DESC:Field of the invention
The present invention relates to improved reversed-phase liquid chromatographic (RP-LC) methods for the quantitative determination of Febuxostat. The present inventions further provide stability indicating analytical methods using the samples generated from forced degradation studies.

Background of the invention
Febuxostat is chemically known as 2-[3-Cyano-4-(2-methylpropoxy)phenyl]-4-methyl-5-thiazolecarboxylic acid.
Febuxostat is a non-purine selective inhibitor of xanthine oxidase. It works by non-competitively blocking the molybdenum pterin center which is the active site on xanthine oxidase. Xanthine oxidase is needed to successively oxidize both hypoxanthine and xanthine to uric acid. Hence, febuxostat inhibits xanthine oxidase, therefore reducing production of uric acid. Febuxostat inhibits both, oxidized as well as reduced form of xanthine oxidase because of which febuxostat cannot be easily displaced from the molybdenum pterin site.

Structure of Febuxostat

The product mixture of a reaction rarely is a single compound pure enough to comply with pharmaceutical standards. Side products and byproducts of the reaction and adjunct reagents used in the reaction will, in most cases, be present. At certain stages during processing of the febuxostat contained in the product mixture into an active pharmaceutical ingredient (“API”), the febuxostat must be analyzed for purity, typically by UPLC, HPLC or GC analysis, to determine if it is suitable for continued processing or ultimately for use in a pharmaceutical product.

The U.S. Food and Drug Administration’s Center for Drug Evaluation and Research (CDER) has promulgated guidelines recommending that drug applicants identify organic impurities of 0.1% or greater in the active ingredient. “Guideline on Impurities in New Drug Substances,” 61 Fed. Reg. 371 (1996); “Guidance for Industry ANDAs: Impurities in Drug Substances,” 64 Fed. Reg. 67917(1999). Unless an impurity has been tested for safety, is in a composition proven to be safe in clinical trials, or is a human metabolite, the CDER further recommends that the drug applicant reduce the amount of the impurity in the active ingredient to below 0.1%. In order to obtain marketing approval for a new drug product, manufacturers must submit to the regulatory authority evidence that the product is acceptable for administration to humans. Such a submission must include, among other things, analytical data showing the impurity profile of the product to demonstrate that the impurities are either absent, or present in a negligible amount. Therefore, there is a need for analytical methods to detect impurities to identify and assay those impurities.

Generally, impurities (side products, byproducts, and adjunct reagents) are identified spectroscopically and by other physical methods and then the impurities are associated with a peak position in a chromatogram (or a spot on a TLC plate). Thereafter, the impurity can be identified by its position in the chromatogram, which is conventionally measured in minutes between injection of the sample on the column and elution of the particular component through the detector, known as the “retention time” (“Rt”). This time period varies daily based upon the condition of the instrumentation and many other factors. To mitigate the effect that such variations have upon accurate identification of an impurity, practitioners use “relative retention time” (“RRt”) to identify impurities.

Summary of the invention
In one aspect, the present invention provides a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of febuxostat.

In another aspect, the present invention provides an HPLC method for febuxostat containing less than about 5% area by HPLC, preferably less than about 3% area by HPLC, more preferably less than 1% area by HPLC, of total impurities.
In another aspect, the present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
In yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating a high performance liquid chromatography (HPLC) method for the determination of febuxostat in the presence of degradation products.

In one aspect, the HPLC method described in the present invention has the following advantages when compared with prior art methods for determining the febuxostat and its related impurities:
i) All the impurities were well separated with a minimum resolution 1.5 (limit: Not less than 1.2);
ii) Gradient profile to elute all related impurities and organic phase is 70% which ensure the elution and detection of non polar impurities forming during the process or stress study;
iii) The present method mobile phase pH is about 2.5 which is more stable in all C18 HPLC columns;
iv) Consistency in specificity, precision & reproducibility with good peak shape; and
v) The degradation impurities from stress studies are well separated from the known impurities.

Brief description of drawings
Fig. 1 illustrates the HPLC chromatogram of spiked (Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X spiked in febuxostat) sample.

Detailed description of the invention
As used herein, “limit of detection (LOD)” refers to the lowest concentration of analyte that can be clearly detected above the base line signal, is estimated is three times the signal to noise ratio.

