RP-HPLC Method for estimation of Alogliptin and Glibenclamide in Synthetic mixture

 

Godge R. K.*, Shinde G. S, Bhosale M. S

 

ABSTRACT:

The objective of the current study was to develop simple, accurate, precise and rapid RP-HPLC method with subsequently validate as per ICH guidelines for the determination of Alogliptin benzoate and Glibenclamide hydrochloride using mobile phase [mixture of Acetonitrile: Phosphate buffer pH-3.6 and in the ratio of 65:35.The proposed method involves the measurement of Retention time at selected analytical wavelength. 240.0 nm. The retention time of Alogliptin benzoate (ALO) and Glibenclamide was found to be 5.055 and 2.838 respectively. The linearity of the proposed method was investigated in the range of 1-5 μg/ml (r = 0.9998) for ALO and 10-50μg/ml (r = 0.9999) for MET respectively. The method was statistically validated for linearity, accuracy and precision. Both inter-day and intra-day variation was found to be showing less % RSD (Relative Standard Deviation) value indicating high grade of precision of the method.

 

 

INTRODUCTION:

GLP-1 and glucose-dependent insulinotropic polypeptide (also known as gastric inhibitory peptide) belong to the incretin class of gastrointestinal hormones. Incretins stimulate a decrease in blood glucose levels by causing increased postprandial insulin release from the beta cells of the pancreas¹. GLP-1 also suppresses glucagon secretion and exhibits other glucoregulatory actions after secretion in the gut.¹¹ DPP-4 is an enzyme that rapidly degrades, and thereby inactivates, both GLP-1 and gastric inhibitory peptide. DPP-4 inhibitors prolong the endogenous plasma levels and hence the activity of both of these key hormones.

 

[^2] Alogliptin, a potent and highly selective DPP-4 inhibitor, is the fourth DPP-4 inhibitor to be introduced in Canada, following the approval of sitagliptin, saxagliptin, and ALOgliptin^3,4.

 

Fig 1: Structure of Alogliptin

 

Glibenclamide (GBC) is an oral hypoglycemic drug that stimulates the pancreatic beta cells to secrete insulin and is often used to treat diabetes, including diabetes during pregnancy. Mechanism (s) of action and therapeutical indications of the sulfonylurea compound glibenclamide, which is a cardinal drug in the treatment of type 2 diabetes mellitus. Data produced in our own laboratory over the past 15 years will be presented, along with reference to the main literature in the field. As pharmacokinetics is concerned, special emphasis will be placed on the detrimental effect of hyperglycemia in the intestinal absorption of this class of drugs. Both beta-cell and extrapancreatic effects of glibenclamide will be highlighted. The mechanism of action of the drug consists in the inhibition of the ATP-sensitive K+ channels, which leads to depolarization of the cells and insulin secretion. Based on the same mechanism are also the extrapancreatic action of the drug at the liver, skeletal muscle, heart muscle and smooth muscle sites. The newly discovered possible physiological actions of the C-peptide molecule [suggesting a stimulatory effect of C-peptide on the Na+, K+ (ATPase) pump and on diabetic complications], cast a new light on all therapeutic approaches (like sulfonylurea class of compounds and whole pancreas or islet of Langerhans transplantation), which induce/replace both insulin and C-peptide secretion.

 

Fig 2: Structure of Glibenclamide

 

 

MATERIALS AND METHODS:

Analytically pure sample of Alogliptin and Glibenclamide with purities greater than 99% were kindly gifted by Cadila Zydus. Ahmadabad, India .

 

Table.No.1. List of instruments used.

