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Title:
REFERENCE STANDARDS FOR DETERMINING THE PURITY OF TELITHROMYCIN AND PROCESSES THEREFOR
Document Type and Number:
WIPO Patent Application WO/2007/069264
Kind Code:
A2
Abstract:
A method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin, which method comprises assaying the said sample for the presence of compound A, B and D, wherein compound A is represented by following structural formula (I); Compound A compound B is represented by following structural formula (II); and compound B and compound D is represented by following structural formula (III) and the compounds A, B and D as well as their uses as reference markers for testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

Inventors:
DESHPANDE PANDURANG BALWANT (IN)
LUTHRA PARVEN KUMAR (IN)
NAUTIYAL RAJESH (IN)
SAHASRABUDDHEY BHUSHAN (IN)
PATEL MANISH KANCHANBHAI (IN)
DAVADRA MAHESH PRAVINCHANDRA (IN)
Application Number:
PCT/IN2006/000356
Publication Date:
June 21, 2007
Filing Date:
September 11, 2006
Export Citation:
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Assignee:
ALEMBIC LTD (IN)
DESHPANDE PANDURANG BALWANT (IN)
LUTHRA PARVEN KUMAR (IN)
NAUTIYAL RAJESH (IN)
SAHASRABUDDHEY BHUSHAN (IN)
PATEL MANISH KANCHANBHAI (IN)
DAVADRA MAHESH PRAVINCHANDRA (IN)
International Classes:
G01N33/50; C07H17/08
Domestic Patent References:
WO2005105821A22005-11-10
WO1997031929A11997-09-04
WO2005108984A22005-11-17
WO2002054062A22002-07-11
Foreign References:
EP1170588A12002-01-09
Other References:
KANFER I ET AL: "Analysis of macrolide antibiotics" JOURNAL OF CHROMATOGRAPHY A, ELSEVIER, AMSTERDAM, NL, vol. 812, no. 1-2, 3 July 1998 (1998-07-03), pages 255-286, XP004127049 ISSN: 0021-9673
Attorney, Agent or Firm:
MAJUMDAR, Subhatosh et al. (5 Harish Mukherjee Road, Calcutta 5, IN)
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Claims:

CLAIMS

1. A method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin, which method comprises assaying the said sample for the presence of compound A, B and D,

wherein compound A is represented by following structural formula;

compound B is represented by following structural formula;

and compound D is represented by following structural formula

2. A method according to claim 1 for testing the purity of a sample of Telithromycin, which comprises steps of: (i) dissolving a sample of reference standard (Telithromycin) in a solvent (diluent) to produce a standard solution (STD) (ii) dissolving a sample of Telithromycin 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 Telithromycin.

3. A compound which is 2'-Hydroxy-ll-amino-ll-N-[4-[4-(3-pyridyl) imidazol-1- yl]butyl]-l l-deoxy-5-O-desosaminyl-6-O-methylerythronolide A 11, 12-cyclic carbamate of formula A

4. A sample of compound as claimed in claim 3 which is in substantially pure form. 5; A sample according to claim 4 which has a purity level of 99% or above.

6. A process for producing compound A as defined in claim 3 comprises hydrolyzing 2'-O-benzoyl-l 1-amino-l l-N-[4-[4-(3-pyridyl)imidazol-l-yl]butyl]-l l-deoxy-5-O- desosaminyI-6-O-methylerythronolide A 11,12-cyclic of formula (VI) in an alcohol.

Formula Vl

7. A process for producing compound A as claimed in claim 6, wherein an alcohol is selected from group comprising of methanol, ethanol, n-propanol, isopropanol, tert- butanol, n-butanol or mixtures thereof.

8. A process for producing compound A as claimed in claim 7, wherein an alcohol is methanol.

9. A process for producing compound A as claimed in claim 6, wherein the reaction is optionally carried out in presence of mineral acid.

