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 capillary electrophoresis method for the
quantitative determination of Pentosan polysulphate sodium.
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 Pentosan polysulphate sodium content (Purity) by quantitative determination by using Capillary Electrophoresis in correlation with internal standards method calculation.
BACKGROUND OF THE INVENTION.
Pentosan polysulphate sodium is a semi-synthetically produced heparin-like macro
molecular carbohydrate derivative, which chemically and structurally resembles
glycosaminoglycans. Pentosan polysulphate sodium is based on a natural product xylan.
extracted from the bark of beech wood tree. Pentosan polysulphate sodium is chemically
known as [(2S,3R,4S,5R)-5-hydroxy-2-{[(3R,4S,5R,6R)-6-hydroxy-4,5-
bisfsulfooxy)oxan-3-yl]oxy}-4-(sulfooxy)oxan-3-yl]oxidanesulfonate sodium . It is a white odorless powder, slightly hygroscopic and soluble in water to 50% at pH 6. It has a molecular weight of 4000 to 6000 Dalton with the following structural formula:
Structure of Pentosan polysulfate sodium

Pentosan polysulphate sodium is currently marketed under the trade name Elmiron by Ortho Mcneil in the form of oral 100 capsules for the treatment of interstitial cystitis.
US 2689848 discloses process for the preparation of Pentosan polysulphate sodium

From xylan.Xylan is a polysaccharide found in plant cell walls., is toxic and hence not suitable for pharmacological usage. Degree of polymerization is a measure of toxicity and hence is directly related to molecular size and configuration.
WO 2007/123800 discloses qualitative analysis of Pentosan polysulphate sodium by capillary electrophoresis wherein, substantially bell shaped curve in the electropherogram corresponding to the presence of Pentosan polysulphate sodium in a graph of charge-in-absorption-versus-time. The bell shaped curve has a first half corresponding to an earlier absorption and a second half corresponding to a later absorption. Though this application explains CE absorption curve with a bell shape defining the area percent of pentosan polysulphate sodium, it does not disclose any quantitative analysis for assessing the purity of each peak obtained in the electropherogram of Pentosan polysulphate sodium.
WO 2008/107906 claims amorphous pentosan polysulphate sodium with molecular weight of range 3000 to 10000 Dalton, wherein PENTOSAN POLYSULPHATE SODIUM is subjected to crystallization in alcohol or lyophilized to obtain amorphous pentosan polysulphate sodium. This patent application also does not reveal any analytical method for determining the quality control limits of pentosan polysulphate sodium.
Journal of Pharmaceutical and Biomediacal analysis 31 (2003) 133-141 discloses the varied therapeutic uses of pentosan polysulphate sodium. It has been investigated for its effectiveness against osteoarthritis, interstitial cystitis, and myocardial ischemia. Also pentosan polysulphate sodium has been found to possess anticoagulant properties helpful in preventing brain paralysis and also a potent substituent for relatively costlier heparin. Furthermore, pentosan polysulphate sodium along with other sulfated polyanions, have shown beneficial effects in treatment of Alzheimer's cerebral ischemia, HIV related encephalopathy or prion diseases. This journal discloses capillary electrophoresis method of analysis to characterize glycosaminoglycans. However the disclosed analytical method does not mention the quantitative aspects of pentosan polysulphate sodium.