Sr. No	Instrument no	Model number
1	HPLC	HPLC 3000 series P-3000-M reciprocating (binary pump) UV-3000-M (UV-Visible Detecter.)
2	Weighing balance	PGB 100
3	Uv-spectrophotometer and software	UV2450 UV Probe v 2.3.3.
4	Ultra sonicator	WUC-4L

 

Selection of wavelength:

10 mg of Alogliptin and Glibenclamide was dissolved in mobile phase. The solution was scanned from 200-400 nm the spectrum was obtained. The overlay spectrum of alogliptin and Glibenclamide was obtained and the isobestic point of Alogliptin And Glibenclamide showed absorbance’s maxima at 240 nm. The spectrums are shown in Fig. 1

 

Fig. 1. Overlay spectra of GLI (229.7 nm) and ALO (275.7 nm)

 

The chromatographic method development for the simultaneous estimation of Alogliptin and Glibenclamide were optimized by several trials for various parameters as different column, flow rate and mobile phase, finally the following chromatographic method was selected for the separation and quantification of Alogliptin and Glibenclamide in API and pharmaceutical dosage form by RP-HPLC method. The separation of the drugs was achieved on Water X bridge C18 column (4.6×150mm) 5μ particle size). The mobile phase consists of a mixture of Methanol: Acetonitrile pH 3.0 (130: 870 % v/v) at a flow rate of 1.0 ml/minute and the volume injected was 10 μl for every injection .the detection wavelength was set at 240 nm.

Preparation of Buffer solution: (Mobile phase A):

Add 0.25 g of Sodium 1-octane Sulfonate monohydrate in 1000 mL of water, mix and filterthe solution through 0.45µ Nylon membrane disc filter, to this add 2.0 mL of Triethylaminemix and adjust the pH 3.0 ± 0.1 with Ortho-phosphoric acid Mix and degas

 

1.    Preparation of Mobile Phase B:

Mix Acetonitrile and Methanol in the proportion of 870:130 v/v respectively and use as mobile phase B.

 

2.    Preparation of Mobile Phase:

Prepare the mixture of Mobile phase A: Mobile Phase B in the ratio of 85:15 v/v respectively.

 

3.    Preparation of Standard stock solution:

Weigh accurately about 25 mg of Alogliptin and Glibenclamide and transfer into  50 mL volumetric flask. Add about 30 mL of diluent, sonicate to dissolve and make up to volume with diluent and mix.

 

4.    Preparation of Sample Stock solution:

Weigh and transfer 10 tablets into 1000 mL volumetric flask. Add about 700 mL of diluent, sonicate for 30 minutes with intermittent shaking. Allow it to cool to room temperature and make up to volume with diluent and mix. Let the solution stand for 5 minutes.  Filter the sample solution through 0.45µ Nylon membrane syringe filter. Discard first 3 mL of filtrate.

 

RESULTS AND DISCUSSION:

Method Development:

A Reverse phase HPLC way was developed keeping in mind the system suitability parameters i.e. resolution factor (Rf) between peaks, tailing factor (T), number of theoretical plates (N), runtime and the cost effectiveness. The optimized method developed resulted in the elution of Alogliptin at 6.2 min and Glibenclamide at 2.6 min.

 

Fig 2: Typical chromatogram of the mixture of the standard  sample.

 

System‐suitability tests are an integral part of method development and are used to ensure adequate performance of the chromatographic system. Retention time (Rt), number of theoretical plates (N), peak resolution (Rs) and peak Tailing factor (T) were evaluated for six replicate injections of the standards at working concentration. The results given in Table 2were within acceptable limits.

 

Table 2: System suitability studies:

Parameters*	Alogliptin	Glibenclamide
Retention time (min)	6.2	2.6
Number Of Theoretical plates (N)	4423	6049
Tailing factor (T)	1.3	1.2

 

Method validation:

Validation of the analytical method is the process that establishes by laboratory studies in which the performance characteristics of the method meet the requirements for the intended analytical application. The RP-HPLC method developed was validated according to International Conference on Harmonization10 guidelines for validation of analytical procedures. The method was validate for the parameters in terms of system suitability, selectivity, linearity, accuracy, precision, ruggedness, robustness, limit of detection(LOD) and limit of quantitation(LOQ).

 

Specificity:

The system suitability for specificity was carried out to determine whether there is any interference of any impurities in retention time of analytical peak. The study was performed by injecting blank. The chromatograms are shown in Fig.No.5

 

Linearity:

The linearity study was performed for the concentration of 10 ppm to 50 ppm Alogliptin and 20 ppm to 100 ppm Glibenclamide level. Each level was injected into chromatographic system. The area of each level was used for calculation of correlation coefficient. The results aretabulated in Table.4-5. Calibration graph for Alogliptin and Glibenclamide are shown in Fig.No.6 and Fig 7.

