Development and Validation of a Liquid Chromatographic Method for Estimation of Vildagliptin in Tablet Dosage Form.

 

Vaishali V. Karkhanis*, Dr. (Mrs). Anandkumari D. Captain

A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy V.V. Nagar- 388120 Gujarat

*Corresponding Author E-mail: vesli@rediffmail.com

 

An accurate, sensitive and precise RP-HPLC method has been developed and validated for the estimation of Vildagliptin (VIL) from bulk drug and Pharmaceutical Dosage form. The separation was achieved by Hypersil C18 column (250mm X 4.6mm, 5μm) in isocratic mode, with mobile phase comprises of Acetonitrile : Buffer in proportion of 25:75v/v, buffer was 0.02M Potassium Di-hydrogen Phosphate (pH 4.5 adjusted with Ortho Phosphoric Acid). The flow rate of mobile phase was 1.0ml/min and employing UV detection with 215nm wavelengths. The retention time of VIL was 4.353 min.The calibration curve was found to be linear within the concentration range of 10µg/ml to 30µg/ml. The regression data for calibration curve shows good linear relationship with r² = 0.9997. The method was validated in accordance with the requirements of ICH guidelines. Moreover, the proposed analytical method was applied to monitor the formulation commercially available.

 

INTRODUCTION:

Vildagliptin (VIL), S-1-[N-(3-hydroxy-1-adamantyl) glycyl] pyrrolidine-2carbonitrile is an oral antihyperglycemic of the new dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. Vildagliptin inhibits the inactivation of GLP-1 and GIP by DPP-4, allowing GLP-1and GIP to potentiate the secretion of insulin in the beta cells and suppress glucagon release by the alpha cells of the islets of Langerhans in the pancreas. Vildagliptin has been shown to reduce hyperglycemia in type 2diabetes mellitus¹. Literature survey revealed that few analytical methods are used for estimation of Vildagliptin^2-4. So the aim of the present work was to develop a reversed-phase liquid chromatographic(RP-LC) method that would be suitable for the determination of VIL from its pharmaceutical dosage form. The proposed method is simple, accurate, reproducible and suitable for routine determination of Vildagliptin from its pharmaceutical dosage form.

 

Figure 1

 

MATERIALS AND METHODS:

1. Material: The HPLC system consisted of following components: Shimadzu- Model LC20AT. Rhenodyne valve with 20μl fixed loop, isocratic system pump, Chromatographic analysis was performed on Hypersil BDS C18 column 250×4.6 mm, 5μm particle size. Analytically pure vildagliptin was procured as gift samples from Apollo Life Sciences Ltd, India. All other chemicals and reagents used were analytical grade and purchased from Merck Chemicals, India. Tablets were procured from the local market.

 

2. Methods:

a. Preparation of standard stock solution and solutions for calibration curve: Stock solutions of Vildagliptin were prepared by dissolving 20 mg of Vildagliptin in 100 ml of volumetric flask with diluent. Aliquot of 1.0ml of the standard stock solution of vildagliptin was transferred into 10 ml volumetric flask and from that appropriate aliquots were taken to give concentration range of 10-30µg/ml for calibration curve.

 

b. Chromatographic conditions: Chromatographic estimation was performed using an Hypersil BDS C18 column (250mm×4.6mm i.d.), mobile phase consisting of Acetonitrile : Buffer in proportion of 25:75v/v, buffer was 0.02M Potassium Di-hydrogen phosphate (pH 4.5 adjusted with Ortho Phosphoric Acid 5%). Detection was done at wavelength of 215nm. The sample was injected using a 20μl fixed loop, flow rate 1ml/min and the total run time was 7minutes.

 

c. Validation: The method was validated as per the ICH guideline⁵.

 

i. Regression analysis- Regression of analytical method is expressed in terms of correlation co-efficient of the regression analysis. Accuracy- For determination of Accuracy, recovery study was carried out. That was performed by standard addition method at three different levels (80%, 100%, and 120%), to the pre-analyzed samples and the subsequent solutions were re-analyzed. At each level, three determinations were performed.

 

ii. Precision- The precision of analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple samplings of homogeneous samples. Intraday precision- Intraday variance for the Vildagliptin was done at the interval of 3 hrs. Interday precision- Interday variance for the Vildagliptin was done at the interval of one day.

 

iii. Limit Of Detection (LOD)- LOD was found out based on the standard deviation of the response and the slope method. Limit Of Quantification (LOQ) - LOQ was foundout based on the standard deviation of the response and the slope method. Specificity- Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.

 

iv. The robustness of the method was established by making deliberate minor variations in mobile phase composition,pH of buffer and flow rate.

 

d. Determination of Vildagliptin in Tablet Dosage Form: Twenty tablets were weighed, finely powdered, and an accurately weighed sample of powdered tablets equivalent to 20mg of Vildagliptin was treated with mobile phase in a 100mL volumetric flask using ultra sonicator. This solution was filtered through 0.45 μm filter paper. Suitable aliquot of the filtered solution was added to a volumetric flask and make up to volume with mobile phase to get appropriate concentration in range.

