This document describes the development and validation of a novel RP-HPLC method for the quantification of Icatibant in formulations. A simple isocratic elution method was developed using a C18 column with methanol, acetonitrile and water as the mobile phase. The method was validated according to ICH guidelines and showed good linearity, precision, accuracy and robustness. The developed method was successfully applied to analyze Icatibant tablet formulations and bulk drug samples.

1. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16
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.
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3. B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16
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 16249.92
Intercept 23355.9
CC 0.999127
Table 2: Linearity results of Icatibant
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1200000
1000000
800000
600000
400000
200000
0
0 10 20 30 40 50 60 70
-200000
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
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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 0.22
116201 0.84
3 PH 5.6 115620 0.33
5.2 115976 0.64
4 WL 222nm 116097 0.74
226nm 116382 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.
CONC INJECTION NO PEAKS AREA R.S.D
SAMPLE
(PPM) (Acceptance
criteria ≤
2.0%)
1 116421
2 115986
10ppm 3 116106
Icatibant 0.167061
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
Icatibant
% Target Conc., Spiked conc, Final Conc, Conc., % of Recovery
Recovery (ppm) (ppm) (ppm) Obtained
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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