2 hours at 60°C), base (1N Na OH for 2 hours at 60°C), oxidation (10% peroxide for 30minat 60°C), and humidity (exposed to 85% RH for 72 hours).

 

Precision:

The precision of the DAPA and SAXA was checked by injecting six individual standard preparations of 100% concentration and calculated %RSD of each compound. The intermediate precision of the method was also assessed using different analysts and a different instrument in the same laboratory.

 

Limits of Detection (LOD) and Quantification (LOQ):

LOD and LOQ of DAPA and SAXA were determined at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentrations. Precision study was also carried out at LOQ level and result was calculated.

 

Linearity:

Linearity examination was prepared by diluting stock solution to the required concentrations. The solutions were prepared at six concentration. The peak area versus concentration in µg/mL data was subjected to method of least squares linear regression analysis.

 

Accuracy:

Accuracy of the method was evaluated by using concentration levels of 50%, 100% and 150% DAPA and SAXA. Standard addition and recovery experiments were conducted to determine accuracy. The percentages of recoveries DAPA and SAXA were calculated.

 

Robustness:

To examine the robustness of the developed method, experimental conditions were deliberately changed (flow rate, column temperature and mobile phase composition) and the resolution between DAPA, SAXA tailing factor, and theoretical plates were evaluated.

 

RESULTS AND DISCUSSION:

Optimization of Chromatographic Conditions:

After conducting several trials sharp peak shapes were observed with the BDS (C18 150 x 4.6mm, 5.0m) column and mobile phase of Ammonium acetate buffer : ACN (40:60%v/v). The flow rate was 1.0mL/min with column ambient temperature and detection was carried at 220nm (Isobestic point), injection volume 10µl was fixed for achieving good elution of eluents. The optimized chromatogram of Dapagliflozin and Saxagliptin was shown in image 2.

 

Image 2 standard chromatogram

 

METHOD VALIDATION:

The developed method was validated for system suitability, specificity, accuracy, precision, linearity, robustness, ruggedness, solution stability, LOD, and LOQ and stability as per ICH guidelines.

 

System Suitability:

The percentage area of Relative Standard deviation (RSD) from six replicate injections was found below 2.0%. Results were presented in image 2 and Table1. As seen from this data all the system suitability parameters are within the limits.

 

Precision:

The six homogeneous test solutions of % RSD data of DAPA, SAXA was within 2.0%. The results are shown in Tables 1. The % RSD for intermediated precision for DAPA and SAXA were found to be 0.3 and 0.2 respectively.

 

Table 1 Results of Precision and System Suitability

S. No	DAPAGLIFLOZIN				SAXAGLIPTIN
	Rt	Area	USP plate Count	USP Tailing	Rt	Area	USP plate Count	USP Tailing
1	2.201	187719	8953	1.34	2.888	95598	11218	1.25
2	2.202	189673	9282	1.35	2.893	95860	11611	1.20
3	2.203	188213	9648	1.37	2.894	95927	11862	1.19
4	2.204	188513	9575	1.37	2.895	95632	12451	1.24
5	2.207	190464	8695	1.42	2.898	95832	12301	1.17
6	2.208	190814	9104	1.47	2.901	95320	12619	1.18
Mean		189233				95695
Std. Dev		1269				225.3
% RSD		0.7				0.2

 

Table 2 Results of Accuracy

Drug	%	TRIAL	AREA (y)	M	c	y-c	Total Conc	Added Conc	Std Conc	Amt Rec	% Rec	AVG %Rec
Dapagliflozin	50%	1	283883	18921	824.12	283058.9	14.96	5	10	4.96	99.20	99.21
		2	283197	18921	824.12	282372.9	14.92	5	10	4.92	98.48
		3	284593	18921	824.12	283768.9	14.99	5	10	4.99	99.95
	100%	1	380660	18921	824.12	379835.9	20.07	10	10	10.07	100.75	100.89
		2	379150	18921	824.12	378325.9	19.99	10	10	9.97	99.95
		3	383009	18921	824.12	382184.9	20.19	10	10	10.19	101.99
	150%	1	476266	18921	824.12	475441.9	25.12	15	10	15.12	100.85	101.05
		2	477288	18921	824.12	476463.9	2.18	15	10	15.18	101.21
		3	476979	18921	824.12	476154.9	25.16	15	10	15.1	101.10
SSaxagliptin	50%	1	149891	19953	555.38	149334.6	7.49	2.5	5	2.49	99.64	99.67
		2	150185	19953	555.38	149628.6	7.50	2.5	5	2.50	100.23
		3	149647	19953	555.38	149090.6	7.47	2.5	5	2.47	99.15
	100%	1	250137	19953	555.38	200390.6	10.05	5	5	505	101.04	100.19
		2	200039	19953	555.38	199482.6	10.00	5	5	5.00	100.13
		3	199310	19953	555.38	198753.6	9.97	5	5	4.97	99.40
	150%	1	250137	19935	555.38	249580.6	12.51	7.5	5	7.51	100.26	100.21
		2	250883	19935	555.38	250326.6	12.55	7.5	5	7.55	100.76
		3	249175	19935	555.38	248618.6	12.47	7.5	5	7.47	99.62