As used herein, “limit of quantization (LOQ)” refers to the lowest concentration of analyte that can be quantified with suitable precision and accuracy, is estimated as ten times the signal to noise ratio.
As used herein, “gradient elution” refers to the change in the composition of the gradient eluent over a fixed period of time, stepwise or at a constant rate of change, as the percentage of the first eluent is decreased while the percentage of the second eluent is increased.
As used herein, “gradient eluent” refers to an eluent composed of varying concentrations of first and second eluent.
The eight main known impurities of febuxostat are:

(i) 2-[3-Carbamoyl-4-(2-methyl propoxy) phenyl]-4-methylthiazole-5-carboxylic acid (Impurity-I )which has the following structure:


The impurity-I is detected and resolved from febuxostat by HPLC with a relative retention time (hereafter referred as RRT) of 0.38.

(ii) 2-[3-Carboxy- 4-(2-methyl propoxy) phenyl]-4-methylthiazole-5- carboxylic acid (Impurity-II), which has the following structure:

Impurity-II is detected and resolved from febuxostat by HPLC with an RRT of 0.45.

(iii) (2-[3-Formyl- 4-(2-methyl propoxy) phenyl]-4-methylthiazole-5-carboxylic acid (Impurity-III), which has the following structure:

The impurity-III is detected and resolved from febuxostat by HPLC with an RRT of 1.11.

(iv) 2-[ 4-(2-methylpropoxy) phenyl]-4-methylthiazole-5-carboxylic acid (Impurity-IV), which has the following structure:

The impurity-IV is detected and resolved from febuxostat by HPLC with an RRT of 1.43.

(v) 2-[3-(hydroxyimino) methyl-4-(2-methyl propoxy) phenyl]-4-methylthiazole-5-carboxylic acid (Impurity-V), which has the following structure:

The impurity- V is detected and resolved from febuxostat by HPLC with an RRT of 0.76

(vi) Ethyl 2-[3 – Cyano - 4 - (2-methylpropoxy) phenyl]- 4 –methyl–5–thiazole carboxylate (Impurity-VI) , which has the following structure:

The impurity-VI is detected and resolved from febuxostat by HPLC with an RRT of 2.46

(vii) Ethyl 2-[3-hydroxyiminomethyl-4-(2-methylpropoxy) phenyl]-4-methyl-5-thiazole carboxylate (Impurity-VII) which has the following structure:


Impurity-VII is detected and resolved from febuxostat by HPLC with an RRT of 2.11

(viii) (Ethyl 2-[3–Formyl–4-(2-methylpropoxy)phenyl]-4-methyl-5-thiazole carboxylate (Impurity-VIII) which has the following structure:

Impurity-VIII is detected and resolved from febuxostat by HPLC with an RRT of 2.69

(ix) (2-[3-Cyano-4-propoxyphenyl]-4-methyl-5-thiazolecarboxylic acid (Impurity-IX) which has the following structure

Impurity-IX is detected and resolved from febuxostat by HPLC with an RRT of 0.72

(x) 2-(3-cyano-4-hydroxyphenyl)-4-methyl-1,3-thiazole-5-carboxylic acid (Impurity-X) which has the following structure


Impurity-X is detected and resolved from febuxostat by HPLC with an RRT of 0.28

According to one aspect of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method for quantifying, by area percent, the amounts of febuxostat and all impurities, preferably, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X present in a sample of febuxostat.

According to another aspect of the present invention, there is provided a stability indicating analytical method using the samples generated from forced degradation studies.

According to another aspect of the present invention, there is provided an accurate and well-defined stability indicating HPLC method for the determination of febuxostat in the presence of degradation products.

Preferably, the method for determining the amount of impurities in a febuxostat sample comprises the steps of:
a) combining a Febuxostat sample with buffer and acetonitrile in the ratio of 40:60 (v/v) to obtain a solution;
b) injecting the sample solution into a 250 mm x 4.6 mm column with 5 µm Kromasil 100-5C18 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 50:50 (v/v) remained up to 8 minutes and progressively increased to 20:80(v/v) with in 12 minutes , further it remained 20:80(v/v) for 15 minutes.
d) Preparing Eluent A by dissolving 1.4 g of sodium perchlorate hydrate in 1000 mL of water. Adjust pH to 2.5 + 0.05 with perchloric acid. and filter through 0.22 micron filter paper.
e) Measuring of the amounts of febuxostat and each impurity at 316 nm wavelength with a UV detector (having an appropriate recording device).

