Analytical Method Development & Validation for Simultaneous Estimation of Saxagliptin & Metformin in Combined Pharmaceutical Dosage Form By RP-HPLC

Saxagliptin  HCl         (SAG), (Fig. 1a)          is chemically named as (1S,3S,5S)-2-[(2S)-2- amino-2-(3-hydroxy-1-adamantyl)acetyl]- 2-azabicyclo[3.1.0]hexane-3-carbonitrile hydrochloride [1]. Saxagliptin  HCl  (SAG)  is  an oral hypoglycemic (anti-diabetic drug) of the dipeptidyl peptidase-4 (DPP-4) inhibitor class of drugs. SAG is used as monotherapy or in combination with other medicines for the treatment of type II diabetes. The drug works to competitively inhibit a protein/enzyme, dipeptidyl peptidase 4 (DPP-4), that results in an increased amount                  of active incretins; Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1  (GLP-1),  reduced  amount  of

release of glucagon and increased release of insulin [2], [3].

       
           
       
Fig. No: 1 Structure of Saxagliptin

       
           
       
Fig. No: 2 Structure of Metformin

Metformin hydrochloride (MET), (Fig. 2b) is chemically designated as 1,1- Dimethyl biguanide hydrochloride [1]. Metformin  hydrochloride  (MET)  is a biguanide antidiabetic. It is given orally

MATERIALS AND METHODS

in the treatment of type II diabetes mellitus; a disease characterized by defects in both insulin secretion and insulin sensitivity, and is the drug of first choice in overweight patients [2]. SAG is not an official drug in any pharmacopeia. While MET was determined by British Pharmacopoeia (BP) [4] and United  States Pharmacopeia (USP) [5], both suggest a nonaqueous titration method for the assay of MET using anhydrous formic acid as a solvent and 0.1 M perchloric acid as a titrant. The end point is determined potentiometrically.

Table 1: Drug details

Table 2: Instruments and Equipment

Preparation of sample stock solution: Weighed 50mg of Emtricitabine, 6.25mg of tenofovir, and 12.5mg of Bictegravir working std 50ml volumetric flask, add 1oml of diluent, sonicate for 10min and make up to the final volume with diluents.(1000?g/Emtricitabine and 125 ?g/ml and Tenofovir and 250?g/ml of Bictegravir) Preparation of sample stock solution: 5 tablets of average equivalent average taken in 100ml of volumetric flask add diluents sonicate filter it (1000?g/mlEmtricitabine and 125 ?g/ml Tenofovir and 250?g/ml of Bictegravir.

Table 3: Chemicals and Reagents

RESULTS AND DISCUSSION:

Method Development

Table 5: Observation of Optimized Chromatogram

Table 8: Linearity Observation of Saxagliptin

       
           
       
Fig No: 6 Calibration curve for Metformin

Optimized chromatographic conditions

Table 6: Shows Optimized chromatographic conditions

• From the above experiment it was found that Saxagliptin and Metformin can effectively be analyzed by using the RP- HPLC method with Mobile phase at a flow rate of 1 ml/min and detection wavelength of 215nm.

CONCLUSION:

The analytical method was developed by studying different parameters. First of all, the maximum absorbance was found to be at 250nm and the peak purity was excellent. Injection volume was selected to be 10µl which gave a good peak area. The column used for the study was X terra C18 because it gave a good peak. 35 º C temperatures were found to be suitable for the nature of the drug solution. The flow rate was fixed at 1.0ml/min because of the good peak area and satisfactory retention time. The mobile phase is ACN, Methanol, and Water (35:50:15 v/v) was fixed due to a good symmetrical peak. So this mobile phase was used for the proposed study. The run time was selected to be 8 min because the analysis gave a peak. In the present investigation, a simple, sensitive, precise, and accurate RP-HPLC method was developed for the quantitative estimation of Metformin and Saxagliptin in bulk drug and pharmaceutical dosage forms. This method was simple since diluted samples are directly used without any preliminary chemical derivatization or purification steps. Metformin and Saxagliptin were freely soluble in ACN and methanol. ACN, Methanol, and Water (35:50:15 v/v) were chosen as the mobile phase. The solvent system used in this method was economical. The % RSD values were within 2 and the method was found to be precise. The results expressed in the Tables for the RP-HPLC method were promising. The RP-HPLC method is more sensitive, accurate, and precise compared to the Spectrophotometric methods. This method can be used for the routine determination of Metformin and Saxagliptin in bulk drugs and Pharmaceutical dosage forms.