Chromatographia 2003, 58,343-348 discloses analytical method for pentosan polysulphate sodium using fourier transform infrared spectroscopy, wherein the mid -TR region is found to give accurate peaks relating to pentosan polysulphate sodium. This journal article discloses comparison data of different samples of pentosan polysulphate sodium by using mid-IR region. However even this article does not disclose any quantitative analytical method for pentosan polysulphate sodium.
Journal of urology Vol 175, 1143-1147 discloses the level of pentosan polysulphate sodium in urine which correspond to low molecular weight fractions of pentosan polysulphate sodium. High molecular weight fractions do not get absorbed by the gastrointestinal tract and hence not excreted out but instead get accumulated in the body. This difference is because commercial pentosan polysulphate sodium is a heterogeneous mixture of various molecular sizes. Therefore the molecular size of pentosan polysulphate sodium affects the urine excretion.
Hence the polydisperse nature of pentosan polysulphate sodium necessitates in the art for an effective reproducible method of quantitative analytical procedure that detects variations in active pharmaceutical ingredient relating to size, degree of sulfation. Also there exists a need in the art for reproducible quality control method to assess different batches of pentosan polysulphate sodium provided by potential suppliers thereby purity of pentosan polysulphate sodium can be optimized.
Surprisingly, the present inventors have found a qualitative analytical procedure wherein pentosan polysulphate sodium purity is optimized and peaks corresponding to pentosan polysulphate sodium are characterized. This analytical method will help to obtain pentosan polysulphate sodium with minor variations in molecular size thereby reducing the toxic effects due to polydisperse nature of pentosan polysulphate sodium as described above.
The present invention relates to Pentosan polysulphate sodium content (Purity) by quantitative determination by using Capillary Electrophoresis in correlation with internal standards method calculation.

The following are the salient features of said invention with adaption of analytical methodology:
■ Quantification of Marker peaks (Fingerprints peaks) obtained in the Electropherogram.
■ Identification of finger prints by Relative Migration time-RMT
■ Quantification of RMT by RMF using internal standard method.
■ Purity by area % of all secondary peaks by CE using internal standard with quantification.
OBJECT OF THE INVENTION:
The main object of the present invention is to provide pentosan polysulphate sodium quantification by capillary electrophoresis, wherein the pre apex portion of the bell curve exhibits multitude of peaks (1-9) are quantifying by using halide internal standard.
Another object of the present invention is to provide pentosan polysulphate sodium quantification by capillary electrophoresis, wherein sum area percentage of all secondary peaks at the ascending of bell curve are not more than 15 % of total area under bell curve and secondary peaks.
Another object of the present invention is to provide pentosan polysulphate sodium identification by capillary electrophoresis, wherein 9 major secondary peaks before apex of bell curve are identifying with relative migration time from 1,1 to 2.2 with respect to internal standard peak.
SUMMARY OF THE INVENTION:
The present invention provides reproducible method of analysis for pentosan polysulphate sodium containing significantly homogenous molecular weights.

Samples of Pentosan polysulphate sodium were analyzed by using capillary electrophoresis methodology. Separation of components with respect to migration time during capillary electrophoresis may be based on differences in various components' charge to mass ratio. A homogeneous buffer solution may be used .A constant electric field may be applied. The process of capillary electrophoresis may be depending on pH of buffer. The analytical capability of capillary electrophoresis methodology on a Pentosan polysulphate sodium in accordance with various embodiments of the invention allows quantification of sulphate in the presence or absence of a chloride ; quantification of 8 major secondary peaks of lower molecular weight pentosan polysulphate sodium.
The main aspect of the present invention is to provide pentosan polysulphate sodium Quantified by capillary electrophoresis, wherein the pre-apex bell curve exhibits major secondary peaks (1-9) are quantifying with respect to halide internal standard and post apex bell curve exhibits no secondary peaks.
Yet another aspect of present invention is to provide pentosan polysulphate sodium quantification by capillary electrophoresis methodology, wherein the area percentage of sum area of all secondary peaks is not more than 15 % of total area of all secondary peaks and area under bell curve.
Brief description of the Drawings:
Figure 1: Electropherogram for standard sodium sulphate and Sodium bromide as internal standard
Figure 2a: Electropherogram for Pentosan polysulphate sodium sample diluted with internal standard

