You are hereDEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR THE SIMULTANEOUS ESTIMATION OF SITAGLIPTIN PHOSPHATE AND METFORMIN HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM

DEVELOPMENT AND VALIDATION OF ANALYTICAL METHODS FOR THE SIMULTANEOUS ESTIMATION OF SITAGLIPTIN PHOSPHATE AND METFORMIN HYDROCHLORIDE IN PHARMACEUTICAL DOSAGE FORM


Table: 11. Recovery Studies

Drug

% Recovery

% RSD*

50% level

100% level

50% level

100% level

Sitagliptin Phosphate

98.7

98.6

0.23

0.31

Metformin  Hydrochloride

101.73

101.4

0.41

0.29

* RSD of three Observations

4. Robustness:  the optimized conditions such as change in mobile phase & slight variation in pH  i.e.,±2% in ratio of acetonitrile in mobile phase, ±0.2 units in pH of buffer.The response factors for these changed chromatographic parameters were almost same as that of the fixed chromatographic parameters and hence developed method is said to be robust as shown in Fig.6&7, Fig.8&9 .

System Suitability Studies
The system suitability studies were carried out as specified in USP. These parameters include column efficiency, resolution, peak asymmetry factor, capacity factors, peak tailing factor and percentage co-efficient of variation for peak area on height of repeatable injection.Although USP requires only two of these criteria for method validation, parameters like column efficiency (N), capacity factor (k) and peak asymmetry factor (As).

Table: 12. System Suitability Studies

Drug

Rs

N

k

a

As

Tailing

Sitagliptin Phosphate

1.9

5951.54

0.6


4.16

1.21

1.3

Metformin Hydrochloride

3568.372

2.5

1.30

1.02

STABILITY STUDIES
Stability studies were performed on Sitagliptin Phosphate and Metformin Hydrochloride to prove the stability indicating property of the method. The stress conditions employed for degradation study includes light exposure, acid hydrolysis (0.1N Hcl), base hydrolysis (0.1N NaoH), water hydrolysis, oxidation (1% hydrogen peroxide), and UV light exposure.The duration of time selected for degradation studies [17] was 6 hours. The photolytic degradation was performed by exposing the solid drugs to sunlight for 6 hours. The solution containing the concentration of 100 µg/ml of each of Sitagliptin Phosphate and Metformin Hydrochloride were prepared using respective solvents (NaoH, Hcl, water and Hydrogen peroxide) separately. For each hour solution containing final concentration of 40µg/ml of Sitagliptin and 5µg/ml Metformin were prepared from the above mentioned stock solutions using the mobile phase and analyzed in the HPLC. The %degradation of both the drugs was calculated and the degradation patterns were much more prominent in alkali hydrolysis & the results shown in the Fig :13 & table : 13.

Table: 13. RESULTS OF STABILITY INDICATING STUDIES FOR SITAGLIPTIN & METFORMIN RESPECTIVELY

Conditions

Amount found (µg/ml)

% Degraded

Duration of time in hours

0 hr

1 hr

2hr

3 hr

4hr

5hr

6 hr

Water (60?c,6hrs)

99.89

99.89

98.91

99.10

98.77

98.16

98.62

98.14

97.67

97.76

97.40

97.74

97.01

96.97

2.99

3.01

0.1% Hcl (60?c,6hrs)

99.81

99.80

99.29

98.64

98.71

96.13

98.64

95.96

98.41

95.91

97.81

94.94

96.94

94.93

3.06

5.07

0.1%NaoH

(60?c,6hrs)

98.97

98.91

95.53

98.91

92.67

97.19

91.10

91.94

87.06

87.68

84.44

83.27

79.14

76.33

20.86

23.67

1%  H2O2

(6 hrs)

99.91

99.91

99.74

99.88

99.72

99.87

99.61

99.19

98.99

98.76

98.73

98.39

98.01

97.91

1.99

2.09

UV light 

(6 hrs)

99.91

99.82

99.86

99.73

99.84

99.42

99.67

99.126

99.21

98.96

99.09

98.94

98.94

98.94

1.06

1.06

Sunlight 

 (6 hrs)

99.95

99.89

-

-

-

-

-

97.99

98.24

2.01

1.76


Fig.13: Chromatogram of mixture of Metformin and Sitagliptin degraded with 0.1N NaoH at 6th hour.

RESULTS AND DISCUSSION

UV SPECTOSCOPIC METHOD: A second order UV derivative spectroscopic method was developed for the determination of Sitagliptin Phosphate and Metformin Hydrochloride in tablet dosage form. In this method, second derivative spectra of Sitagliptin and Metformin were recorded in water. From the spectra, 249nm and 278nm were selected as the zero crossing points. At 249nm Sitagliptin showed zero absorbance but Metformin had the considerable absorbance. Similarly Metformin had no absorbance but Sitagliptin had a considerable absorbance. A calibration curve was plotted for both Sitagliptin and metformin in the concentration range of 5 to 50µg/ml. The slope, intercept and correlation coefficient values were found to be 0.000082, 0.000018 and 0.998 for Sitagliptin and 0.00031, 0.00016 and 0.998 for Metformin respectively. The formulation was extracted, diluted and spectra was recorded and processed to get derivative spectrum. The 2nd derivative UV absorbances were noted and the amounts were calculated.

HPLC METHOD: The retention times of Sitagliptin and Metformin were found to be 4.08 and 2.13 minutes respectively.The developed method was validated as per ICH guidelines.  Calibration graphs were potted using standard peak areas vs. concentration of standard solutions.  The slope, intercept and correlation coefficient values were found to be 1449, 22710 and 0.998 for Metformin respectively.  Sitagliptin was found to be linear in the range of 10 to 100µg/ml and Metformin was found to be linear in the range of 5 to 50µg/ml.  The LOD of Sitagliptin and Metformin were found to be 0.4µg/ml and 0.251µg/ml respectively.  The LOQ of Sitagliptin and Metformin were found to be 0.90µg/ml and 0.6µg/ml respectively.  Precision of the developed method was studied under intraday precision, interday precision and repeatability of the injection.  Low % RSD values indicate that the method is precise.  The developed method was found to be robust.  System suitability parameters like number of theoretical plates (N), Peak asymmetry factor (As), Capacity factor (K), selectivity factor (α) and Resolution (Rs) were studied. Stability studies were carried out under various stress conditions like acid hydrolysis, base hydrolysis, water hydrolysis, oxidation, photolytic degradation and under UV-light. Both the drugs Sitagliptin and metformin were degraded more in the alkaline condition.

SUMMARY AND CONCLUSION
The direct UV method development for the simultaneous analysis of Sitagliptin and metformin can be applied for the routine analysis of formulation.Incomparision with direct UV method, the second order derivative UV spectroscopic method eliminates the interference from UV-absorbing excipients. This method was found to be fast and can be used for formulation screening.

The developed RP-HPLC method offers simplicity, selectivity, precision and accuracy. In this proposed method symmetrical peaks with good resolution were obtained. Out of all the methods developed, the RP-HPLC method was more sensitive and precise. However, both of these methods can be used for the simultaneous analysis of Sitagliptin and Metformin in formulation.

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