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A simple, rapid and accurate RP-HPLC method was developed for the quantification of
isoproterenol HCl in bulk and its formulation by RP-HPLC method using C18 ODS column
(Phenomenex Luna, 250 x 4.6 mm, 5μm) in isocratic mode. The mobile phase consisted of
methanol and 0.1% triethylamine buffer (PH7) in the ratio of 20:80 (v/v) was used. The flow rate
was maintained at 1.0 mL/min and the injection volume was 20μL . Detection wavelength with
UV detector at 279 nm and run time was kept 10 min. The retention time of isoproterenol HCl
was 3.3 min. The method was linear over the concentration range 10-60 μg/ml. The recovery was
found to be 99.82 0%. The validation of method was carried out utilizing ICH-guidelines. The
described HPLC method was successfully employed for the analysis of pharmaceutical
Key words: HPLC method, Isoproterenol HCl, Method development, ICH-guidelines
Isoproterenol hydrochloride chemically 3,4-dihydroxy-α-[(isopropylamino)methyl]benzyl
alcohol hydrochloride. In addition, isoproterenol acts on beta-2 adrenergic receptors in
bronchiolar and vascular smooth muscle, thereby causing smooth muscle relaxation.
Isoproterenol exerts its effect on the beta-1 adrenergic receptors in the myocardium, thereby
increasing heart rate and cardiac output. Cardiac output is increased because of the positive
inotropic and chronotropic effects of the drug in the face of diminished peripheral vascular
resistance. The cardiac effects of isoproterenol may lead to palpitations, sinus tachycardia, and
more serious arrhythmias; large doses of isoproterenol may cause myocardial necrosis in
animals1,2,3. The chemical structure of isoproterenol hydrochloride was shown in figure 1.

Fig 1: Chemical structure of Isoproterenol hydrochloride

Literature survey revealed that quantification methods have been reported for the estimation
isoproterenol hydrochloride in pharmaceutical dosage forms. Bio analytical methods (LC-
MS/MS) have been reported for the quantification of isoproterenol hydrochloride in biological
fluids4, 5,6,7,8
. There is no chromatographic method have been reported for the analysis of
isoproterenol hydrochloride in single analyte. The present work aims at developing simple,
accurate and reproducible RP-HPLC method for the estimation of isoproterenol in bulk and its
formulation according to ICH guidelines9.


