INTRODUCTION:

Saxagliptin , is chemically (1S,3S,5S)-2-[(2S)-2-Amino-2-(3hydroxytricyclo[3.3.1.13,7]dec-1-yl)acetyl]-2-azabicyclo[3.1.0]hexane 3carbonitrile previously identified as BMS-477118, is a new oral hypoglycaemic agent (antidiabetic drug) of the new peptidyl peptidase-4 (DPP-4) inhibitor class of drugs1-8(figure1). Saxagliptin recently approved for the treatment of type-2 diabetes mellitus(2). Literature survey reveals that the drug has been studied for pharmacokinetic parameters(3-5) and can be estimated only by LC-MS/MS(6), Spectrophotometric method(7) and one HPLC(8) method have been reported. The present study describes a simple, sensitive, accurate and precise HPLC method for the estimation of Saxagliptin in bulk and pharmaceutical dosage forms.

Figure1

EXPERIMENTAL:

1. Material:

The HPLC system consisted of following components: Shimadzu- Model LC20AT . Rheodyne 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 saxagliptin was procured as gift samples from Triveni Chemicals, Gujarat, 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 Saxagliptin were prepared by dissolving 20 mg of Saxagliptin in 100 ml of volumetric flask with diluent. Aliquot of 5.0ml of the standard stock solution of saxagliptin was transferred into 10 ml volumetric flask and from that appropriate aliquots were taken to give concentration range of 50-150µg/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 60:40v/v, buffer was 0.02M Potassium Di-hydrogen phosphate (pH 4.5 adjusted with Ortho Phosphoric Acid 5%). Detection was done at wavelength of 220nm. The sample was injected using a 20μl fixed loop, flow rate 1ml/min and the total run time was 06 minutes.

c. Validation:

The method was validated as per the ICH guideline (9).

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 Saxagliptin was done at the interval of 3 hrs. Interday precision- Interday variance for the Saxagliptin 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.

d. Determination of Saxagliptin in Tablet Dosage Form: Twenty tablets were weighed, finely powdered, and an accurately weighed sample of powdered tablets equivalent to 10mg of Saxagliptin 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.

RESULTS AND DISCUSSION:

Several mobile phase compositions were tried to resolve the peak of SAX. The mobile phase containing Acetonitrile: Buffer in proportion of 30:70v/v, buffer was 0.02M Potassium Di-hydrogen (pH 4.5 adjusted with Ortho Phosphoric Acid) was found ideal to resolve the peak of SAX. Retention time of SAX was 3.487 min [Figure 2]. Linear regression data showed a good liner relationship over a concentration range of 50-150μg/ml for SAX. The correlation coefficients (r2) was 0.9999 [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 1.79615µg/ml and 5.44288µg/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].Robustness was studied by changing different parameters [Table 4]. The assay result was repeated for three times which was found to be 99.8 % of labeled claim [Table 5].

CONCLUSIONS:

A method of quantitative determination of Saxagliptin 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 thankful to Triveni Chemicals, Gujarat (India) for providing gift sample of drugs. We 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.

Figure 1 : Chromatogram of SAX Standard

TABLE 1: System suitability Parameters

Retention times (RT)	3.487
Theoretical plates (N)	4671
Tailing factor (AS)	1.542
%RSD(n=5)	0.24

TABLE 2 : Recovery Study Data For SAX

Level	Area	Amount recovered(µg/ml)	% recovery	% RSD
80	3722.735	79.85	99.81	1.27
100	4689.767	100.71	100.71	1.22
120	5582.79	119.98	99.99	0.83

TABLE 3 : Precision Data

	Intraday			Interday
concentration	50(µg/ml)	100(µg/ml)	150(µg/ml)	50(µg/ml)	100(µg/ml)	150(µg/ml)
Mean	2324.822	4655.882	6965.095	2328.714	4663.616	6979.058
S.D	25.77	58.68	80.46	29.19	37.70	66.62
% RSD	1.10	1.26	1.15	1.25	0.80	0.95

TABLE 4 : Robustness Data

Robust condition	Area	%RSD
Mobile Phase (buffer/ACN)(72:28)	4662.065 ± 49.14119	1.054065
Mobile Phase (buffer/ACN)(68:32)	4657.443 ± 56.56846	1.214582
Flow rate +2ml/min	4427.708±33.4309	0.755038
Flow rate -2ml/min	4912.598±40.4521	0.82343
pH of buffer+2	4662.129±70.69115	1.51628
pH of buffer-2	4641.892±62.89708	1.35498

Table 5: Assay Result

Formulation	Mg/ tablet	% Assay
Tablet	5	99.8

Figure 2: Calibration Curve of SAX

REFERENCES:

1). http://www.drugbank.ca/drugs/DB06335.

2) Deanna S. Kania, Jasmine D. Gonzalvo, and Zachary A.Weber.., Saxagliptin: A Clinical Review in the Treatment of Type 2 Diabetes Mellitus.Clinical Therapeutics 2011,Vol.33, 1005-1022.

3) David W Boulton, Angela Tang, Chirag Patel, Lorna Castaneda, Uli Frevert, Li Li, David M Kornhauser. A Comparision of the single-dose pharmacokinetics and safety of Saxagliptin insubjects with hepatic impairment and in healthy subjects. European Journal of InternalMedicine 20S (2009), S1–S283.

4) Fura, Khanna, Vyas, Koplowitz, Shu-Ying Chang, Caporuscio, et al, Pharmacokinetics of the Dipeptidyl Peptidase 4 inhibitor Saxagliptin in Rats, Dogs, and Monkeys and Clinical Projections. Drug Metab Dispos. 2009; 37: 1164-1171.

5) Chirag G Patel, Li Li, Suzette Girgis, David M Kornhauser, Ernest U Frevert, David W Boulton.., Twoway pharmaco kinetic interaction studies between saxagliptin and cytochrome P450 substrates or inhibitors: simvastatin, diltiazem extended-release, and ketoconazole. Clinical Pharmacology: Advances and Applications 2011:2,13–25.

6) Cornelius Hess, Frank Musshoff and Burkhard Madea.., Simultaneous identification and validated quantification of 11 oral hypoglycaemic drugs in plasma by electrospray ionisation liquid chromatography–mass spectrometry, Analytical and Bioanalytical Chemistry 2011, 400: 33-41.

7) Kalaichelvi R, Jayachandran E, Validated Spectroscopic method for the estimation of Saxagliptin in pure and from tablet formulation. Int J Pharm Pharm Sci. 2011; 3: 179-180

8) Srikanth Inturi,Ravikanth Inturi, Israelkumar Tagaram Validated novel LC determination of Saxagliptin in pure and Pharmaceutical dosage forms. International Journal of Pharmaceutical Research aand Development. 2011; 3(8): 45-52.

[9] 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 30.04.2013 Modified on 20.05.2013

Asian J. Research Chem. 6(6): June 2013; Page 552-554