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 FINGOLIMOD 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 fingolimod hydrochloride. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
BACKGROUND OF THE INVENTION
Fingolimod hydrochloride is chemically known as 2-Amino-2-[2-(4-octylphenyl)ethyl]-l,3-propanediol hydrochloride. Fingolimod is a sphingosine-1 -phosphate receptor modulator. In the body, fingolimod is metabolized by sphingosine kinase to the active metabolite fingolimod phosphate.

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

In another aspect, the present invention provides an HPLC method for fmgolimod 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.
Yet another aspect, the present invention provides a simple, accurate and well-defined stability indicating and high performance liquid chromatography (HPLC) method for the determination of fmgolimod hydrochloride in the presence of degradation products.
In one aspect, the HPLC method described in the present invention has the following advantages for determining the fmgolimod hydrochloride and its related impurities:
i) All the impurities were well separated with a minimum resolution 1.91.
ii) Gradient profile to elute all related impurities and organic phase is 75% 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 CI 8
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-IV, impurity-V, impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII spiked in fmgolimod hydrochloride) sample.

Fig. 2 Spiked solution (Fingolimod hydrochloride + 0.15% of each Known impurity spiked solution) @ 215 nm.
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 Fingolimod hydrochloride are:
(i) 2-Amino-2-[2-(4-hexylphenyl)ethyl]-l,3-propanediol (Impurity-I) which has the following structure:

The impurity-I is detected and resolved from fingolimod hydrochloride by HPLC with a
relative retention time (hereafter referred as RRT) of 0.40.

(ii) 2-Amino-2-[2-(4-heptylphenyl)ethyl]-l,3-propanediol (Impurity-II), which has the following structure:

Impurity-II is detected and resolved from fingolimod hydrochloride by HPLC with a RRT 0.77.
(iii) 2-Amino-2-[2-(4-decylphenyl)ethyl]-l,3-propanediol (Impurity-IV), which has the following structure:

Impurity-IV is detected and resolved from fingolimod hydrochloride by HPLC with an RRT of
1.37.
(iv) N-[l-(hydroxymethyl)-3-(4-octylphenyl)propyl]acetamide (Impurity-V), which has the
following structure:

Impurity-V is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of
1.63.
(v) 2-Ethylamino-2-[2-(4-octylphenyl)ethyl]-l,3-propanediol (Impurity-VI) , which has the
following structure:

Impurity-VI is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of 1.14.

(vi) 1, 3-propanediyl-2-acetamido-2-[2-(4-octylphenyl) ethyl] ylidenediacetate (Impurity-VII) which has the following structure:

Impurity-VII is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of
2.04.
(vii) 2-Chloro-l-(4-octylphenyl) ethanone (Impurity-IX) which has the following structure:

Impurity-IX is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of
2.14.
(viii) 2-Acetylamino-2-[2-(4-octanoyl-phenyl)ethyl] propane-1,3-dioate (Impurity-X) which has
the following structure:

Impurity-X is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of 2.11.
(ix) 2-Acetylamino-2-[2-(4-octyIphenyI) ethyl] propane-1,3-dioate (Impurity-XI) which has the following structure:

Impurity-XI is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of 2.26.

(x) 2-Acetylamino-2-[2-(4-octylphenyl) ethyl]- 1,3-propanediol (Impurity-XII) which has the following structure:

Impurity-XII is detected and resolved from fingolimod hydrochloride by HPLC with a RRT of
1.60.
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 fingolimod
hydrochloride and all impurities, preferably, impurity-I, impurity-II, impurity-IV, impurity-V,
impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII present in a
sample of fingolimod 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 fingolimod hydrochloride in the presence of degradation products
Preferably, the method for determining the amount of impurities in a fingolimod hydrochloride sample comprises the steps of:
a) Combining a fingolimod hydrochloride sample with water and acetonitrile in the ratio of about 50:50 (v/v) to obtain a solution;
b) injecting the sample solution into a 150 mm x 3.9 mm, column with 5(im Symmetry C18 column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 65:35 (v/v) initial and progressively increased to 25:75(v/v) in 40 minutes .

