DEVELOPMENT AND VALIDATION OF A REVERSED-PHASE HPLC METHOD FOR ASSAY OF AZITHROMYCIN IN POWDER FOR ORAL SUSPENSION

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Results and discussion

Optimization of the chromatographic conditions
During the analysis of basic drugs like azithromycin  one of the well known problem is peak tailing. Since these compounds strongly interact with polar ends of HPLC column packing materials, causing severe peak asymmetry and low separation efficiencies. High purity silica backbone and advances in bonding technology have alleviated the tailing problem of polar compounds in HPLC to a significant extent. During the optimization of the method, different columns (Inertsil C8, 250 mm × 4.6 mm, 5 μm; Zorbax C18 250 mm × 4.6 mm, 5 μm; Symmetry C18 250 mm × 4.6 mm, 5 μm) and two organic solvents (acetonitrile and methanol) were tested. The chromatographic conditions were also optimized by using different buffers like phosphate, acetate and citrate for mobile phase preparation. After a series of screening experiments, it was concluded that phosphate buffers gave better peak shapes than their acetate and citrate counterparts. The chromatographic separation was achieved on a  Asahipak ODP 4E (250 mm × 4.6 mm, 5 μm)  column, by using a mixture of ammonium dihydrogen phosphate–methanol (0.05M) (30:70, v/v) as mobile phase. Asahipak  columns are having high pH (9–13) and temperature (20–60 °C) stability. About mobile phase, due to the lack of other chromophore than the ester group in azithromycin and, therefore, the need to work at a low wavelength (210 nm), methanol was considered as organic solvent instead of acetonitrile . At 30°C column temperature and pH 9.0 of mobile phase, the peak shape of azithromycin was found symmetrical. The flow rate kept was 1.5mL/min to achieve adequate retention time of  azithromycin peak  (Fig. 1-2)

Validation of method

Specificity
The specificity of the HPLC method is illustrated in (Figs. 3-7) where complete separation of azithromycin was noticed in presence of impurities.  In addition there was no interference at  analysis with photo diode detector, purity angle was less than purity threshold for the analyte. he retention time of azithromycin in the chromatogram of placebo solution. In peak  purity This shows that the peak of analytes was pure and excipients in the formulation did not interfere the analyte

Selectivity
Neither formulation ingredients nor degradation products interfered with quantitation of azithromycin. All samples and placebo were analyzed using the assay chromatographiccondition described. No evidence of interactive  degradation products was seen during evaluation. However  azithromycin  was observed to be susceptible to acidic and oxidative condition. So avoid acidic condition during analysis.Mild degradation was observed in base , water, thermal, and UV degradation.Selectivity was demonstrated showing that azithromycin peak was free of interference of degradation products indicating that the proposed method is stability indicating.

Accuracy
Accuracy of the method was calculated by recovery studies at six levels for 50% and 150% level and three levels for 75%, 100%, and 125%. (Table 1).The mean percentage recovery obtained for azithromycin was found to be in between 99.90 and 101.0%  respectively.

Table 1

Results of the recovery analysis of  azithromycin

S.No.

% spike level

Amount added(mg)

Amount recovered (mg)

%Recovery

%Mean Recovery

% RSD

1.

50

93.71

93.60

99.9

100.2

0.4

2.

93.91

93.63

99.7

3.

93.79

94.32

100.6

4.

93.72

94.18

100.5

5.

93.65

94.04

100.4

6.

93.62

94.00

100.4

1.

75

148.01

147.96

100.0

99.9

0.2

2.

148.19

148.20

100.0

3.

148.16

147.74

99.7

1.

100

198.68

199.09

100.2

100.2

0.1

2.

198.73

199.42

100.3

3.

198.23

198.37

100.1

1.

125

243.69

245.84

100.9

101.0

0.2

2.

243.75

246.56

101.2

3.

243.57

245.87

100.9

1.

150

299.84

302.39

100.9

100.9

0.3

2.

299.48

301.45

100.7

3.

299.51

302.65

101.1

4.

299.80

302.24

100.8

5.

299.85

301.51

100.6

6.

299.84

303.82

101.3

 

Precision
The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing five replicate analyses of the same working solution. The relative standard deviation (R.S.D.) obtained for  azithromycin was  0.2. (Table2).

Table 2

 System suitability parameters

Parameters

Azithromycin

Theoritical Plates

8335

Peak Symmetry

0.9

%RSD

0.2

The intra- and inter-day variability or precision data are summarized in (Table3). The intra-day precision of the developed LC method was determined by preparing the samples of the same batch. 6. 4 g of sample weighed  into six separate 250 mL volumetric flask ,and made upto  the mark with diluent . Filtered   about  10 mL  of  above solution through 0.45µm nylon syringe filter by discarding first 4 mL solution. Injected  blank,  and six replicate injections of repeatability solutions.The %R.S.D,% assay of the assay results was used to evaluate the method precision. The inter-day precision was also determined by the same procedure. The results indicated the good precision of the developed method (Table3).

Table .3

Intra -and inter- day assay precision data(n=12)

Sample No.

Method Precision

Intermediate Precision

Over all % RSD (n=12)

1

98.3

101.8

 

 

 

1.0

 

 

 

2

100.2

101.3

3

100.1

98.6

4

99.9

100.2

5

99.9

100.3

6

100.5

101.1

Mean

99.8

100.6

% RSD

0.8

1.1

View Within Article Linearity
Linearity was determined for azithromycin in the range of 382–1208 μg/mL. The correlation coefficient (‘r’) value for the drug was >0.999. Typically, the regression equation for the calibration curve was found to be y = 966.6x − 8620.8 for azithromycin.

Robustness
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.

Robustness of the method was investigated under a variety of conditions including changes of pH of the mobile phase, flow rate, percentage of methanol in the mobile phase and column oven temperature. The  standard solution is injected in five replicates and sample solution of 100% concentration is prepared and injected  for every condition and % R.S.D. of assay was calculated for each condition. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 4)

Table 4

Results of robustness study.

Robustness parameter

Level

% RSD of Results

pH of mobile phase

8.8  ( Low pH)

0.1

9.2 ( High pH)

0.1

organic composition

High % organic strength

0.1

Low % organic strength

1.1

flow rate

1.4 mL/min

0.2

1.6 mL/min

0.1

column oven temperature

25

0.1

35

0.3

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