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, 5m 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 20l 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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B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 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-esteraseinhibitor 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. 2504.6mm, Electronic balance-DENVER (SI234), manual Rheodyne injector with a 20 l 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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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, 5) was used. The instrument is equipped with a LC 20AT pump for solvent delivery and variable wavelength programmable LC 7000 UV-detector. A 20L 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 l 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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B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16

Api Concentration Mobile Phase Wavelength Column PH Concentration Retention Time Run Time Area Th. Plates Tailing Factor Pump Pressure 10ppm MeoH:ACN:H2O 57:30:13 224nm C18 Column 5.4 10ppm 9.89 10min 115233 4453 0.91 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 1 2 3 4 5 6 Concentration (g/ml) 10 20 30 40 50 60 Slope Intercept CC Table 2: Linearity results of Icatibant Area 115233 300426 458762 621482 799879 953210 16249.92 23355.9 0.999127

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

1200000 1000000 800000 600000 400000 200000 0 0 -200000 Figure 3: Calibration curve of Icatibant 10 20 30 40 50 60 70

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 (g/ml) 1 2 3 4 5 6 RSD

Area 115568 115798 116029 115867 114972 116003 0.342387

Table 3: Intraday Precision Results for Icatibant

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

Sample (g/ml) 1 2 3 4 5 6 RSD Area 114213 115689 115982 119689 115867 116021 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 Limit of Quantification Limit of Detection Measured Value 2.5ppm 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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B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16

S.NO 1 2 Parameter Standard MP Change Methanol:ACN:water 57:30:13 47:40:13 3 4 PH WL 5.6 5.2 222nm 226nm 115489 116201 115620 115976 116097 116382 0.22 0.84 0.33 0.64 0.74 0.99 Area 115233 % of Change

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 SAMPLE (PPM)

INJECTION NO

PEAKS AREA

R.S.D (Acceptance criteria 2.0%)

1 2 10ppm Icatibant 3 4 5 6

116421 115986 116106 115938 116036 115874 0.167061

Table 7: Ruggedness results of Icatibant

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B.Lakshmi et al., IJSIT, 2012, 1(1), 08-16 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 % Recovery 50% 50% 50% 100% 100% 100% 150% 150% 150% Target Conc., (ppm) 10ppm 10ppm 10ppm 20ppm 20ppm 20ppm 40ppm 40ppm 40ppm Spiked conc, (ppm) 10ppm 10ppm 10ppm 20ppm 20ppm 20ppm 40ppm 40ppm 40ppm Final Conc, (ppm) 30ppm 30ppm 30ppm 40ppm 40ppm 40ppm 60ppm 60ppm 60ppm Conc., Obtained 29.84 30.28 30.51 39.32 40.27 40.61 59.28 60.41 60.53 99.46 100.9 101.7 98.3 100.6 101.5 98.8 100.6 100.8 % of Recovery

Table 8: Recovery results of Icatibant Formulation INTELENCE Dosage 100mg Concentration 120ppm Table 9: Formulation Analysis Amount found 119.80 % Assay 99.83

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

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