AN ISOCRATIC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF RABEPRAZOLE AND MOSAPRIDE IN TABLET DOSAGE FORMS BY USING RP-HPLC

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3. Precision: It is a measure of degree of repeatability of an analytical method under normal operation and it is normally expressed as % of relative standard deviation (% RSD). The standard solution was injected for five times and measured the area for all five injections in HPLC. The % RSD for the area of five replicate injections was found to be within the specified limits. The data are represented in table no. 3 and 4.

Table no. 3: It shows precision results for Rabeprazole

Injection

Peak Areas of

Rabeprazole sodium

% Assay

1

2

3

4

5

6

Mean

SD

% RSD

2120053

2120201

2120451

2120304

2120409

2120059

2120246

170.9976

0.01

99.98

99.99

100.00

99.99

100.00

99.98

99.99

0.008

0.01

Table no. 4: It shows precision results for Mosapride

Injection

Peak Areas of

Mosapride

% Assay

1

2

3

4

5

6

Mean

SD

% RSD

1440041

1440420

1440984

1440452

1440657

1440064

1440436

358.9054

0.02

99.97

100.01

100.04

100.00

100.01

99.97

100

0.026

0.02

Acceptance Criteria:The %RSD for the area of all the five injections should not be more than 2%.

4. Intermediate Precision / Ruggedness: To evaluate the intermediate precision (also known as Ruggedness) of the method, precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and measured the area for all five injections in HPLC. The % RSD for the area of five replicate injections was found to be within the specified limits. The data are represented in table no. 5 and 6.

Table no. 5: It shows ruggedness results for Rabeprazole

Injection

Peak Areas of

Rabeprazole sodium

 

% Assay

1

2

3

4

5

6

Mean

SD

% RSD

2120451

2120304

2120409

2120059

2120054

2120199

2120246

170.8801

0.01

100.00

99.99

100.00

99.98

99.98

99.99

99.99

0.008

0.01

Table no. 6: It shows ruggedness results for Mosapride

Injection

Peak Areas of

Mosapride

% Assay

1

2

3

4

5

6

Mean

SD

% RSD

1440984

1440452

1440657

1440064

1440309

1440379

1440474

315.7286

0.02

100.04

100.00

99.97

99.97

99.99

99.99

99.99

0.02

0.02

Acceptance Criteria:The %RSD for the area of all the five injections should not be more than 2%.

5. Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and value found. The standard solution with Accuracy -50 %, Accuracy -100 % and Accuracy -150 % were injected into chromatographic system and calculated the amount found and amount added for Rabeprazole and Mosaprideand further calculated the individual recovery and mean recovery values. The data are represented in table no. 7 and 8.

Table No. 7: It shows accuracy results for Rabeprazole

Concentration

% of spiked level

Amount added

(mg)

Amount found

(mg)

% Recovery

Statistical Analysis of % Recovery

50 %

50 %

50 %

 

20

20

20

20.00

19.99

20.01

100.00

99.99

100.03

MEAN

100.00

 

 

% RSD

 

0.02

100 %

100 %

100%

 

40

40

40

39.99

39.99

39.99

99.98

99.98

99.99

MEAN

% RSD

99.98

0.01

150 %

150 %

150 %

60

60

60

59.99

59.99

59.99

99.99

99.99

99.98

MEAN

%RSD

99.98

0.01

Table No. 8: It shows accuracy results for Mosapride

Concentration

% of spiked level

Amount added

(mg)

Amount found

(mg)

% Recovery

Statistical Analysis of % Recovery

50 %

50 %

50 %

20

20

20

19.99

19.99

20.01

99.98

99.98

100.06

MEAN

%RSD

100.00

0.04

100 %

100 %

100 %

40

40

40

40.00

39.99

40.00

100.00

99.97

100.00

MEAN

%RSD

99.99

0.01

150%

150 %

150 %

60

60

60

60.01

59.99

60.01

100.02

99.99

100.02

MEAN

%RSD

100.01

0.01

Acceptance Criteria:The %Recovery for each level should be between 98.0 to 102.0 %.

6. Linearity:It is the ability of the method to elicit test result that is directly proportional to analytic concentration within a given range. It is generally reported as variance of slope or regression line. It is determined by series of three to six injections of five of more standards. Different levels of solution were prepared and injected to the chromatographic system and the peak area was measured.Plotted a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient. The calibration curve was represented in fig. no. 3 and 4. The data are represented in table no. 5 and 6.

Table no. 9: It shows linearity results for Rabeprazole

Concentration (µg/mL)

Average    Area

Statistical Analysis

20

30

40

50

60

70

80

1060367

1590454

2120164

2650519

3180207

3710698

4240367

Slope

y-Intercept

Correlation Coefficient

53002

301.7

1

Table no. 10: It shows linearity results for Mosapride

Concentration (µg/mL)

Average      Area

Statistical Analysis

20

30

40

50

60

70

80

720468

1080643

1440364

1800465

2160760

2520583

2879905

Slope

y-Intercept

Correlation Coefficient

35995

707.7

1

Fig. no. 5: It shows calibration curve for Rabeprazole

Fig. no. 6: It shows calibration curve for Mosapride

7. Limit of Detection: The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value.

Limit of Detection for Rabeprazole and Mosapride: The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of detection is the lowest concentration of the substance that can be detected, not necessarily quantified by the method. (Regression statistics)The minimum concentration at which the analyte can be detected is determined from the linearity curve by applying the following formula.

Where S – slope of the calibration curve
σ – Residual standard deviation

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