FORM2THE PATENTS ACT, 1970(39 of 1970)&The Patents Rules, 2003COMPLETE SPECIFICATION(See section 10; rule 13)1. Title of the invention. - NOVEL COMPOUND USEFUL AS REFERENCE MARKER FOR ROXITHROMYCIN2. Applicant(s)(a) NAME : ALEMBIC LIMITED(b) NATIONALITY: An Indian Company.(c) ADDRESS : Alembic Campus, Alembic Road, Vadodara - 390 003, Gujarat, India.3. PREAMBLE TO THE DESCRIPTIONThe following specification particularly describes the invention and the manner in which it is to be performed:

Field of the Invention
Present invention relates to novel compound of formula (II) which is a useful reference marker for analysis of Roxithromycin of formula (I). Further it discloses method for analyzing purity of Roxithromycin samples for its formulation dosage forms.

CH3 CH3
Formula (I) Formula (II)
Background of the Invention
Roxithromycin is a semi-synthetic macrolide antibiotic chemically know as 9-(0-((2-Methoxyethoxy)methyl)oxime)erythromycin. It is used to treat respiratory tract, urinary and soft tissue infections. Roxithromycin is derived from erythromycin, containing the same 14-membered lactone ring. However, an N-oxime side chain is attached to the lactone ring.
Roxithromycin is first disclosed in US patent no. 4349545. The process for preparation of Roxithromycin is exemplified in synthetic scheme as shown in Scheme-I

Acetone, NaHC03, H3C
CH3 (methoxyethoxymethyl chloride, inert atmosphere, reflux
H,C
CH,
,CL ,0.
L^-CH3

CH3
Erythromycin 9-oxime

Roxithromycin

The process involves reaction of Erythromycin 9-oxime with methyoxyethoxy methyl chloride in presence of base.
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.
To obtain marketing approval for a new drug product, it is required to submit data to the regulatory authorities proving the safety and efficacy of the drug. This data include analytical data which shows that (i) drug is free from impurities or impurities are present only in negligible amount and (ii) shelf-life or storage stability of drug is acceptable. In order to generate this data the drug is tested against an external standard or reference marker.
Impurities generally found in pharmaceutically active agents and formulations containing them include residual amounts of synthetic precursors to the active agent, by-products which arise during synthesis of the active agent, residual solvent, isomers of the active agent, contaminants which were present in materials used in the synthesis of the active agent or in the preparation of the pharmaceutical formulation, and unidentified adventitious substances. Other impurities which may appear on storage include substances resulting from degradation of the active agent, for instance by oxidation or hydrolysis.

The present invention provides compound of formula (II), which is a useful reference marker for testing the purity of samples of Roxithromycin and also its pharmaceutical dosage forms.
Objects of the invention
It is an object of the present invention to provide novel compound of formula (II) useful as reference markers for the analysis of Roxithromycin and pharmaceutical formulations thereof.
Another object of the present invention is to provide a method of testing the purity of a sample of Roxithromycin or a pharmaceutical dosage form comprising Roxithromycin.
Summary of the invention
Accordingly, there is provided novel compound of formula (II) which is useful as reference markers for the analysis of Roxithromycin and pharmaceutical composition thereof. Also, there is provided process for preparation compound of formula (II).
The present invention also provides method of testing the purity of a sample of Roxithromycin or a pharmaceutical dosage form comprising Roxithromycin comprising assaying the said sample for the presence of compound of formula (II).
Detailed description
The compound of formula (II) is chemically defined as 9-[0-[(2-methoxyethoxy)ethoxymethyl]oxime]. 1 -(chloromethoxy)-2-(2-methoxyethoxy)ethane is an impurity which is present in 2-methoxy ethoxy methyl chloride (MEM-CI), which is used for synthesis of Roxithromycin (Scheme-I). Thus, during synthesis of Roxithromycin when MEM-CI is reacted with 9-[0-[(2-methoxyethoxy)ethyl]oxime], compound of formula (II) is obtained as the by-product.
Compound of formula (II) is obtained by reacting Erythromycin A oxime with 1-(chloromethoxy)-2-(2-methoxyethoxy)ethane in presence of solvent and base. The

General Scheme of reaction is shown in Figure-1. The solvent can be polar solvent selected from group comprising of acetone, ethyl acetate, DMF, DMSO, HMPT or Ether solvents like ethyl ether, THF or dioxane or mixtures thereof.
.OH A .CL ,-\ /\ ,0.