Figure 2b: Overlaid electropherogram for Pentosan polysulphate sodium six replicates
Figure 3: Electropherogram for standard sodium bromide as internal standard
Figure 4: Electropherogram for Pentosan polysulphate sodium sample diluted with internal standard with integration of bell curve
DETAILED DESCRIPTION OF THE INVENTION:
Various embodiments of the present invention are directed to quantitative analysis of pentosan polysulphate sodium. The pentosan polysulphate sodium is used as an anticoagulant to treat osteoarthritis, interstitial cystitis, and transmissible spongiform encephalopathy. Pentosan polysulphate sodium due to its varied pharmacological activities demands an effective quantitative analytical method to differentiate fine structural details of polysaccharides and should exhibits uniformity in all production batches. The natural product xylan can vary from batch to batch and hence may give rise to structural differences in the product obtained from such batches. Therefore there exists a need for an efficient quantitative analytical method for optimizing the purity of pentosan polysulphate sodium.
The main embodiment of the present invention relates to process optimization of pentosan polysulphate sodium quantified by capillary electrophoresis. Electrophoresis is an electro-analytical technique used for the separation of large molecules from small molecules. The differential movement of molecules through a capillary under the influence of an electric field is called capillary electrophoresis. The main components of capillary electrophoresis are
1) Source vial, destination vial and capillary tube filled with electrolyte buffer solution.
2) Capillary is introduced into the sample vial and then returned to source vial.

3) Migration of analytes initiated by electric field (E) applied between the source & destination vial.
4) All ions (+)ve or (-)ve are pulled through capillary in same direction by electro osmotic flow.
5) Separated analytes get detected near the outlet end of the tube which is sent to data output device and displayed as electropherogram as peaks with different migration times.
The detectors can be either UV-Vis absorbance, fluorescence detection or coupled with MS or surface enhanced raman spectroscopy.
Pentosan polysulphate sodium is a semi-synthetic polysulfated oligosaccharide comprising a mixture of multiply charged anionic polysaccharides. After injection of a sample into the capillary tube, the right end of the tube and an electrode are placed in a buffer reservoir. A voltage is applied between the electrode and cathode. The bulk fluid in the capillary flows with the positively charged, hydrated cations in a process called electro-osmosis. Other positively charged analytes move in the same direction towards the cathode, if electro osmotic flow exceeds electrophoretic mobility for a group of anions, the anions will move in the other direction. The polarity of the electrodes may be reversed to ensure that the negatively charged anions in the sample pass the detector.
Pentosan polysulphate sodium and internal standard have anionic ends and is non -light absorbing. As the Pentosan polysulphate sodium sample and buffer move through the capillary , the similarly charged , non light absorbing sample displaces the light absorbing buffer, creating a zone of reduced buffer concentration .The sample and internal standard passes the detector window , wherein the detector will detect a reduction in light absorption EOF of buffer solution & electrophoretic mobilities of different anions will enable different components of Pentosan polysulphate sodium to travel the capillary tube at different rates which are seen at the detector. Therefore small and multiple charged anions move quickly and are detected at early times. Homogeneity of Pentosan polysulphate sodium may be detected by calculating relative size of small

oligosaccharides peaks as % of total Pentosan polysulphate sodium peaks in e lectropherogram.
Relative migration time is calculated by time required for migration by Pentosan polysulphate peaks in relation to halide internal standard peak.
In another embodiment, the present invention provides a pharmaceutical composition that includes a therapeutically effective amount of pure Pentosan polysulphate sodium and its salts prepared according to the processes of the present invention and one or more pharmaceutically acceptable carriers, excipients or diluents.
Accordingly, the pharmaceutical composition comprising pure Pentosan polysulphate sodium or their pharmaceutically acceptable salts along with one or more pharmaceutically acceptable carriers of this invention may further be formulated as solid oral dosage forms.
Instrumentation:
The analysis was performed on the Beckman Coulter CE instrument, equipped with a UV diode array detector; a polyamide coated fused silica capillary with an effective length of 37 cm was used. The wavelength was set at 217 nm. 32Carat software installed on a computer allowed for instrumental operation and integration strategies.
Solution Preparation:
1) Buffer preparation (8.75 mmol/ liter; pH 4.9)
Background electrolyte: 183.9 mg of Benzene -1,2,4-tricarboxilic acid dissolved in 50 ml
milli-Q water. A freshly prepared 0.1N NaOH was added to obtain a pH of 4.9.This
solution was then made to 100 ml with milli-Q water. The final concentration of 8.75
mmol/lit was obtained for the Benzene -1,2,4-tricarboxilic acid solution,
Internal standard solution (25 ppm as Bromide):