10. A process for producing compound A as claimed in claim 9, wherein mineral acid is selected from group comprising of HCl, H 2 SO 4 . 11. A process for producing compound A as claimed in claim 6, wherein compound A is optionally purified by recrystallization from ketonic solvent, preferably acetone. 12. A compound which is 2'-hydroxy-l 1-amino-l l-N-[4-[4-(3-pyridyl) imidazol-1-yl] Butyl]-l l-deoxy-5-0-desosaminyl-3-[(methylthio)methoxy]-6-0-methyl erythronolide A ll, 12-cyclic carbamate of formula B

13. A sample of compound as claimed in claim 12 which is in substantially pure form.

14. A sample according to claim 13 which has a purity level of 95% or above.

15. A compound which is N-oxide of Telithromycin i.e. 2'-hydroxyl-ll-amino-ll-N- [4-[4-(3-pyridyl) imidazol-1-yl] butyl] - ll-deoxy-5-O-[3'-N-oxide-5 '-methyl tetrahydro 2H-pyran-2'-ol]-6-0-methyl erythronolide A 11, 12 - cyclic carbamate of formula D

D

16. A sample of compound as claimed in claim 15 which is in substantially pure form.

17. A sample according to claim 16 which has a purity level of 99% or above.

18. A process for producing compound D as defined in claim 15 comprises reacting Telithromycin of formula (I) with oxidizing agent, in a suitable solvent.

Formula (I)

19. A process for producing compound D as claimed in claim 18, wherein oxidizing agent is hydrogen peroxide.

20. A process for producing compound D as claimed in claim 18, wherein suitable solvent is methanol.

21. A method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin, which method comprises determination of reference maker A, B and D in the said sample by HPLC method.

22. A method according to claim 21 for testing the purity of a sample of Telithromycin, which comprises steps of:

(i) dissolving a sample of compound A, B and D alone or in any combination in a solvent (diluent) to produce a system suitability solution (SST); (ii) dissolving a sample of reference standard (Telithromycin) in a solvent (diluent) to produce a standard solution (STD); (iii) dissolving a sample of Telithromycin 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 Telithromycin sample.

23. Use of compound A as reference maker for testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

24. Use of compound B as reference maker for testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

25. Use of compound D as reference maker for testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

Description:

NOVEL COMPOUNDS

Field of invention:

The present invention relates to novel compounds useful as reference markers for the analysis of Telithromycin of formula (I) and pharmaceutical formulations thereof. It also discloses method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

Background of the invention: Telithromycin is chemically known as l l,12-dideoxy-3-de[(2,6-dideoxy-3-C-methyl-3- O-methyl-α-L-ribohexopyranosyl)oxy]-6-O-methyl-3-oxo-12,ll- (oxycarbonyl[4-[4-(3- pyridinyl)-lH-imidazol-l-yl]butyl]imino)-erythromycin. It is marketed under brand name "Ketek" and is indicated for the treatment of bacterial infections. Telithromycin of formula (I) is a ketolide which differs chemically from the macrolide group of antibacterials by the lack of α-L-cladinose at position 3 of the erythronolide A ring, resulting in a 3-keto function. It is further characterized by a C 11 -C 12 carbamate substituted by an imidazolyl and pyridyl ring through a butyl chain. Telithromycin exhibits antibacterial activity and is used for treatment of community acquired pneumonia, acute exacerbation of chronic bronchitis, acute sinusitis, tonsillitis/pharyngitis.

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.

It has been observed by inventors of the present invention that Telithromycin contain five known impurities. These impurities are:

(i) 2'-Hydroxy-l 1-amino-l l-N-[4-[4-(3-pyridyl) imidazol-l-yl]butyl]-l 1-deoxy- 5-O-desosaminyl-6-O-methylerythronolide A 11, 12-cyclic carbamate (Compound A)

(ii) 2'-hydroxy-l 1-amino-l l-N-[4-[4-(3-pyridyl) imidazol-1-yl] Butyl]- 11 -deoxy- 5-O-desosaminyl-3-[(methylthio) methoxy]-6-O-rnethylerythronolide A 11,

