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 LINEZOLID.
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 linezolid. The present invention further provides a stability indicating analytical method using the samples generated from forced degradation studies.
BACKGROUND OF THE INVENTION
Linezolid is chemically known as N-[[(5S)-3-[3-Fluoro-4-(4-morpholinyl)phenyl]-2-oxo-5-oxazolidinyl]methyl]acetamide. Linezolid is a synthetic antibiotic, the first of the oxazolidinone class, used for the treatment of infections caused by multi-resistant bacteria including streptococcus and methicillin-resistant Staphylococcus aureus (MRSA). The drug works by inhibiting the initiation of bacterial protein synthesis..

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 Linezolid contained in the product mixture into an active pharmaceutical ingredient ("API"), the Linezolid 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 spectrascopically 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 linezolid.
In another aspect, the present invention provides an HPLC method for linezolid 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 and high performance liquid chromatography (HPLC) method for the determination of linezolid in the presence of degradation products.
In one aspect, the HPLC method described in the present invention has the following advantages for determining the linezolid 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 55 % 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 4.0 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-B, impurity-C, impurity-D, impurity-I, impurity-II spiked in linezolid) sample.
Fig. 2 illustrates the spiked solution (linezolid + 0.15% of each Known impurity spiked solution) @ 250 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 five main known impurities of linezolid are:
(0 N-[[(5S)-3-[4-(4-morpholinyl) phenyl]-2-oxo-5-oxazolidinyl] methyl] acetamide (Impurity-B) which has the following structure:

The impurity-B is detected and resolved from linezolid by HPLC with a relative retention time (hereafter referred as RRT) of 0.81.
(ii) Bis (N-[[(5S)-3-[3-fluoro-4-(4-morpholinyI) phenyl]-2-oxo-5-oxazolidinyl] methyl] }acetamide_(Impurity-C), which has the following structure:


Impurity-C is detected and resolved from linezolid by HPLC with a RRT of 1.78.
(iii) (5S)-5-(amino methyl)-4-(3-fluoro-4-morpholin-4-ylphenyl)-l, 3-oxazolidin-2-one (Impurity-D), which has the following structure:

Impurity-D is detected and resolved from linezolid by HPLC with an RRT of 0.51.
(iv) (R)-[N-3- (3-Fluoro-4-rnorpholinylphenyl) -2-oxo-5-oxazolidinyl] methyl azide (Impurity-I), which has the following structure:

Impurity-I is detected and resolved from linezolid by HPLC with a RRT of 1.57.
(v) (R)- [N-3- (3-Fluoro-4-morpholinylphenyl) -2-oxo-5- oxazolidinyl] methyl methanesulfonate (Impurity-II) , which has the following structure:

Impurity-II is detected and resolved from linezolid by HPLC with a RRT of 1.29.
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

linezolid and all impurities, preferably, impurity-B, impurity-C, impurity-D, impurity-I and impurity-II present in a sample of linezolid.
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 linezolid in the presence of degradation products.
Preferably, the method for determining the amount of impurities in a linezolid sample comprises the steps of:
a) Combining a linezolid sample with water and acetonitrile in the ratio of about 60:40 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mm x 4.6mm, column with 5μm Symmetry C18_column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 85:15 (v/v) initial and progressively increased to 45:55(v/v) in l5minutes.
d) Eluent A is buffer (Dissolve about 2.72 g of potassium dihydrogen phosphate in 1000 mL of water. Adjust the pH 4.0 with 5% orthophosphoric acid. Filter it through 0.2 μm filter paper (UltiporR N66R Nylon 6,6-membrane , 47 mm, PIN-NR047100; Make:PALL Life Sciences)
e) Preparing Eluent B by mixing acetonitrile and methanol in the ratio of 50:50 (v/v).
Preferably, the initial ratio of A Eluent and B Eluent in step-(c) may be changed linearly to 45:55 (v/v) within 15 minutes followed by same ratio for 5 minutes. After 5 minutes the initial gradient of 85:15 is for 5 minutes to be conditioned for every analysis. The column temperature may be maintained at about 30°C.

The LOD /LOQ values of linezolid and its related impurities. impurity-B, impurity-C, impurity-D, impurity-I, impurity-II are summarized in Table 1.
Table 1

S.No Components LOD (%) LOQ (%)
1 Impurity-D 0.0043 0.0129
2 Impurity-B 0.0031 0.0095
3 Impurity-II 0.0019 0.0057
4 Impurity-I 0.0022 0.0067
5 Impurity-C 0.0047 0.0142
6 Linezolid 0.0033 0.0100
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 linezolid, Intentional degradation was attempted to stress conditions of acid hydrolysis (using 1M HC1), base hydrolysis (using 1M NaOH), and oxidative degradation (using 3.0% H2O2). to evaluate the ability of the proposed method to separate linezolid from its degradation products. To check and ensure the homogeneity and purity of linezolid 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 (2.72 g of potassium dihydrogen phosphate in 1000 mL of water. Adjust the pH 4.0 with 5% orthophosphoric acid. Filter it through 0.2 μm filter paper (UltiporR N66R Nylon 6,6-membrane , 47 mm, P/N-NR047100; Make:PALL Life Sciences)), methanol and acetonitrile. In the optimized conditions, the linezolid,

