A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT IN FORMULATIONS

B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16

IJST (www.ijsit.com), Volume 1, Issue 1, September-October 2012

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A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT

IN FORMULATIONS

B.Lakshmi, Kallam Haranadhreddy Institute of Technology, Guntur

Prof T.V.Reddy , Prof Malla reddy College of Engineering, Secunderabad.

ABSTRACT

A simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of Icatibant

tablet dosage form. Isocratic elution at a flow rate of 1ml/min was employed on a symmetry Chromosil C18

(250x4.6mm, 5µm in particle size) at ambient temperature. The mobile phase consisted of Methanol:

Acetonitrile: water 57:13:30 v/v,. The UV detection wavelength was 224nm and 20µl ss was injected. The

retention time for Icatibant was 9.89min. The percentage RSD for precision and accuracy of the method was

found to be less than 2%. The method was validated as per the ICH guidelines. The method was successfully

applied for routine analysis of Icatibant tablet dosage form and bulk drug.

Key Words: Icatibant, RP-HPLC, UV detection, recovery, precise, 224nm.



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INTRODUCTION

Icatibant (trade name Firazyr) is a peptidomimetic drug consisting of ten amino acids, which is selective and

specific antagonist ofbradykinin B2 receptors. It has been approved by the European Commission for the

symptomatic treatment of acute attacks,[1][2] of hereditary angioedema (HAE) in adults (with C1-esterase-

inhibitor deficiency).

Bradykinin is a peptide-based hormone that is formed locally in tissues, very often in response to a trauma. It

increases vessel permeability, dilates blood vessels and causes smooth muscle cells to contract. Bradykinin

plays an important role as the mediator of pain. Surplus bradykinin is responsible for the typical symptoms of

inflammation, such as swelling, redness, overheating and pain. These symptoms are mediated by activation of

bradykinin B2 receptors. Icatibant acts as a bradykinin inhibitor by blocking the binding of native bradykinin

to the bradykinin B2 receptor.

EXPERIMENTAL

Materials:

Working standard of Icatibant was obtained from well reputed research laboratories. HPLC grade

water, Methanol was purchased from E. Merck (Mumbai, India).

Apparatus:

A Series HPLC [6-11] system PEAK LC 7000 isocratic HPLC with PEAK 7000 delivery system. Rheodyne

manual sample injector with switch (77251), Analytical column Chromosil C18. 250×4.6mm, Electronic

balance-DENVER (SI234), manual Rheodyne injector with a 20 μl loop was used for the injection of sample.

PEAK LC software was used. UV 2301 Spectrophotometer was used to determine the wavelength of maximum

absorbance.

Determination of wavelength of maximum absorbance:

The standard solutions of Icatibant were scanned in the range of 200 -400 nm against mobile phase

as a blank. Icatibant showed maximum absorbance at 224nm. So the wavelength selected for the

determination of Icatibant was 224nm.

http://en.wikipedia.org/wiki/File:Icatibant.png



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Chromatographic equipment and conditions:

To develop a High Pressure Liquid Chromatographic method for quantitative estimation of

ICATIBANT an isocratic PEAK HPLC instrument with Zodiac C18 column (250 mm x 4.6 mm, 5μ) was used.

The instrument is equipped with a LC 20AT pump for solvent delivery and variable wavelength

programmable LC – 7000 UV-detector. A 20μL Rheodyne inject port was used for injecting the samples. Data

was analyzed by using PEAK software.

The mobile phase consisted of Methanol: Acetonitrile: water 57:30:13 v/v,. Injections were carried

out using a 20 μl loop at room temperature (20 + 2 °C) and the flow rate was 1 ml/min. Detection was

performed at 224nm with 10min runtime.

Standard and sample solutions:

A 10 mg amount of Icatibant reference substance was accurately weighed and dissolved in 10 ml

mobile phase in a 10 ml volumetric flask to obtain 1000 ppm concentrated solution. Required concentrations

were prepared by serial dilution of this solution.

A composite of 20 (INTELENCE) tablets was prepared by grinding them to a fine, uniform size

powder. 10 mg of Icatibant was accurately weighed and quantitatively transferred into a 100 ml volumetric

flask. Approximately 25 ml mobile phase were added and the solution was sonicated for 15 min. The flask

was filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted

with mobile phase to a concentration of 10ppm.

Method validation:

Method validation was performed following ICH specifications for specificity, range of linearity,

accuracy, precision and robustness.

RESULTS AND DISCUSSION

System Suitability:

Having optimized the efficiency of a chromatographic separation, the quality of the chromatograph

was monitored by applying the following system suitability tests: capacity factor, tailing factor and theoretical

plates. The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0,

tailing factor ≤2.0 and theoretical plates >2500. In all cases, the relative standard deviation (R.S.D) for the

analytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from reference

substance solution is presented. System suitability parameters were shown in Table.1. Standard

chromatogram was given in Figure.2



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Api Concentration 10ppm

Mobile Phase MeoH:ACN:H2O

57:30:13

Wavelength 224nm

Column C18 Column

PH 5.4

Concentration 10ppm

Retention Time 9.89

Run Time 10min

Area 115233

Th. Plates 4453

Tailing Factor 0.91

Pump Pressure 8.2 MPa

Table1: System suitability parameters of ICATIBANT

Figure 2: Standard chromatogram of Icatibant

Range of linearity:

Standard curves were constructed daily, for three consecutive days, using seven standard concentrations in a

range of 10, 20, 30, 40, 50, and 60ppm for Icatibant. The linearity of peak area responses versus

concentrations was demonstrated by linear least square regression analysis. The linear regression equation

was y = 9304+ 3500x (r= 0.999). Linearity values can show in Table 2.

