Biomedical Research

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Research Article - Biomedical Research (2022) Volume 33, Issue 2

Method development and validation of Ipratropium bromide by HPLC.

Landage SS*, Shembade SH, Tamboli AM

Department of Pharmaceutical Chemistry, Sahyadri College of Pharmacy, Methvade, Maharashtra, India

Corresponding Author:
Landage SS
Department of Pharmaceutical Chemistry
Sahyadri College of Pharmacy

Accepted date: February 25, 2021

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The present work was focused on the development and validation of High-Performance Liquid Chromatography (HPLC) method which is simple, rapid, precise, accurate, sensitive, and economical for the quantitation of Ipratropium bromide in bulk and capsule dosage form has been validated. The chromatographic separation was attained on Agilent zorbax bonus-RP column with dimensions (250 × 4.6 mm, 5μ) particle size employing 0.1% Trifluoroacetic acid and Acetonitrile (ACN) in the ratio of 70:30% v/v as mobile phase, which was pumped at a rate of 1.0 ml/min and detected at a wavelength of 210 nm. The linearity of the method was demonstrated in the concentration range of 24-56 μg/ml for Ipratropium bromide with a correlation coefficient (r²) of 0.999. Percentage drug recovery was found to be 99.03%-100.08%, and percentage relative standard deviation was <2%. Limit of detection and limit of quantification values were found to be 1.15 μg/ml and 3.49 μg/ml, respectively, and assay of marketed capsule formulation was found to be 99.44%. The developed HPLC method was found to be simple, specific, sensitive, rapid, linear, accurate, precise, and economical and could be used for regular quality control of Ipratropium bromide in bulk and capsule formulation.


Ipratropium bromide, High-performance liquid chromatography, Validation, ICH guidelines.


Ipratropium bromide chemically known as (1R,3R,5S,8R)- 3-(3-hydroxy-2-phenylpropanoyl) oxy)-8-methyl-8-(propan-2- yl)-8-azabicyclo (3.2.1) octan-8-ium bromide is a muscarinic antagonist structurally related to atropine but often considered safer and more effective for inhalation use. It is used for various bronchial disorders, in rhinitis, and as an antiarrhythmic. It blocks muscarinic cholinergic receptors, without specificity for subtypes, resulting in a decrease in the formation of cyclic Guanosine Monophosphate (cGMP). It is freely soluble in water and methanol, sparingly soluble in ethanol, and insoluble in lipophilic solvents such as ether, chloroform and fluorocarbons [1- 7]. The combination preparation Ipratropium bromide/ salbutamol is a formulation containing Ipratropium bromide and salbutamol sulphate used in the management of Chronic Obstructive Pulmonary Disease (COPD) and asthma. An extensive literature survey revealed few RP-HPLC methods for routine quality control analysis, related substances and impurity determinations in dosage forms containing Ipratropium bromide and salbutamol sulphate. An LC-MS/MS7 method was also reported for the simultaneous determination of albuterol sulphate and Ipratropium bromide in rat plasma. An attempt has been made to develop a new RP-HPLC method for simultaneous determination of Ipratropium bromide and salbutamol sulphate in inhalations which could also provide the stability related information [8-12].

Materials and Methods

Ipratropium bromide was procured as a gift sample from Vamsi Labs Ltd. Used in the study. The pharmaceutical dosage form used in the study was Ipravent rotacaps labeled to contain 40 mcg. Ipravent rotacaps was purchased from the local pharmacy, sangola. All other chemicals and reagents used were of analytical grade.

Chromatographic condition

Chromatographic separation was performed using an Agilent zorbax bonus-RP column dimension (250 × 4.6 mm, 5μ) was used for the separation. The elution was carried out gradient at flow rate 1.0 ml/min using mobile phase consisted of 0.1% Trifluoroacetic acid: ACN (70:30 v/v).

Preparation of standard solution

Standard Stock Solution-I (SSS-I)

• Initially Prepare a Standard Stock Solution (SSS-I) of by adding 5 mg of Ipratropium bromide in 10 ml volumetric flask and add 5 ml diluents, mix for 2 minutes and make the volume to 10 ml with diluents. (Conc. of Ipratropium bromide=500 μg/ml).

• Then add 0.8 ml of SSS-I in 10 ml volumetric flask and add 5 ml diluents and vortex and make up the volume with diluents. (Conc. of Ipratropium bssromide=40 μg/ml).

Selection of wavelength

The sample was scanned from 200-400 nm with PDA detector. The Wavelength selected for analysis chosen was 210 nm on basis of appropriate intensity of Ipratropium bromide (Table 1) (Figures 1 and 2).

Column temperature 30º C
Flow rate 1.0 ml/min
Mobile phase 0.1% Trifluoroacetic acid : Acetonitrile (70:30 v/v)
Runtime 10 minutes
Injection volume 10 µl
Wavelength 210 nm
Diluent 0.1% TFA:ACN
Column Agilent zorbax bonus-RP
RT of Ipratropium bromide 2.50 Minutes

Table 1: Chromatographic condition.

Figure 1: Chromatogram of standard ipratropium bromide.

Figure 2: Chromatogram of sample of ipratropium bromide.

Sample preparation for assay

Capsule Sample Solution (CSS)

• 20 Capsule contents were weighed and average weight was calculated. And the content was mixed in mortar and pestle.

• Powder Weight equivalent to 400 μg Ipratropium Bromide was weighed into 10 ml volumetric flask and add 5 ml diluent, sonicate for 10 minutes and make the volume to 10 ml with diluent. (Conc. of Ipratropium Bromide=40 μg/ml).

