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 Ivabradine hydrochloride.
2. Applicant(s)
(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 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 Ivabradine hydrochloride. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
Background of the invention
Ivabradine hydrochloride is chemically known as 3-[3-({[(7S)-3,4-dimethoxybicyclo [4.2.0]octa-l,3,5-trien-7-yl]methyl}(methyl)amino)propyl]-7,8-dimethoxy-l,3,4,5-tetrahydro-2H-3-benzazepin-2-one hydrochloride.
The first selective and specific 1(f) inhibitor, for the symptomatic treatment of chronic stable angina pectoris in patients with normal sinus rhythm who have a contraindication or intolerance to beta-blockers. Ivabradine is the first pure heart rate-lowering agent and acts by selective inhibition of the cardiac pacemaker 1(f) current, which controls spontaneous diastolic depolarization in the sinus node and regulates heart rate. Ivabradine's effects are selective to the sinus node; it has no effect on intracardiac conduction, myocardial contractility or ventricular depolarization. Unlike beta-blockers, Ivabradine is free from sexual disturbances, respiratory side effects caused by constriction or spasm of the airways, bradycardia or rebound phenomena.
Structure of Ivabradine hydrochloride

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 Ivabradine hydrochloride contained in the product mixture into an active pharmaceutical ingredient ("API"), the Ivabradine hydrochloride 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 Ivabradine hydrochloride

In another aspect, the present invention provides an HPLC method for Ivabradine 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,
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 High performance liquid chromatography (HPLC) method for the determination of ivabradine hydrochloride 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 ivabradine
hydrochloride and its related impurities:
i) All the impurities were well separated with a minimum resolution 2.0
ii) Gradient profile to elute all related impurities and organic phase is 20% 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 7.5 which is more stable in most of the
CI8 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 ivabradine hydrochloride) 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 ten main known impurities of ivabradine hydrochloride are:
(i) l-[(75)-3,4-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]-N-
methylmethanamine hydrochloride (Impurity-I) which has the following structure:

The impurity-I is detected and resolved from ivabradine hydrochloride by HPLC with an relative retention time (hereafter referred as RRt) of 0.21.
(ii) l-(3,4-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl)methanamine hydrochloride (Impurity-II), which has the following structure:

Impurity-II is detected and resolved from ivabradine hydrochloride by HPLC with an RRT of 0.18.

(iii)3-(3-chloropropyl)-7,8-dimethoxy-l,3,4,5-tetrahydro-2H-3-benzazepin- 2-one (Impurity-III), which has the following structure:

The impurity-III is detected and resolved from ivabradine hydrochloride by HPLC with an RRTof 0.39.
(iv)N-{[(75)-3,4-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methyl}-TV-methylacetamide (Impurity-IV), which has the following structure:

The impurity-IV is detected and resolved from ivabradine hydrochloride by HPLC with an RRT of 0.60.
(v) 3-(7,8-dimethoxy-2-oxo-l,2,4,5-tetrahydro-3H-3-benzazepin-3-yl)propyl acetate (Impurity-V), which has the following structure:

The impurity- V is detected and resolved from ivabradine hydrochloride by HPLC with an RRT of 0.62.
(vi) 3-(3-chloropropyl)-7,8-dimethoxy-l,3,4,5-tetrahydro-2H-3-benzazepin- 2-one (Impurity-VI), which has the following structure:


The impurity-VI is detected and resolved from ivabradine hydrochloride by HPLC with an RRTof 0.75.
(Vii)3,3'-propane-l,3-diylbis(7,8-dimethoxy-1,3,4,5-tetrahydro-2H-3-benzazepin-2-one) (Impurity-VII) which has the following structure:

Impurity-VII is detected and resolved from ivabradine hydrochloride by HPLC with an RRT of 0.78.
(viii)3-[3-({[(7S)-3,4-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methyl}(methyl)amino)propyl]-7,8-dimethoxy-l,3 -dihydro-2H-3-benzazepin-2-one hydrochloride (Impurity-VIII) which has the following structure:

Impurity-VIII is detected and resolved from ivabradine hydrochloride by HPLC with an RRTof 1.08.
(ix)3-[3-({[(7S)-3,4-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methyl} (methyl) amino) propyl]-7,8-dimethoxy-l,3,4,5-tetrahydro-2H-3-benzazepin-2-one -N-oxide (Impurity-IX) which has the following structure.

Impurity-IX is detected and resolved from ivabradine hydrochloride by HPLC with an RRTof 0.56.
(x) [4,5-dimethoxy-2-(2-{3-(methylamino) {[(7S)-3,4-dimethoxybicyclo [4.2.0] octa-l,3,5-trien-7-yl]methyl}propyl amino}ethyl)phenyl]acetic acid (Impurity-X) which has the following structure.


Impurity-X is detected and resolved from ivabradine hydrochloride by HPLC with an RRT of 0.67.
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 ivabradine hydrochloride 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 ivabradine hydrochloride.
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 ivabradine hydrochloride in the presence of degradation products.
Preferably, the method for determining the amount of impurities in ivabradine hydrochloride sample comprises the steps of:
a) combining a Ivabradine hydrochloride sample with mixture of water and methanol in the ratio of 50:50 (v/v) to obtain a solution;
b) injecting the sample solution into a 250 mm x 4.6 mm column with 5 um X Bridge CI8 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 45 minutes.
d) Preparing Eluent A by mixing buffer and methanol in the ratio of 950:50 (v/v)
e) Measuring of the amounts of ivabradine and each impurity at 230nm wavelength with a UV detector (having an appropriate recording device.

Preferably, the initial ratio of A Eluent and B Eluent in step-(c) may be continued at the same ratio for 5 minutes then changed linearly to 30:70 (v/v) within 40 minutes followed by same ratio for 15 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
45°C.
The LOD /LOQ values of ivabradine hydrochloride 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

S.No Components LOD (%) LOQ (%)
1 Impurity-I 0.0036 0.0108
2 Impurity-II 0.0053 0.0161
3 Impurity-III 0.0039 0.0118
4 Impurity-IV 0.0076 0.0230
5 Impurity-V 0.0060 0.0182
6 Impurity-VI 0.0060 0.0183
7 Impurity-VII 0.0053 0.0162
8 Impurity-VIII 0.0055 0.0166
9 Impurity-IX 0.0056 0.0170
10 Impurity-X 0.0040 0.0122
11 Ivabradine hydrochloride 0.0048 0.0146
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 ivabradine hydrochloride, intentional degradation was attempted using 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 ivabradine hydrochloride from its degradation products. To check and ensure the homogeneity and purity of ivabradine 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 of triethylamine mixed with 1000 mL of water. Adjust the pH to 7.5 ± 0.05 with diluted orthophosphoricacid) and organic solvents namely methanol and acetonitriie. In the optimized conditions ivabradine hydrochloride, 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 2.0 and the typical retention times (RT) of ivabradine hydrochloride,
impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-
VII, impurity-VIII, impurity-IX, and impurity-X were about 34.4, 7.1, 6A, 13.3, 20.7,
21.3, 25.9, 26.9, 37.1, 19.2 and 23.1 minutes respectively, and typically shown in
Figure 1. The system suitability results and the developed LC method was found to be
specific for ivabradine and its ten impurities, namely impurity-I, impurity-II, impurity-
III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-Vlll, impurity-IX,
and impurity-X. The system suitability values and mass numbers of ivabradine
hydrochloride and its impurities were summarized in Table 2.
Table 2

Compound (n=l) Rt Rs N T (m/z)
Impurity-II 6.35 7101 0.97 194.3
Impurity-I 7.09 2.44 8802 0.97 208.2
Impurity-III 13.30 21.88 43126 0.90 280.3
Impurity-IX 19.21 23.29 100440 .0.95 485.6
Impurity-IV 20.73 5.82 95525 0.92 250.3
Impurity-V 21.30 2.16 115259 0.91 322.3

