METHOD DEVELOPMENT AND VALIDATION FOR ESTIMATION OF NAPROXEN IN BULK SAMPLES AS WELL AS IN TABLET DOSAGE FORMS BY USING RP-HPLC

 

Fig. No. 3 A typical chromatogram for sample drug

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 within the specified limits. The data are represented in table no. 2 and 3.

Table no. 2: Precision results for Naproxen (System Precision)

Injection

Peak Areas

% Assay

1

4435.56

100.56

2

4437.58

100.88

3

4435.56

100.78

4

4440.15

100.06

5

4445.13

101.02

Mean

4438.796

100.06

SD

62.64

52.3

% RSD

1.23

0.09

Table no. 3: Precision results for Naproxen (Method Precision)

Injection

Peak Areas

% Assay

1

4437.5151

100.86

2

4439.6279

100.91

3

4437.5151

100.86

4

4440.1612

100.92

5

4446.1712

100.06

6

4445.1312

101.03

Mean

4448.67

100.77

SD

58.90

44.5

% RSD

1.56

0.08

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 within the specified limits. The data are represented in table no. 4.

Table no. 4: Ruggedness results for Naproxen

Injection

Peak Areas

% Assay

1

4434.01

100.54

2

4436.79

100.86

3

4439.451

100.12

4

4442.512

100.56

5

4448.112

100.04

6

4461.012

101.26

Mean

4448.98

100.88

SD

78.90

67.0

% RSD

1.5

0.8

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, 100 and 150 % were injected into chromatographic system and calculated the amount found and amount added for Naproxen and further calculated the individual recovery and mean recovery values. The data are represented in table no. 5.

Table No. 5: Accuracy results for Naproxen

Concentration

% of spiked level

Amount added

(mg)

Amount found

(mg)

% Recovery

Statistical Analysis of % Recovery

50%

Sample 1

24.98

25.02

99.82

MEAN

99.82

50%

Sample 2

23.89

24.15

98.89

 

 

50%

Sample 3

24.89

25.14

98.98

%RSD

0.82

100 %

Sample 1

50.47

49.54

100.92

MEAN

100.3

100 %

Sample 2

50.45

50.03

100.83

 

 

100%

Sample 3

51.46

51.2

100.56

%RSD

1.62

150%

Sample 1

76.03

74.99

101.38

MEAN

101.5

150%

Sample 2

75.78

74.66

101.50

 

 

150%

Sample 3

75.86

74.79

101.42

%RSD

0.62

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. 4. The data are represented in table no. 6.

Table no. 6: Linearity results for Naproxen

Concentration (µg/mL)

Average

Area

Statistical Analysis

20

1621.89

 

Slope

y-Intercept

Correlation Coefficient

 

190.9x

-2361

0.999

40

5141.73

50

7066.67

60

9054.19

70

10980.2

80

13060.1

Fig. No. 4 Calibration curve for Naproxen

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 Naproxen: 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

8. Limit of Quantification: It is defined as lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy and reliability by a given method under stated experimental conditions. LOQ is expressed as a concentration at a specified signal to noise ratio.

Limit of Quantification for Naproxen: The lowest concentration of the sample was prepared with respect to the base line noise and measured the signal to noise ratio. Limit of Quantification is the lowest concentration of the substance that can be estimated quantitatively. It can be determined from linearity curve by applying the following formula

9. Robustness: As part of the Robustness, deliberate change in the flow rate, mobile phase composition, temperature variation was made to evaluate the impact on the method. The standard and samples of Naproxen were injected by changing the conditions of chromatography. There was no significant change in the parameters like resolution, tailing factor, asymmetric factor, and plate count. The data are represented in table no. 7 and fig. no. 5, 6 and 7.

Table No. 7: System Suitability Results for Naproxen (Change in Flow Rate)

Flow 0.8 mL/min.

Std. Area

Tailing factor

Flow 1.0 mL/min.

Std. Area

Tailing factor

Flow 1.2 mL/min.

Std. Area

Tailing factor

 

6079.40

1.106

 

4882.35

1.110

 

4076.02

1.123

5895.63

1.110

4970.64

1.112

4167.62

1.125

5935.37

1.112

4900.20

1.110

4138.32

1.124

6056.36

1.118

4924.73

1.111

4140.31

1.124

6059.63

1.117

4781.37

1.112

4098.21

1.123

Avg

6005.081

1.112

Avg

4891.86

1.111

Avg

4124.10

1.1238

SD

74.977

0.0044

SD

62.697

0.00089

SD

32.683

0.0007

% RSD

1.248

0.4003

% RSD

1.281

0.0804

% RSD

0.7925

0.0065

                   

Fig. no. 5: A typical chromatogram for robustness with flow rate (for 0.8 mL/min flow)

 

Fig. no. 6: A typical chromatogram for robustness with flow rate (for 1.0 mL/min flow)

 

Fig. no. 7: A typical chromatogram for robustness with flow rate (for 1.2 mL/min flow)

RESULTS AND DISCUSSION
To optimize the mobile phase, various proportions of buffer (pH 4.0) with methanol [HPLC Grade] were tested. The use of buffer (pH 4.0) and methanol [HPLC Grade] in the ratio of 40:60 (v/v) resulted in peak with good shapes and resolution. A flow rate of 1.0 mL /min was found to be optimum in the 0.4-1.5 mL/min range resulting in short retention time, baseline stability and minimum noise.

By applying the proposed method, the retention time of Naproxen was observed at 3.063 min at 210 nm. Quantitative linearity was obeyed in the concentration ranges of 20-80 µg/mL for Naproxen. The relevant regression equation was y = 86.83x + 57.31
(R² = 0.999) (where y is the peak area ratio and x is the concentration of Naproxen (µg/mL)). The intra-day and inter-day drugs variations by the proposed method showed an RSD less than 2 %, indicating that the method is precise. The corresponding mean recovery was 98.89- 101.50 %. This reveals that the method is quite accurate. The tailing factor and USP plate count were 1.108 and 10198; which were within the acceptance limits. The limits of detection for Naproxen obtained by the proposed method was 0.13 µg/mL and limits of quantification for Naproxen obtained by the proposed method was 0.40 µg /mL, which indicate the sensitivity of the method. The method tolerated minor variations in optimized chromatographic conditions indicating good robustness, which indicate the efficient performance of the column. 

No interfering peaks were found in the chromatograms indicating that the excipients used in tablet formulations did not interfere with the estimation of the drug by the proposed HPLC method.

CONCLUSION
The proposed HPLC method was found to be simple, precise, accurate and sensitive for the determination of Naproxen. The method was validated as per ICH guidelines and all the parameters met within the acceptance criteria. Applicability of this method for simultaneous estimation of Naproxen from tablet dosage forms was confirmed. Hence, this  method  is  specific and  can  be  successfully  used  for  the  estimation of Naproxen in bulk drug samples, pharmaceutical dosage forms. Hence, this method can be easily and conveniently adopted for routine quality control analysis of the above drug.

ACKNOWLEDGEMENT: The authors greatly acknowledge M/s. Pharma Train, Hyderabad, Telagana, India for providing the gift sample of Naproxen.

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