 

 

Accuracy:

Recovery of DAPA and SAXA was found to be 98.0% to 102.0%. The summary of % recovery for individual data was mentioned in Table 2.

 

Linearity

Linear calibration plots are tested at different concentration levels. The correlation coefficient obtained was 0.999 for both the components. Results were mentioned in table 3 and image 3.

 

Table 3 Linearity of Dapagliflozin and Saxagliptin

S. No	Conc of dapa	Avg area of dapa	Conc of saxa	Avg area of saxa
1	0	0	0	0
2	2.5	48156	1.25	48853
3	5	96853	2.5	98040
4	7.5	140843	3.5	141769
5	10	191556	5	193697
6	12.5	239070	6.25	236543
7	15	282649	7.5	283843

 

 

Image 3 Linearity of Saxagliptin

 

Limit of Detection (LOD) and Limit of Quantification (LOQ)

Limit of detection result for DAPA and SAXA were found to be 21.6 and 11.5 respectively and were within the limits. LOQ for DAPA and SAXA were found to be 9.2 and 9.1 respectively and were within the limits.

 

Robustness:

Robustness studies were performed by changing the flow rate (+ 2 ml/min), column temperature (+5^oC) and mobile phase ratio. No significant effect was observed on system suitability parameters of DAPA and SAXA. Thus, the method was found to be robust with respect to variability in applied conditions.

 

Assay:

The assay results were found to be 99.65 % w/w and 99.01% w/w for DAPA and SAXA respectively. The results were within the limits as per the ICH guidelines.

 

Specificity / Stability:

Blank, placebo and degradation samples were analyzed with the above mentioned HPLC conditions using a PDA detector to monitor the homogeneity and purity of the DAPA and SAXA. Blank, Placebo, Individual impurities of DAPA, SAXA were verified and proved to be non-interfering with each other thus proving the specificity of the method, retention time of DAPA and SAXA, Degradation was not observed in photolytic stress, humidity and thermal stress studies. Significant degradation was observed in oxidative conditions. It was interesting to note that all the peaks due to degradation were well resolved from the peaks of DAPA and SAXA. The verification of peak purity indicates that there is no interference from degradants, facilitating error-free quantification of DAPA and SAXA. Hence the method is considered to be “stability-indicating.” The specificity results were shown in Tables 4.

Table 4 Results of stability studies

S. No	Condition	DAPA Area	SAXA Area	DAPA % Recovery	SAXA% Recovery	% Degradation DAPA	% Degradation SAXA
1	Acid	172823	91286	92.06	94.98	7.94	5.02
2	Base	180601	93948	96.20	97.75	3.80	2.25
3	Peroxide	171827	92804	91.53	96.56	8.47	3.44
4	Thermal	182209	94557	97.06	98.38	2.94	1.62
5	UV	183000	94557	97.48	98.38	2.52	1.62
6	Humid	185856	95881	99.00	99.76	1.00	0.24

 

 

CONCLUSIONS:

The HPLC method developed and validated for the simultaneous determination of DAPA and SAXA in pharmaceutical dosage form was precise, accurate, and specific. The method is validated as per ICH guidelines and found to be specific, precise, linear, accurate, rugged, and robust. The developed method can be used for the regular analysis and stability analysis of DAPA and SAXA either individually or in their combination dosage forms.

 

ACKNOWLEDGMENTS:

The authors wish to thank the management of Vels University, for supporting this work. The authors wish to acknowledge the Hetero labs for providing the samples for their research. They would also like to thank colleagues for their support to complete research work.

 

CONFLICT OF INTEREST:

The authors have no conflict of interests to disclose other than what has been acknowledged above.

 

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Received on 05.01.2020            Modified on 28.02.2020

Accepted on 17.04.2020         © RJPT All right reserved