Methods for Analyzing Cysteamine Compositions

Abstract

Methods of analyzing purity of compositions comprising cysteamine and detecting impurities in cysteamine compositions are described.

What is claimed is: 
     
         1 . A method of analyzing purity of a composition comprising cysteamine comprising:
 (i) injecting a sample solution comprising cysteamine onto a reverse-phase HPLC column;   (ii) eluting the sample from the column using a mobile phase comprising an alkyl sulfonic acid, a buffer, acetonitrile, and methanol; and   (iii) measuring the eluted sample using a UV detector at a wavelength of about 170 nm to about 250 nm.   
     
     
         2 . The method of  claim 1 , further comprising dissolving a cysteamine sample in a solvent having an acidic pH to form the sample solution comprising cysteamine. 
     
     
         3 . A method of analyzing purity of enteric-coated cysteamine beads comprising:
 (i) grinding enteric-coated cysteamine beads;   (ii) dissolving the ground beads in a solvent having an acidic pH to form a sample solution comprising cysteamine;   (iii) injecting the sample solution onto a reverse-phase HPLC column;   (iv) eluting the sample from the column using a mobile phase comprising an alkyl sulfonic acid, a buffer, acetonitrile, and methanol; and   (v) measuring the eluted sample using a UV detector at a wavelength of about 170 nm to about 250 nm.   
     
     
         4 . The method of  claim 2  or  3 , wherein the solvent comprises an alkyl sulfonic acid, a buffer, acetonitrile, and methanol. 
     
     
         5 . The method of any of the preceding claims, wherein the HPLC column is selected from the group consisting of a C18 column, a C8 column, a silica column, a cyano-bonded silica column, and a phenyl-bonded silica column. 
     
     
         6 . The method of any of the preceding claims, wherein the HPLC column has a packing material particle size of about 1 μm to about 10 μm in diameter. 
     
     
         7 . The method of any of the preceding claims, wherein the HPLC column has a packing material particle size of about 2 μm to about 5 μm in diameter. 
     
     
         8 . The method of any of the preceding claims, wherein the HPLC column has an internal diameter of about 0.1 mm to about 10 mm. 
     
     
         9 . The method of any of the preceding claims, wherein the HPLC column has an internal diameter of about 1 mm to about 5 mm. 
     
     
         10 . The method of any of the preceding claims, wherein the HPLC column has a length of about 5 mm to about 500 mm. 
     
     
         11 . The method of any of the preceding claims, wherein the HPLC column has a length of about 50 mm to about 250 mm. 
     
     
         12 . The method of any of the preceding claims, wherein the eluted sample is measured using a UV detector at a wavelength of about 180 nm to about 230 nm. 
     
     
         13 . The method of any of the preceding claims, wherein the eluted sample is measured using a UV detector at a wavelength of about 190 nm to about 210 nm. 
     
     
         14 . The method of any of the preceding claims, wherein the mobile phase comprises:
 about 5% to about 95% by volume of an aqueous solution having a pH of about 2.0 to 3.0, the aqueous solution comprising an alkyl sulfonic acid and a phosphate buffer,   about 1% to about 30% by volume of acetonitrile, and   about 5% to about 85% by volume of methanol.   
     
     
         15 . The method of  claim 14 , wherein the aqueous solution comprises:
 about 15 mM to about 300 mM of an alkyl sulfonic acid, and   about 15 mM to about 200 mM phosphate buffer.   
     
     
         16 . The method of any of the preceding claims, wherein the sample is eluted using a gradient elution. 
     
     
         17 . The method of  claim 16 , comprising gradient eluting the sample using first and second mobile phases,
 wherein the first mobile phase comprises:   about 75% to about 95% by volume of an aqueous solution having a pH of about 2.0 to 3.0, the aqueous solution comprising an alkyl sulfonic acid and a phosphate buffer,   about 1% to about 8% by volume of acetonitrile, and   about 5% to about 20% by volume of methanol; and   the second mobile phase comprises:   about 5% to about 30% by volume of an aqueous solution having a pH of about 2.0 to 3.0, the aqueous solution comprising an alkyl sulfonic acid and a phosphate buffer,   about 8% to about 30% by volume of acetonitrile, and   about 50% to about 85% by volume of methanol.   
     
