Simultaneous determination of Saxagliptin Hydrochloride and Glibenclamide in Synthetic Mixture using Spectrophotometric technique (First order Derivative Method)

Pranali S. Sisode¹ , Hasumati A. Raj¹, Vineet C. Jain²

¹Department of Quality Assurance, Shree Dhanvantary Pharmacy College, Kim, Surat, Gujarat, India.

²Department of Pharmacognosy, Shree Dhanvantary Pharmacy College, Kim, Surat, Gujarat, India.

*Corresponding Author E-mail: pranalisisode19@gmail.com,drharaj@yahoo.com

ABSTRACT:

A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Saxagliptin Hydrochloride and Glibenclamide in synthetic mixture using first order derivative zero-crossing method. Saxagliptin Hydrochloride showed zero crossing point at 315.00nm while Glibenclamide showed zero crossing point at 229.40nm. The dA/dλ was measured at 229.40nm for Saxagliptin Hydrochloride and 315.00nm for Glibenclamide and calibration curves were plotted as dA/dλ versus concentration, respectively. The method was found to be linear (r2>0.9995) in the range of 5-25μg/ml for Saxagliptin Hydrochloride at 229.40nm. The linear correlation was obtained (r2>0.9994) in the range of 5-25 μg/ml for Glibenclamide at 315.00nm. The limit of determination was 0.243μg/ml and 0.317μg/ml for Saxagliptin Hydrochloride and Glibenclamide, respectively. The limit of quantification was 0.738μg/ml and 0.960μg/ml for Saxagliptin Hydrochloride and Glibenclamide respectively. The accuracy of these method were evaluated by recovery studies and good recovery result were obtained greater than 99% shows first order derivation zero crossing. The method was successfully applied for simultaneous determination of Saxagliptin Hydrochloride and Glibenclamide in binary mixture.

KEYWORDS: Saxagliptin Hydrochloride, Glibenclamide, First Derivative Method, Spectroscopic method.

INTRODUCTION:

Saxagliptin is a potent, selective dipeptidyl peptidase-4(DPP-4) inhibitor, specifically designed for extended inhibition of the DPP-4 enzyme.DPP-4 inhibitors enhance the levels of the glucoregulatory hormones glucagon-like peptide 1(GLP-1) and glucose-dependent insulin tropic peptide (GIP), thereby acting to promote insulin synthesis and release, and suppress glucagon secretion, among other important glucoregulatory effects.DPP-4 inhibition are associated with a favourable safety profile, including a low risk of hypoglycaemia because of the glucose-dependent nature of incretin hormone activity, a neutral effect on body weight and potential for improved B cell function.

Chemical name: (1 S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1Adamantyl)acetyl]-2-azabicyclohexane-3-carbonitrile.Saxagliptin hydrochloride appears as white or almost white Solid powder. The drug is slightly soluble in DMSO and in methanol. Saxagliptin hydrochloride melts at 96-102°C. The pKa value of saxagliptin hydrochloride is 7.90. The molecular formula of saxagliptin hydrochloride is C₁₈H₂₅N₃O₂. It’s molecular weight is 351.87 gm/mol. The structural formula is shown in Figure 1.

[Figure 1: Chemical structure of saxagliptin hydrochloride]

Glibenclamide include: Stimulation of insulin release from the B2 cells of the pancreas by blocking the ATP-sensitive K+ channels, resulting in depolarization and Ca²⁺ influx. Reduction in hepatic glucose production. Increase in peripheral insulin sensitivity. The chemical name: 5-Chloro-N-[4-(cyclohexyl ureidosulfonyl) phenethyl]-2 methoxy benzamide. Glibenclamide is a white crystalline powder that is freely soluble in Methanol and Insoluble in water. Glibenclamide melts at 173-175°C. The pKa value of Glibenclamide is 5.11. The molecular formula of glibenclamide is C₂₃H₂₈CIN₃O₅S. Its molecular weight is 494.0 g/mol. The structural formula is shown in Figure 2.

[Figure 2: Chemical structure of glibenclamide]

The review of literature regarding quantitative analysis of Saxagliptin Hydrochloride and Glibenclamide revealed that no Simultaneous Equation method attempt was made to develop analytical methods for Saxagliptin Hydrochloride and Glibenclamide. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual and combination of drugs. The focus of the present study was to develop and validate a rapid, stable, specific, and economic Spectroscopic method for the estimation of Saxagliptin Hydrochloride and Glibenclamide in Synthetic Mixture.