Impurity-X 23.06 6.85 137620 0.92 487.5
Impurity-VI 25.93 11.03 152337 0.94 298.3
Impurity-VII 26.92 3.81 190898 0.93 483.7
Ivabradine 34.36 26.93 213307 1.07 469.5
Impurity-VIII 37.07 9.21 276885 0.84 467.7
*n=l: determination, Rt: retention time, Rs: USP resolution, N: number of theoretical plates (USP tangent method), T: USP tailing factor, m/z: mass number.
Degradation in test solution was observed using 3% hydrogen peroxide at 60°C for 6 hours, 1M sodium hydroxide at 60°C for 6 hours and 1M HCL at 60°C for 6 hours. Impurities observed in stress condition using PDA detector. Major degradants was impurities-DC and impurity-X .Other unknown were also specific in this method .The peak test results obtained from PDA & LC-MS/MS confirm that the ivabradine peak is homogeneous and pure in all analyzed stress samples.
The percentage recovery of Ivabradine 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 a Waters X Bridge C18 250 mm><4.6 mm column with 5 um particles. The mobile phase-A consists buffer (1 mL of triethylamine mixed with 1000 mL of water adjusted pH-7.5 with orthophosphoricacid) and methanol. Mobile phase-B consists of methanol and acetonitrile. The flow rate of the mobile phase was kept

at 1.0 ml/min. Beginning with the gradient ratio of mobile phase-A and mobile phase-B 80:20 (v/v), system was continued at the same ratio for 5 minutes. The ratio was changed linearly 80:20(v/v) within 40 minutes and again system was continued at the same ratio for 15 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 45°C and the wavelength was monitored at a wavelength of 230 nm. The injection volume was 10 uL for related substances determination. Water and methanol in the ratio of 50:50(v/v) was used as diluent during the standard and test samples preparation.
Preparation of reference solution-l:
7.5 mg of impurity-VIII was accurately weighed and transferred to the lOOmL volumetric flask(BOROSIL-Class-A) , separately; 50ml of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.
Preparation of reference solution-2.
7.5 mg of each Impurity-I, impurity-II, impurity-III, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-VIII, impurity-IX, impurity-X, and 5.0 mg Ivabradine hydrochloride standards were accurately weighed and transferred to the lOOmL volumetric flask(BOROSIL-Class-A), separately; 20ml of methanol was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent. Pipette out 2.0mL from solution and transferred in to a 50mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.
A working solution of 2000ug/ml was prepared for related substances determination
analysis.

We Claim,
1. A HPLC method for analyzing Ivabradine 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 and methanol.
3. A HPLC method according to claim 1, wherein the first eluent B is methanol and acetonitrile.
4. A HPLC method according to claim 1, wherein gradient of A eluent and B eluent in the ratio of 80:20(v/v) initial and progressively increased to 30:70(v/v) in 45 minutes.
5. A HPLC method according to claim 1, wherein UV detector is set to 230nm wavelength.
6. A HPLC method according to claim 1, wherein impurities are separated with a minimum resolution 2.0
7. A HPLC method according to claim 2, wherein buffer islml of triethylamine mixed with 1000ml of water and pH about 7.5.
8. A HPLC method according to claim 2, wherein buffer and methanol in the ratio of 950:50 (v/v).
9. A HPLC method for Ivabradine 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.

10. A HPLC method determining the amount of impurities in ivabradine hydrochloride sample comprises the steps of:
a) combining a Ivabradine hydrochloride sample with mixture of water and methanol in the ratio of 50:50(v/v) to obtain a solution;
b) injecting the sample solution into a 250mmx 4.6mm column with 5um X Bridge CI8 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 45 minutes.
d) Preparing eluent A by mixing buffer and methanol in the ratio of 950:50 (v/v)
e) Measuring of the amounts of ivabradine and each impurity at 230nm wavelength with a UV detector (having an appropriate recording device).