FIELD OF THE INVENTION
It relates to an ultraviolet spectroscopy method for the determination of sevelamer through a phosphate binding assay wherein the method involves the step of derivatisation using ammonium molybdate reagent.
BACKGROUND OF THE INVENTION
Sevelamer is an aliphatic amine polymer indicated for the control of serum level of phosphate in chronic kidney disease. The protonated amine in sevelamer binds phosphate through ionic and hydrogen bonding in the gastrointestinal tract, without concomitantly increasing the absorption of any clinically undesirable material, particularly calcium or aluminium. Sevelamer is known chemically as poly (allylamine-co-N, N'-diallyl-1, 3-diamino-2-hydroxypropane).
Use of Sevelamer and its pharmaceutical composition, process for its preparation is disclosed in US 5,496,545. Currently, Sevelamer hydrochloride and Sevelamer carbonate are being marketed under the trade name of Renagel ™ and Renvela ™ respectively.
Sevelamer is highly insoluble drug, due to which it is difficult to carry out assay of sevelamer. Also, there is no direct method reported for the assay of sevelamer. The important aspect of analytical determination of sevelamer is an in vitro phosphate binding assay. It is an indirect method for the determination of sevelamer. Moreover, in vitro phosphate binding assay is required for measuring the efficacy of sevelamer.
Several attempts have been made in the prior art to develop analytical method for determining phosphate binding assay for sevelamer.
An article published in Journal of pharmaceutical and biomedical analysis Vol. 19 (1999), 911-915 by Jeffery R Mazzeo et al discloses a phosphate binding assay for sevelamer hydrochloride using ion chromatography.
Another article published in Journal of pharmaceutical and biomedical analysis Vol. 35 (2004) 753-760 discloses the determination of the binding parameter constant of Renagel™ capsules and tablets by high performance capillary electrophoresis.
We have now developed an ultraviolet spectroscopy method for the determination of sevelamer through a phosphate binding assay.

Ultraviolet spectroscopy based on derivatisation is simple and cost effective analytical method as it does not require use of any sophisticated instrument. Also, it is less time consuming as compare to other methods known in the prior art for phosphate binding assay of sevelamer.
SUMMARY OF THE INVENTION
The present invention relates to method for determination of sevelamer by ultraviolet spectroscopy through a phosphate binding assay.
According to one of the aspect, there is provided an ultraviolet spectroscopy method for the determination of sevelamer through a phosphate binding assay wherein the method involves the step of derivatisation using ammonium molybdate reagent.
In other aspect, there is provided an ultraviolet spectroscopy method for the determination of sevelamer in a pharmaceutical composition through a phosphate binding assay wherein the method involves the step of derivatisation using ammonium molybdate reagent.
According to one of the aspect, there is provided an ultraviolet spectroscopy method for the determination of sevelamer comprises the steps of:
a Mixing sevelamer with a solution of known phosphate concentration; b. Filtering off sevelamer-phosphate complex formed in step a);
c Adding ammonium molybdate to the filtrate obtained in step b) containing unbound phosphate leading to formation of yellow colored complex;
d. Measuring absorbance of yellow colored complex formed in step c) by ultraviolet
spectroscopy;
e. Calculating the phosphate binding capacity of sevelamer by measuring difference between
the concentration of phosphate solution of step a) and concentration of unbound phosphate
of step c).
DETAILED DESCRIPTION
The term 'Sevelamer' as used herein includes hydrated or anhydrate form of Sevelamer. The pharmaceutically acceptable salts thereof include hydrochloride, carbonate, hydrobromide, phosphate, sulfate and the like.

