Saxagliptin was obtained as a gift sample from Mylan laboratories Ltd. (Hyderabad, India). Metformin was obtained as a gift sample from Aurobindo Pharmaceuticals Ltd. (Hyderabad, India). Kombiglyze XR (Saxagliptin- 5 mg and Metformin-1000 mg) tablets and Onglyza (saxagliptin-5 mg) tablets manufactured by Bristol-Mayerss- quibb, (USA) were purchased. Potassium dihydrogen orthophosphate (KH₂PO₄) of AR grade was purchased from Merck and HPLC grade solvents like Methanol and Acetonitrile were purchased from SD fine chemicals Ltd., India.

It consists of Ultra Fast Liquid Chromatograph of Shimadzu (Kyoto, Japan) equipped with binary pump (LC 20AD), UV/Visible detector (LC 20A) and Rheodyne injector port. The output signal was monitored and pro- cessed using Lab solutions software. The stationary phase employed is Enable C₁₈ G column (250 × 4.6 mm; 5 μm particle size).

Stock solution A was prepared by dissolving accurately weighed Saxagliptin (5 mg) and Metformin (1000 mg) in 100 mL Methanol, which results in concentration of Saxagliptin and Metformin as 50 µg/mL and 10,000 µg/mL, respectively. From this stock solution 0.4 ml was taken in 10 ml volumetric flask and volume was made up to the mark with Methanol, which results in concentration of Saxagliptin and Metformin as 2 µg/mL and 400 µg/mL, respectively (Stock solution B).

Ten tablets of KOMBIGLYZE were weighed and then finely powdered. An accurately weighed portion of the powder equivalent to 2000 mg of Metformin and 10 mg of Saxagliptin was transferred to 100 mL volumetric flask and 50 ml Methanol was added and sonicated for 25 min. Then the volume was made up to 100 ml with Methanol and was filtered. Serial dilutions were made from this solution to obtain required concentrations.

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc. It can be performed by various methods and among them one is analyzing the sample after subjecting to degradation studies, which results in analyte degradation up to 10% to 30%. The Forced Degradation studies were performed by exposing drug solution to stress conditions like acidic (0.1 N HCl, 1 N HCl), basic (0.1 N NaOH, 1 N NaOH), peroxide (H₂O₂ 3% v/v) and photo induced degradation (direct sunlight for 24 Hrs). The resulting solutions were analyzed for analyte peak and unknown degradants generated by stress induced degradation.

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantified as an exact value. LOD was calculated using the following formula:

where σ is the standard deviation of the response and S is the slope of the calibration curve.

The Limit of detection (LOD) for Saxagliptin and for Metformin are determined.

The quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. LOQ was calculated using the following formula;

where σ is the standard deviation of the response and S is the slope of the calibration curve.

The limit of quantification for Saxagliptin and for Meftormin are determined.

Accuracy is calculated as the percentage of recovery by the assay of known added amount of analyte in the sample, or as the difference between the mean and the accepted true value, together with the confidence intervals. ICH documents recommend that accuracy should be assessed using a minimum of nine determinations i.e., three replicates at three concentrations across the specified range of the procedure. Acceptance criteria is the mean % recovery at each spiked level should be not less than 98% and not more than 102%.

The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. It can be determined by measuring repeatability, intermediate precision and reproducibility. ICH guidelines recommend that repeatability should be assessed using a minimum of nine determinations covering the specified range of the procedure (i.e., three replicates of three concentrations) or using a minimum of six determinations at 100% of the test concentration. Acceptance criteria are % RSD of the peak areas and %assay of six injections should be not more than 2.0%.

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The variable method parameters may involve small variations such as temperature (±5˚C), KH₂PO₄ buffer pH (±0.5), ionic strength of buffers, level of additives to mobile phase, flow rate (±0.2 mL/min), wavelength (±2 nm). Acceptance criteria are %RSD of peak areas and %assay of main peaks should be not more than 2.0%.

During the trials with 0.02 M KH₂PO₄ buffer (pH 4.5):Acetonitrile (80:20 %v/v) mobile phase with a flow rate 1 mL/min, resulted in single Metformin peak and no Saxagliptin peak was observed. 0.05 M KH₂PO₄ buffer (pH: 4.5):Methanol (70:30 %v/v) with a flow rate of 1 mL/min, resulted in no resolution between Metformin and Saxagliptin peaks. 0.05 M KH₂PO₄ buffer (pH 4.5):Methanol (60:40 %v/v) with flow rate of 1 mL/min was resulted in fronting and broadening of Saxagliptin peak. Hence further trials were continued and at last an optimized method was developed, which employs 0.05 M KH₂PO₄ buffer (pH 4.5):Methanol:Acetonitrile (60: 20:20 %v/v) as mobile phase with a flow rate of 0.6 mL/min. The optimized chromatographic conditions were shown in Table 2. The standard solution (Metformin and Saxagliptin) was analyzed by the developed HPLC method and the chromatogram in both normalized and zoom conditions was shown in Figure 1(a) and Figure 1(b), respectively. The retention time of (Rt) Metformin and Saxagliptin were found as 4.3 min and 6.9 min, respectively.

The standard and test solutions of Metformin and Saxagliptin were analyzed by the HPLC method and the chromatograms were shown in Figure 2(a) and Figure 2(b), respectively. The % assay of Metformin and Saxagliptin were found to be 99.66% and 99.04%, respectively. As the % assay results were found to be within the limits i.e., 98% - 102% for both the drugs, the developed method can be routinely used for the simultaneous estimation of Metformin and Saxagliptin in the formulations.

As per ICH guidelines the method was validated for various parameters which includes Specificity, Linearity, LOD, LOQ, Accuracy, Precision and Robustness.