Methods for analyzing cysteamine compositions
View Patent ↗Methods of analyzing purity of compositions comprising cysteamine and detecting impurities in cysteamine compositions are described.
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;
wherein the sample is eluted using a gradient elution, the elution 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.
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;
wherein the sample is eluted using a gradient elution, the elution 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.
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 claim 1 or 3 , wherein the HPLC column has a packing material particle size of about 1 μm to about 10 μm in diameter.
6. The method of claim 1 or 3 , wherein the HPLC column has an internal diameter of about 0.1 mm to about 10 mm.
7. The method of claim 1 or 3 , wherein the eluted sample is measured using a UV detector at a wavelength of about 180 nm to about 230 nm.
8. The method of claim 1 or 3 , 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.
9. The method of claim 1 or 3 , 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.
10. The method of claim 1 or 3 , wherein the sample is eluted using a flow rate of about 0.001 mL/min to about 2 mL/min.
11. The method of claim 1 or 3 , wherein the sample solution comprises cysteamine bitartrate.
12. The method of claim 3 , wherein the beads are ground to form a powder.
13. The method of claim 3 , wherein the beads are ground to form a paste.
14. 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.
15. The method of claim 14 , 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
16. The method of claim 14 , wherein the HPLC column has a packing material particle size of about 3.5 μm in diameter.
17. The method of claim 14 , wherein the HPLC column has a length of about 150 mm and an internal diameter of about 4.6 mm.
18. The method of any one of claim 1 , 3 , or 14 , comprising injecting at least 30 μg of cysteamine onto the column.