 

Figure-3: Calibration graph of Alogliptin

 

Figure-4: Calibration graph of Glibenclamide

 

The linearity study was performed for concentration range of 50μg -150μg Alogliptin and 50μg - 150 μg Glibenclamide and the correlation coefficient was found to be 1.000 and 1.000.(NLT 0.999)respectively.

 

Accuracy:

The accuracy study was performed for 50%, 100% and 150 % for Alogliptin and Glibenclamide. Each levelwas injected in triplicate into chromatographic system. T he area of each level was used for calculation of % recovery. The results are tabulated in Table.No.3

 

Table-3: Accuracy results for Alogliptin and Glibenclamide

Sr. No.	Drug	%Concentration (at specification level)	Amount Taken (mg)	Amount added (mg)	Average area	Recovery	Mean recovery
1	Alogliptin	50%	20	10	1324983	99.63%	99.48%
2		100%	20	20	1456275	99.7%
3		150%	20	30	1702141	99.13
1	Glibenclamide	50%	10	5	824323	100.6%	101.00%
2		100%	10	10	971433	101.4%
3		150%	10	15	1203301	101.0%

The accuracy study was performed for % recovery of Alogliptin and Glibenclamide The % recovery was found to be 98.48% and 101.00% respectively.

 

Robustness:

Table 4: Robustness for Allogliptin and  Glibenclamide

Drug	Changes in parameters	Values	Retention time	Theoretical Plates	% RSD of standard area	% Assay
Alogliptin	*Control	As per method	6.2	11090	0.23	99.9
	Flow rate (1 ml/min)	0.8	6.32	11280	0.21	99.3
		1.2	6.12	10104	0.24	98.3
	Wavelength (240nm)	241	6.21	11228	0.27	100.6
		242	6.21	11505	0.32	100.6
	Column temperature (25°C)	20°C	6.2	9470	0.19	98.2
	**Control	As per method	6.2	9014	0.33	102.4
	Column temperature	30°C	6.24	12433	0.14	101.3
Glibenclamide	*Control	As per method	2.6	12607	0.35	101.7
	Flow rate (1 ml/min)	0.8	2.81	33843	0.16	101.2
		1.2	2.57	31641	0.21	100.1
	Wavelength (240nm)	241	2.62	26141	0.24	102.4
		242	2.62	51821	0.29	102.4
	Column temperature ( 25°C)	20°C	2.62	268604	0.15	99.9
	**Control	As per method	2.62	218640	0.2	100.6
	Column temperature	30°C	2.57	331128	0.2	99.4

 

CONCLUSION:

HPLC has gained the valuable position in the field of analysis due to ease of performance, specificity, sensitivity and the analysis of sample of complex nature. This technique was employed in the present investigation for estimation of Alogliptin and Glibenclamide tablet formulation. Waters  Xbridge C18 (150 x 4.6 mm), 3µ was used for the study. The standard and sample solution of Alogliptin and Glibenclamide prepared in diluent.Different pure solvents of varying polarity in different proportions were tried as mobile phase for development of the chromatogram.

 

The mobile phase that was found to be most suitable was Alogliptin and Glibenclamide. The wavelength 235nm were selected for the evaluation of the chromatogram of Alogliptin and Glibenclamide respectively. The selection of the wavelength was based on the λmax obtained by scanning of standard laboratory mixture in water: methanol. This system gave good resolution and optimum retention time with appropriate tailing factor (<2). After establishing the chromatographic conditions, standard laboratory mixture was prepared and analysed by procedure described under Materials and methods. It gave accurate, reliable results and was extended for estimation of drugs in tablet formulation. The results from table clearly indicate that the RP-HPLC technique can be successfully applied for the estimation of above-mentioned drugs in their formulation.

 

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Accepted on 30.09.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(2):555-559.