5. Results and discussion5.1. Optim

5. Results and discussion
5.1. Optimization of chromatographic techniques
Throughout the method development [9e14], numerous trails have been carried out to confirm optimized chromatographies condition. At first binary solvents as phosphate buffer pH 3.0: methanol (45:55%v/v), phosphate buffer pH 3.0: methanol (60:40%v/v), phosphate buffer pH 4.0: acetonitrile
(60:40%v/v), phosphate buffer pH 3.0: acetonitrile (40:60%v/v) at 208 nm were attempted however did not get clear conclusion due splitting of peak, asymmetric peak and lack of resolution among the three drugs. Another major drawback arises throughout methodology development to acquire resolution between saxagliptin and sitagliptin since each drug area unit eluted at the same time from the column. This challenge was met by using completely different mobile phase in three different ratios as phosphate buffer pH 4.0: methanol:acetonitrile (40:10:50%v/v), phosphate buffer pH 4.0: methanol:acetonitrile (60: 10: 30 %v/v). Correspondingly totally different make of C18 and C8 column as inspire, Xterra, Intersil ODS etc., were used throughout the method development. Yet fail to obtain clear results due lack of resolution among the drugs. The primary target in developing this stability-indicating RP-HPLC method is to achieve the resolution among the all the drugs and its degradation products. A chromatographic separation of the three drugs was achieved with a Inertsil C18 (4.6 250 mm, 5 mm) analytical column using buffer potassium dihydrogen phosphate adjusted pH 4 with orthophosphoric acid: methanol:acetonitrile (70:10:20%v/v) in isocratic mode at a flow rate of 1 mL/min, column at ambient temperature and detection of all the drugs were monitored at 215 nm using a PDA detector.
5.2. Analysis of formulations
Three totally different brands (Istamet, Janumet, Kombiglyze) were analyzed by this developed technique and percentage of the assay were calculated. Results were found within ICH limit and summarized, in (Table 2).
5.3. Validation of chromatographic systems
5.3.1. Linearity
Linearity was performed at five different concentrations for all titled drugs. The described method shows outstanding linearity over a range of 100, 150 , 200, 250, 300 mg/mL for Met, 1,1.5,2,2.5,3.0 for Saxa and 10, 15, 20, 25, 30 mg/mL for Sita with excellent correlation coefficient (0.999) [15]. The intercept and slope values were computed (Fig. 3).
5.3.2. Recovery study
Recovery study was conducted at three different levels and mean recovery values for the Met, Saxa and Sita were found to be 100.34, 99.64 and 99.52 % respectively. The result was shown (Table 3).
5.3.3. Precision
Precision and intermediate precision/ruggedness of theanalyticalmethodwasestablishedforbothsystemand method/procedure. The relative standard deviation was found less than 2. The result was displayed (Table 4).
5.3.4. Specificity
It had been found that chromatogram of the working placebo solution, not shown any interference at the retention time of the Met, Saxa and Sita. Consequently, it can be concluded that main excipients as crystalline cellulose, hypromellose microcrystalline cellulose, polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl fumarate that were present in the formulations as excipients does not interfere with the analytical method for the determinations of Met, Saxa and Sita. The specificity of the method was also confirmed by forcing degradation study. In peak purity study with a photo diode detector, purity angle was lower than the purity threshold for both the analytes (Figs. 1 and 2).
5.3.5. Limit of detection (LOD) and limit of quantification ( LOQ )
The limit of detection of the Met, Saxa, Sita 0.03 ,
0.0017, 0.025 mg/mL and limit of quantification 0.068 , 0.013, 0.084 mg/mL respectively indicating out method was extremely rapid and sensitive.
5.3.6. System suitability study
System suitability was achieved by checking various parameters and found within the ICH limit. Robustness study.