 

TABLE 1: SYSTEM SUITABILITY PARAMETERS

RESULTS AND DISCUSSION:

Several mobile phase compositions were tried to resolve the peak of VIL. The mobile phase containing Acetonitrile : Buffer in proportion of 25:75v/v, buffer was 0.02M Potassium Di-hydrogen (pH 4.5 adjusted with Ortho Phosphoric Acid) was found ideal to resolve the peak of VIL. Retention time of VIL was 4.353 min [Figure 2]. Linear regression data showed a good liner relationship over a concentration range of 10-30μg/ml for VIL. The correlation coefficients (r2) was 0.9997 [Figure 3]. The system suitability parameters are shown in [Table 1]The accuracy of the method was evaluated by carrying out recovery studies, were performed by standard addition method at three different levels I, II and III (80%, 100%, and 120%), to the pre-analyzed samples and the subsequent solutions were re-analyzed. At each level, three determinations were performed [Table 2].The limit of detection and limit of quantification were found to be 0.673µg/ml and 2.04µg/ml respectively. The intra-day and inter day precision was determined by analyzing standard solution of 50, 100 and 150 µg/mL and the results are reported in terms of relative standard deviation. [Table 3].Data derived from robustness study is depicted . [Table 4]. The assay result was repeated for three times which was found to be 99.76 % of labeled claim [Table 5].

 

FIGURE 2 : CHROMATOGRAM OF VIL STANDARD

 

FIGURE 3 : CALIBRATION CURVE OF VIL

 

TABLE 2 : RECOVERY STUDY DATA FOR VIL

Level	Sets	Area	Amount added (µg/ml)	Amount Recovered (µg/ml)	% Recovery	Mean %  Recovery	% RSD
80	1	4941.642	16	15.886	100.425	100.492	0.702
80	2	4981.198	16	16.014	101.225
80	3	4911.978	16	15.791	99.826
100	1	6251.683	20	20.122	101.519	100.721	1.211
100	2	6239.457	20	20.083	101.321
100	3	6115.864	20	19.683	99.323
120	1	7413.461	24	23.879	100.249	100.432	0.379
120	2	7467.639	24	24.054	100.979
120	3	7426.989	24	23.922	100.432

 

TABLE 3 : PRECISION DATA

Concentration	Intraday			Interday
	10(µg/ml)	20(µg/ml)	30(µg/ml)	10(µg/ml)	20(µg/ml)	30(µg/ml)
Area	3076.098	6223.045	9304.347	3096.208	6179.804	9248.771
	3114.815	6087.601	9332.153	3086.908	6167.497	9230.228
	3056.031	6260.264	9137.978	3105.483	6145.78	9082.71
Mean	3082.314	6190.303	9258.159	3096.199	6164.360	9187.236
S.D	29.881	90.868	105.004	9.2875	17.2275	90.996
% RSD	0.96	1.46	1.13	0.29	0.27	0.99

 

TABLE4: ROBUSTNESS DATA

Robust condition	Area	%RSD
Mobile Phase (buffer: ACN)77:23	6179.862 ± 77.42679	1.253
Mobile Phase (buffer: ACN)73:27	6173.681 ± 81.04008	1.313
Flow rate +0.2ml/min	5879.385±55.71154	0.947
Flow rate -0.2ml/min	6515.741±65.97053	1.012
pH of buffer+0.2	6188.081±65.45686	1.058
pH of buffer-0.2	6203.504±85.36253	1.376

 

Table 5 : ASSAY RESULT

Formulation	Amount(ug/ml)	%Assay
Tablet	20	99.76

 

CONCLUSIONS:

A method of quantitative determination of Vildagliptin using HPLC has been developed. The validation results have demonstrated that this method is accurate, precise and linear. The method can also be applied for drug content in pharmaceutical preparations.

 

ACKNOWLEDGEMENTS:

The authors are also thankful to the Principal, A.R. College of Pharmacy and G.H. Patel Institute of Pharmacy, who had helped us throughout the entire work.

 

REFERENCES:

1.       http://www.drugbank.ca/drugs/DB04876

2.       Ramzia I. El-Bagary, Ehab F. Elkady, Bassam M. Ayoub. LiquidChromatographic Methods for the Determination of Vildagliptin in the Presence of its Synthetic Intermediate and the Simultaneous Determination of Pioglitazone Hydrochloride and Metformin Hydrochloride, International Journal of BiomedicalScience 7(3), 201-208, (2011)

3.       Amanda Thomas Barden, BárbaraSalamon, Elfrides Eva Sherman Schapoval, and Martin Steppe;Stability-Indicating RP-LC Method for the Determination of VildagliptinandMass Spectrometry Detection for Main Degradation Product, J Chromatogr Sci 50(5): 426-432,(2012).

4.       Aparajita Malakar, Bishwajit Bokshi, Dilruba Nasrin, Development and Validation of RP-HPLC method for estimation of Vildagliptin from tablet dosage form. International journal of Pharmaceutical and Life Sciences Vol, 1, Issue 1, Serial2, August 2012

5.       International Conference of Harmonisation (ICH) of Technical Requirement for the registration of Pharmaceuticals for human use. Validation of Analytical Procedures Methodology, ICH-Q 213, Geneva(1996)

 

 

 

Received on 31.07.2013       Modified on 02.10.2013

Accepted on 11.10.2013      © AJRC All right reserved