Method for determining specific groups constituting heparins or low molecular weight heparins

Abstract

The invention provides methods for analysing heparins, low-molecular-weight heparins, ultralow-molecular-weight heparins, and oligosaccharides by high performance liquid chromatography a stationary phase dynamically coated with a quaternary ammonium salt. The methods of the invention may be used to analyse samples without pre-treatment or to analyse samples that have been partially or exhaustively depolymerised and, optionally, reduced. Specific saccharides can be detected.

1 . A method of assaying a sample chosen from heparin, low-molecular-weight heparin, ultra low molecular weight heparin, and oligosaccharides, wherein the sample is analysed using a reversed phase column coated with a quaternary ammonium salt for chromatographic separation and analysis of a complex mixture of oligosaccharides.  
     
     
         2 . The method according to  claim 1 , wherein the reversed phase column is a C 8  or C 18  column.  
     
     
         3 . The method according to  claim 1 , wherein all of the oligosaccharides from disaccharides to dodecasaccharides are detected.  
     
     
         4 . The method according to  claim 2 , wherein the sample is analysed without pre-treatment using the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt.  
     
     
         5 . The method according to the  claim 2 , wherein the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is a cetyl trimethylammonium-strong anion exchange (CTA-SAX) column.  
     
     
         6 . The method according to  claim 2 , wherein the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is a CTA-SAX column.  
     
     
         7 . The method according to  claim 2 , wherein the sample is fractionated by Gel Permeation Chromatography (GPC) prior to the chromatographic separation using the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt.  
     
     
         8 . The method according to  claim 7 , wherein the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is a CTA-SAX column.  
     
     
         9 . The method according to  claim 2 , wherein the sample is depolymerized either partially or totally prior to the chromatographic separation using the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt.  
     
     
         10 . The method according to  claim 2 , wherein the sample is reduced after it is depolymerised and prior to the chromatographic separation using the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt.  
     
     
         11 . The method according to the  claim 9 , wherein the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is a CTA-SAX column.  
     
     
         12 . The method according to the  claim 10 , wherein the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is a CTA-SAX column.  
     
     
         13 . A method of assaying a sample chosen from heparins, low-molecular-weight heparins, ultra low molecular weight heparins, and oligosaccharides comprising: 
 (a) depolymerizing the sample by enzymatic depolymerization    (b) reducing the depolymerized sample; and    (c) assaying the sample of step (a) and/or step (b) by CTA-SAX chromatography.    
     
     
         14 . The method according to  claim 13 , wherein the sample is enzymatically depolymerised using at least one heparinase.  
     
     
         15 . The method according to  claim 14 , wherein the at least one heparinase is chosen from heparinase 1 (EC 4.2.2.7.), heparinase 2 (heparin lyase II), and heparinase 3 (EC 4.2.2.8.).  
     
     
         16 . The method according to  claim 14 , wherein the sample is enzymatically depolymerised using a mixture of heparinases comprising heparinase 1 (EC 4.2.2.7.), heparinase 2 (heparin lyase II), and heparinase 3 (EC 4.2.2.8.).  
     
     
         17 . The method according to  claim 10 , wherein the sample is reduced by NaBH 4  or by an alkali metal salt of the borohydride anion  
     
     
         18 . The method according to  claim 1 , wherein the low-molecular weight heparin is enoxaparin sodium.  
     
     
         19 . The method according to  claim 10 , wherein the sample is enoxaparin sodium and said sample is reduced to reduce the reducing ends of said enoxaparin sodium which are not in the 1,6-anhydro form.  
     
     
         20 . The method according to  claim 1 , wherein the chromatographic separation uses a mobile phase which is transparent to UV light having wavelengths in the range of about 200 nm to about 400 nm.  
     
     
         21 . The method according to  claim 1 , wherein the chromatographic separation uses a mobile phase comprising methane sulfonate salts.  
     
     
         22 . The method according to  claim 1 , wherein the chromatographic separation uses a mobile phase comprising ammonium methane sulfonate or sodium methane sulfonate.  
     
     
         23 . The method according to  claim 2 , wherein chromatography on the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is performed at a pH of about 2.0 to about 7.0  
     
     
         24 . The method according to  claim 2 , wherein chromatography on the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is performed at a pH of about 2.5 to about 3.0.  
     
     
         25 . The method according to  claim 2 , wherein chromatography on the C 8  or C 18  reversed phase column coated with a quaternary ammonium salt is performed in a mobile phase comprising water adjusted to about pH 3 by adding methane sulfonic acid and/or 2 M ammonium methane sulfonate, at about pH 2.5.  
     
     
         26 . The method according to  claim 1 , further comprising a step for detecting the presence of oligosaccharide chains whose end is modified with a 1,6-anhydro bond.  
     
     
         27 . The method according to  claim 1 , further comprising a step for detecting acetylated sugars.  
     
     
         28 . The method according to  claim 27 , wherein acetylated sugars are detected by subtracting an absorbance measured at a wavelength at which both acetylated and nonacetylated sugars absorb from an absorbance measured at a wavelength at which acetylated but not nonacetylated sugar absorbs.  
     
     
         29 . The method according to  claim 28 , wherein acetylated sugars are selectively detected.  
     
     
         30 . The method according to  claim 28 , wherein the acetylated sugars detected are at least one acetylated oligosaccharide chosen from ΔIVa, ΔIIa, ΔIIIa, ΔIa, ΔIIa-IVs glu , and ΔIIa-IIs glu    
     
     
         31 . The method according to  claim 1 , wherein the low-molecular weight heparin is any LMWH seeking approval by a regulatory authority pursuant to an application citing Lovenox®/Clexane® (enoxaparin sodium injection) as the listed drug.  
     
     
         32 . The method of  claim 1  wherein said analysis leads to detection of at least one saccharide chosen from any of the following four saccharides  
       
         
           
           
               
               
           
         
       
     
     
         33 . The method of  claim 1  wherein said analysis leads to detection of  
       
         
           
           
               
               
           
         
       
     
     
         34 . The method of  claim 1  wherein said analysis leads to detection of at least one saccharide chosen from any of the following saccharides: