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ABSTRACT
An accurate, sensitive, precise, rapid and isocratic Reversed-Phase HPLC, (RP-HPLC) method
for simultaneous estimation of Ipratropium bromide and Levosalbutamol in the bulk drug and
in the pharmaceutical metered dose inhalers has been developed and validated. The best
separation was achieved on a 250 mm 4.6 mm i.d., 5 m particle, Inertsil ODS 3V-RP C18
column with Acetonitrile as the organic modifier and Di-Potassium Hydrogen Phosphate
[0.03M] in water with pH 3.2 adjusted with Ortho-Phosphoric Acid (0.1% v/v) in the
proportion of [30:70 v/v] as mobile phase at a flow rate of 0.8 mL min-1. UV detection was at
242 nm. Retention times were found to be 5.206 min. for Ipratropium bromide and 7.016 min.
for Levosalbutamol.The response was a linear function of concentration over the range of 2.00
to 6.00 g/ml, and 5.00 to 15.00 g/ml respectively with correlation coefficient of 1.000 for
Ipratropium Bromide and 0.994 for Levosalbutamol respectively. The percentage assay of
Ipratropium Bromide and Levosalbutamol were found to be 99.87 %, and 101.42 %
respectively. The Limit of Detection (LOD) for Ipratropium bromide and Levosalbutamol were
found to be 1.27 g/ml and 4.41g/ml respectively. The Limit of Quantification (LOQ) for
Ipratropium bromide and Levosalbutamol were found to be 3.81g/ml and 13.23g/ml
respectively. The excipients present in the formulation were not interfered with the assay. The
method is suitable for application in quality-control laboratories, because it is simple and rapid
with good accuracy and precision.
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Fig. 2: Levosalbutamol
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Table 5: Results from testing of the Robustness of the method (n=3, 100% of the Working Standard Solution contains Ipratropium Bromide
7.2g/mL and Levosalbutamol 10.8 g/mL
Mean Peak Areas S.D % RSD Mean Retention Time (in min) S.D
Condition Studied in Modification In
Ipratropium Ipratropium Ipratropium
Robustness OFAT analysis. Levosalbutamol Levosalbutamol Levosalbutamol
Bromide Bromide Bromide
5572554
240 57494262 186637.6 0.56 1.18 5.2210.84 7.0591.18
2445.9
Detector Wavelength 5522311
242 57455798 222989.5 0.821 1.28 5.2330.43 7.0560.94
in (nm) 12380.4
5509629
244 57755569 54908.3 0.789 0.921 5.2170.42 7.0570.58
31681.8
5514140
3.0 57361218 182324.6 0.921 0.825 5.2360.65 7.0340.32
7821.9
5502051
Mobile Phase 3.2 57250000 239193.4 0.619 0.625 5.2190.81 7.0561.18
5689.8
pH
5512170
3.4 57551700 94432.3 0.527 0.825 5.2340.94 7.0631.27
6437.5
5510819
68:32 57241699 96350.4 0.661 1.47 5.2350.63 7.0620.54
18130.9
Mobile Phase 5527875
70:30 57390457 89445.8 0.982 1.19 5.2281.04 7.0680.76
Composition in (v/v) 9995.2
5519419
72:28 57318435 268422.6 1.292 0.73 5.2371.13 7.0720.54
2206.4
5492663
0.6 56848412 112360.2 0.654 0.58 5.2290.76 7.0830.69
7830.6
Flow rate in 5537009
0.8 57726939 162129.1 0.563 1.26 5.2340.78 7.0540.26
mL/minute. 3745.3
5487356
1.0 56969661 93318.2 0.76 1.58 5.2280.65 6.8770.82
5689.7
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