Method Validation

The methods were validated as per the ICH guidelines on analytical process validation. The linearity, accuracy, precision, and specificity of the method were validated as per ICH guidelines [13-15].


Stock solution of Ipratropium bromide at a concentration of 60%-140% was prepared in the mobiles phase. From the stock several dilutions between 24-56 μg/ml were prepared and injected into the column at a rate of 1 ml/min at injection volume of 10 μL. The calibration curve was plotted using area of retention time versus concentrations μg/ml. linear regression analysis was used to assess the linearity using least square regression method.


The assay precision was carried out by interday and intraday study and the process was evaluated for solutions (24,32,40,48,56 μg/ml) and were analyzed at different time points. The RSD (Relative Standard Deviation) was calculated for the methods.


Samples were prepared of 80%, 100% and 120% concentration by spiking the same amount of concentration given in Tables for Ipratropium Bromide. Samples were injected in duplicate to calculate % RSD and % recovery was also calculated.

System suitability

A single sample was prepared as described and 5 injections were made from same sample and checked for system suitability. System suitability parameters are as below

1. Retention time,

2. Theoretical plates,

3. Asymmetry (Tailing factor),

4. Resolution.

Limit of detection and limit of quantification

The Limit of Detection (LOD) and Limit of Quantification (LOQ) were determined according to ICH guidelines by the below mentioned formulae (Table 2).

Sr. No. % Level Volume of Ipratropium bromide stock solution to be taken (ml) Concentration of Ipratropium bromide (µg/ml) Diluted to volume (ml)
1. 60 0.6 24 10
2. 80 0.8 32 10
3. 100 1.0 40 10
4. 120 1.2 48 10
5. 140 1.4 56 10

Table 2: Linearity dilutions.




SE=Standard Error of Y intercept; A=Slope of the calibration curve.

Results and Discussion

The percentage assay of Ipratropium bromide was found to be 99.44% as shown in Table 3. Linearity was studied by plotting a graph of area vs. concentration. The calibration curve for the method obtained was linear over the concentration of Ipratropium bromide 24-56 μg/ml. The correlation coefficient obtained was 0.999, thus indicating excellent correlation between peak areas. Calibration data is presented in Table 4 and calibration curve shown in Figure 3 it proves the linearity over the concentration range 24 to 56 μg/ml. The precision study of same dilutions (40 μg/ml) of Ipratropium bromide solutions in 0.1% Trifluoroacetic acid: Acetonitrile (70:30 v/v) showed % RSD less than 2% as shown in Table 5. A good accuracy was verified with a mean recovery which was not less than 99.03% and not more than 100.08% and the range of % RSD is not less than 0.09 and not more than 0.22 for study of the different dilutions of Ipratropium bromide in 0.1% Trifluoroacetic acid: Acetonitrile (70:30 v/v) solutions as shown in Table 6. The parameter of system suitability is Retention time, Therotical plates, Assymmetry (Tailing factor) and Resolution as shown in Table 7. The lowest amount of analyte in a sample that can be detected but not necessarily quantitated LOD (Limit of Detection) and the lowest amount of analyte in a sample that can be determined with accepTable precision and accuracy under the stated experimental conditions or quantitation limit LOQ (Limit of Quantitation) was determined. The LOD and LOQ were found to be 1.15 μg/ml and 3.49 μg/ml as shown in Table 8. The significantly low value of LOD and LOQ proved the sensitivity of the process.

Ipratropium bromide
Sample Working standard Drug product
Area 704752 700786
Assay - 99.44

Table 3: Assay data of Ipratropium bromide.

% Level Concentration (ug/ml) Area
60 24 429123
80 32 559039
100 40 704242
120 48 837803
140 56 974718

Table 4: Linearity data of Ipratropium bromide.

Ipratropium bromide
Concentration of sample (µg/ml) Sample ID Area
40 Rep 1 704242
40 Rep 2 706398
40 Rep 3 708856
40 Rep 4 702490
40 Rep 5 701774
Average 704752
STDEV 2906.775
RSD 0.41

Table 5: Precision data of Ipratropium bromide.

Sample ID Reps Spiked conc.
Area Amt. Recovered
% Recovery Average STDEV RSD
80% Rep 1 31.99 559039 31.7233 99.16 99.03 0.1758 0.18
Rep 2 31.99 557637 31.6437 98.91
100% Rep 1 39.99 704242 39.9630 99.93 100.08 0.2163 0.22
Rep 2 39.99 706398 40.0854 100.23
120% Rep 1 47.99 837803 47.5421 99.07 99.13 0.0938 0.09
Rep 2 47.99 838925 47.6058 99.20

Table 6: Accuracy data of Ipratropium bromide by HPLC method.

Parameter Ipratropium bromide
Retention time 2.50 minute
Therotical plates 10005
Asymmetry (Tailing factor) 1.07
Resolution 0.00

Table 7: System suitability parameters.

Drug LOD (µg/ml) LOQ (µg/ml)
Ipratropium bromide 1.15 (µg/ml) 3.49 (µg/ml)

Table 8: LOD and LOQ of Ipratropium bromide.

Figure 3: Linearity graph of ipratropium bromide.


The developed HPLC method is able to determine Ipratropium bromide in raw materials and in pharmaceutical dosage forms; this can be attributed to the good separation and resolution of the chromatographic peaks with optimum retention time of 2.50 minutes. The results were in good conformity with the affirmed statistical and pharmacopeial contents. Hence, this method can be considered to be precise, reliable, rapid, simple, sensitive and economical nature.


The authors extend a deep sense of gratitude to management and principal of Sahyadri College of Pharmacy, methvade, sangola for providing all necessary support and facility to complete this research work.


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