content_image/932529.jpg content_image/932530.jpg
almost no degradation (0.2%) was observed after 26 hours in a solvent mixture at 4 ° C. using a nitrate buffer ( see Example 6, Table 12). 

[0045] A desired counter anion may be introduced, e.g., by an ion exchange process, which can include, e.g., adding desired counter anions to the system to exchange with other anions utilized in the separation process, which may associate with one or more of the (IR, 1 ’R)-atracurium isomers. Any suitable ion exchange methods may be utilized as well, e.g., a suitable ion exchange resin. 

[0046] The isolation of the cisatracurium from the elution liquid mixture can be carried out by any suitable method such as Solid Phase Extraction (SPE). A non-limiting example of a method for isolating the cisatracurium solution from the elu› tion liquid is by SPE. For example, the isolation can be effected by successively transferring a cisatracurium besylate sample solution ( containing the aqueous phase and the organic solvent) and water through a C18 sorbent, which causes the separation of the cisatracurium besylate from the buffer species. The cisatracurium besylate retained by the sorbent can be then removed, e.g., by eluting from the sorbent with methanol. The evaluation of the anions is preferably carried out by HPLC, and the cations are preferably evaluated indirectly (since the cations and the anions are removed at about the same rate). Recovery of the isolated material and the anions can be checked after performing each step of the SPE method. 

[0047] Thus, the present invention provides a SPE method for isolating a product (e.g., an isomer of (lR,l’R)-actracu› rium besylate) from an HPLC elution liquid mixture, which method preferably includes: 

[0048] successively transferring the sample solution and water through a sorbent; 

[0049] eluting the product from the sorbent with an organic solvent, e.g., methanol; and 

[0050] methanol. 

[0051] The SPE method of the present invention can be utilized for isolating the 1 R-cis, 1 ’R-cis isomer from a HPLC elution liquid mixture thereof. In one embodiment, the method includes: 

[0052] optionally evaporating at least a portion of the organic solvent from an aqueous HPLC elution liquid con› taining an organic solvent; 

[0053] adding an organic solvent and separating the phases and optionally washing the organic layer; 

[0054] optionally changing the anion using a suitable ion exchange method; and 

[0055] isolating the product from the aqueous phase by spray-drying or freeze-drying. 

[0056] The organic solvent added in the SPE method of the present invention can include, for example, ethyl acetate, n-propyl acetate, isopropyl acetate, n-butyl acetate, isobutyl acetate, toluene, chloroform, dichloromethane, and the like, and mixtures thereof. A preferred solvent for the SPE method of the present invention is dichloromethane. 

[0057] The present invention further provides a pharma› ceutical composition comprising substantially pure cisatra› curium besylate, which can be obtained in accordance with the present invention, and can be employed, e.g., in surgery as a neuromuscular blocking agent as a formulation for admin› istration, e.g., by injection or infusion. 

[0058] FIG. 3 depicts the chromatogram of a reference sample obtained by diluting a lyophilized atracurium besylate 

washing the sorbent with an organic solvent, e.g., 

sample, containing, inter alia, approximately 54% lR-cis, 1 ’R-cis, 34% lR-cis,1 ’R-trans and 6% IR-trans,! ’R-trans iso› mers (hereinafter the "reference sample"), and a small amount of benzenesulfonic acid in the corresponding aque› ous phase, and injecting the mixture into the HPLC system, equipped with a C18 stationary phase according to a gradient method in which the eluent contains a mixture of an aqueous phase and at least one solvent. FIG. 4 depicts the chromato› gram ofan exemplary starting sample of (IR, 1 ’R)-atracurium besylate isomer mixture (hereinafter "sample 1 "), and FIG. 5 depicts the chromatogram of a cisatracurium besylate (NIM› BEXfi) buffer solution (hereinafter "sample 2"). 

[0059] The following examples further illustrate the inven› tion but, of course, should not be construed as in any way limiting its scope. 

[0060] Cisatracurium besylate was analyzed by High Per› formance Liquid Chromatography (HPLC). Exemplary HPLC separations were performed, e.g., using the following conditions: Colunm and packing-Hypersil Hyperprep HS C18, 250x21.2 mm, 15µ, P.N. 37115-125; UV detection› UV operated at 280 nm; flow rate: 13 ml/min; Mobile phase: Eluent A: 20 mM NaNO$_{3}$, pH adjusted to 2.0 with HNO$_{3}$ . Eluent B: methanol. The gradient elution is as detailed in Table 2: 

<img src='content_image/932529.jpg'>

Another example ofHPLC separation using the same colunm as in the previous example is: UV detection at 280 nm; flow rate: 13 ml/min; Mobile phase: Eluent A: 40 mM buffer solution containing diethylamine (DEA), pH adjusted to 3.5 with formic acid Eluent B: methanol. The gradient elution is as detailed in Table 3: 

<img src='content_image/932530.jpg'>

The chromatogram corresponding to the separation of (IR, 1 ’R)-atracurium besylate, according to the method using the gradient detailed in Table 3, is depicted in FIG. 6. 

[0061] This example demonstrates a Reverse Phase proce› dure for isolating cisatracurium besylate on C18 stationary phase using different aqueous phases. 

[0062] Atracurium besylate reference sample (10 mg/ml) was analyzed on a C18 stationary phase by gradient elution, using different aqueous phases in the solvent mixtures with methanol. Colunm and packing: Alltech, Altima C18, 250x 4.6x5µ, Cat. No. 88056; UV detection: 280 nm; flow rate: 1 ml/min; The results, including the gradient elutions, are detailed in Table 4: 

## EXAMPLES 

## Example 1 

