FIELD OF THE INVENTION:

The present invention relates to a sensitive reversed phase liquid chromatographic (RP-LC) method for determination of 4-Hydrazinobenzoic acid, a genotoxic impurity in Deferasirox using High Performance Liquid Chromatography method.
BACKGROUND OF THE INVENTION:

Deferasirox, as the current line available iron chelator approved by the FDA and brand name Exjade listed for the treatment of transfusion-dependent chronic iron overload (transfusional hemosiderosis disease). The structural formula for Deferasirox and 4-Hydrazinobenzoic acid is as shown below:
Deferasirox:

4-Hydrazinobenzoic acid:

4-Hydrazinobenzoic acid is one of the key starting materials used in the final step synthesis of Deferasirox. 4-Hydrazinobenzoic acid is identified as genotoxic impurity as per the structural alerts. As per recent FDA and other guidelines genotoxic impurities needs to be controlled as per TTC approach. 4-Hydrazinobenzoic acid needs to be controlled to a level of less than 0.5 ppm as per the maximum daily dose of Deferasirox.

The present invention discloses using of base deactivated octadecyl silica column as stationary phase, and mobile phase is a gradient elution using ion pair phosphate buffer and methanol with UV detection. Preparation of standard and sample solutions is through solvent extraction. The proposed extraction procedure is simple and removes the Deferasirox completely in the organic layer during sample preparation.

SUMMARY OF THE INVENTION:

The present invention provides a sensitive method for the determination of 4-hydrazinobenzoic acid, a genotoxic impurity in Deferasirox using High Performance Liquid Chromatography method with UVdetection.
One aspect of the present invention is to provide a method for determination of 4-Hydrazinobenzoic acid using HPLC; selection of base deactivated octadecyl silica as stationary phase, and mobile phase is a gradient elution using ion pair phosphate buffer and methanol thereof.

Another aspect of the present invention is to provide process for the preparation of standard and sample solution using solvent extraction by organic and aqueous solvents.

Another aspect of the present invention is to provide UV detection for the determination for 4-Hydrazinobenzoic acid.

ADVANTAGES OF THE PRESENT INVENTION:

1. Method is sensitive with cost effective and easy for operation.

2. Enhancing sensitivity of method by increasing the sample concentration,

3. Loading of Deferasirox on High Performance Liquid Chromatography column will be reduced to get the better column life.

BRIEF DESCRIPTION OF THE FIGURES:

Fig 1: Illustrates the chromatogram for blank.

Fig 2: Illustrate the chromatogram for standard solution.

Fig 3: Illustrate the chromatogram for sample solution.

Fig 4: Illustrate the chromatogram for sample solution spiked with 4-Hydrazinobenzoic acid.

Fig 5: Illustrate the chromatogram for LOQ solution of 4-Hydrazinobenzoic acid.

Fig 6: Illustrate the chromatogram for LOD solution of 4-Hydrazinobenzoic acid.

DETAILED DESCRIPTION OF THE INVENTION:

The present invention provides a sensitive method for determination of 4-hydrazinobenzoic acid, a genotoxic impurity in Deferasirox using HPLC-UV detection.

In one embodiment of the present invention provides the HPLC method for determination of 4-Hydrazinobenzoic acid is as a genotoxic impurity in Deferasirox,

Wherein uses the mobile phase comprises of two or more liquids, including first eluent-A and second eluent-B; the concentration of eluents will change using a gradient. Wherein the first eluent-A is buffer with methanol and the second eluent-B is only methanol.

In another embodiment of the present invention provides preparation of the standard solution comprises by dissolving 4-Hydrazinobenzoic acid in diluent and transferring 5 ml of the same in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane added, shake for few minutes and keep a side for separation of layers. Collected the aqueous layer for injection.

In yet another embodiment of the present invention provides a preparation of sample solution comprises by dissolving 500 mg in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane and 5 ml of diluent added, shake for few minutes and keep a side for separation of layers. Collected the aqueous layer for injection.

