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 L-Serine content in D-serine of Lacosamide
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 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 L-serine content in D-Serine of Lacosamide.
Background of the invention
Lacosamide (R)-2-acetamido-N-benzyl-3methoxypropionamide, has the following formula:

It is a drug that has been used in the treatment of epilepsy. Lacosamide is marketed under the trade name Vimpat® by UCB. It was approved by the FDA as an adjunctive therapy for partial-onset seizures in October 2008.
D-Serine is the key starting material of lacosamide, used in the manufacturing process of lacosamide. L-Serine is the unwanted isomer of D-Serine. D-Serine has non chromophoric structure; hence it is non UV active compound. To enhance the response of the compound and its other isomer in UV, derivatization method has been applied to prepare its derivatives by using Na -5-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) as derivatisation agent.
Structure of D-Serine


The product mixture of a reaction rarely is a single compound. 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 D-Serine , D-Serine 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 our desired reaction.
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
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 amount of L-Serine present in a sample of D-Serine.
In another aspect, the present invention provides an HPLC method for D-Serine containing less than about 5% area by HPlA preferably less than about 3% area by HPLC, more preferably less than 1% area by PPLC, of L-Serine.
In yet another aspect, the present invention provides a simple, accurate and well-defined high performance liquid chromatography (HPLC) method for the determination of L-Serine content in D-Serine.
In one aspect, the present invention provided a reversed-phase liquid chromatographic (RP-LC) method for the quantitative determination of L-Serine content in D-Serine of lacosamide.
In one aspect, the HPLC method described in the present invention has the following
advantages:
i) L-Serine and D-Serine of non chromophoric compound are well separated by UV
detection with a resolution not less than 2.5 . ii) Gradient profile to elute D-Serine and organic phase is 80% which ensure the elution
and detection of unwanted impurities. iii) The present method mobile phase pH is about 3.0 which is more stable in all CI8
HPLC columns; iv) Consistency in specificity, precision & reproducibility with good peak shape.

Brief description of drawings
Fig. 1 depicts an UV spectrum of derivatized D-serine.
Fig. 2 depicts a HPLC chromatogram of spiked (L-Serine in D-Serine) sample.
Fig. 3 depicts a Spiked solution (D-Serine + 0.1% of L-Serine spiked solution) @ 340 nm.
Detailed description of the invention
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.
Reaction


determine enantiomeric amino acids. The reagent reacts stoichiometrically with the amino group of enantiomeric amino acids to produce stable diastereomeric derivatives, which can readily be separated by reverse-phase HPLC. The dinitrophenyl alanine amide moiety strongly absorbs at 340 nm (є = 30,000 M- l- cm-1), allowing detection in the subnanomolar range by reacting mole ratio of D-serine and Marfey's reagent in 1:1.2 by derivatisation technique.
(2S)-2-ammo-3-hydroxypropanoic acid (L-Serine) has the following structure

The derivatized L-Serine is detected and resolved from derivatized D-Serine by HPLC with a relative retention time (hereafter referred as RRT) of 0.92.
According to one embodiment of the present invention, there is provided a reversed-phase liquid chromatographic (RP-LC) method for quantifying, by area percent, the amount of L-Serine present in a sample of D-Serine.
Preferably, the method for determining the amount of L-Serine in D-Serine in sample comprises the steps of
a) Combining a D-Serine sample with water and then mixing with Na -5-fluoro-2,4-dinitrophenyl-5-L-alanine amide and sodium carbonate and heated for 40 min at 60°C to obtain a solution;
b) injecting the sample solution into a 250 mm x 4.6 mm, Inertsil ODS-3 column with 5μm column;
c) gradient eluting the sample with a mixture of buffer and acetonitrile in the ratio of 85:15 (v/v) initial and progressively increased to 20:80(v/v) in 20 minutes .
d) Preparing eluent-A by transferring 7 mL of triethyl amine in 1000 ml of water, and adjust pH - 3.0 with orthophosphoricacid solution. Filter it through 0.45 Μ membrane filter and degas.