129.1 mg of sodium bromide dissolved in milli-Q water and diluted up to 100 ml with milli-Q, further 25 ml diluted to 1000 ml with milli-Q water.
Standard Sulphate solution (10 ppm as sulphate):
147.6 mg of sodium sulphate dissolved in dissolved and diluted up to 100 ml with 25 ppm bromide internal standard solution, further 2 ml diluted to 200 ml with 25 ppm bromide internal standard solution.
Sample preparation:
10 mg of Pentosan polysulphate sodium sample dissolved and diluted to 10ml with 25
ppm bromide internal standard.
Method development:
All solution was filtered through 0.22 micron syringe filter. The sample was introduced at the cathode hydrodynamicaly at an optimal setting of 20 seconds at 0.5 psi. The optimal voltage applied was -20 kV. The temperature for the optimum analysis was held at 25°C .Before its first use , the capillary was flushed with freshly prepared 1.0 N NaOH for 1 hour. Equilibrium was performed for 1 hour by flushing background electrolytes (BGE)at 25 °C. This was the effect of protonating the silanol sited on the capillary surface. The capillary was rinsed between each injection for 5 minutes with BGE. After each sequence or where the capillary was not used for extended periods, the capillary was rinsed with milli-Q water for 30 min and dried by flushing with air for 30 min.
Pentosan polysulphate sodium samples were run using the above conditions and electropherogram for various samples were obtained .Buffer additives may include any of the following organic salts, organic solvents, urea, sulfonic acids, cationic surfactants, cellulose derivatives, amines, organic acids and organic polymers. The analytic capability of capillary electrophoresis on a Pentosan polysulphate sodium in accordance with various embodiments of the invention allows quantification of sulfate in the presence or absence of a known amount of chloride; quantification of total Pentosan polysulphate sodium.

An electropherogram for Pentosan polysulphate sodium is prepared using capillary electrophoresis in a manner that satisfies a peak resolution standard, the electropherogram comprising a change in absorption-versus-time graph. Figure 2 depicts electropherogram of Pentosan polysulphate sodium and internal standard , wherein the pre apex bell curve exhibits a bromide peak and multitude of secondary peaks (1-9) and post apex bell curve exhibits no secondary peaks. The bell curve generally appears in the middle of the electrophero gram.
Methodology used for thelnvention
Capillary electrophoresis system, Make: Beckman Coulter; Model: P/ACE MDQ
Operating software: 32 carat
Microbalance, Make: Mettter-Toledo, Model: UMX-2
Method parameters:

Column Fused silica capillary column
42 cm x 50 μ , effective length 34 cm
Current 300 μA
Sample cooler 25°C
Cartridge temp 25 °C
Absorbance Signal Indirect
Equilibration period & pressure 2 min at 30 psi with rinsing buffer
Injection 20 second at 0.5 psi
Voltage 20 KV
Runtime 12 min.
Polarity Reverse
Detection wavelength 217nm
Buffer Preparation Dissolve 183.9 mg of 1, 2, 4-benzene tricarboxilic in 60 ml milli-Q water, adjust pH 4.90 with 1 M NaOH solution and make to 100 ml with milli-Q water.
a) All weightings should be done using microbalance only.
b) All measuring glassware should be class A only.