d) Eluent A is buffer (5.52 gm (0.04 M) of Sodium phosphate monobasic monohydrate in l000 mL of water. Add 3.0mL of Triethylamine and dissolve it. Adjust the pH to 2.5 + 0.05 with orthophosphoric acid solution. Filter it through 0.45 μ membrane filter and degas. (PALL : Ultipor N Nylone 6,6,membrane)
e) Preparing Eluent B by mixing acetonitrile and methanol in the ratio of 90:10 (v/v) Preferably, the initial ratio of A Eluent and B Eluent in step-(c) may be continued at the same ratio for 12 minutes then changed linearly to 25:75 (v/v) within 28 minutes followed by same ratio for 15 minutes. After 3 minutes the initial gradient of 65:35 is for 7 minutes to be conditioned for every analysis. The column temperature may be maintained at about 35°C.
The LOD /LOQ values of fingolimod hydrochloride and its related impurities, impurity-I, impurity-II. impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII are summarized in Table 1.
Table 1

S.No Components LOD (%) LOQ (%)
1 Impurity-1 0.0061 0.0184
2 Impurity-II 0.0125 0.0380
3 Impurity-VI 0.0073 0.0220
4 Impurity-IV 0.0078 0.0237
5 Impurity-XII 0.0098 0.0297
6 Impurity-V 0.0109 0.0330
7 Impurity-VII 0.0124 0.0375
8 Impurity-X 0.0114 0.0344
9 Impurity-IX 0.0107 0.0325
10 Impurity-XI 0.0159 0.0482
11 7ingolimod hydrochloride 0.0059 0.0180

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 fingolimod hydrochloride, Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1M HCI), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H2O2), to evaluate the ability of the proposed method to separate fingolimod hydrochloride from its degradation products. To check and ensure the homogeneity and purity of fingolimod 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. 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 (5.52 gm ( 0.04 M) of Sodium phosphate monobasic monohydrate in l000 mL of water. Add 3.0mL of Triethylamine and dissolve it. Adjust the pH to to 2.5 + 0.05 with orthophosphoric acid solution. Filter it through 0.45 μ membrane filter and degas. (PALL : Ultipor N Nylone 6,6,membrane), methanol and acetonitrile. In the optimized conditions, the fingolimod hydrochloride , impurity-I, impurity-II, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII were well separated with a resolution of 1.91 and the typical retention times (RT) of fingolimod hydrochloride, impurity-I, impurity-II, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII were about 19.46, 7.79, 15.04, 26.58, 31.65, 22.20, 39.66, 41.61, 41.02, 44.01 and 31.07 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for fingolimod hydrochloride and its ten impurities, namely impurity-I, impurity-II, impurity-IV, impurity-V, impurity-VI, impurity-VII, impurity-IX, impurity-X, impurity-XI and impurity-XII.

The system suitability values of fingolimod hydrochloride and its impurities were summarized in Table 2
Table 2

