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Cisatracurium Besylate
(sis'' at ra kure' ee um bes' i late).
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C65H82N2O18S2 1243.48
Isoquinolinium, 2,2¢-[1,5-pentanediylbis[oxy(3-oxo-3,1-propanediyl)]]bis[1-[(3,4-dimethoxyphenyl)methyl]-1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-, dibenzenesulfonate, [1R-[1,2(1¢R*,2¢R*)]]-;    
(1R,2R)-2-(2-Carboxyethyl)-1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium benzenesulfonate, pentamethylene ester     [96946-42-8].
DEFINITION
Cisatracurium Besylate contains NLT 97.0% and NMT 102.0% of cisatracurium besylate (C65H82N2O18S2), calculated on the anhydrous and solvent-free basis.
IDENTIFICATION
•  B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
ASSAY
•  Procedure
Buffer:  33.3 g/L of ammonium formate prepared as follows. Dissolve 32.8 g of ammonium formate in 984 mL of water, and add 16 mL of anhydrous formic acid.
Mobile phase:  Acetonitrile, methanol, and Buffer (20:20:60)
Diluent:  Acetonitrile, methanol, and water (20:20:60). Add 0.4 mL of anhydrous formic acid per 1 L.
System suitability solution:  0.7 mg/mL of USP Cisatracurium Besylate System Suitability Mixture RS in Diluent
Standard solution:  0.7 mg/mL of USP Cisatracurium Besylate RS in Diluent
Sample solution:  0.7 mg/mL of Cisatracurium Besylate in Diluent
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 280 nm
Column:  4.6-mm × 25.0-cm; 5-µm packing L1
Flow rate:  1.5 mL/min
Injection volume:  20 µL
Run time:  NLT 2.5 times the retention time of cisatracurium
System suitability 
Samples:  System suitability solution and Standard solution
[Note—See Table 1 for relative retention times. ]
Suitability requirements 
Resolution:  NLT 2.0 between the peaks for R-cis-R¢-trans-atracurium and cisatracurium, System suitability solution
Tailing factor:  NMT 1.7 for cisatracurium, System suitability solution
Relative standard deviation:  NMT 1.5%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of cisatracurium besylate (C65H82N2O18S2) in the portion of Cisatracurium Besylate taken:
Result = (rU/rS) × (CS/CU) × 100

rU== peak response from the Sample solution
rS== peak response from the Standard solution
CS== concentration of USP Cisatracurium Besylate RS in the Standard solution (mg/mL)
CU== concentration of Cisatracurium Besylate in the Sample solution (mg/mL)
Acceptance criteria:  97.0%–102.0% on the anhydrous and solvent-free basis
IMPURITIES
•  Residue on Ignition 281: NMT 0.1%
•  Organic Impurities
Mobile phase, Diluent, System suitability solution, Standard solution, Sample solution, Chromatographic system, and System suitability:  Proceed as directed in the Assay.
Analysis 
Sample:  Sample solution
Calculate the percentage of each impurity in the portion of Cisatracurium Besylate taken:
Result = (rU/rT) × 100