c) Milli-Q water should be used fresh only.
d) Use 0.22 urn filter before injecting any solution.
Solutions preparation:
I. Internal standard solution preparation (25 ppm Bromide standard solution. Use as
diluent) [Solution A]:
Weigh and transfer 128.75 mg of sodium bromide (crystalline >99.99% trace metals
basis.
Make: Sigma Aldrich, Cat. No. 229881-10 G) standard into a 100 mL volumetric flask,
dissolve and dilute to volume with fresh Milli-Q water. Dilute 25 mL of this solution to
1000 mL with fresh Milli-Q water.
II. Standard Sulphate solution (10 ppm sulfate solution)[Solution-B]:
Weigh and transfer 147.84 mg of sodium sulphate(>99.99% trace metals basis, Make: Sigma Aldrich. Cat. No. 204447-10 g) standard into a 100 mL volumetric flask, dissolve and dilute to volume with solution-A. Dilute 2 mL of this solution to 200 mL with solution-A.
III. Standard preparation:
Weigh and transfer 10 mg API standard into a 10 mL volumetric flask, dissolve and dilute to volume with solution-A. Prepare in duplicate. (Label as standard preparation-1 and standard preparation-2).
IV. Sample preparation:
Weigh and transfer 10 mg of test sample into a 10 mL volumetric flask, dissolve and dilute to volume with solution-A. Prepare in duplicate.(Label as sample preparation-1 and sample preparation-2).
Evaluation of system suitability:
Procedure for conditioning the new column
Install the new column cartridge in CE system. Rinse column with milli-Q water for 30

minutes at 30 psi pressure, followed by 1 M NaOH solution for 12 hours at 20 psi. Further rinse with water for 30 minutes at 30 psi .Rinse with buffer for 30 min at 30 psi. and equilibrate the column at 20 KV (reverse polarity) for 1 hour.
Column regeneration:
Install the column cartridge in CE system for regeneration. Rinse column with milli-Q water for 10 minutes at 30 psi pressure. Rinse with the 1M NaOH solution for 15 minutes at 30 psi. Rinse with buffer for 30 min at 30 psi. For conditioning, equilibrate the column at 20 KV (reverse polarity) for 10 minutes.
Column regeneration for used column:
Install the used column cartridge in CE system. Rinse column with milli-Q water for 10
minutes at 30 psi pressure. Rinse with buffer for 10 min at 30 psi. and equilibrate the
column at 20KV (reverse polarity) for 10 minutes.
Inject blank (Milli-Q), solution-A and solution-B using above conditions. (Order of
injection is given below). The system is suitable for analysis, if and only if, the
resolution between peaks due to sulphate and bromide obtained from soIution-(b) should
not be less than 2.0. The % RSD of relative response of sulphate to that of bromide
obtained from six replicate of solution-(b) should not be more than 5.0
Procedure:
If system suitability passes, inject standard preparation-1, standard preparation-2, sample preparation-1 and sample preparation-2 followed by standard preparation-1 (Order of injections given below). Determine the following:
a) Determine the relative migration time and relative response of peaks namely PI, P2, P3, P4, P5; P6, P7, P8 and P9 for six injections of standard preparation-1 with respect to the respective bromide peak.
b) Determine the range (minimum value and maximum values) for relative migration time and relative response of these nine peaks from the six injections of standard preparation-1.

c) Determine the relative migration time for all nine peaks from six injections of standard preparation-1 and two injections of standard preparation-2 against the average bromide migration time.
d) Determine the range (minimum to maximum value)for relative migration time of nine peaks from six injections of standard preparation-1 and two injections of standard preparation-2.
e) Determine the relative response for all nine peaks from six injections of standard preparation-1 and two injections of standard preparation-2 against the response of bromide peak in the respective injection.
Specification criteria:
I. The response of individual peak should not be less than 95.0 % with respect to
standard. U- The response of total bell curve should not be less than 95.0% with respect to
standard.
III. The percentage of all secondary peaks area (Except sulphate peak) against bell
curve area (total area) should be between 11,0%to 16.0%.
IV. There should not be post apex peak in bell curve.
V. Number of major secondary peaks should be eight and minor peaks should be
seven.
Order of injections:

Sr. No. Preparation No. of injections
1 Blank(Milli-Q) 2
2 Solution-A 1
3 Solution-B 2
4 Standard preparation-1 6
5 Standard preparation-2 2
6 Sample preparation-1 1
7 Sample preparation-2 1
8 Standard preparation-1 2

Calculation:
(A) Determination of relative response of sulphate to bromide peak in solution-(b)

(B) Determination of relative migration time of individual secondary peaks (Label as peak 1 to peak 9) in standard solution-1, standard solution-2 and test preparation with respect to bromide peak in each injection
Migration time of respective peak in standard /test

(D) Determination of % of individual peak in Test preparation:

(C) Determination of relative response of individual secondary peaks (Label as peak 1 to peak 9) in standard solution-1, standard solution-2 and test preparation with respect to bromide peak

Art: Relative response of respective peak in test preparation
Ars: Average relative response of respective peak obtained from six injections of

standard solution-1 and two injections of standard solution-2. Ws: Weight of API in standard solution in (mg). Wt: Weight of test sample in test preparation in (mg). P : Potency of API in-house reference standard. LOD: Loss on drying of test sample (%w/w)
* The relative migration time of the individual peak in test preparation should fall in the range for relative migration time obtained from six injections of standard preparation-1 and two injections of standard preparation-2.
(E) Area percentage of all secondary peaks (Label as peak 2 to peak 9) except sulphate peak against total area under bell curve.

Asp: Sum of area counts of all secondary peaks in test preparation. Atp: Total area counts under bell curve in test preparation.
(F) Percentage of area under bell curve of test preparation against that of standard

Art: Area under bell curve in test preparation.
Ars: Average area under bell curve in standard preparation-1.
Ws: Weight of API in standard solution in mg.
Wt: Weight of test sample in test preparation in mg.
P : Potency of API in-house reference standard.
LOD: Loss on drying of test sample (%w/w)
Table-1: Migration times of major secondary peaks and internal standard for six replicates of standard preparation

Injection No.
Bromide MT P1 MT P2 MT P3 MT P4
1 2.046 2.396 2.996 3.192 3.450
2 2.050 2.400 3.004 3.200 3.458
3 2.054 2.408 3.013 3.208 3.471
4 2.058 2.408 3.017 3.217 3.479
5 2.063 2.417 3.029 3.229 3.492
6 2.067 2.421 3.038 3.237 3.504
7 2.071 2.429 3.050 3.250 3.521
8 2.075 2.433 3.063 3.263 3.533
AVG 2.061 2.414 3.026 3.225 3.489
Min 2.046 2.396 2.996 3.192 3.450
Max 2.075 2.433 3.063 3.263 3.533

Injection No. MT P5 MT P6 MT P7 MT P8 MT P9
1 3.675 3.858 4.013 4.142 4.250
2 3.683 3.867 4.021 4.150 4.263
3 3.700 3.888 4.042 4.171 4.283
4 3.708 3.896 4.050 4.183 4.296
5 3.725 3.913 4.067 4.204 4.317
6 3.737 3.929 4.088 4.225 4.338
7 3.754 3.950 4.108 4.246 4.362
8 3.767 3.962 4.125 4.263 4.379
AVG 3.719 3.908 4.064 4.198 4.311
Min 3.675 3.858 4.013 4.142 4.250
Max 3.767 3.962 4.125 4.263 4.379
Table-2 : Relative migration times (RMT) of major secondary peaks with respect to internal standard for six replicates of standard preparation

Injection
No. RMT PI RMT P2 RMT P3 RMT P4 RMT P5 RMT P6 RMT P7 RMT P8 RMT P9
1 1.17 1.46 1.56 1.69 1.80 1.89 1.96 2.02 2.08
2 1.17 1.47 1.56 1.69 1.80 1.89 1.97 2.03 2.08
3 1.18 1.47 1.57 1.70 1.81 1.90 1.98 2.04 2.09
4 1.18 1.47 1.57 1.70 1.81 1.90 1.98 2.04 2.10
5 1.18 1.48 1.58 1.71 1.82 1.91 1.99 2.05 2.11
6 1.18 1.48 1,58 1.71 1.83 1.92 2.00 2.07 2.12
7 1.19 1.49 1.59 1.72 1.83 1.93 2.01 2.08 2.13
8 1.19 1.50 1.59 1.73 1.84 1.94 2.02 2.08 2.14
AVG 1.180 1.479 1.576 1.705 1.818 1.910 1.986 2.052 2.107
Min 1.17 1.46 1.56 1.69 1.80 1.89 1.96 2.02 2.08