Preferably, the initial ratio of eluent A and eluent B in step-(c) may be continued at the same ratio for 8 minutes then changed linearly to 20:80 (v/v) with in 12 minutes followed by same ratio for 15 minutes. After 3.0 minutes the initial gradient of 40:60 is for 7.0 minutes to be conditioned for every analysis. The column temperature may be maintained at about 30°C.
The LOD /LOQ values of febuxostat and its related impurities, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X are summarized in Table 1.

Table 1

Components LOD (%) LOQ (%)
Impurity-I 0.0017 0.0051
Impurity-II 0.0022 0.0066
Impurity-III 0.0046 0.0140
Impurity-IV 0.0016 0.0049
Impurity-V 0.0016 0.0049
Impurity-VI 0.0019 0.0057
Impurity-VII 0.0025 0.0076
Impurity-VIII 0.0020 0.0061
Impurity-IX 0.0018 0.0053
Impurity-X 0.0016 0.0047
Febuxostat 0.0020 0.0060

Specificity is the ability of the method to measure the analyte response in the presence of its potential impurities and degradation products. The specificity of the LC method for febuxostat Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1.0M HCl), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H2O2), thermal and photo degradation to evaluate the ability of the proposed method to separate febuxostat from its degradation products. To check and ensure the homogeneity and purity of febuxostat peak in the stressed sample solutions, PDA-UV detector was employed.

Preferably, the limit of detection (LOD) and limit of quantification (LOQ) were estimated by signal to noise ratio method, by injecting a diluted solution with known concentration.
The accuracy of the related substances method with the spiked impurities was evaluated at 0.15 % concentration level,
According to another aspect of the present invention, there is provided a chromatographic method to get the separation of all impurities and stress studies degradants from analyte peak. Satisfactory chromatographic separation was achieved using the mobile phase consists of buffer (Eluent A by dissolving 1.4 g of sodium perchlorate hydrate in 1000 mL of water. Adjust pH to 2.5 + 0.05 with perchloric acid) In the optimized conditions the febuxostat, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X were well separated with a resolution of 1.5 and the typical retention times (RT) of febuxostat, Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X were about 10.5, 4.0, 4.7, 11.7, 15.1, 8.0, 25.9, 22.2, 28.3, 7.6 and 3.0 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for febuxostat and its ten impurities, namely Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X. The system suitability values and mass numbers of febuxostat and its impurities were summarized in Table 2.
Table 2
Compound (n=1) Rt Rs N T (m/z)
Impurity-I 4.01 6.84 10628.4 1.14 334.3
Impurity-II 4.72 4.22 11240.6 1.13 335.3
Impurity-III 11.69 2.97 11603.1 319.3
Impurity-IV 15.09 7.18 17254.4 1.59 291.3
Impurity-V 7.97 1.33 13591.5 334.3
Impurity-VI 25.89 12.15 98369.7 1.000 344.4
Impurity-VII 22.16 18.67 103937.4 1.00 362.4
Impurity-VIII 28.29 6.28 71478.2 0.98 347.4
Impurity-IX 7.59 12.09 11396.4 302.3
Impurity-X 2.97 - 7750.9 0.99 260.2
Febuxostat 10.52 8.51 17947.9 1.12 316.3

*n=1: determination, Rt: retention time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.

High level of degradation in test solution was observed using 1M sodium hydroxide at 60°C for 6 hours. Impurities observed in stress condition using PDA detector. Major degradants was impurity-I .Other unknown were also specific in this method .The peak test results obtained from PDA & LC-MS/MS confirm that the febuxostat peak is homogeneous and pure in all analyzed stress samples.

The percentage recovery of febuxostat of its impurities in bulk drug samples was done at 0.15 %. The percentage recovery of Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX and impurity-X in bulk drugs samples was ranged from 90.00 to 110.00.
In deliberate varied chromatographic conditions (pH and column), the robustness of the method is confirmed.

Experimental
The LC system, used for method development and forced degradation studies and method validation was Waters-Alliance (manufactured by Waters India Ltd) LC system with a photo diode detector. The out put signal was monitored and processed using Empower software system (designed by Waters India) on IBM computer (Digital Equipment Co).
The chromatographic column used was an Akzo Nobel make Kromasil 100-5C18 (250 mm x 4.6 mm) with 5.0µm particles. The mobile phase consists buffer (1.4 g of sodium perchlorate hydrate in 1000 mL of water. Adjust pH to 2.5 + 0.05 with perchloric acid)), and solvent is acetonitrile. The flow rate of the mobile phase was kept at 1.0 ml/min. beginning with the gradient ratio of mobile phase buffer and solvent 40:60(v/v), system was continued at the same ratio for 8 minutes. The ratio was changed linearly 20:80(v/v) within 12 minutes and again system was continued at the same ratio for 15 minutes. After 3.0 minutes the initial gradient of 40:60 is for 7 minutes to be conditioned for every analysis. The column temperature was maintained at 30°C and the wavelength was monitored at a wavelength of 316 nm. The injection volume was 10 µL for related substances determination. Buffer was used as diluent during the standard and test samples preparation.

Preparation of reference solution-1:
Weigh and transfer about 7.5 mg of each of impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI (Stage-I), impurity-VII (Stage-I-insitu), impurity-VIII (KSM), impurity-IX (n-propyl febuxostat), impurity-X (Desisobutyl impurity) and 5 mg of febuxostat standard into a 100 mL volumetric flask, add about 10 mL of methanol and 80 mL of acetonitrile. Sonicate to dissolve and make up the volume with diluent. Mix well and transfer 1 mL of this solution into a 10 mL volumetric flask. Add 8 mL of Acetonitrile mix well and make up the volume with diluent.
Weigh and transfer about 5 mg of febuxostat standard into a 10 mL volumetric flask, add 1 mL of above system suitability stock solution and 8 mL of Acetonitrile. Sonicate to dissolve and make up the volume with diluent and mix well.

Reference solution-(a) preparation (Impurity-I to impurity-X @ 0.15% and API @ 0.1% level):
Transfer 1 mL of system suitability stock solution in to a 10 mL volumetric flask. Add 8 mL of
Acetonitrile, makeup the volume with diluent and mix well

Reference solution-(b) preparation (API @ 0.1 % level):
Weigh and transfer about 25 mg of febuxostat standard into a 50 mL volumetric flask, add 45 mL of acetonitrile and sonicate to dissolve. Make up the volume with diluent and mix well. Transfer 1 mL of this solution in to a 100 mL volumetric flask. Add 90mL of acetonitrile and mix well. Makeup the volume with diluent and mix well. Transfer 1 mL of the resulting solution into a 10 mL volumetric flask. Add 8 mL of acetonitrile and mix well. Makeup the volume with diluent and mix well.

,CLAIMS:Claims:

1. A reversed-phase liquid chromatographic (RP-LC) method for analyzing Febuxostat,wherein the mobile phase comprises of two or more liquids,including a first eluent A and a second eluent B, and the relative concentration of the liquids is varied to the predetermined gradient.

2. A reversed-phase liquid chromatographic (RP-LC) method according to claim 1, wherein the first eluent A is buffer.

3. A reversed-phase liquid chromatographic (RP-LC) method according to claim 1, wherein the first eluent B is Acetonitrile.

4. A reversed-phase liquid chromatographic (RP-LC) method according to claim 1, wherein gradient A Eluent and B Eluent in the ratio of 50:50 (v/v) initial,and progressively increased to 20:80(v/v) for 15 minutes.

5. A reversed-phase liquid chromatographic (RP-LC) method according to claim 2, wherein buffer is 1.4 g of sodium perchlorate hydrate in 1000 mL of water and pH about 2.55.

6. A reversed-phase liquid chromatographic (RP-LC) method for Febuxostat containing less than about 5 % area by RPLC,preferably less than about 3 % area by RPLC,more preferably less than about 1 % area by RPLC, of total impurities.

7. A reversed-phase liquid chromatographic (RP-LC) method for determining the amount of impurities in febuxostat sample comprises the steps of :
a) combining a Febuxostat sample with buffer and acetonitrile in the ratio of 40:60 (v/v) to obtain a solution;
b) injecting the sample solution into a 250 mm x 4.6 mm column with 5 µm Kromasil 100-5C18 column;

c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 50:50 (v/v) remained up to 8 minutes and progressively increased to 20:80(v/v) with in 12 minutes , further it remained 20:80(v/v) for 15 minutes.
d) Preparing Eluent A by dissolving 1.4 g of sodium perchlorate hydrate in 1000 mL of water. Adjust pH to 2.5 + 0.05 with perchloric acid. and filter through 0.22 micron filter paper.
e) Measuring of the amounts of febuxostat and each impurity at 316 nm wavelength with a UV detector (having an appropriate recording device).