impurity-B, impurity-C, impurity-D, impurity-I, and impurity-II were well separated with a minimum resolution of 2.0 and the typical retention times (RT) of linezolid , impurity-B, impurity-C, impurity-D, impurity-I, and impurity-II were about 10.12, 8.22, 17.97, 5.16, 15.88 and 13.04 minutes respectively, and typically shown in Figure 1. The system suitability results and the developed LC method was found to be specific for linezolid and its five impurities, namely impurity-B, impurity-C, impurity-D, impurity-I, and impurity-II.
The system suitability values of linezolid and its impurities were summarized in Table 2.
Table 2

Compound (n=l) Rt Rs N T m/z(M+H)
Impurity-D 5.16 44819 1.25 296.3
Impurity-B 8.22 12.82 142862 1.28 320.5
Linezolid 10.12 8.27 196948 1.28 338.5
Impurity-11 13.04 12.29 318230 1.25 375.7
Impurity-1 15.88 11.74 446304 1.20 322.6
Impurity-C 17.97 8.07 461634 1.25 617.2
*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 20 min. at 60°C, 1M sodium hydroxide 10 min. RT and 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 linezolid 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 CIS (150mm x 4.6 mm), column with 5 urn particles. The mobile phase consists buffer (Dissolve about 2.72 g of potassium dihydrogen phosphate in 1000 mL of water. Adjust the pH 4.0 with 5% orthophosphoric acid. Filter it through 0.2 μm filter paper (UltiporR N66R Nylon 6,6-membrane , 47 mm, PIN-NR047100; Make:PALL Life Sciences)), and solvents are acetonitrile and methanol. Eluent-A is Buffer and Eluent-B is Acetonitrile: Methanol :: 50 : 50 (v/v).
The flow rate of the mobile phase was kept at l.Oml/min. Beginning with the gradient ratio of A Eluent and B Eluent in the ratio of 85:15, then changed linearly to 45:55 (v/v) within 15 minutes followed by same ratio for 5 minutes. After 5 minutes the initial gradient of 85:15 is for 5 minutes to be conditioned for every analysis. The column temperature was maintained at 30°C and the wavelength was monitored at a wavelength of 250 nm. The injection volume was 10μL for related substances determination. Water : Acetonitrile :: 60 : 40 (v/v) was used as diluent during the standard and test samples preparation.
Preparation of system suitability stock solution preparation:
10.5 mg each of impurity-B, impurity-C, impurity-D, impurity-I, impurity-II and 7.0 mg
of Linezolid standards standard was accurately weighed and transferred to the l00mL
volumetric flask(BOROSIL-CIass-A) . separately; add 50 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 5 mL of this solution into a 10 mL volumetric flask and make up
the volume with diluent.
Preparation of system suitability solution preparation.
35 mg of linezolid working standard was accurately weighed and transferred to the 50mL volumetric flask(BOROSIL-Class-A) , separately; add 1.0 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.
Reference solution-(a) preparation:
Dilute 1.0 mL of above system suitability stock solution to 50 mL with diluent and mix
well.
Reference solution-(b) preparation:
35 mg of Linezolid 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 1.0 mL from solution and transferred in to a l00mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent. Further Pipette out 1.0 mL from the resulting solution and transferred in to a 10 mL volumetric flask (BOROSIL-Class-A), and made up to mark with diluent.
A working solution of 700μg/ml was prepared for related substances determination analysis.

We Claim,
1. A HPLC method for analyzing Linezolid, 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 mixing acetonitrile and methanol in the ratio of 50:50 (v/v).
4. A HPLC method according to claim 1, wherein gradient of A eluent and B eluent in the ratio of 85:15 (v/v) initial and progressively increased to 45:55(v/v) in 15 minutes.
5. A HPLC method according to claim 2, wherein buffer is about 2.72 g potassium dihydrogen ortho phosphate in 1 liter of water and pH about 4.0.
6. A HPLC method for Linezolid 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 Linezolid sample comprises the steps of:

a) Combining a linezolid sample with water and acetonitrile in the ratio of about 60:40 (v/v) to obtain a solution;
b) injecting the sample solution into a 150mm x 4.6mm, column with 5μm Symmetry C18_column;
c) gradient eluting the sample with a mixture of A Eluent and B Eluent in the ratio of 85:15 (v/v) initial and progressively increased to 45:55(v/v) in15minutes.

d) Eluent A is buffer (dissolve about 2.72 g of potassium dihydrogen phosphate in 1000 mL of water and pH 4.0.
e) Preparing Eluent B by mixing acetonitrile and methanol in the ratio of 50:50 (v/v).; and
f) Measuring of the amounts of Linezolid and each impurity at 250nm wavelength.