12-cyclic carbamate (Compound B) (iii) Epimeric Telithromycin (Compound C) (iv) N-oxide Telithromycin (Compound D)

(v) 2'-benzoyl-l 1-amino-l l-N-[4-[4-(3-pyridyl)imidazol-l-yl]butyl]-l l-deoxy-5- O-desosaminyl-6-O-methylerythronolide A 11, 12-cyclic carbamate

(Compound E)

Out of these, compounds A, B, C and D are impurities arising from side reactions during the synthesis of Telithromycin and compound E is synthetic precursor of the active agent. The inventors of present invention have found that out of these five impurities, compound A 5 B and D can be used as reference markers for the analysis of Telithromycin or of pharmaceutical dosage forms comprising Telithromycin.

Object of the invention:

It is an object of the present invention to provide novel compounds useful as reference markers for the analysis of Telithromycin and pharmaceutical formulations thereof.

Another object of the present invention is to provide a method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin.

Yet another object of the present invention is to provide novel compounds, Compound A, B and D, which are useful as reference markers for the analysis of Telithromycin and pharmaceutical formulations thereof.

Further object of the present invention is to provide Telithromycin having purity greater than about 98%, more preferably greater than about 98.5%.

Summary of the invention:

Accordingly, there is provided novel compounds A, B and D, which are useful as reference markers for the analysis of Telithromycin and pharmaceutical composition thereof. Also, there is provided process for preparation of these novel compounds.

The present invention also provides method of testing the purity of a sample of Telithromycin or a pharmaceutical dosage form comprising Telithromycin comprising assaying the said sample for the presence of a compound selected form Compounds A, B and D.

Another aspect of the present invention provides Telithromycin having purity greater than about 98%, more preferably greater than about 98.5%.

Detailed description of the invention:

The synthesis of Telithromycin is in accordance with PCT application No. PCT/IN05/00125 which is incorporated herein as a reference and depicted below in synthetic scheme.

2'-Hydroxy-l 1-amino-l l-N-[4-[4-(3-pyridyl) imidazol-l-yl]butyl]-l l-deoxy-5-O- desosaminyl-6-O-methylerythronolide A 11, 12-cyclic carbamate (Compound A) is formed due to side reactions occurring during the synthesis of Telithromycin.

2'-O-benzoyl-l 1-amino-l l-N-[4-[4-(3-pyridyl)imidazol-l-yl]butyl]-l l-deoxy-5-O- desosaminyl-6-O-methylerythronolide A 11,12-cyclic carbamate of formula (VI) is oxidized to 2'-benzoyl-l 1-amino-l l-N-[4-[4-(3-pyridyl)imidazol-l-yl]butyl]-l 1-deoxy- 5-O-desosaminyl-6-O-methylerythronolide A 11,12-cyclic carbamate of formula (VII). Further hydrolysis of compound of formula (VII) results in Telithromycin of formula (I). During the step of oxidation some amount of the starting material i.e. compound of formula (VI) remains unreacted, and then in next step of hydrolysis it gets converted to compound A.

Compound A is prepared by carrying out hydrolysis of 2'-O-benzoyl-l 1-amino-l l-N-[4- [4-(3-pyridyl)imidazol-l-yl]butyl]-ll-deoxy-5-0-desosaminyl- 6-0-methylerythronolide A 11,12-cyclic carbamate of formula (VI) in alcohol. The alcohol is selected from group comprising of methanol, ethanol, n-propanol, isopropanol, tert-butanol, n-butanol or mixtures thereof, preferably methanol. The reaction is carried out at a temperature of 0 0 C to 100°C and preferably at 20 0 C to 7O 0 C. The reaction can also be carried out in presence of mineral acid selected from group comprising of HCl, H 2 SO 4 and like.

Compound A can be further purified by recrystallization from a solvent preferably acetone. .

Compound A is substantially pure and having purity at least 99%.

