FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention - An improved method for the quantitative determination of Prasugrel
HC1.
2. Applicants)
(a) NAME : ALEMBIC PHARMACEUTICALS LIMITED
(b) NATIONALITY: An Indian Company.
(c) ADDRESS: Alembic Campus, Alembic Road,
Vadodara-390, 003, Gujarat, India
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed:

Field of the invention
The present invention relates to an improved reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of Prasugrel HC1. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
Background of the invention
Prasugrel HC1 is chemically known as 2-Acetoxy-5-(a-cyclopropylcarbonyl-2-fluorobenzyl)-4,5,6,7-tetrahydrothieno [3,2-c] pyridine hydrochloride
Prasugrel is a prodrug, oxidation by intestinal and hepatic cytochrome P-450 enzymes convert prasugrel into its active metabolite. Prasugrel has a rapid and almost complete absorption after oral ingestion of a loading dose. Its active form binds irreversibly to the adenosine diphosphate (ADP) P2Y12 receptor on platelets for their lifespan, thereby inhibiting their activation and decreasing subsequent platelet aggregation.
Prasugrel has a greater antiplatelet effect than clopidogrel because it is metabolized more efficiently. Some of the differences in metabolism between clopidogrel and prasugrel may be explained by genetic polymorphisms affecting the cytochrome P-450 system.

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 Prasugrel HC1 contained in the product mixture into an active pharmaceutical ingredient ("API")) the Prasugrel HC1 must be analyzed for purity,

typically by 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 Prasugrel HC1.
In another aspect, the present invention provides an HPLC method for Prasugrel HCI 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 an Ultra performance liquid chromatography (UPLC) method for the determination of Prasugrel HCI 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 Prasugrel HCI and its related impurities: i) All the impurities were well separated with a minimum resolution 3.0 (limit: Not less
than 1.5); 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 normal
C8 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 chromatograms of spiked (SC-III, PTSA, Oxo compound, MC-I, MC-II, MC-III and SC-II spiked in Prasugrel HC1) samples.
Detailed description of the invention
As used herein, "limit of detection (LOD)" refers to the lowest concentration of analyze 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 eluents.
The five main known impurities of Prasugrel HC1 are:
(i) 2-0x0-2,4,5,6,7,7a-hexahydrothieno[3,2-c]pyridine-p-toluenesulfonate (Side chain Stage-Ill) , which has the following structure:

The Side chain Stage-Ill is detected and resolved from Prasugrel HC1 by HPLC with an relative retention time (hereafter referred as rrt) of 0.13.

(ii) p-Toluenesulfonic acid monohydrate (PTSA), which has the following structure:

The PTSA is detected and resolved from Prasugrel HC1 by HPLC with an rrt of 0.65.
(iii) 2-Oxo-5-(a-cyclopropylcarbonyl-2-fluorobenzyl)2,4,5,6,7,7a-hexahydro thieno[3,2-cjpyridine. (Oxo Compound), which has the following structure:

The Oxo Compound is detected and resolved from Prasugrel HC1 by HPLC with an rrt of 1.46 and 1.52.
(iv) Cyclopropyl-2-fluorobenzyl ketone (MC-I), which has the following structure:

The impurity MC-I is detected and resolved from Prasugrel HC1 by HPLC with an rrt of 1.62.
(v) a-Cyclopropylcarbonyl-2-fluoro benzylbromide (MC-II), which has the following structure:


The impurity MC- II is detected and resolved from Prasugrel HCI by HPLC with an RRt of 1.91.
(vi) 2-(Tert-Butyldimethylsilyloxy)-5-(α-cyclopropylcarbonyJ-2-fluorobenzy])- 4,5,6,7-tetrahydrothieno[3,2-c] pyridine. (MC-III)_, which has the following structure:

The impurity MC- III is detected and resolved from Prasugrel HCI by HPLC with an RRt
of 2.26.
(vii) 5-Triphenylmethyl-2-oxo-2, 4, 5,6,7,7a-hexahydro thieno[3,2-c]pyridine
(SC-II) which has the following structure:

The impurity SC- II is detected and resolved from Prasugrel HCI by HPLC with an RRt of 2.93.
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 Prasugrel HCI and all impurities, preferably, SC-III,PTSA,Oxo compound, MC-I,MC-II,MC-III and SC-II present in a sample of Prasugrel HCI.
According to another aspect of the present invention, there is provided a stability indicating analytical method using the samples generated from forced degradation studies.