Sevelamer may be present in the pharmaceutical composition. The term 'pharmaceutical composition' as used herein includes solid dosage forms such as tablet, capsule, pill and the like
Phosphate binding assay is used to determine the binding capacity of sevelamer for phosphate ions. It is an indirect method for the assay of sevelamer. In this assay, the concentration of derivatized unbound phosphate ions is quantitavely determined by ultraviolet spectroscopy and accordingly binding capacity of sevelamer is calculated.
Ultraviolet spectroscopy is used to determine the concentration of unbound phosphate ions. The phosphate ions are derivatized using ammonium molybdate to render them ultraviolet active. The absorbance of phosphate ion is measured at lambda max (Amax) 380nm.
Unbound phosphate ions as used herein include those excess phosphate ions which are not bound by sevelamer.
Ammonium molybdate reagent is used as derivatising agent. It forms soluble yellow colored complex of ammonium molybdophosphate with phosphate ions. This reagent is prepared by dissolving sufficient amount of ammonium molybdate in sulphuric acid to make 1-10%w/v ammonium molybdate solution, in particular 1-4%w/v.
A known concentration of phosphate stock solution is added to sevelamer solution wherein sevelamer binds to phosphate ions based on its binding capacity. Phosphate stock solution is prepared by dissolving known quantity of mono basic potassium phosphate (KH2P04) in water.
Sample dispersion is prepared by dispersing known quantity of sevelamer in excess of phosphate solution. Tablets containing sevelamer may be crushed just before analysis.
Binding capacity of sevelamer is calculated from the difference of concentration of phosphate stock solution and concentration of unbound phosphate as determined by ultraviolet spectroscopy method. To comply with official requirements the binding capacity of sevelamer may be in the range of 4.77 to 6.38 mMole/gm.
Sevelamer is hygroscopic, therefore loss on drying may be noted before analysis. It may be noted by using any of the method known in the prior art like thermo gravimetric analysis (TGA), electric oven, karl fischer titration and the like. Also sevelamer is a hydrogel, absorbs around ~ 6 -9 times its weight of water. Therefore, the ratio of solution volume to mass may be kept high to avoid changes to solution concentration. The ratio of solution to mass may be in the range of 0.5 - 1.0.
Binding capacity of sevelamer is influenced by pH, therefore the pH of phosphate stock solution may be adjusted to 6.5-7.5, in particular 7. At this pH, monovalent and divalent anions of

phosphate stock solution would exist in equilibrium. Also, Sevelamer increases the pH of the unbuffered phosphate stock solution. Therefore, to prevent any change in pH a buffer namely BES buffer (N, N-Bis (hydroxyethyl)-2-aminoethanesulphonic acid) is added.
Phosphate binding assay for sevelamer by ultraviolet spectroscopy was validated. The method demonstrated excellent accuracy and precision. In case of sevelamer API accuracy was in the range of 99-102%, and precision was found to be 1.1%. Incase of tablets accuracy was found to be 99% and precision was found to be 1.6%.
In order to further illustrate the present invention, a detailed prototype analysis method is provided below. However, the prototype is for the purpose of illustration and should not be construed as limiting on the scope of the present invention.
Method for the determination of sevelamer API through a phosphate binding assay
1 Preparation of Phosphate Standard Stock solution (Phosphate SS) 2.72gm of KH2PO4 and 21.32 gm of BES buffer were dissolved, in about 700 mL of water; pH was adjusted to around 7 with 1N NaOH and diluted to 1000 mL with water.
2. Preparation of derivatising reagent
A 2% solution of ammonium molybdate was prepared in 1N H2SO4
3. Preparation of standard solution
5 ml of Phosphate SS was diluted to 50 ml with water. 5 ml of this solution along 10 ml of derivatising reagent was transferred to volumetric flask, and mixed till a yellow colored complex is formed, and then volume was made up to 50 ml with water.
4. Preparation of sample solution
(a) Binding Step: About 140 mg of Sevelamer HCI sample was added to 100 mL of Phosphate
stock solution and mixed. The dispersion was then filtered to remove, sevelamer phosphate
complex.
(b) Derivatisation step: 5 mL of filtrate obtained in step a) was diluted to 50 mL with water. 5 mL
of this solution along with 10 mL of derivatising reagent was added into a volumetric flask and
mixed till a yellow colored complex is formed, and volume was made up to 50 ml water.
5. Preparation of Standard blank:
5 mL of water was added along with 10 mL of derivatising reagent into a volumetric flask, mixed, and then volume was made up with water up to 50 ml

6. Preparation of Sample blank
(a) Binding Step: About 140 mg of Sevelamer HCI sample was added to 100 mL of water, mixed
and then filtered
(b) Derivatisation step: 5 ml of filtrate obtained in step a) was diluted to 50 mL with water. 5 mL
of this solution was added along with 10 mL of derivatising reagent into a volumetric flask, and
then volume was made up to 50ml with water.
Procedure
UV absorbance of standard and sample in duplicate was measured at 380 nm using standard
blank and sample blank respectively, as reference.
Calculation
(i) Cone, of Phosphate standard stock solution (mMole/ litre)
a 1
A = -— x 1000000 x
b Mol. Wt. of KH2PO4
Where
a = Weight of potassium dihydrogen phosphate (gm)
b = Dilution of standard in mL
Mol. Wt. of KH2PO4 = 136.09
(ii) Cone, of unbound phosphate (mMole/litre)
RxA
C
-
S Where
R = Average absorbance of Phosphate from sample.
S = Average absorbance of phosphate from standard solution.
A Concentration of Phosphate standard stock solution.
(iii) Phosphate binding capacity (mMole/ gm)
(A-C)X0.1
Binding Capacity =
Wx[(100-LOD)/100]