2'-hydroxy-l 1-amino-l l-N-[4-[4-(3-pyridyl) imidazol-1-yl] Butyl]-1 l-deoxy-5-O- desosaminyl-3-[(methylthio) methoxy]-6-0-methylerythronolide A 11, 12-cyclic

carbamate (Compound B) is also formed due to side reactions occurring during the synthesis of Telithromycin.

During oxidation of compound of formula (VI) to compound of formula (VII) by Corey- Kim oxidation method using N-cholorsuccinimide and dimethyl sulphide in presence of triethyl amine, compound of formula (Via) is obtained as impurity. This compound is carried forward in last step of hydrolysis and gets converted to Compound B.

Compound B is isolated by carrying of preparative HPLC of the crude Telithromycin. Compound B obtained by this method is substantially pure and has purity at least 95%.

N-oxide Telithromycin (Compound D) is also formed due to side reactions occurring during the synthesis of Telithromycin.

Telithromycin obtained in accordance with Scheme-I is further purified by treating Telithromycin with halogenated solvent to obtain solution and adding an anti-solvent to said solution to obtain pure Telithromycin.

The halogenated solvent is selected from group comprising of methylene dichloride, ethylene dichloride, chloroform, carbon tetrachloride or like, preferably methylene dichloride. The anti-solvent is selected from group comprising of methyl tertbutyl ether, diethyl ether, diisopropyl ether, cyclohexane, n-heptane, n-hexane or like, preferably methyl tertbutyl ether.

Ethereal solvents usually contain some content of peroxide in it. Thus when used for purification of Telithromycin, it causes oxidation of Telithromycin resulting in Compound D.

Compound D can thus easily be prepared by reacting Telithromycin with oxidizing agent, preferably hydrogen peroxide in a suitable solvent, preferably methanol. The reaction is carried out in temperature range of 2O 0 C to reflux temperature of the solvent for about 10 to 20 hours.

Compound D obtained by method described above is substantially pure and has purity at least 99%.

When compounds A and B are used as reference markers they must be in a suitably pure form.

The test sample of drug substance (Telithromycin) or drug product (pharmaceutical dosage form of Telithromycin) 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 A or B and D.

The content or each compound in the test sample can then be determined.

In one aspect of the present invention, the method for testing the purity of a sample of

Telithromycin by determination of Related Compounds comprises steps of:

(i) dissolving a sample of reference standard (compound A and D) in a solvent

(diluent) to produce a system suitability solution (SST);

(ii) dissolving a sample of reference standard (Telithromycin) in a solvent (diluent) to produce a standard solution (STD);

(iii) dissolving a sample of Telithromycin 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 Telithromycin the sample.

In this further aspect it is necessary to run a system suitability solution (SST) through the HPLC column prior to step (i) in order to determine the resolution factor between compound A and D.

In another aspect of the present invention, the method for testing the purity of a sample of Telithromycin by Assay comprises steps of:

(i) dissolving a sample of reference standard (Telithromycin) in a solvent (diluent) to produce a standard solution (STD)

(ii) dissolving a sample of Telithromycin 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 Telithromycin.

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-I Preparation of 2'-hydroxyl-ll-amino-ll-N-[4-[4-(3-pyridyl) imidazol-1-yl] butyl] - ll-deoxy-5-0-[3 '-N-oxide-5 '-methyl tetrahydro 2H-pyran-2'-ol]-6-O-metkyl erythronolide A 11, 12 - cyclic carbamate (Compound D).