S.No Concentration (µg/ml) Area

1 10 115233

2 20 300426

3 30 458762

4 40 621482

5 50 799879

6 60 953210

Slope

Intercept

CC

16249.92

23355.9

0.999127

Table 2: Linearity results of Icatibant



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Figure 3: Calibration curve of Icatibant

Precision:

To study precision, six replicate standard solutions of Icatibant (10ppm) were prepared and

analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses

was calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%.

Results of system precision studies are shown in Table.3 and Table.4.

Sample (µg/ml) Area

1 115568

2 115798

3 116029

4 115867

5 114972

6 116003

RSD 0.342387

Table 3: Intraday Precision Results for Icatibant

-200000

0

200000

400000

600000

800000

1000000

1200000

0 10 20 30 40 50 60 70



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Sample (µg/ml) Area

1 114213

2 115689

3 115982

4 119689

5 115867

6 116021

RSD 1.565656

Table 4: Inter day Precision results of Icatibant

Limit of Detection and Limit of Quantification:

To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injected

until peak was disappeared. After 0.83ppm dilution Peak was not clearly observed, based on which 0.83ppm

is considered as Limit of Detection and Limit of Quantification is 2.5ppm.

Parameter Measured Value

Limit of Quantification 2.5ppm

Limit of Detection 0.83ppm

Table 5: LOD and LOQ results of Icatibant

Robustness:

Typical variations in liquid chromatography conditions were used to evaluate the robustness of the

assay method. The robustness study was performed by slight modification in flow rate of the mobile phase,

composition of the mobile phase and wavelength of the detector. Icatibant at standard concentration was

analyzed under these changed experimental conditions. It was observed that there were no marked changes

in chromatograms, which demonstrated that the developed method was robust in nature. The robustness

acceptance criteria set in the validation were the same established on system suitability test describe above.

Results were shown in table 6.



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S.NO Parameter Change Area % of Change

1 Standard ……………… 115233 …………

2 MP

Methanol:ACN:water

57:30:13

47:40:13

115489

116201

0.22

0.84

3 PH 5.6

5.2

115620

115976

0.33

0.64

4 WL 222nm

226nm

116097

116382

0.74

0.99

Table 6: Robustness results of Icatibant

Ruggedness:

Ruggedness was performed by using six replicate injections of standard and sample solutions of

concentrations which were prepared and analyzed by different analyst on three different. Ruggedness also

expressed in terms of percentage relative standard deviation.

SAMPLE

CONC

(PPM)

INJECTION NO

PEAKS AREA

R.S.D

(Acceptance

criteria ≤

2.0%)

Icatibant

10ppm

1 116421

0.167061

2 115986

3 116106

4 115938

5 116036

6 115874

Table 7: Ruggedness results of Icatibant



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Recovery:

The accuracy of the method was determined by standard addition method. A known amount of standard drug

was added to the fixed amount of pre-analyzed tablet solution. Percent recovery was calculated by comparing

the area before and after the addition of the standard drug. Recovery test was performed at 3 different

concentrations i.e. 90ppm, 120ppm, 150ppm. The percent recovery was calculated and results are presented

in Table. Satisfactory recoveries ranging from 98.07 to 101.14 were obtained by the proposed method. This

indicates that the proposed method was accurate. Results are given in table.8

%

Recovery

Icatibant

Target Conc.,

(ppm)

Spiked conc,

(ppm)

Final Conc,

(ppm)

Conc.,

Obtained

% of Recovery

50% 10ppm 10ppm 30ppm 29.84 99.46

50% 10ppm 10ppm 30ppm 30.28 100.9

50% 10ppm 10ppm 30ppm 30.51 101.7

100% 20ppm 20ppm 40ppm 39.32 98.3

100% 20ppm 20ppm 40ppm 40.27 100.6

100% 20ppm 20ppm 40ppm 40.61 101.5

150% 40ppm 40ppm 60ppm 59.28 98.8

150% 40ppm 40ppm 60ppm 60.41 100.6

150% 40ppm 40ppm 60ppm 60.53 100.8

Table 8: Recovery results of Icatibant

Formulation Dosage Concentration Amount found % Assay

INTELENCE 100mg 120ppm 119.80 99.83

Table 9: Formulation Analysis

CONCLUSION

The proposed method for the assay of Icatibant in tablets or capsules is very simple and rapid. It

should be emphasized it is isocratic and the mobile phase do not contain any buffer. The method was

validated for specificity, linearity, precision, accuracy and robustness. Although the method could effectively

separate the drug from its products, further studies should be performed in order to use it to evaluate the

stability of pharmaceutical formulations.



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A NOVEL RP-HPLC METHOD FOR THE QUANTIFICATION OF ICATIBANT IN FORMULATIONS

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