Max
1.19 1.50 1.59 1.73 1.84 1.94 2.02 2.08 2.14
Table-3 : Area response of major secondary peaks for six replicates of standard preparation

Inject ion
No. Brom ide Resp
onse P1 Resp
onse
P2 Resp
onse P3 Resp
onse P4 Resp
onse
P5 Resp
onse P6 Resp
onse
P7 Resp
onse
P8 Resp
onse
P9
1 8995 6341 888 2909 3369 5443 4866 4992 3388 2501
2 8934 6428 945 2849 3366 5576 5030 5099 3373 2478
3 8868 6344 946 2861 3308 5561 4900 4943 3476 2431
4 8851 6385 958 2890 3265 5649 4972 5088 3579 2438
5 8890 6350 1006 2876 3397 5790 5025 5039 3650 2475
6 8999 6368 922 2906 3384 5738 5044 4962 3579 2543
7 9032 6380 915 2943 3427 5602 5063 4965 3697 2471
8 9118 6318 949 2810 3484 5528 5093 4784 3535 2536
AVG 8960. 9 6364. 3 941.1 2880. 5 3375. 0 5610. 9 4999. 1 4984.
0 3534. 6 2484. 1
Min 8851 6318 888 2810 3265 5443 4866 4784 3373 2431
Max 9118 6428 1006 2943 3484 5790 5093 5099 3697 2543
Table-4 : Relative response of major secondary peaks with respect to internal standard peak response for six replicates of standard preparation

Injection
No. RR P1 RR P2 RR P3 RR P4 RR
P5 RR
P6 RR P7 RR P8 RRP9
1 0.70 0.10 0.32 0.37 0.61 0.54 0.55 0.38 0.28
2 0.72 0.11 0.32 0.38 0.62 0.56 0.57 0.38 0.28
3 0.72 0.11 0.32 0.37 0.63 0.55 0.56 0.39 0.27
4 0.72 0.11 0.33 0.37 0.64 0.56 0.57 0.40 0.28
5 0.71 0.11 0.32 0.38 0.65 0.57 0.57 0.41 0.28
6 0.72 0.10 0.33 0.38 0.65 0.57 0.56 0.40 0.29
7 0.72 0.10 0.33 0.39 0.63 0.57 0.56 0.42 0.28
8 0.71 0.11 0.32 0.39 0.62 0.57 0.54 0.40 0.29
AVG 0.715 0.106 0.324 0.379 0.630 0.562 0.560 0.397 0.279
Min 0.70 0.10 0.32 0.37 0.61 0.54 0.54 0.38 0.27
Max 0.72 0.11 0.33 0.39 0.65 0.57 0.57 0.42 0.29

We Claim,
1. A capillary electrophoresis method is quantifies pentosan polysulphate sodium.
2. A capillary electrophoresis method as per claim 1 quantifies pentosan polysulfate sodium with internal standard calculation.
3. A capillary electrophoresis method as per claim 1 quantifies the all secondary major peaks including sulfate peak.
4. A capillary electrophoresis method as per claiml quantifies all major secondary peaks of lower molecular weights by using halide salt as internal standard
5. A capillary electrophoresis method as per claiml quantifies secondary peaks of lower molecular weights before apex of bell curve in electropherogram of pentosan polysulphate sodium
6. A capillary electrophoresis method quantifies as per claiml the contribution of secondary peaks of relative migration times from 1.1 to 2.2 are not more than 15 % of total area under bell curve.
7. A capillary electrophoresis method as per claiml quantifies total area under bell curve in electropherogram of pentosan polysulphate sodium.
8. A capillary electrophoresis method as per claiml identifies the secondary peaks in terms of relative migration time with respect to halide peak in pentosan polysulphate sodium.
9. A capillary electrophoresis method as per claiml calculates the relative response of major secondary peaks of pentosan polysulphate sodium in terms of percentage area with respect to halide peak.

10. A capillary electrophoresis method as per claiml quantifies sulfate content in pentosan polysulphate sodium in terms of percentage area with respect to halide peak.