MATERIALS AND METHODOLOGY:

Apparatus:

A double beam UV/Visible spectrophotometer (Shimadzumodel2450, Japan) with spectral width of 2nm, 1 cm quartz cells was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe system software.

Reference samples:

• Saxagliptin raw material was received as gift sample from CTX life science, Sachin, Surat.

• Glibenclamide raw material was received as gift sample from Prudence Pharma Chem. GIDC Estate, Ankleshwer.

Materials and reagents:

Methanol AR grade (Rankem).

FIRST DERIVATIVE CONDITIONS¹³

Ø Mode : Spectrum

Ø Scan speed :Fast

Ø Wavelength range: 200-400 nm

Ø Derivative order: first

Ø Scaling factor: 50

PREPARATION OF STANDARD SOLUTION AND SYNTHETIC MIXTURE:

Preparation of stock solution for Saxagliptin Hydrochloride:

An accurately weighed quantity equivalent to 10mg of Saxagliptin Hydrochloride was transferred into 100 ml volumetric flask dissolved it into 25ml of methanol than sonicate for 15min and make up the volume upto the mark with the methanol to obtain standard solution having concentration of SAXA(100μg/ml).

Preparation of Standard stock solution for Glibenclamide:

An accurately weighed quantity of Glibenclamide (10mg) was transferred to a separate 100 ml volumetric flask and dissolved it into 25ml methanol than sonicate for 15min and diluted upto the mark with methanol to obtain standard solution having concentration of GLB (100μg/ml).

Preparation of Standard Mixture Solution of Saxagliptin Hydrochloride and Glibenclamide (1:1):

1 ml of working standard stock solution of SAXA (100μg/ml) and 1ml of standard Stock solution of GLB (100μg/ml) were pipetted out into 10ml volumetric flask and volume was adjusted upto the mark with methanol to get 10μg/ml of SAXA and 10μg/ml of GLB.

Preparation of Test Solution:

The preparation of synthetic mixture was as per patent:

Saxagliptin Hydrochloride………. 100mg

Glibenclamide……………………… 100mg

Excipients…………………………… qs.

Take Synthetic powder equivalent to10mg of SAXA in 100ml volumetric flask. Dissolve it in 25ml Methanol sonicate for 15 min and make upto the mark with Methanol. Shake vigorously and filter the solution. Finally the solution had the concentration 100μg/ml and 100μg/ml respectively for SAXA and GLB. After that from this solution 1ml was pipette out and diluted up to 10 ml with Methanol. So the concentration was 10μg/ml and 10μg/ml for SAXA and GLB respectively.

Procedure:

Selection of wavelength and method development for determination of Saxagliptin Hydrochloride and Glibenclamide

Figure 3: Overlain Zero Order spectra of SAXA and GLB in ratio (1:1)

Figure 4 Overlain First Order spectra of SAXA and GLB in ratio (1:1)

First order derivative Method is based on measurement of absorbance at 229.40nm (ZCP of GLB) as wavelengths for quantification of Saxagliptin where no interference due to Glibenclamide was observed, similarly absorbance at 315nm (ZCP of SAXA) is selected for quantification of Glibenclamide, where no interference due to Saxagliptin was observed.

Calibration curves were plotted at 229.40 nm for SAXA and 315 nm for GLB. Regression equations were determined from the calibration curves.

Calibration curves for Saxagliptin Hydrochloride:

This series consisted of five concentrations of standard SAXA solution ranging from 5-25μg/ml. The solutions were prepared by pipetting out Standard SAXA stock solution (0.5ml, 1.0ml, 1.5ml, 2.0ml, and 2.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order derivative spectrum, measured the absorbance at 229.40nm against a reagent blank solution (Methanol).

Calibration curve for Glibenclamide:

This series consisted of five concentrations of standard GLB solution ranging from 5-25μg/ml. The solutions were prepared by pipetting out Standard GLB stock solution (0.5ml, 1.0ml, 1.5ml, 2.0ml and 2.5ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with Methanol. A zero order derivative spectrum measured the absorbance at 315nm against a reagent blank solution (Methanol).

RESULTS AND DISCUSSION:

Validation Parameters:¹⁴

Linearity:

Five point calibration curves were obtained in the concentration range of 5-25μg/ml for Saxagliptin Hydrochloride and 5-25μg/ml for Glibenclamide. The response of drug was found to be linear in investigation range and the regression equations was found to be y = -0.028x - 0.0254 for SAXA(n=5) and y = -0.019x - 0.008 for GLB (n=5), with the correlation coefficient 0.9995 and 0.9994 (n=5) respectively, is listed in Table 1.