As used herein, "Limit of Detection (LOD) refers to the lowest concentration of analyte that can be clearly detected above the base line signal with a signal to noise ratio between 3 or 2:1

As used herein, "Limit of Quantitation (LOQ) refers to the lowest concentration of analyte that can be clearly quantified above the base line signal with a signal to noise ratio of 10:1.

As used herein, "Gradient elution" refers to the change in the composition of eluents over a period of time.

As used herein, "Specificity" refers to the ability to assess unequivocally the analyte in the presence of components which may be expected to be present

Experimental procedure :

The method for determination of 4-Hydrazinobenzoic acid is validated and comprises the steps there of:

Preparation of buffer: Dissolve about 3.4 g of potassium dihydrogen ortho phosphate and 1.0 g of octane-1-sulphonic acid sodium salt in 1000 ml of water. Adjust pH to 3.0 with orthophosphoric acid.

Mixture of buffer and methanol in the ratio of 72.5:27.5(%v/v) used as eluent A Preparation of Mobile phase B: Methanol used as eluent B.

Injecting 50 \xL of sample and standard solutions on to a Hypersil BDS C18 column with 250 mm length, 4.6 mm internal diameter and a particle size of 5 |im.

Gradient elution as initially 100% of eluent A up to 12 minutes and changing to 50% of eluent B up to 15 minutes and holding eluent B up to 25 minutes. Then equilibrating the column with initial ratio.

Measurement of levels of 4-Hydrazinobenzoid acid was done at a wavelength of 265 nm using UV detector.
A diluent of 0.5%v/v solution of orthophosphoric acid in water used during the standard and sample preparation.

Standard preparation comprises of dissolving 4-Hydrazinobenzoic acid in diluent and taking 5 ml of the same in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane added, shake for few minutes and keep a side for separation of layers. Collected the aqueous layer for injection.

Sample preparation comprises of dissolving 500 mg of sample in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane and 5 ml of diluent added, shake for few minutes and keep a side for separation of layers. Collected the aqueous layer for injection.

Preferably, specificity of this method was observed with the listed impurities of Deferasirox.

Preferably, LOD and LOQ values were estimated and tabulated in the following table no:l

Preferably, Accuracy (% Recovery) of 4-Hydrazinobenzoic acid in presence of Deferasirox were found in the range of 50 to 120% of the target value(0.5 ppm) and cumulative average values were tabulated in the following Table No: 2

CLAIMS:

1. HPLC method for determination of 4-Hydrazinobenzoic acid, genotoxic impurity in Deferasirox, wherein uses the mobile phase comprises of two or more liquids, including, first eluent- A and second eluent -B, and the concentration of eluents will change using a gradient.

2) Accordingly to claim I, wherein the first eluent-A is ion pair phosphate buffer with methanol and second eluent-B is methanol.

3) Accordingly to claim 1, the gradient elution of gradient elution as initially 100% of eluent A up to 12 minutes and changing to 50% of eluent B up to 15 minutes and holding eluent B up to 25 minutes.

4)A HPLC method for determining 4-Hydrazinobenzoic acid where in uses the standard preparation comprises of dissolving 4-Hydrazinobenzoic acid in diluent and taking 5 ml of the same in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane added and shake for few minutes to isolate the aqueous layer for injection.

5)A HPLC method for determining 4-Hydrazinobenzoic acid where in uses sample preparation comprises of dissolving 500 mg in a separating funnel which is having 10 ml of tetrahydrofuran, then 40 ml of dichloromethane and 5mL of diuent added and shake for few minutes to isolate the aqueous layer for injection.

6)Accordingly to preceding claims, wherein the diluent used as 0.5%v/v orthophosphoric acid in water; buffer as dissolve about 3.4 g of potassium dihydrogen ortho phosphate and 1.0 g of octane-1-sulphonic acid sodium salt in 1000 ml of water (adjust pH to 3.0 with orthophosphoric acid)

7) HPLC method accordingly to preceding claims, where in uses UV detection of 265 nm; wherein uses injection volume > 50 \xL and column oven temperature of 40° C ± 2° C.