ผลลัพธ์ (อังกฤษ) 2:

5. Results and discussion
5.1. Optimization of chromatographic Techniques
Throughout the method Development [9e14], have been carried out numerous Trails to Confirm Optimized Chromatographies condition. At First binary solvents as phosphate buffer pH 3.0: methanol (45: 55% v / v), phosphate buffer pH 3.0: methanol (60: 40% v / v), phosphate buffer pH 4.0: acetonitrile
(60: 40% v /. v), phosphate buffer pH 3.0: acetonitrile (40: 60% v / v) at 208 nm were attempted however did not get clear conclusion due splitting of peak, asymmetric peak and lack of resolution among the three drugs. Another major drawback arises throughout methodology development to acquire resolution between saxagliptin and sitagliptin since each drug area unit eluted at the same time from the column. This challenge was met by using completely different mobile phase in three different ratios as phosphate buffer pH 4.0: methanol: acetonitrile (40: 10: 50% v / v), phosphate buffer pH 4.0: methanol: acetonitrile (60: 10: 30%. v / v). Correspondingly totally different make of C18 and C8 column as inspire, Xterra, Intersil ODS etc., were used throughout the method development. Yet fail to obtain clear results due lack of resolution among the drugs. The primary target in developing this stability-indicating RP-HPLC method is to achieve the resolution among the all the drugs and its degradation products. A chromatographic separation of the three drugs was achieved with a Inertsil C18 (4.6 250 mm, 5 mm) analytical column using buffer potassium dihydrogen phosphate adjusted pH 4 with orthophosphoric acid: methanol: acetonitrile (70: 10: 20% v / v) in. isocratic mode at a flow rate of 1 mL / min, at Ambient Temperature column and detection of all the Drugs were monitored at 215 NM using a PDA Detector.
5.2. Analysis of formulations
Three totally different brands (Istamet, Janumet, Kombiglyze) were analyzed by Technique and developed this percentage of the assay were calculated. Found Within ICH Limit and Summarized results were, in (Table 2).
5.3. Validation of chromatographic Systems
5.3.1. Linearity
Linearity was performed at Five different concentrations for all titled Drugs. The described method shows outstanding linearity over a range of 100, 150, 200, 250, 300 mg / mL for Met, 1,1.5,2,2.5,3.0 for Saxa and 10, 15, 20, 25, 30 mg / mL for. Sita with excellent correlation coefficient (0.999) [15]. The Slope and intercept values ​​were computed (Fig. 3).
5.3.2. Study Recovery
Recovery Study was conducted at Three different levels and Recovery Mean values ​​for the Met, Saxa and Sita were Found to be 100.34, 99.64 and 99.52% respectively. The Result was shown (Table 3).
5.3.3. Precision
Precision and Precision Intermediate / ruggedness of Theanalyticalmethodwasestablishedforbothsystemand method / procedure. Relative standard deviation was less than the Found 2. The Result was displayed (Table 4).
5.3.4. Specificity
It had been working placebo Found that chromatogram of the Solution, not shown any interference at the retention time of the Met, Saxa and Sita. Consequently, it can be concluded that main excipients as crystalline cellulose, hypromellose microcrystalline cellulose, polyvinylpyrrolidone, sodium lauryl sulfate, and sodium stearyl fumarate that were present in the formulations as excipients does not interfere with the analytical method for the determinations of Met, Saxa and. Sita. The specificity of the method was also confirmed by forcing degradation study. Study with a purity in Peak Detector Photo diode, was Angle Lower purity than the purity threshold for both the analytes (Figs. 1 and 2).
5.3.5. Limit of detection (LOD) and Limit of quantification (LOQ)
The Limit of detection of the Met, Saxa, Sita 12:03,
0.0017, 0.025 mg / mL and Limit of quantification 0.068, 0.013, 0.084 mg / mL respectively Indicating out method was extremely. Rapid and sensitive.
5.3.6. SUITABILITY System Study
System SUITABILITY was achieved by checking Various Parameters and Found Within the ICH Limit. Robustness study.