<-"3
Erythromycin A oxime Formula (II)
Figure-1
Base can be selected from group comprising of Alkali metal or alkaline earth metal carbonate, bicarbonates or alkoxides like sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, calcium carbonate, barium carbonate or methoxide, ethoxide, propoxide or butoxide of sodium or potassium.
The reaction is generally carried out at room temperature or at reflux temperature of solvent used.
The test sample of drug substance (Roxithromycin) or drug product (pharmaceutical dosage form of Roxithromycin) to be analyzed may be assayed by one or more conventional analytical techniques. The analytical technique includes high performance liquid chromatography (HPLC). The results obtained are compared with the results obtained from testing a substantially pure reference sample of compound of formula (II).
In one aspect of the present invention, the method for testing the purity of a sample of Roxithromycin by Assay comprising steps of:
(i) dissolving a sample of reference standard (Roxithromycin) in a solvent
(diluent) to produce a standard solution (STD) (ii) dissolving a sample of Roxithromycin in a solvent (diluent) to produce a sample solution

(iii) injecting the diluent, STD and sample solution on to an HPLC column, and determining the area of each peak and calculating the assay of Roxithromycin.
Another aspect of the present invention provides method of testing the purity of a sample of Roxithromycin or a pharmaceutical dosage form comprising Roxithromycin, which method comprises determination of reference maker Compound (II) in the said sample by HPLC method. The said method comprising steps of:
(i) dissolving a sample Compound (II) in a solvent (diluent) to produce a system
suitability solution (SST);
(ii) dissolving a sample of reference standard (Roxithromycin) in a solvent
(diluent) to produce a standard solution (STD);
(iii) dissolving a sample of Roxithromycin in a solvent (diluent) to produce a
sample solution;
(iv) injecting the diluent, SST, standard solution and sample solution on to an HPLC column and determining the area of each peaks and calculating related compound in the Roxithromycin sample.
The following examples illustrate the invention further. It should be understood, however, that the invention is not confined to the specific limitations set forth in the individual examples but rather to the scope of the appended claims.
Example-1
Preparation of Erythromycin 9-[0-[(2-methoxyethoxy)ethoxymethyl]oxime]
(Compound II)
Erythromycin A oxime base (100 gm, 0.1335 moles) was dissolved in 460 ml acetone. The solution was stirred for 30-35 min at ambient temperature and further cooled to 0-5°C. Sodium methoxide (10 gm, 0.185 moles) and 1-(chloromethoxy)-2-(2-methoxy ethoxy)ethane (18.8 gm, 0.111moles) was added to the reaction mass. The reaction mixture was stirred for 30-45 min and completion of reaction was monitored by TLC for the absence of Erythromycin A oxime.
The temperature of reaction mass was then raised to ambient and 200 ml of water was added over the period of 1 hour. Further the reaction mass was stirred for 30 min. For

complete precipitation excess 440 ml of water was added by slow addition. The slurry was stirred for 2.0 hrs and then the crystalline precipitate was collected by filtration and thoroughly washed with water (100 ml) and further dried at 55°C. Erythromycin 9-[0-[(2-methoxyethoxy)ethoxymethyl]oxime] (27 gm; m.p.-101.7°C.)
Purity of the product was determined by HPLC as 91.99%. The HPLC conditions were as follows:
Chromatographic system:

Column : Column, 15 cm long with an internal diameter of 0.46 cm, packed
with 5 m Octadecylsilyl silica gel (C18 Symmetry, Waters) or
equivalent.
Temperature : 15° C
Pump : Gradient
Flow rate : 1.1 ml / minute
Injector : Autoinjector or Injection valve
(maintained at 8°C)
Injection volume : 20 ml
Detector Type : Spectrophotometric detector
Wavelength : 205 nm
Run time : 100 minutes
Data processor : Set at area percent mode
Mobile Phase:
Ammonium Dihydrogen Phosphate Solution-I:
Weigh accurately 59.7 g of Ammonium Dihydrogen Phosphate and transfer it in a suitable glass beaker. Add about 800 ml of water and sonicate till a clear solution is obtained. Transfer the contents of the beaker in a 1000 ml volumetric flask, and make up the volume with water and mix. Adjust pH 4.3 with dilute sodium hydroxide solution.
Mobile Phase A:
Measure 740 ml of Ammonium Dihydrogen Phosphate solution-l in a suitable measuring cylinder and transfer to a suitable glass beaker. Measure 260 ml of acetonitrile in a