Where
W =Sample weight (gm)
LOD = Loss on Drying (% w/w)
A = Concentration of Phosphate standard stock solution
C = Concentration of Unbound Phosphate.
0.1 = Dilution factor in Litre
Method for the determination of sevelamer in sevelamer tablet through a phosphate binding assay
1. Preparation of Phosphate Standard Stock solution (Phosphate SS)
2.72 gm of KH2PO4 and 21.32 gm of BES buffer and 4.68 gm of NaCI were dissolved in about 700 mL of water, and pH was adjusted around 7 with 1N NaOH and diluted to 1000 mL with water.
2. Preparation of derivatisinq reagent
A 3 % Ammonium Molybdate solution was prepared in 1N H2S04.
3. Standard solution and Standard blank were prepared similarly as that of API.
4. Preparation of sample solution
(a) Binding Step: Average weight of 20 tablets was determined. Tablet powder equivalent to 0.14
g of Sevelamer HCI was added along with 100 mL of Phosphate stock solution into a flask and
mixed. The dispersion was then filtered to remove sevelamer phosphate complex.
(b) Derivatisation step: 5 mL of filtrate obtained in step a) was diluted to 50 mL with water. 5 mL
of this solution was added along with 10 mL of derivatising reagent into a volumetric flask, mixed
till a yellow colored complex is formed and then volume was made up to 50ml with water.
5. Preparation of Sample blank
(a) Binding Step: Tablet powder equivalent to 0.14 g of Sevelamer HCI was added to 100 ml of water and filtered.

(b) Derivatisation step: 5 ml of filtrate obtained in step a) was diluted to 50 ml with water. 5 mL of this solution was added along with 10 ml of derivatising reagent into a volumetric flask, and volume was made up to 50 ml with water.
Procedure
UV absorbance of standard and sample in duplicate was measured at 380 nm using standard
blank and sample blank respectively, as reference.
Calculation:
(i) Concentration of Phosphate standard stock solution (mMole/ litre) and (ii) Concentration of unbound phosphate (m Moles/litre) were calculated in a similar manner as that given for API.
(iii) Phosphate binding capacity on dried basis (m Moles/ g)
(A-C) x 0.1 x Avg. wt. of Tablets (g)
Binding Capacity (on dried basis) =
W x [(100- LOD)/100] x Amt of sevelamer in the tablet (g) Where
W = Sample weight (g)
LOD = Loss on Drying (% w/w)
A = Concentration of Phosphate standard stock solution
C = Concentration of Unbound Phosphate.
0.1 = Dilution factor in Litre

Example 1
Two batches of sevelamer API were analyzed by above method and binding capacity was determined (Table 1). Also, binding capacity determined through ultraviolet spectroscopy was compared vis-a-vis with ion chromatography.
Table 1: Binding capacity of two batches of sevelamer API.

(Table Removed)

WE CLAIM :
1. An ultraviolet spectroscopy method for the determination of sevelamer through a phosphate
binding assay wherein the method involves the step of derivatisation using ammonium
molybdate reagent.
2. The ultraviolet spectroscopy method according to claim 1 wherein said method comprises
the steps of:
a. Mixing sevelamer with a solution of known phosphate concentration;
b. Filtering off sevelamer-phosphate complex formed in step a);
c. Adding ammonium molybdate to the filtrate obtained in step b) containing unbound
phosphate leading to formation of yellow colored complex;
d. Measuring absorbance of yellow colored complex formed in step c) by ultraviolet
spectroscopy;
e. Calculating the phosphate binding capacity of sevelamer by measuring difference
between the concentration of phosphate solution of step a) and concentration of
unbound phosphate of step c).
3. The ultraviolet spectroscopy method according to claim 1 wherein said sevelamer is
sevelamer hydrochloride.
4. The ultraviolet spectroscopy method according to claim 1 wherein said ammonium
molybdate reagent is present in the concentration of about 1-10%w/v.
5. The ultraviolet spectroscopy method according to claim 2 wherein said ammonium
molybdate reagent is used to derivatise unbound phosphate ions.
6. The ultraviolet spectroscopy method according to claim 2 wherein said phosphate binding
capacity of sevelamer on dried basis is in the range of 4.77 to 6.38 mMole/gm.
7. The ultraviolet spectroscopy method according to claim 2 wherein said phosphate solution of
step a) comprises buffering agent.
8. The ultraviolet spectroscopy method according to claim 2 wherein pH of said phosphate
solution of step a) is in the range of 6.5-7.5.
. The ultraviolet spectroscopy method according to claim 2 wherein said absorbance of yellow
colored complex of step d) is measured at Amax 380nm.
10. The ultraviolet spectroscopy method as described herein.