e) Measuring of the amounts of D-serine and L-Serine at 340 nm wavelength with a UV detector (having an appropriate recording device).
Preferably, the initial ratio of eluent A and acetonitrile in step-(c) is changed linearly to 70:30 (v/v) within 15 minutes again changed linearly to 20:80 (v/v) within 20 minutes, After 4 minutes the initial gradient of 85:15 is achieved and further conditioned for 6 minutes. The column temperature to be maintained at about 35°C. To check and ensure the homogeneity and purity of D-Serine peak in the sample solutions, PDA-UV detector was employed.
According to another embodiment of the present invention, there is provided a chromatographic method to get the separation and detection of D-Serine and L-Serine peaks. Satisfactory chromatographic separation was achieved using the mobile phase consists of buffer (7 mL of triethyl amine mixed with 1000 mL of water and adjusted the pH to 3.0 with orthophosphoric acid) In the optimized conditions D-Serine and L-Serine were well separated with a resolution not less than 2.5 and the typical retention times (RT) of D-Serine and L-Serine were about 15.20 and 13.92 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for D-Serine and L-Serine.
The system suitability values of D-Serine and L-Serine were summarized in Table 2.
Table 2

Compound (n=l) Rt Rs N T
L-Serine 13.92 40036 1.05
D-Serine 15.20 4.59 49991 1.09
*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.

Experimental
The LC system, used for method development 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 Inertsil ODS-3 (250 mm x 4.6 mm), column with 5 urn particles. The mobile phase consists buffer (7 mL of triethyl amine mixed with 1000 mL of water), and solvent is acetonitrile. The flow rate of the mobile phase was kept at 1 .Oml/min. Beginning with the gradient ratio of mobile phase buffer and solvent (acetonitrile) 85:15 (v/v), the ratio was changed linearly 70:30(v/v) within 15 minutes and the ratio was again changed linearly 20:80(v/v) within 20 minutes and after 4 minutes the initial gradient of 85:15 is for 6 minutes to be conditioned for every analysis. The column temperature was maintained at 35oC and the wavelength was monitored at a wavelength of 340 nm. The injection volume was 10 uL for L-Serine content determination. Eluent A and acetonitrile was used as diluent during the standard and test samples preparation.
Example 1
Preparation of Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide solution
50mg of Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide was accurately weighed and transferred into a lOmL volumetric flask (BOROSIL-Class-A), 5-7 mL of acetonitrile was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with acetonitrile.(This solution to be store in refrigerator when it is not in use).

Example 2
Preparation of Sodium carbonate solution (5%)
2.5g of sodium carbonate was weighed and transferred in to 50mL volumetric flask, 35-40 mL of water was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with water.
Example 3
Preparation of stock solution-1 preparation
5mg of L-Serine standard was accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A), 5-7 ml of water 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 lOmL volumetric flask (BOROSIL-Class-A), and made up to mark with water.
Example 4
Preparation of stock solution-2 preparation
200mg of D-Serine standard was accurately weighed and transferred to the 10mL volumetric flask(BOROSIL-Class-A) , 5-7 ml of water was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with diluent.

Example 5
Preparation of system suitability solution preparation
Transfer 100 μL of each stock solution-1 and stock solution-2, 500 μL of Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide solution and 20 μL of sodium carbonate solution transferred to the lOmL volumetric flask (BOROSIL-Class-A) , closed and heated at 60°C for 40 min. and allow to cool at room temperature and made up to mark with diluent.
Example 6
Preparation of reference solution-(a)
100 μL of stock solution-1, 500 μL of Na-5-fluoro-2,4-dinitrophenyl-5-L-alanine amide solution and 20 μL of sodium carbonate solution transferred to the lOmL volumetric flask(BOROSIL-Class-A) , closed and heated at 60°C for 40 min. and allow to cool at room temperature and made up to mark with diluent. A working solution of 200μg/ml was prepared for L-Serine content analysis.

We Claim,
1. A HPLC method for analyzing L-serine content in D-Serine of Lacosamide, 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 7 mL of triethyl amine mixed with 1000 mL of water pH-2.5.
5. A HPLC method for D-Serine 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 L-Serine.
6. A HPLC method determining the amount of L-serine content in D-Serine of
Lacosamide sample comprises the steps of:
a) Combining a D-Serine sample with water and then mixing with Na -5-fluoro-2,4-dinitrophenyl-5-L-alanine amide and sodium carbonate and heated for 40 min at 60°C to obtain a solution;
b) injecting the sample solution into a 250 mm x 4.6 mm, Inertsil ODS-3 column with 5 urn column;
c) gradient eluting the sample with a mixture of buffer and acetonitrile in the ratio of 85:15 (v/v) initial and progressively increased to 20:80(v/v) in 20 minutes
d) Preparing eluent-A by transferring 7 mL of triethyl amine in 1000 ml of water, and adjust pH = 3.0 with orthophosphoricacid solution
e) Measuring of the amounts of D-serine and L-Serine at 340 nm wavelength with a UV detector (having an appropriate recording device).