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 determination of average
molecular weight of Pentosan polysulfate sodium using Dextran calibration standard by GPC
2. 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 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 method of Gel Permeation Chromatographie (GPC) for the determination of average molecular weight of Pentosan polysulfate sodium.
Background of the invention
Pentosan polysulfate sodium [37319-17-8] is chemically known as [(2S,3R,4S,5R)-5-hydroxy-2-{[(3R,4S,5R,6R)-6-hydroxy-4,5-bis(sulfooxy)oxan-3-yl]oxy}-4-(sulfooxy)oxan-3-yI]oxidanesulfonic acid having molecular weight 4000 to 6000 Dalton.
The current pharmaceutical product containing Pentosan polysulfate sodium salt is being sold by Ortho McNeil under the trade name Elmiron ® in the form of capsules which is used for the treatment of bladder infection, interstitial cystitis and tumors.
The structural formula is as shown below.

Pentosan polysulfate was disclosed in US patent no. 2689848. The process wherein Xylan was treated with chlorosulfonic acid in the presence of pyridine to obtain sulfuric acid ester salt of xylan followed by oxidative depolymerisation in acidic or neutral aqueous medium to obtain depolymerised product which was dialyzed and

followed the fractionation process to obtain desired product. However, the isolation of sulfuric acid ester salt of xylan provides sticky material. Moreover, this process did not provide end product with desired molecular weight.
The product mixture is a single compound pure enough to comply with pharmaceutical standards. The pentosan polysulfate sodium must be analyzed for average molecular weight determination, typically by GPC method, to determine if it is suitable for continued processing or ultimately for use in a drug product.
Summary of the invention
It is therefore an object of the present invention to provide an improved method of Gel permeation chromatographic (GPC) for the determination of average molecular weight of pentosan polysulfate sodium.
Another object of the present invention provides a GPC method for Pentosan polysulfate sodium containing average molecular weight between 4000 to 6000 Dalton by GPC.
A furthermore object of the present invention is to provide a simple, accurate and well-defined GPC method for the determination of average molecular weight of Pentosan polysulfate sodium using dextran calibration standard.
Brief description of the drawings
Fig. 1 shows the chromatogram of Mobile phase (Blank reading)
Fig. 2 shows the chromatogram of standard solution of Pentosan polysulfate sodium
Fig 3 shows the chromatogram of Overlay of Dextran calibration standards
Fig 4 shows the chromatogram of Test sample

Detailed description of the invention:
According to present aspect, present invention provides an improved process for the preparation of Pentosan polysulfate of formula (I) or salt thereof, which is operationally simple, easy to handle and applicable at an industrial scale.
According to another aspect of the present invention is to provide precise method for determining of average molecular weight of Pentosan polysulfate sodium of formula (I) or salt thereof, by using dextran calibration standard comprising steps of:
a) Injecting the calibration standard and sample solution into a Ultrahydrogel ™ 250 (300mm X 7.8mm), make: Waters (Part No WAT011525).
b) Preparing Buffer ( Mobile phase) by adding Sodium chloride and sodium dihydrogen phosphate dihydrate in water and pH adjusted to 6.50 with sodium hydroxide solution
c) Measuring of the retention time of Dextran calibration standard and test sample of pentosan polysulfate sodium with a Refractive index detector (having an appropriate recording device).
d) Combining a Pentosan polysulfate Sodium and Dextran calibration standard in the buffer.
e) The buffer is prepared by mixing about one liter water with about 20gm sodium chloride and 15.6gm sodium dihydrogen phosphate dihydrate.
f) The column temperature should be maintained at about 35 C.
The average molecular weight of Pentosan polysulfate sodium of formula (I) or salt thereof, obtained as per present invention by using LC system, used for method development was Shimadnzu LC10 CHT (manufactured by Shimandzu corp. Ltd) system with a Refractive index detector. The out put signal was monitored and processed using Class VP and GPC software (designed by shimandzu).
The chromatographic column used was a Waters Ultrahydrogel ™ 250 (300mm X 7.8mm). The mobile phase consists of 20gm Sodium chloride and \5.6gm sodium

dihydrogen phosphate dihydrate in one liter of water after adjusting pH 6.50 with sodium hydroxide solution. The flow rate of the mobile phase was kept at l.Oml/min. The column temperature was maintained at 35 C and the detection was monitored by refractive index detector. The injection volume was 20 µL for calibration standard and test sample after dissolving in mobile phase.
The process of the present invention is described by the following examples, which are illustrative only and should not be construed so as to limit the scope of the invention in any manner.
Example-1 Preparation of Calibration standard solution
50mg of each of Dextran standards with molecular weight ranging from 1500 Daltons to 21500 Daltons were accurately weighed and transferred to the lOmL volumetric flask separately; 5ml of mobile phase was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with same and filter through 0.22micron filter paper.
A working solution of 5mg/ml was prepared for the determination of average molecular weight analysis.
Example-2 Preparation of Test sample solution-
50mg of test sample was accurately weighed and transferred to the lOmL volumetric flask; 5ml of mobile phase was added in to the flask and shaken for five minutes in an ultrasonic bath and made up to mark with same.
A working solution of 5mg/ml was prepared for the determination of average molecular weight analysis.

We Claim,
1. A method to determine average molecular weight of pentosan polysulfate sodium by gel permeation chromatographic (GPC).
2. A method according to claim 1 wherein, pentosan polysulfate sodium molecular weight is determine by gel permeation chromatographic (GPC) containing average molecular weight between 4000 to 6000 Dalton by GPC.
3. A method according to claim 1 wherein, buffer of mobile phase is solution of sodium chloride and sodium dihydrogen phosphate dihydrate in water where pH is 6.50.
4. A method according to claim 1 wherein, pentosan polysulfate sodium molecular weight is determine by gel permeation chromatographic (GPC) using dextran calibration standard.
5. A method according to claim 1 wherein, dextran calibration standard and test sample of pentosan polysulfate sodium is prepared in buffer of water sodium chloride and sodium dihydrogen phosphate dihydrate.
6. A method according to claim 5 wherein, buffer is prepared by mixing one liter water with about 20gm sodium chloride and 15.6gm sodium dihydrogen phosphate dihydrate.
7. A method according to claim 5 wherein, dextran calibration standard and test sample of pentosan polysulfate sodium is injected into Ultrahydrogel M 250 (300mm X 7.8mm), make: Waters (Part No WAT011525).
8. A method according to claim 3 wherein, dextran calibration standard and test sample of pentosan polysulfate sodium retention time is measured by with a Refractive index detector.

9. An method for determining of average molecular weight of Pentosan polysulfate sodium of formula (I) or salt thereof, by using dextran calibration standard comprising steps of:
a) Injecting the calibration standard and sample solution into Ultrahydrogel ™ 250 (300mm X 7.8mm), make: Waters (Part No WAT011525).
b) Preparing Buffer (Mobile phase) by adding Sodium chloride and sodium dihydrogen phosphate dihydrate in water and pH adjusted to 6.50 with sodium hydroxide solution
c) Measuring of the retention time of Dextran calibration standard and test sample of pentosan polysulfate sodium with a Refractive index detector (having an appropriate recording device).
d) Combining a Pentosan polysulfate Sodium and Dextran calibration standard in the buffer.
e) The buffer is prepared by mixing about one liter water with about 20gm sodium chloride and 15.6gm sodium dihydrogen phosphate dihydrate.
f) The column temperature should be maintained at about 35 C.