US 8,461,338 B2  6  5  sirable, especially on large scale. Therefore, there is a need for  improved methods, that avoid using strong acids, for separat› ing the ( 1 R, 1 ’R  )-atracurium besy late isomer mixture, particu› larly on a large scale. The present invention provides such  methods.  The present invention provides an improved chromato› graphic method of separating the isomers of (lR,1 ’R)-atracu- rium salts (e.g. the besylate salt), i.e., (lR-cis,l’R-cis), (lR› cis,l’R-trans), and (lR-trans,lR-trans) isomers, which avoids  the use of strong acids. Good separation as well as improved  stability of the lR-cis,1 ’R-cis cisatracurium besylate isomer  can be achieved in accordance with the present invention. The  method of chromatographically separating the (lR,1 ’R)-atra› curium salts ( e.g. the besylate salt) isomer mixture uses mix› tures of an organic solvent, a polar aprotic co-solvent and a  weak acid as eluent. In a preferred embodiment of the present  invention, the weak acid is an organic acid. The improved  chromatographic method of separating the (lR,1 ’R)-atracu› rium salts (e.g., the besylate salt) isomer mixture provides  also the elimination of other substances.  The method of separating the isomers of (lR,1 ’R)-atracu- rium salts ( e.g., the besylate salt) preferably includes the steps  of:  (a) dissolving an isomer mixture of (lR,1 ’R)-atracurium  salt (e.g., the besylate salt) in at least one organic solvent;  (b) applying the isomer mixture solution to an HPLC col› unm containing a suitable stationary phase;  (c) eluting the colunm with a mobile phase containing an  organic solvent, a polar aprotic co-solvent and a weak acid;  ( d) collecting the fractions containing the 1 R-cis, 1 ’R-cis  isomer;  ( e) optionally performing an ion exchange step to intro› duce the besylate anion; and  (f)  isolating the lR-cis,l’R-cis isomer (e.g., cisatracurium  besylate).  The present invention additionally provides a method for  isolating lR-cis,1 ’R-cis isomer of atracurium besylate  obtained in accordance with the separation process of the  present invention. The method for isolating the 1 R-cis, 1 ’R-cis  isomer of atracurium besylate preferably includes the steps  of:  (a) combining eluted fractions, which preferably contain at  least 90% of the lR-cis,l’R-cis isomer;  (b) washing the combined eluted fractions with acidic  brine and separating the phases;  ( c) drying the organic phase and evaporating the solvent to  obtain a residual oil;  ( d) dissolving the oil in a solvent; and  (e) optionally isolating the product, e.g., by lyophilizing  the solution or by precipitation.  The method for isolating the 1 R-cis, 1 ’R-cis isomer of cisa› tracurium besylate further optionally includes performing an  ion exchange, e.g., via a strong anion exchange colunm or  cartridge (hereinafter SAX).  A particularly preferred embodiment includes performing  high-performance liquid chromatography (HPLC) separation  using a colunm packed with silica, eluting with a mobile  phase, which includes a mixture of an organic solvent in  combination with a polar aprotic co-solvent and a weak acid,  e.g., dichloromethane, DMSO and formic acid. Preferably,  the dichloromethane:DMSO:formic acid ratio in the eluent  mixture is about 70-85 (dichloromethane):8-20 (DMSO):5- (f)  isolating the 1 R-cis, 1 ’R-cis isomer ( e.g., cisatracurium  besylate).  ( e) optionally performing an ion exchange step as needed  to introduce the besylate anion; and  (d) collecting the fractions containing the lR-cis,l’R-cis  isomer;  (c) eluting the colunm with a mobile phase containing an  organic solvent, a polar aprotic co-solvent and a weak acid;  (b) applying the ( 1 R, 1 ’R  )-atracurium salt (e.g., the besy late  salt) isomer mixture to an HPLC colunm containing a suitable  stationary phase;  (a) dissolving the (lR,l’R)-atracurium salt (e.g., the besy› late salt) isomer mixture in at least one organic solvent;  In one embodiment, the present invention provides a  method of separating isomers of (lR,1 ’R)-atracurium salt  (e.g., the besylate salt), which includes the steps of:  The term "isomeric purity" as used herein, refers to the area  percent of the peak corresponding to the 1 R-cis, 1 ’R-cis iso- mer relative to the total area percent of the (lR-cis,l ’R-cis),  (lR-cis,1 ’R-trans) and (lR-trans,1 ’R-trans) isomers.  The term "(lR,l’R) atracurium besylate isomer mixture"  refers to a mixture of the (lR-cis, 1 ’R-cis), (lR-cis,1 ’R-trans)  and (lR-trans,l’R-trans) isomers, while the term "cisatracu› rium besylate" refers to the lR-cis,l’R-cis isomer.  The term "strong acids" refers to acids that dissociate prac› tically completely (>99%) in aqueous solutions at standard  temperature and pressure, such as benzenesulfonic acid, hav› ing a pKa of0 and lower, while the term "weak acids" refers  to acids that do not dissociate completely in aqueous solu› tions, such as acetic acid, having a pKa value of2.5 or higher.  The inventors of the present invention have found that the  1 R-cis, 1 ’R-cis isomer, obtained by HPLC separation as  described in U.S. ’510 and U.S. ’978, is unstable in the eluent  mixture and leads to the formation of decomposition prod› ucts. Surprisingly, it has been further found by the inventors  of the present invention, that good separation as well as  improved stability of the 1 R-cis, 1 ’R-cis isomer can be  achieved when the eluent contains a polar aprotic co-solvent  and a weak acid, which is preferably an organic acid, thus the  use of strong acid can be avoided.  The present invention provides an improved chromato› graphic method of separating the (lR,1 ’R)-atracurium salts  ( e.g., the besylate salt) isomer mixture, which avoids the use  of strong acids in the eluent mixture. The method of the  present invention allows for the separation of the 1 R-cis, I ’R› eis isomer from an isomer mixture of (lR,l’R)-atracurium  salt (e.g., the besylate salt) and other compounds present.  FIG.  3  depicts the chromatogram of (lR,l’R)-atracurium  besylate isomer mixture using N-methyl-2-pyrrolidone as  polar aprotic co-solvent in the eluent mixture.  FIG.  2  depicts the chromatogram of (lR,l’R)-atracurium  besylate isomer mixture prepared in accordance with the  present invention.  FIG.  1  depicts the stability of the lR-cis,1 ’R-cis isomer in  the eluent mixture at 25°  C. as a function of time.  In accordance with the present invention, the desired  1 R-cis, 1 ’R-cis isomer can be obtained in high isomeric purity,  e.g., greater than 99.5%.  15 (formic acid). Exemplary eluent mixtures contain dichlo› romethane, DMSOandformic acid in ratios of e.g., 75:15:10,  78:13:9 and 80:10:10.  BRIEF SUMMARY OF THE INVENTION  BRIEF DESCRIPTION OF THE DRAWINGS  DETAILED DESCRIPTION OF THE INVENTION 