Table 1. Calibration data for SAXA at ZCP 315nm and GLB at ZCP 229.40 nm and 229.00nmrespectively. *(n=5)

Sr. no.	Conc.(μg/ml) at 229.40 nm	Absorbance* ±S.D	%RSD	Conc.(μg/ml) at 315 nm	Absorbance*±S.D.	%RSD
1	5	-0.173±0.001	0.851	5	-0.102±0.001	0.876
2	10	-0.305±0.002	0.854	10	-0.203±0.001	0.724
3	15	-0.458±0.002	0.445	15	-0.288±0.002	0.972
4	20	-0.611±0.002	0.446	20	-0.395±0.002	0.599
5	25	-0.740±0.003	0.430	25	-0.483±0.002	0.441

Figure 5 Calibration curve of SAXA at ZCP 315nm

Figure 6 Calibration curve of GLB at ZCP 229.40nm

Precision:

The precision of the method was evaluated in terms of inter-day and intra-day by carrying out independent assays of three concentrations chosen from range of the standard curves (15, 20 and 25μg/ml of SAXA and GLB respectively) and the %RSD of assay (inter-day and intra-day) was calculated. The results of study are shown in Table 2 and 3.

Accuracy:

The accuracy of the method was determined by spiking of SAXA and GLB to pre quantified sample solutions of SAXA (10μg/ml) and GLB (10μg/ml) in triplicate at three concentration level of 80, 100, 120% of the specified limit. The percentage recoveries of SAXA and GLB were calculated and the result is nearer to 100% shown in Table 4 and 5.

Table 2 Intraday precision data for estimation of SAXA and GLB*(n=3)

Saxagliptin Hydrochloride and Glibenclamide Synthetic Mixture
Sr. No.	Conc. (µg/ml) At 229.40nm	Absorbance. at 229.40nm±SD	%RSD	Conc. (µg/ml) At 315nm	Absorbance. at 315nm±SD	%RSD
1	10	-0.305±0.002	0.655	10	-0.204±0.001	0.490
2	15	-0.457±0.002	0.455	15	-0.288±0.529	0.529
3	20	-0.607±0.003	0.501	20	-0.393±0.001	0.388

Table 3 Interday precision data for estimation of SAXA and GLB*(n=3)

Saxagliptin Hydrochloride and Glibenclamide Synthetic Mixture
Sr. no.	Conc. (µg/ml) at 229.40nm	Absorbance at 229.40nm±SD	%RSD	Conc. (µg/ml)at 229.40nm	Absorbance at 315nm±SD	%RSD
1	10	-0.304±0.002	0.826	10	-0.205±0.001	-0.742
2	15	-0.456±0.003	0.703	15	-0.287±0.001	-0.531
3	20	-0.601±0.001	0.253	20	-0.395±0.002	-0.506

Table 4 Data for Quantity of Synthetic Mixture and API (n=3)

Sr. no.	Level of Recovery	Quantity of formulation (mg)		Quantity of API(mg)		Total concentration(mg)
		SAXA	GLB	SAXA	GLB	SAXA	GLB
1	Control	10	10	-	-	10	10
2	80%	10	10	8	8	18	18
3	100%	10	10	10	10	20	20
4	120%	10	10	12	12	22	22

Table 5 Recovery Data for Accuracy (n=3)

Sr. no.	Level of Recovery	Amount found (µg/ml)		% recovery		S.D		% RSD
		SAXA	GLB	SAXA	GLB	SAXA	GLB	SAXA	GLB
1	Control	9.962	9.993	99.62	99.936	0.367	0.431	0.368	0.431
2	80%	17.971	18.013	99.637	100.166	0.339	0.427	0.341	0.426
3	100%	19.982	20.021	99.823	100.200	0.490	0.386	0.491	0.386
4	120%	22.023	22.052	100.194	100.001	0.453	0.437	0.452	0.437

3.4 Limit of Detection and Limit of Quantification:

The limit of detection (LOD) and limit of quantitation (LOQ) of the method were evaluated by standard deviation of response and slope method. LOQ and LOD were calculated by the equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where “N” is standard deviation of the absorbance, and “B” is the slope of the corresponding calibration curve. The limit of detection (LOD) were found to be 0.243μg/ml for SAXA and 0.738μg/ml for GLB respectively and limit of quantitation (LOQ) were found to be 0.317μg/ml for SAXA and 0.960μg/ml for GLB presented in Table 6.

Robustness and Ruggedness:

· Robustness and Ruggedness of the method was determined by subjecting the method to slight change in the method condition, individually, the :

· Different stock solution preparation

· Change in instrument (UV-Vis Spectrophotometer model 1800 and 2450).

· Three replicates were made for the same concentration

· % RSD was calculated mentioned in Table No.7

Table 6 LOD and LOQ data of SAXA and GLB*(n=10)

Sr. no	Drugs	LOD (µg/ml)	LOQ (µg/ml)
1	Saxagliptin Hydrochloride	0.243	0.738
2	Glibendamide	0.317

Table 7 Robustness and Ruggedness data of SAXA and GLB *(n=3)

Saxagliptin Hydrochloride and Glibenclamide in Synthetic Mixture
Change in parameter		At Wavelength 229.40 (n=3)		At Wavelength 315 (n=3)
Robustness
		Abs.*	% RSD	Abs.*	% RSD
Change in Instrument	INS 1 : UV-2450	-0.317	0.315	-0.204	0.746
	INS 2 : UV-1800	-0.312	0.489	-0.202	0.754
Change in Analyst	Analyst-1	-0.310	0.491	-0.199	0.766
	Analyst-2	-0.319	0.313	-0.207	0.966
Ruggedness
Change in Solvent	2% Ethanol in Methanol	-0.287	0.531	-0.196	0.510
	5% Ethanol in Methanol	-0.304	0.683	-0.137	0.729
DRUGS		SAXA		GLB
Change in Ratio	1:2(SAXA-5,GLB-10)	-0.222	0.686	-0.214	0.214
	2:1(SAXA-10,GLB-5)	-0.322	0.473	-0.120	0.833

4. APPLICATION OF THE PROPOSED METHOD FOR ANALYSIS OF SAXA AND GLB SYNTHETIC MIXTURE:

A zero order spectrum of the sample solution containing 10µg/ml of SAXA and 10µg/ml of GLB was recorded and the absorbance at 229.40nm and 315.00nm were noted for estimation of SAXA and GLB, respectively. The concentration of SAXA and GLB in mixture was determined using the corresponding calibration graph. The results from the analysis of synthetic mixture containing Saxagliptin Hydrochloride(10mg)and Glibenclamide(10mg) in combination are presented in Table 8. The percent assay shows that there is no interference from excipients and the proposed method can successfully applied to analysis of commercial formulation containing SAXA and GLB.The %assay values are tabulated in Table 8.

Table 8 %Assay Recovered

Sr. No.	Drugs	% Assay
1	Saxagliptin Hydrochloride	100.356±0.350
2	Glibenclamide	99.933±0.430

Table. 9 Summary of Validation Parameters

Sr. No.	Parameter	Saxagliptin Hydrochloride	Glibenclamide
1	Wavelength Max.	229.40nm	315.00nm
2	Linearity (µg/ml) (n=6)	5-25 µg/ml	5-25 µg/ml
3	Regression equation	y = -0.028x - 0.0254	y = -0.019x - 0.008
4	Correlation coefficient (r^2)	0.9995	0.9994
5	Accuracy(%Recovery) (n=3)	99.818%	100.075%
6	Precision Intra-day (%RSD)(n=3) Inter-day (%RSD)(n=3) Repeatability(%RSD)(n=3)	0.253-0.554 0.221-0.634 0.248-0.864	0.388-0.728 0.389-0.707 0.344-0.751
7	LOD (µg/ml) (n=10)	0.243	0.317
8	LOQ (µg/ml) (n=10)	0.738	0.960
9	Robustness (%RSD)	0.313-0.686	0.467-0.966
10	Assay	100.356%	99.933%

5. CONCLUSION:

All the parameters are validated as per ICH guidelines for the method validation and found to be suitable for routine quantitative analysis in pharmaceutical dosage forms. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition. Assay results obtained by proposed method are in fair agreement.

6. CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

7. ACKNOWLEDGEMENT:

We are sincerely thankful to Shree Dhanvantary Pharmacy College, Kim, Surat, for providing us Infrastructure facilities and moral support to carry out this research work. We are also thankful to SDPARC for giving us their special time and guidance for this research work. We also thank our colleagues for their helping hand.

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Received on 04.03.2016 Accepted on 10.04.2016

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2016; 6(2): 77-82.

DOI: 10.5958/2231-5675.2016.00012.0