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described above, using strong acids (having pKa value of 0 and lower) such as benzenesulfonic acid. 

[0027] The term "buffered" mobile phase, as described herein, refers to a mobile phases containing an aqueous phase, which includes a weak organic acid and its conjugate salt, such as formic acid and ammonium formate 

[0028] As used herein the term "substantially free of other geometrical and optical isomers" means that no other geo› metrical and optical can be detected within the limits of the HPLC method. 

[0029] The term "isomeric purity" as defined herein, refers to the area percent of the peak corresponding to the lR-cis, 1 ’R-cis isomer relative to the area percent of the (lR-cis,1 ’R› cis isomer), (lR-cis,1 ’R-trans) and (lR-trans,1 ’R-trans) iso› mers. Isomeric purity may be expressed in terms of the following equation: 

wherein, A=% area of the lR-cis, 1 ’R-cis isomer, 8=% area of the lR-cis,1 ’R-trans isomer and C=% area of the IR-trans, 1 ’R-trans isomer. 

[0030] Thus, the present invention provides a chromato› graphic method for separating the isomers of (IR, 1 ’R)-atra› curium salt (e.g., the besylate salt), which uses a Reverse Phase HPLC (RP HPLC) method for obtaining a highly pure product, that is lR-cis,l’R-cis isomer salt (e.g., the besylate salt), having isomeric purity higher than 99.5%. 

[0031] According to one embodiment of the present inven› tion, separating the desired 1 R-cis, 1 R’-cis isomer from a mix› ture of isomers by a method, which includes: 

[0032] loading a solution of (IR, 1 ’R)-atracurium salt ( e.g., the besylate salt) into a Reverse Phase HPLC system, equipped with a suitable separating colunm comprising a stationary phase; 

[0033] eluting the colunm with an eluent mobile phase that includes an aqueous phase, an organic solvent or a mixture thereof; 

[0034] collecting at least one fraction comprising the desired product; and 

[0035] isolating the product. [0036] The lR-cis,1 ’R-cis isomer obtained in accordance with the present invention preferably is substantially free of other geometrical and optical isomers. As is evident from the experimental section of the present application, the obtained lR-cis,l’R-cis isomer salt (e.g., the besylate salt) contains less than about 1 % of the lR-trans,1 ’R trans and/or lR-cis, 1 ’R-trans isomer, preferably less about than 0.1 % of the lR-trans,l’R-trans and/or lR-cis,l’R-trans isomer. Thus, the highly pure 1 R-cis, 1 ’R-cis isomer is obtained having isomer purity of at least about 98.5%, and preferably having an isomer purity higher than 99.5%. 

[0037] In accordance with the present invention, a suitable RP stationary phase column can include Cl stationary phase, C3 stationary phase, C4 stationary phase, CS stationary phase, C 14 stationary phase, C 18 stationary phase, other polymeric packing, e.g., polyamide, polymethacrylate, poly› styrene, and the like. 

[0038] The RP HPLC separation method of the present invention can be utilized on analytical, semi-preparative and preparative scales. Preferred organic bonded reverse phases for obtaining the lR-cis-l’R-cis isomer include Cl, C4 and Cl 8 phases. The results of exemplary RP methods for isolat- 

<img src='content_image/943717.jpg'>

ing the lR-cis-l’R-cis isomer from the (lR,l’R)-atracurium besylate isomer mixture on different stationary phases are provided in Table 1 below. 

<img src='content_image/943718.jpg'>

[0039] According to a preferred embodiment of the present invention, the eluent includes an aqueous phase that contains at least one organic solvent. The aqueous phase preferably comprises an aqueous mixture of an acid and optionally also an inorganic salt ( e.g. NaCl) or an amine ( e.g., triethylamine ). 

[0040] In a preferred embodiment, the aqueous phase fur› ther includes a buffer, e.g., a mixture of a weak acid and its conjugate salt (e.g., acetic acid and sodium acetate or citric acid and sodium citrate). Suitable buffers include, for example, mixtures of acetic acid and sodium acetate, citric acid and sodium citrate, formic acid and ammonium formate, and the like. The acid can be either an organic or inorganic acid. Preferred organic acids include, for example, acetic acid, citric acid, formic acid, camphoric acid, adamanta› neacetic acid and the like, and combinations thereof. Pre› ferred inorganic acids include, for example, hydrochloric acid, hydrobromic acid, phosphoric acid, boric acid, nitric acid, and the like, and combinations thereof. 

[0041] Suitable organic solvents, which may be included in the aqueous mobile phase in accordance with the present invention, include, for example, acetonitrile, methanol, etha› nol, isopropyl alcohol, tetrahydrofuran (THF), and the like, and mixtures thereof. 

[0042] In one embodiment, the (lR,l’R)-atracurium besy› late isomers are separated by eluting with an aqueous phase that contains a salt of an acid, or an acid and a conjugate salt of an acid ( e.g., nitric acid and sodium nitrate), at a pH of from about 1.0 to about 5.5, and more preferably at a pH of from about 3.0 to about 3.5. Suitable acid salts can include, for example, ammonium formate, sodium formate, ammonium acetate, sodium acetate, sodium nitrate, sodium chloride, potassium chloride, barium chloride, sodium bromide, cal› cium bromide, monopotassium dihydrogenphosphate, mono› sodium dihydrogenphosphate, and the like, and combinations thereof. 

[0043] Preferred buffer concentrations, for isolating the 1 R-cis, 1 ’R-cis isomer, range from about 20 mM to about 40 mM. In some instances, a buffer concentration in the higher range increases the retention time of the lR-cis,l’R-cis iso› mer, as depicted in FIG. 1, and also improves the isomer’s separation, as depicted in FIG. 2. 

[0044] The cisatracurium salt is substantially stable under the separation conditions of the present invention. For instance, the extent of cisatracurium degradation is only slightly (less than 2%) after 26 hours at room temperature in a solvent mixture, e.g., at pH 2, using nitrate buffer, and 