     
         18 . The method of  claim 17 , wherein the aqueous solution of the first mobile phase comprises:
 about 15 mM to about 50 mM of an alkyl sulfonic acid, and   about 15 mM to about 100 mM phosphate buffer.   
     
     
         19 . The method of  claim 17 , wherein the aqueous solution of the second mobile phase comprises:
 about 100 mM to about 300 mM of an alkyl sulfonic acid, and   about 100 mM to about 200 mM phosphate buffer.   
     
     
         20 . The method of any of the preceding claims, wherein the sample is eluted using a flow rate of about 0.001 mL/min to about 2 mL/min. 
     
     
         21 . The method of any of the preceding claims, wherein the sample is eluted at a column temperature of about 20° C. to about 80° C. 
     
     
         22 . The method of any of the preceding claims, wherein the sample is eluted at a column temperature of about 30° C. to about 50° C. 
     
     
         23 . The method of any of the preceding claims, wherein the sample solution comprises cysteamine bitartrate. 
     
     
         24 . The method of  claim 3 , wherein the beads are ground to form a powder. 
     
     
         25 . The method of  claim 3 , wherein the beads are ground to form a paste. 
     
     
         26 . The method of any of the preceding claims, wherein the alkyl sulfonic acid is selected from the group consisting of ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, pentanesulfonic acid, hexanesulfonic acid, heptanesulfonic acid, octanesulfonic acid, nonanesulfonic acid, and decanesulfonic acid. 
     
     
         27 . The method of any of the preceding claims, wherein the alkyl sulfonic acid is selected from the group consisting of 1-hexanesulfonic acid and 1-octanesulfonic acid. 
     
     
         28 . A method of analyzing purity of compositions comprising cysteamine comprising:
 (i) dissolving a cysteamine sample in a solvent having an acidic pH to form a sample solution comprising cysteamine;   (ii) injecting the sample solution onto a C18 reverse-phase HPLC column;   (iii) gradient eluting the sample from the column using first and second mobile phases,   wherein the first mobile phase comprises: about 85% by volume of an aqueous solution having a pH of about 2.6, the aqueous solution comprising about 23.6 mM 1-octanesulfonic acid sodium and about 29 mM sodium phosphate; about 3% by volume of acetonitrile; and about 12% by volume of methanol, and   the second mobile phase comprises: about 10% by volume of an aqueous solution having a pH of about 2.6, the aqueous solution comprising about 0.2 M 1-octanesulfonic acid sodium and about 0.1 M sodium phosphate; about 18% by volume of acetonitrile; and about 72% by volume of methanol; and   (iv) measuring the eluted sample using a UV detector at a wavelength of about 210 nm or less.   
     
     
         29 . The method of  claim 28 , comprising gradient eluting at a flow rate of about 1.0 mL/min according to the following profile: 
       
         
           
                 
                 
               
                     
                     
                 
                     
                   HPLC Gradient 
                 
                 
                 
                 
               
                   Time (min) 
                   First Mobile Phase (%) 
                   Second Mobile Phase (%) 
                 
                     
                 
                 
                 
                 
               
                   0.0 
                   100 
                   0 
                 
                   2.0 
                   100 
                   0 
                 
                   20.0 
                   60 
                   40 
                 
                   25.0 
                   60 
                   40 
                 
                   25.1 
                   100 
                   0 
                 
                   40.0 
                   100 
                   0 
                 
                     
                 
             
                
                
               
            
             
                
                
               
            
             
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         30 . The method of  claim 28  or  29 , wherein the HPLC column has a packing material particle size of about 3.5 μm in diameter. 
     
     
         31 . The method of any one of  claims 28  to  30 , wherein the HPLC column has a length of about 150 mm and an internal diameter of about 4.6 mm. 
     
     
         32 . The method of any of the preceding claims, comprising injecting at least 30 μg of cysteamine onto the column.