Hydrogen peroxide solution (30% w/w) (5.5 ml) was added to a solution of Telithromycin

(2.0 g) in methanol (8.0 ml) at ambient temperature. After stirring at room temperature for 24 hrs, the resulting solution was extracted with chloroform. The chloroform layer was separated, washed with brine and dried on sodium sulfate. Solvent was evaporated from chloroform layer to give residue. Diisopropyl ether (14 ml) was added to the residue, stirred for 2 hour and filtered to give solid. The solid was dried under vacuum for

4 hrs at room temperature. (1.6 gm)

Purity of the product was determined by HPLC as 99.25 %. The HPLC condition were as follows

Column: Phenomenex Luna C-18, 5μm, 4.6 x 150 mm

Mobile phase: (A) Buffer* & (B) Acetonitrile

*Buffer Preparation: Dissolve accurately weighed about 2.28g OfKH 2 PO 4 and 0.103Og of

NH 4 Cl, adjust pH 8.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

Gradient Profile:

Temperature: Ambient

Flow rate: 1.0 ml//minute

Wavelength: 263 nm

Injection volume: 20 μl

Diluent: Use buffer of pH:7.0 # and acetonitrile in ratio(53: 47).

Buffer Preparation pH:7.0 # : Dissolve accurately weighed about 2.28g of KH2PO4 and

0.103Og of NH 4 Cl, adjust pH 7.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

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 D solution was injected and found the purity by area normalization method. The product had the following characteristics Molecular formula: C 43 H 65 N 5 O 11 Molecular wt: 828

TLC (silica gel with ethyl acetate: toluene: methanol: ammonia (40:25:20:2)

IR (in KBr): 3413, 2973,1551,1456,1710,1746,1320,1110 cm '1 . 1 H nmr (in CDCl 3 / 300 MHz): 8.93 (lH,s); 8.41 (lH,d); 8.06 (lH,m); 7.51 (lH,d); 7.32

(lH,d); 7.28 (lH,m); 4.9 (lH,dd); 4.36 (lH,d); 4.22 (lH,d); 4.01 (2H,t); 3.86 (lH,qt); 3.7

(5H,m); 3.55 (lH,m); 3.41 (2H,m); 3.12 (9H,m); 2.58 (6H,s); 1.9 (6H,m); 1.6 (5H,m);

1.46 (3H,s); 1.31 (15H,m); 1.16 (3H,d); 1.0 (3H,d); 0.83 (3H,t) δ ppm.

Mass spectra: m/z= 828.8 (M+l) In the mass spectra, Telithromycin N- Oxide gives the M+l at 828.8

Molecular ion peak was observed to be m/z = 828.8.

EXAMPLE-2

Preparation of 2'-Hydroxy-ll-amino-ll-N-[4-[4-(3-pyridyl) imidazol-l-yl]butyl]-ll- deqxy-5-O-desosammyl-6-O-methylerythronolide A 11, 12-cyclic carbamate (Compound A)

14Og 2'-O-benzoyl-l 1-amino-l l-N-[4-[4-(3-pyridyl)imidazol-l-yl]butyl]-l l-deoxy-5-O- desosaminyl-6-O-methylerythronolide A 11, 12-cyclic carbamate (compound VI) was added to methanol (1400ml) and heated to reflux for 8 hrs. The reaction was monitored on TLC. After completion of reaction, the solvent was distilled off under vacuum to give residue which is stripped with acetone to remove traces of methanol. Acetone (280ml) was added to the residue and heated to reflux, cooled to 15°C and stirred for 1 hrs. The solid was filtered and washed with chilled 2x70ml acetone to get the title compound A (84 gm). Purity of the product was determined by HPLC as 99.49 %. The HPLC conditions were as follows.

Column: Phenomenex Luna C-18, 5μm, 4.6 x 150 mm

Mobile phase : (A) Buffer* and (B) Acetonitrile

*Buffer Preparation : Dissolve accurately weighed about 2.28g Of KH 2 PO 4 and 0.103Og

Of NH 4 Cl, adjust pH 8.0 by diluted KOH solution and filter through 0.45 μ membrane filter paper.

Gradient Profile:

Temperature: Ambient

Flow rate: 1.0 ml//minute

Wavelength: 263 nm

Injection volume: 20 μl

Diluent: Use buffer of pH:7.0 # and acetonitrile in ratio(53: 47).

Buffer Preparation pH:7.0 : Dissolve accurately weighed about 2.28g of KH 2 PO 4 and

0.103Og of NH 4 Cl, adjust pH 7.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

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 A solution was injected and found the purity by area normalization method.