Instrumentation and Materials
The liquid chromatographic system consists of shimadzu LC Solutions- 20 AD UFLC with UV-
VIS detector, binary pump and septum injector valve with 20 μl fixed loop. The analytes were
monitored at 279 nm. Chromatographic analysis was performed on Phenomenex Luna C 18 ODS
column having 250 mm× 4.6 mm i.d. and 5 μm particle size.
Materials used:
API of isoproterenol hydrochloride was procured as a gift sample by Mylan laboratories,
Bangalore, Karnataka. Water was distilled and purified with Merck Millipore system.
Formulation of isoproterenol hydrochloride (ISOLIN 2mg/1ml) was purchased from local
Chromatographic Conditions
The Phenomnex C18 column ODS (250 x 4.6mm, 5µm) equilibrated with mobile phase
methanol and 0.1%triethylamine buffer in the ratio of 20:80 (v/v) was used and the flow rate was
maintained at 1 mL/min. Detection wavelength with UV detector at 279 nm, and the injection
volume was 20 μL and run time was kept 10 min.
Preparation of mobile phase
The mobile phase was prepared by taking 20% Methanol and 80% 0.1%triethylamine Buffer .It
was filtered through 0.45µm membrane filter and degassed under ultrasonic bath prior use. The
mobile phase was pumped through the column to stabilize the column.
Preparation of stock solution
10 mg isoproterenol HCl drug was weighed accurately and it was dissolved in the mobile phase
and after complete dissolution the volume was made up to 10ml. The stock solution was
The specificity of the proposed method was determined by comparing the results obtained by
running the standard solution and with blank.
Linearity and Range
The linearity was determined for isoproterenol HCl. Solution of the drug at six different
concentrations was analyzed and calibration curve was constructed by plotting mean response
factor against the respective concentration. The method was evaluated by determination of the
correlation coefficient and intercept value. Isoproterenol follows linearity in the concentration
range of 10- 60 µg/mL respectively.
Recovery assessment was obtained by using standard addition technique which was by adding
known quantities of pure standards at three different levels in 50%, 100% and 150% to the pre
analysed sample formulation. From the amount of drug found, amount of drug recovered and
percentage recovery were calculated which sense to conformation that the proposed method was
The robustness was determined by injecting triplicate injections of standard and three-
sample solutions in single at each different condition with respect to control condition.
Robustness of the method was checked by varying the instrumental conditions; flow rate
(±10%mL/min) and temperature (±5˚C). Sample solution was injected in each condition.
System Suitability Parameter
System suitability testing is an integral part of many analytical procedures. The tests are based on
the concept that the equipment, electronics, analytical operations and samples to be analyzed
constitute an integral system that can be evaluated as such. Following system suitability test
parameters were established. The results were shown in table no.5.
Assay procedure
Marketed formulation of isoproterenol HCl is available in injection forms. Drug equivalent to
1mg/ml was transferred into 10ml volumetric flask and dissolved using mobile phase. The
solution was sonicated for 5 mins. From the prepared solution optimized sample solutions were
prepared and calculate the assay of pharmaceutical dosage form.
The comparison of the data of the drug solution before spiking and the spiked drug solution
revealed that there was no significant interference of placebo with the recovery of isoproterenol
HCl, inferring that the method was specific. The results were shown in table no.1.
Linearity and Range
The method was found to be linear. It was found to be In the linearity study, regression equation
and coefficient of correlation for isoproterenol was found to be y = 30386x + 5215.9, r2 =0.999.
linearity data was shown in table no.2 and fig. 2.
The mean recovery was found to be 99.82%. The limit for mean recovery is 90-110%. Thus the
method was found to be accurate. The results were shown in table no.3.
This method is robust for the analysis of isoproterenol HCl within the specified range of
deviations in the experimental conditions. The results were shown in table no.4.
The percent content of isoproterenol HCl formulation was found to be 101.84%.

Table No. 1: Specificity Data

S.No Peak Name Observation

1 Blank Nil

2 Standard Rt = 3.3min λ max =279nm

Table No. 2: Linearity and range

S. No Concentration (µg/ml) Peak Area
10 306975
20 621575
30 910648
40 1219069
50 1521984
60 1832046
Calibration curve of Isoproterenol
y = 30386x + 5215.9
1500000 R² = 0.999

Peak area


0 10 20 30 40 50 60 70
Concentration mcg/ml

Fig 2:Calibration curve of Isoproterenol


Fig 3: Optimized chromatogram of Isoproterenol

Table No.3: Results for accuracy
Spiked Peak area Amount Amount Recovery % Mean
Concentration (n=3) added Found Recovery
(μg/ml) (μg/ml) (μg/ml)

609461 19.98 99.87

20 621575 20.012 20.38 101.86 100.92
616547 20.22 101.03
1238125 40.60 101.44
40 1191981 40.025 39.09 97.66 99.28
1204981 39.51 98.73
1832046 60.08 100.07
60 1805645 60.038 59.21 98.63 99.26
1813891 59.48 99.08

Table No.4: Results for robustness

Flow rate(±10%) Temperature((± 5oC Control 0.9ml/min 1.1ml/min 30 oC 40 oC
1216547 1186482 1235768 1230768 1186482
1219659 1169084 1216786 1216786 1169084
1193981 1216547 1190548 1190548 1204986
1210062 1190704.33 1214367 1212700.66 1186851
11441.97 19605.3592 18540.04 16671.9237 14659.25
0.94 1.64 1.52 1.37 1.23

Table No. 5: Results of System Suitability

Parameter Result Acceptance Limit
Retention time (Rt)* 3.3min --
Resolution factor* NA --
Number of theoretical 4105 More than 2000
plates (N)*
Tailing factor (T)* 1.62 Less than 2
* Number of injections: 6 replicates
The developed method was validated as per ICH guideline and was found to be within the
prescribed limit. It concludes that the developed methods are simple, selective, precise , accurate
and robust and suitable for both authentic and pharmaceutical dosage form.
I would like thank Mylan laboratories for providing isoproterenol hydrochloride working
standard as a gift sample. I also extend my thanks to Director, JNTUA-OTPRI for providing
necessary facilities for carrying out this research work.
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