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. 

## BRIEF SUMMARY OF THE INVENTION 

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- 

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. 

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%. 

## BRIEF DESCRIPTION OF THE DRAWINGS 

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. 

FIG. 2 depicts the chromatogram of (lR,l’R)-atracurium besylate isomer mixture prepared in accordance with the present invention. 

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. 

## DETAILED DESCRIPTION OF THE INVENTION 

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. 

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 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 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 "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. 

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: 

(a) dissolving the (lR,l’R)-atracurium salt (e.g., the besy› late salt) isomer mixture in at least one organic solvent; 

(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; 

(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 lR-cis,l’R-cis isomer; 

( e) optionally performing an ion exchange step as needed to introduce the besylate anion; and 

(f) isolating the 1 R-cis, 1 ’R-cis isomer ( e.g., cisatracurium besylate). 