The product had the following characteristics: Molecular formula: C 4S He 7 NsOjO Molecular mass: 814.02 TLC (silica gel with ethyl acetate: toluene: methanol; ammonia: 40:25:20:2)

IR (in KBr): 3479, 2971,2939,1551,1455,1110,1167,1733 cm "1 .

1 U NMR (in CDCl 3 / 300 MHz) : 8.97 (IH s ); 8.41 (IH d ); 8.09 (IH d );

7.53 (IH s ); 7.41 (IH s); 7.28 (IH m); 5.01 (IH d); 4.39 (IH d); 4.03 (2H m);

3.64 (4H t); 3.48 (3H d); 3.2 (IH t); 3.04 (IH d); 2.78 (3H s); 2.5 (3H m); 2.2 (8H s); 1.93 (5H m); 1.66 (7H m);1.42 (3H s); 1.27 (6H d); 1.21 (5H d); 1.1 (7H d); 1.01 (4H d); 0.8 (4H t) δ ppm. Mass spectra: m/z= 814.8 (M+l)

EXAMPLE-3 Isolation of 2'-hydroxy-ll-amino-ll-N-[4-[4-(3-pyridyl)imidazol-l-yl] Butyl] -11- deoxy-5-0-desosaminyl-3-[(methylthio) methoxy] -6-O-methyIerythronolide A 11,12-cyclic carbamate ( Compound - B)

Compound-B was isolated from crude Telithromycin by Preparative Chromatography using Buffer : Acetonitrile :: 68 : 32 ( Buffer being 50 ml Ammonia in 1000 ml distilled water, pH adjusted to 8.0 with glacial acetic acid). Other chromatographic conditions are Column: X-Terra 20 X 250 mm id; Flow Rate: 3O.ml/min; λ max: 263 nm. ( 20 mg)

The product had the following characteristics: Molecular formula: C 4S H 71 NsO 10 S Molecular mass: 874

IR (in KBr): 3604, 2972, 1506, 1714, 1747, 1317, 1107 cm "1 .

1 H NMR (in CDCI 3 / 300 MHz) : 8.88 (IH s ); 8.38 (IH d ); 8.03 (IH d );

7.54 (IH s ); 7.30 (IH s); 7.24 (IH m); 5.5 (4H s); 4.92 (IH d); 4.79 (IH d); 4.51 (IH d); 4.41 (2H d); 3.99(2H t); 3.72 (3H m); 3.52 (3H m); 3.26 (IH d); 3.17 (IH m); 3.01 (IH m); 2.84 (4H s);2.7 (IH m); 2.5 (IHm); 2.33 (6H s); 2.19 (3H s); 1.98 (3H s); 1.85(5H m); 1.65 (4H, m); 1.56 ( 2H d); 1.45 (2H, m); 1.34 ( 3H s); 1.21 (7H d); 1.16 (3H d); 1.0 (10Hm); 0.74 (4Ht) S ppm. Mass spectra: m/z= 875.8 (M+l) '

EXAMPLE-4

Assay for TelithromycJH by HPLC

Assay for Telithromycin by HPLC comprises following steps:

Preparation of Standard Solution & System Suitability Solution

Accurately weigh and transfer about 125mg of standard into a 50ml volumetric flask, dissolve in 15 ml of diluent and sonicate it for 2 min.; make up the volume with diluent and mix for 2 min. Further dilute 5mL of this solution to 5OmL with diluent and mix well.

Preparation of Sample Solution

Accurately weigh and transfer about 125mg of test into a 50ml volumetric flask, dissolve in 15 ml of diluent and sonicate it for 2 min.; make up the volume with diluent and mix for 2 min. Further dilute 5mL of this solution to 5OmL with diluent and mix well. Prepare test solution in duplicate.