Accoarding to another aspect of the present invention, there is provided an accurate and well-defined stability indicating an HPLC method for the determination of Prasugrel HC1 in the presence of degradation products.
Preferably, the method for determining the amount of impurities in a Prasugrel HC1 sample comprises the steps of:
a) combining a Prasugrel sample with acetonitrile to obtain a solution;
b) injecting the sample solution into a 250mm x4.6 mm column with 5 (im Symmetry C8 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 80:20 (v/v) initial and progressively increased to 30:70(v/v) in 35 minutes .
d) preparing Eluent A by adding 1 ml of Triethylamine in water and the pH adjusted was about 2.5 with orthophosphoric acid solution
e) preparing B Eluent with acetonitrile ; and
f) Measuring of the amounts of Prasugrel and each impurity at 220nm wavelength with a UV detector (having an appropriate recording device.
The buffer in step-(d) may be prepared by mixing about 3 L water with about 1 ml of triethylamine adjusted pH= 2.5 with orthophosphoric acid solution.
Preferably, the ratio of mobile phase buffer and solvent in step-(d) may be continued at the same ratio for 4 minutes then changed linearly to 30:70 (v/v) within 31 minutes followed by same ratio for 25 minutes. After 2 minutes the initial gradient of 80:20 is for 8 minutes to be conditioned for every analysis. The column temperature may be maintained at about 30°C.
The LOD /LOQ values of Prasugrel and its related impurities, SC-III, PTSA, Oxo compound, MC-I, MC-II, MC-III and SC-II are summarized in Table 1.

Table 1

S.No Components LOQ(μg/m]) LOD (ng/ml)
1 SC-III 0.08 0.03
2 PTSA 0.15 0.05
3 Oxo compound peak-1 0.38 0.13
4 Oxo compound peak-2 0.15 0.05
5 MC-I 0.38 0.13
6 MC-II 0.15 0.05
7 MC-III 0.15 0.05
8 SC-II 0.15 0.05
9 Prasugrel 0.15 0.05
Specificity is the ability of the method to measure the analyze response in the presence of its potential impurities and degradation products. The specificity of the LC method for Prasugrel intentional degradation was attempted to stress conditions of acid hydrolysis (using 1.0M HC1), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H202) to evaluate the ability of the proposed method to separate Prasugrel from its degradation products. To check and ensure the homogeneity and purity of Prasugrel 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 % of concentration levels.
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 (1 ml triethylamine in 1000ml of HPLC water pH adjusted was 2.5, mid solvent acetonitrile.
In the optimized conditions the Prasugrel SC-III, PTSA, Oxo compound, MC-I,MC-II,MC-III and SC-II were well separated with a resolution of greater than 3 and the typical retention times (RT) of SC-III,PTSA, Prasugrel Oxo compound, MC-I,MC-II,MC-III and SC-II were about 2.15, 10.59, 16.35 , 23.82& 24.89 ,26.53,31.23,37.02 and 47.89 minutes , and typically shown in Pigure 1. The system suitability results and the developed LC method was found to be specific for Prasugrel and its seven impurities, namely SC-III, PTSA, Oxo compound, MC-I,MC-II,MC-III and SC-II.
The system suitability values and mass numbers of Prasugrel and its impurities were summarized in Table 2.
Table 2