Compound (n=l) Rt Rs N T m/z [M + H]
Impurity-I 7.79 7729 0.99 280.3
Impurity-II 15.04 18.41 20362 0.96 294.3
Fingolimod 19.46 7.98 13234 3.46 308.2
Impurity-VI 22.20 5.81 115812 1.01 336.4
Impurity-IV 26.58 16.90 180334 0.98 336.4
Impurity-XII 31.07 16.14 177069 0.92 350.2
Impurity-V 31.65 1.91 183868 0.99 320.4
Impurity-VII 39.66 26.34 279193 0.92 434.2
Impurity-X 41.02 4.53 323394 0.94 448.6
Impurity-IX 41.61 2.00 320078 0.97 267.1
Impurity-XI 44.01 6.96 208202 0.98 434.2
*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 for 6 hours at 60°C and 1M sodium hydroxide 3 hours RT and no degradation was observed with 1M HC1 for 6 hours at 60°C. Impurities observed in stress condition using PDA detector. Major degradant is an unknown impurity .Other unknown were also specific in this method .The peak test results obtained from PDA confirm that the fingolimod peak is homogeneous and pure in all analyzed stress samples.
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 Symmetry C18 (150mm x 3.9mm), column with 5 μm particles. The mobile phase consists buffer (5.52 gm (0.04 M) of Sodium phosphate monobasic monohydrate in lOOOmL of water. Add 3.0mL of Triethylamine and dissolve it. Adjust the pH to to 2.5 + 0.05 with orthophosphoric acid solution. Filter it through 0.45 μ membrane filter and degas. (PALL : Ultipor N Nylone 6,6,membrane), and solvents are acetonitrile and methanol. Eluent-A is Buffer and Eluent-B is Acetonitrile: Methanol: 90 : 10 (v/v). The flow rate of the mobile phase was kept at 1.5ml/min. Beginning with the gradient ratio of A Eluent and B Eluent in the ratio of 65:35, continued at the same ratio for 12 minutes then changed linearly to 25:75 (v/v) within 28 minutes followed by same ratio for 15 minutes. After 3 minutes the initial gradient
of 65:35 is for 7 minutes to be conditioned for every analysis. The column temperature was maintained at 35°C and the wavelength was monitored at a wavelength of 215 nm. The injection volume was 10 μL for related substances determination. Water: Acetonitrile: 50: 50 (v/v) was used as diluent during the standard and test samples preparation.
Preparation of system suitability stock solution preparation.
7.5 mg each of impurity VI standard was accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A) , separately; 10ml of acetonitrile and 5 mL of diluent was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.
Mix well and transfer 1 mL of this solution into a 10 mL volumetric flask and make up the volume with diluent.
Preparation of system suitability solution preparation.
15 mg of fingolimod hydrochloride working standard was accurately weighed and transferred to the lOmL volumetric flask(BOROSIL-Class-A) , separately; add 1.5 mL system suitability

stock solution 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:
5 mg of fingolimod hydrochloride working standard, weighed and transferred to the 50 mL
volumetric flask(BOROSIL-Class-A), separately; 30 mL 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 1.5 mL from solution and transferred in to a lOOmL volumetric flask (BOROSIL-
Class-A), and made up to mark with diluent.
A working solution of 1500μg/ml was prepared for related substances determination analysis.

We Claims,
1. A HPLC method for analyzing Fingolimod 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 and methanol 90: 10(v/v).
4. A HPLC method according to claim 1, wherein gradient of eluent A and eluent B in the ratio of 65:35 initial and continued at the same ratio for 12 minutes then changed linearly to 25:75 (v/v) within 28 minutes followed by same ratio for 15 minutes. Again changed linearly to initial ratio within 3 minutes followed by same ratio for 7 minutes.
5. A HPLC method according to claim 1, wherein UV detector is set to 215nm wavelength.
6. A HPLC method for Fingolimod 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.
7. A HPLC method determining the amount of impurities in Fingolimod hydrochloride sample comprises the steps of:
i) Combining a fingolimod hydrochloride sample with water and acetonitrile in
the ratio of about 50:50 (v/v) to obtain a solution;

ii) injecting the sample solution into a 150 mm x 3.9 mm, column with 5um
Symmetry C18 column; iii) gradient eluting the sample with a mixture of A Eluent and B Eluent in the
ratio of 65:35 (v/v) initial and progressively increased to 25:75(v/v) in 12
minutes then changed linearly to 25:75 (v/v) within 28 minutes followed by
same ratio for 15 minutes. Again changed linearly to initial ratio within 3
minutes followed by same ratio for 7 minutes, iv) Eluent A comprises of Buffer.
v) Eluent B comprises of acetonitrile and methanol 90: 10 (v/v). vi) Measuring of the amounts of Fingolimod hydrochloride and each impurity at
215 nm wavelength with a UV detector (having an appropriate recording
device).
8. A HPLC method according to previous claims, wherein buffer is 5.52 g Sodium phosphate monobasic monohydrate in l000 mL of water. Add 3.0mL of Triethylamine and dissolve it. Adjust the pH to to 2.5 + 0.05 with 3.0mL of Triethylamine and with orthophosphoric acid solution