measuring cylinder and transfer to a glass beaker containing Ammonium Dihydrogen Phosphate Solution. Sonicate to mix. Filter the mobile phase A through 0.45 |i membrane filter and degas.
Mobile Phase B:
Transfer about 700 ml of Acetonitrile to a suitable glass beaker. Add 300 ml of water to the glass beaker containing Acetonitrile and sonicate to mix. Filter through 0.45 \i membrane filter and degas.
Ammonium Dihydrogen Phosphate Solution-ll:
Weigh accurately 48.6 g of Ammonium Dihydrogen Phosphate and transfer it in a suitable glass beaker. Add about 500 ml of water and sonicate till a clear solution is obtained. Transfer the contents of the beaker in a 1000 ml volumetric flask, and make up the volume with water and mix. Adjust pH 5.3 with dilute sodium hydroxide solution
Solution A:
Transfer 300 ml of Acetonitrile to a suitable glass beaker. Add 700 ml of Ammonium Dihydrogen Phosphate Solution-ll to the glass beaker containing Acetonitrile and sonicate to mix. Filter through 0.45 |u membrane filter and degas.
Blank Solution:
Use solution A as such.

Use following Gradient programme :
Time (min) Mobile phase A(%v/v) Mobile phase B(%v/v) Comments
0-50 100 0 Isocratic
50-51 100-»90 0 -»10 Linear gradient
51-80 90 10 Isocratic
80-81 90^100 10 -*0 Linear gradient
81-100 100 0 Isocratic

Pre-equilibrate the column for about 60 min. with mobile phase or until a stable base line is obtained. When a stable baseline was obtained, the diluent was injected. Compound (II) solution is injected and the purity is found by area normalization method.
The product had the following characteristics Molecular formula: C43H8oN2016 Molecular wt: 881
1H nmr (in CDCI3 / 300 MHz): 5.16-5.18 (2H,2d); 4.87-4.89 (1H,d); 4.40-4.42 (1H,d); 4.33 (1H,s); 3.99-4.20 (2H,d); 3.75-3.78 (3H,m); 3.66-3.69 (4H,m); 3.55-3.58 (3H,t); 3.37 (3H,s); 3.31 (3H,s); 3.12 (1H,s); 3.01 (1H,t); 2.28 (6H,s);1.48 (4H,s); 1.28-1.30 (3H,d); 1.02-1.05 (3H,d); 0.81-0.83 (3H,t) 6 ppm.
Mass spectra: m/z= 881.0 (M)
In the mass spectra, Compound (II) gives the M+1 at 881.8
Molecular ion peak was observed to be m/z =881.8
Example-2
Determination of Related Compound:
Assay for Roxithromyin by HPLC comprises following steps:
Test solution:
Weigh accurately 50 mg of the test sample and transfer to a 25 ml volumetric flask. Add about 15 ml of solution A and shake the contents of the flask. Sonicate to dissolve . Dilute with the solution A to produce 25 ml and mix. Filter this solution through 0.45 \x membrane filter.
Reference solution (a):
Weigh accurately 50 mg of roxithromycin working standard and transfer to a 25 ml volumetric flask. Add about 15 ml of solution A and shake the contents of the flask. Sonicate to dissolve. Dilute with the solution A to produce 25 ml and mix.
Reference solution (b):
Transfer 1 ml of Reference solution (a) to a 100 ml volumetric flask. Dilute to volume
with solution A and mix.
Filter this solution through 0.45 |i membrane filter.

Reference solution (c):
Weigh accurately 5.0 mg of Roxithromycin for system suitability CRS and transfer to a test tube, add 2.5 ml of solution A and Filter this solution through 0.45 ^ membrane filter.
Reference solution (d):
Transfer 1.0 ml of toluene to a 100 ml volumetric flask. Dilute to volume with acetonitrile
and mix.
Further dilute 0.2 ml of this solution to 100 ml with solution A.
Procedure:
Before analysis equilibrate the chromatographic system with mobile phase A till a constant baseline is obtained to eliminate/minimize system noise. Inject the solutions in following sequence, record the chromatograms and interpret the results as mentioned in the following table:

Injection Solutions No. of Injections Interpretation / Calculations
Blank solution 1 Disregard any peak obtained due to Blank
Referencesolution(c) 1 The retention time of Roxithromycin is between 19.8 and 24.2 min. and relative retention time if impurity G with reference to Roxithromycin is about 1.15. Peak to valley ratio minimum 2
Adjust the flow rate of the mobile phase, if necessary, to meet the above requirements.
Referencesolution(d) 1 Identify the peak of toluene and disregard any peak due to toluene in the test solution.
Referencesolution(b) *5 Calculate the mean peak area obtained due to roxithromycin and relative standard deviation using Microsoft Excel or equivalent.The % Relative Standard Deviation obtained for 5 replicate injections is not more than 5.0 %

Test Solution 2 Calculate % Impurity against reference solution (b)Disregard any peak with an area less than 0.05 times that of the principal peak in the chromatogram obtained with reference solution (b).
*5 replicate injections are to be considered to meet the requirements.
Relative retention time with reference to roxithromycin (RT = Between 19.8 and 24.2 min)
Impurity A =about 0.28, impurity B =about 0.31, impurity C = about 0.33, impurity D = about 0.62, impurity E = about 0.67, impurity F = about 0.83, impurity G = about 1.15, impurity H = about 1.85, impurity I = about 3.1, impurity J = about 2.65, impurity K about 1.7, Compound (II) = about 0.91
Calculations:
Formula 1:
% Individual Impurity = Ai x Wsx 1 x25x Px 100
Asx25x100xWtx100
Formula 2:
% Total Impurity = Sum of each individual impurity and unknown impurity obtained
for test solution Where,
Ai = Peak area due to each individual impurity
As = Mean peak area of Roxithromycin peak in the reference solution (b) from the
replicate injections.
Ws = Weight of Roxithromycin working standard taken for preparation of reference
solution (a). Wt = Weight of test sample taken for preparation of test solution. P = Potency of Roxithromycin working standard

We Claim:
1. Erythromycin 9-[0-[(2-methoxyethoxy)ethoxymethyl]oxime] (compound of formula (II))

-CL ^0
H3C
CH3 L^_^-VCH3
\--^\_0H CH3
Formula (II)
2. A process for preparation of compound of formula (II) comprising of reacting Erythromycin 9-oxime with 1-(chloromethoxy)-2-(2-methoxyethoxy)ethane in presence of solvent and base
3. A process claimed in claim 2, wherein said solvent is selected from group comprising of acetone, ethyl acetate, Dimethyl formamide, Dimethylsulphoxide, N,N,N,N,N,N-hexamethylphosphoric acid triamide, ethyl ether, Tetrahydrofuran, dioxane or mixtures thereof.
4. A process claimed in claim 2, wherein said base is selected from group comprising of sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, calcium carbonate, barium carbonate or methoxide, ethoxide, propoxide and butoxide of sodium or potassium.
5. A method of testing the purity of a sample of Roxithromycin or a pharmaceutical dosage form comprising Roxithromycin, the method comprising assaying the said sample for the presence of compound of formula (II).

6. A method according to claim 5 for testing the purity of a sample of Roxithromycin,
comprising steps of:
(i) dissolving a sample of reference standard (Roxithromycin) in a solvent
(diluent) to produce a standard solution (STD) (ii) dissolving a sample of Roxithromycin in a solvent (diluent) to produce a
sample solution (iii) injecting the diluent, STD and sample solution on to an HPLC column, and
determining the area of each peak and calculating the assay of
Roxithromycin.
7. A method of testing the purity of a sample of Roxithromycin or a pharmaceutical dosage form comprising Roxithromycin, the method comprising determination of reference maker Compound (II) in the said sample by HPLC method.
8. A method according to claim 7 for testing the purity of a sample of Roxithromycin, comprising steps of:
(i) dissolving a sample Compound (II) in a solvent (diluent) to produce a
system suitability solution (SST); (ii) dissolving a sample of reference standard (Roxithromycin) in a solvent
(diluent) to produce a standard solution (STD); (iii) dissolving a sample of Roxithromycin in a solvent (diluent) to produce a
sample solution; (iv) injecting the diluent, SST, standard solution and sample solution on to an
HPLC column and determining the area of each peaks and calculating
related compound in the Roxithromycin sample.

A B S TRACT
Present invention relates to novel compound of formula (II) which is a useful reference marker for analysis of Roxithromycin of formula (I). Further it discloses method for analyzing purity of Roxithromycin samples for its formulation dosage forms.