Compound (n=l) Rt Rs N T (m/z)
SC-III 2.15 - 2613 1.00 327
PTSA 10.59 33.06 15869 1.11 190.20
Prasugrel 16.35 14.37 20838 0.69 409.94
Oxo compound peak-1 23.82 22.02 99972 0.95 331.40
Oxo compound peak-2 24.89 3.48 .106752 0.94 331.40
MC-I 26.53 4.58 155023 1.00 178.20
MC-II 31.23 4.14 211061 1.02 257.09
MC-III 37.02 19.31 210551 0.98 445.66
SC-II 47.89 26.51 149859 0.99 397.53
*n=1etical plates (USP tangent method), T: USP tailing factor, m/z: mass number.
High level of degradation in test solution was observed using 3% hydrogen peroxide at 60°C for 3 hrs, 1M sodium hydroxide at 60°C for 3 hrs and 1M HCL at 60°C for 3 hours. Impurities observed in stress condition using PDA detector major degradiant was oxo compound. Other unknown were also specific in this method .The peak test results obtained from PDA & LC-MS/MS confirm that the Prasugrel peak is homogeneous and pure in all analyzed stress samples.

The percentage recovery of Prasugrel of its impurities in bulk drug samples was done at 0.15 %. The percentage recovery of SC-III, PTSA, Oxo compound, MC-I, MC-II, MC-III and SC-II in bulk drugs samples was ranged from 90.00 to 110.00.
In deliberate varied chromatographic conditions (pH and column) the resolution between all the components is not less than 3.0, illustrating the robustness of the method.
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 a Waters symmetry C8 250mmx4.6 mm column with 5 urn particles. The mobile phase consists buffer (1 ml of triethylamine in 1000ml of HPLC water pH-2.5 with ortho phosphoric 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 80:20(v/v), system was continued at the same ratio for 4 minutes. The ratio was changed linearly 30:70(v/v) within 31 minutes and again system was continued at the same ratio for 25 minutes. After 2 minutes the initial gradient of 80:20 is for 8 minutes to be conditioned for every analysis. The column temperature was maintained at 30°C and the wavelength was monitored at a wavelength of 220 nm. The injection volume was 20 μL for related substances determination. Acetonitrile was used as diluent during the standard and test samples preparation.
Preparation of reference solution-1
7.5 mg of each MC-II and SC-II were accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A), separately; 20ml of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with

diluent. Pipette out S.OmL from solution and transferred in to a 50mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.
Preparation of reference solution-2.
7.5 mg of each SC-III, PTSA, Oxo compound, MC-I, ,MC-III and prasugrel were accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A) , separately; 20ml of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Pipette out S.OmL from solution and transferred in to a 50mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.
A working solution of lOOOug/ml was prepared for related substances determination analysis.

We Claim,
1. A HPLC method for analyzing Prasugrel hydrochloride, wherein the mobile phase comprises two or more liquids including a first eluent A and a second eluent B, and the relative concentration of the liquids is varied to a predetermined gradient.
2. A HPLC method according to claim 1, wherein the first eluent A is buffer.
3. A HPLC method according to claim 1, wherein the first eluent B is acetonitrile.
4. A HPLC method according to claim 2, wherein buffer 1 ml of triethylamine in 1000ml of HPLC water pH-2.5.
5. A HPLC method for Prasugrel hydrochloride 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.
6. A HPLC method determining the amount of impurities in Prasugrel hydrochloride sample comprises the steps of:

a) combining a Prasugrel sample with acetonitrile to obtain a solution;
b) injecting the sample solution into a 250mmx4.6 mm column with 5 urn Symmetry C8 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 80:20 (v/v) initial and progressively increased to 30:70(v/v) in 35 minutes.
d) preparing Eluent A by adding 1 ml of Triethylamine in water and the pH adjusted was about 2.5 with orthophosphoric acid solution
e) preparing B Eluent with acetonitrile ; and
f) Measuring of the amounts of Prasugrel and each impurity at 220nm wavelength with a UV detector (having an appropriate recording device).