rU== peak response of each impurity from the Sample solution
rT== sum of all the peak responses from the Sample solution
Acceptance criteria:  See Table 1. Disregard any peak representing less than 0.09% of the area of the major peak.
Table 1
Name Relative
Retention
Time 		Acceptance
Criteria,
NMT (%)
Besylatea 0.10 —
cis-Quaternary acidb 0.14 0.2
(R)-N-Methyllaudanosinec 0.16 0.2
(R)-Laudanosined 0.20 0.6
cis-Quaternary methyl estere 0.23 0.4
cis-Quaternary alcoholf 0.29 0.5
R-trans-R¢-trans-Atracuriumg 0.74 0.2
R-cis-R¢-trans-Atracuriumh 0.87 0.8
Cisatracurium 1.0 —
trans-Monoquaternary
compoundi 		1.17 0.5
trans-Monoacrylatej 1.28 0.5
cis-Monoquaternary
compoundk 		1.39 0.7
cis-cis-Triester analogl 1.46 0.4
cis-Monoacrylatem 1.56 1.0
Any individual unspecified impurity — 0.1
Total impurities — 3.0
a   This peak is due to the counterion and is not to be reported or included in Total impurities.
b   (1R,2R)-2-(2-Carboxyethyl)-1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium benzenesulfonate.
c   (R)-1,2,3,4-Tetrahydro-6,7-dimethoxy-2,2-dimethyl-1-veratrylisoquinolinium benzenesulfonate.
d   (R)-1,2,3,4-Tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinoline.
e   (1R,2R)-1,2,3,4-Tetrahydro-6,7-dimethoxy-2-[2-(methoxycarbonyl)ethyl]-2-methyl-1-veratrylisoquinolinium benzenesulfonate.
f   (1R,2R)-1,2,3,4-Tetrahydro-2-(9-hydroxy-3-oxo-4-oxanonyl)-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium benzenesulfonate.
g   (1R,1¢R,2S,2¢S)-2,2¢-(3,11-Dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) dibenzenesulfonate.
h   (1R,1¢R,2R,2¢S)-2,2¢-(3,11-Dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) dibenzenesulfonate.
i   (1R,1¢R,2S)-2-Methyl-2,2¢-(3,11-dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-1-veratrylisoquinolinium) dibenzenesulfonate.
j   (1R,2S)-2-(3,11-Dioxo-4,10-dioxa-12-tridecenyl)-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) benzenesulfonate.
k   (1R,1¢R,2R)-2-Methyl-2,2¢-(3,11-dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-1-veratrylisoquinolinium) dibenzenesulfonate.
l   (1R,1¢R,2R,2¢R)-2,2¢-(3,7,15-Trioxo-4,8,14-trioxa-heptadecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) dibenzenesulfonate.
m   (1R,2R)-2-(3,11-Dioxo-4,10-dioxa-12-tridecenyl)-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) benzenesulfonate.
•  Limit of Methyl Benzenesulfonate
[Note—Prepare the solutions immediately before use. Methyl benzenesulfonate is slowly hydrolyzed in aqueous solutions. ]
Mobile phase:  Acetonitrile and water (45:55)
Internal standard solution:  1.2 mg/mL of methyl p-toluenesulfonate in acetonitrile
Standard stock solution A:  1.2 mg/mL of methyl benzenesulfonate in acetonitrile
Standard stock solution B:  48 µg/mL of methyl benzenesulfonate from Standard stock solution A in Mobile phase prepared as follows. Transfer 2.0 mL of Standard stock solution A and 5.0 mL of Internal standard solution to a 50-mL volumetric flask, and dilute with Mobile phase to volume.
Standard solution:  2.4 µg/mL of methyl benzenesulfonate from Standard stock solution B in Mobile phase
Sample solution:  Nominally 0.2 g/mL of Cisatracurium Besylate in Mobile phase prepared as follows. Transfer 1.0 g of Cisatracurium Besylate into a suitable separator. Immediately add 25 µL of Internal standard solution, and dissolve the contents in 25 mL of water using vigorous shaking. Add 25 mL of ethyl acetate, and shake vigorously for 2 min. Allow the phases to separate until the aqueous layer is clear and for NMT 2 h. Evaporate the organic layer to dryness in a stream of air. Dissolve the residue in 5.0 mL of Mobile phase by sonication and gentle swirling.
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  LC
Detector:  UV 214 nm
Column:  4.6-mm × 25.0-cm; 5-µm packing L1
Flow rate:  1 mL/min
Injection volume:  20 µL
Run time:  5 times the retention time of methyl benzenesulfonate
System suitability 
Sample:  Standard solution
Suitability requirements 
Resolution:  NLT 3.0 between methyl benzenesulfonate and methyl p-toluenesulfonate
Relative standard deviation:  NMT 2.0% for methyl benzenesulfonate and methyl p-toluenesulfonate
Analysis 
Samples:  Standard solution and Sample solution
Calculate the concentration of methyl benzenesulfonate in the portion of Cisatracurium Besylate taken:
Result = (RU/RS) × (CS/CU)

RU== peak height ratio of methyl benzenesulfonate to the internal standard from the Sample solution
RS== peak height ratio of methyl benzenesulfonate to the internal standard from the Standard solution
CS== concentration of methyl benzenesulfonate in the Standard solution (µg/mL)
CU== concentration of Cisatracurium Besylate in the Sample solution (g/mL)
Acceptance criteria:  NMT 10 ppm
SPECIFIC TESTS
•  Optical Rotation, Specific Rotation 781
Sample solution:  10.0 mg/mL in acetonitrile
Acceptance criteria:  60.0 to 54.0 at 20, calculated on the anhydrous and solvent-free basis
•  pH 791
Sample solution:  7 mg/mL of Cisatracurium Besylate in water
Acceptance criteria:  5.0–6.5
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in tight, light-resistant containers. Store cold, desiccated.
•  USP Reference Standards 11
USP Cisatracurium Besylate RS
USP Cisatracurium Besylate System Suitability Mixture RS
Cisatracurium besylate.
R-trans-R¢-trans-Atracurium:
(1R,1¢R,2S,2¢S)-2,2¢-(3,11-Dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) dibenzenesulfonate.
    C65H82N2O18S2         1243.48
R-cis-R¢-trans-Atracurium:
(1R,1¢R,2R,2¢S)-2,2¢-(3,11-Dioxo-4,10-dioxatridecamethylene)bis(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) dibenzenesulfonate.
    C65H82N2O18S2         1243.48
Other related compounds.
USP38
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