Chromatographic Procedure

The following conditions were used:

Column: Phenomenex Luna C- 18, 5μm, 4.6 x 150 mm

Mobile phase: (A) Buffer* (55%) and (B) Acetonitrile (45%) *Buffer Preparation: Dissolve accurately weighed about 2.28g OfKH 2 PO 4 and 0.103Og of

NH 4 Cl, adjust pH 8.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

Temperature: Ambient

Flow rate: 0.8ml//minute Wavelength: 263 nm

Injection volume: 20 μl

Diluent: Use buffer of pH: 7.0 # and acetonitrile in ratio (55: 45).

Buffer Preparation pH:7.0 # : Dissolve accurately weighed about 2.28g of KH 2 PO 4 and 0.103Og of NH 4 Cl, adjust pH 7.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

Injection Procedure

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 system suitability solution were injected six replicate and the relative standard deviation was calculated and it should not be more than 1.0%.

The standard solution and sample solution were then injected onto the column.

Calculations

From the main peak area of the standard solution the Assay for Telithromycin was calculated as follows:

Ws X At XP

% Telithromycin =

WtXAs where: Ws=weight (mg) of Telithromycin standard taken

Wt=weight (mg) of Telithromycin sample taken

P=% purity of Telithromycin reference standard

As=area of Telithromycin peak in standard solution injection

At=area of Telithromycin peak in sample solutions injection

EXAMPLE-5

Determination of Related Compounds

% of individual reference marker in drug by HPLC comprises following steps:

Preparation of System Suitability Solutions

In a 10ml volumetric flask transfer accurately weighed 2.5mg of Diol (compound A) and N-Oxide (compound D) impurity, add about 5ml of diluent and sonicate to dissolve, makeup the volume with diluent.

Pipette out 0.5mL of Stock solution into a 50ml volumetric flask and makeup the volume with diluent.

Preparation of Standard Solution

Accurately weigh and transfer about 12.5mg of Telithromycin standard into a 25ml volumetric flask, dissolve in 15 ml of diluent and sonicate it for 2 min.; make up the volume with diluent and mix for 2 min.

Preparation of Sample Solution

Accurately weigh and transfer about 12.5mg of sample into a 25ml volumetric flask, dissolve in 15 ml of diluent and sonicate it for 2 min.; make up the volume with diluent and mix for 2 min.Prepare test solution in duplicate

Chromatographic Procedure

The following conditions were used:

Column: Phenomenex Luna C-18, 5μm, 4.6 x 150 mm

Mobile phase: (A) Buffer* and (B) Acetonitrile

*Buffer Preparation: Dissolve accurately weighed about 2.28g OfKH 2 PO 4 and 0.103Og of

NH 4 Cl, adjust pH 8.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

Gradient Profile:

Temperature: Ambient Flow rate: 1.0 ml//minute Wavelength: 263 nm Injection volume: 20 μl

Diluent: Use buffer of pH: 7.0 and acetonitrile in ratio (53: 47).

Buffer Preparation pH:7.0 # : Dissolve accurately weighed about 2.28g of KH 2 PO 4 and 0.103Og of NH 4 Cl, adjust pH 7.0 by diluted KOH solution and filter through 0.45μ membrane filter paper.

Injection Procedure

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 system suitability solution was injected and the resolution factor between Diol and N-Oxide was calculated and it should be more than 6.0.

The standard solution and sample solution were then injected onto the column.

Calculations The content of any component eluting was calculated with respect to the standard Telithromycin as follows:

Ws X Ai

% of individual compound (w/w) =

Wt X As X RRF

where

Ai=area of peak for Individual compound in sample solution injection Ws=weight (mg) of Telithromycin reference standard taken As=area of Telithromycin peak in standard solution injection Wt=weight (mg) of Telithromycin sample taken RRF - relative response factor

TABLE l

Retention Relative Relative Response

Component Time (min.) Retention Time Factor (RRF)

Compound A 3.8 0.5 0.96

Compound B 11.8 1.6 1.14

Compound D 2.2 0.3 1.24

Telithromycin 7.4 1.0 1.00