Add the following:
Cisatracurium Besylate Injection
DEFINITION
Cisatracurium Besylate Injection contains NLT 90.0% and NMT 110.0% of the labeled amount of cisatracurium (C53H72N2O12). Multiple-dose containers may contain benzyl alcohol.
IDENTIFICATION
•  A. Infrared Absorption 197
Standard solution:  3 mg/mL of USP Cisatracurium Besylate RS in methylene chloride
Sample solution 
[Note—If an emulsion forms during the extraction with methylene chloride, use a centrifuge to separate the layers. ]
For Injections that do not contain benzyl alcohol:  Nominally 2 mg/mL of cisatracurium from the Injection in methylene chloride prepared as follows. Transfer an amount of Injection equivalent to 10 mg of cisatracurium into a suitable separator, and extract with 5 mL of methylene chloride. Use the lower methylene chloride layer.
For Injections containing benzyl alcohol:  Nominally 2 mg/mL of cisatracurium from the Injection in methylene chloride prepared as follows. Transfer an amount of Injection equivalent to 10 mg of cisatracurium into a suitable separator, and extract with 20.0 mL of ethyl acetate. Transfer the lower aqueous layer into another separator, and extract with 5 mL of methylene chloride. Use the lower methylene chloride layer.
Analysis 
Samples:  Standard solution and Sample solution
Add several drops of the Standard solution and Sample solution to separate potassium bromide plates. Allot the solvent to evaporate, and analyze the resulting residue.
Acceptance criteria:  Meets the requirements
•  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
Solution A:  Acetonitrile, methanol, water, and anhydrous formic acid (21:21:60:1)
Mobile phase:  19.4 g/L of ammonium formate in Solution A. Filter under vacuum using a suitable filter.
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:  Nominally 0.7 mg/mL of cisatracurium besylate from the Injection in Diluent, equivalent to 0.5 mg/mL of cisatracurium, prepared as follows. Using a “to contain” pipet, transfer a suitable volume of Injection to an appropriate volumetric flask, and dilute with Diluent to volume.
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:  10 µL
System suitability 
Samples:  System suitability solution and Standard solution
[Note—See Table 1 for relative retention times. ]
Suitability requirements 
Resolution:  NLT 2.0 between R-cis-R¢-trans-atracurium and cisatracurium, System suitability solution
Tailing factor:  NMT 1.7 for cisatracurium, Standard solution
Relative standard deviation:  NMT 1.5%, Standard solution
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of cisatracurium (C53H72N2O12) in the portion of Injection taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU== peak response of cisatracurium from the Sample solution
rS== peak response of cisatracurium from the Standard solution
CS== concentration of USP Cisatracurium Besylate RS in the Standard solution
CU== nominal concentration of cisatracurium in the Sample solution (mg/mL)
Mr1== molecular weight of cisatracurium, 929.14
Mr2== molecular weight of cisatracurium besylate, 1243.48
Acceptance criteria:  90.0%–110.0%
OTHER COMPONENTS
•  Benzyl Alcohol Content (if present)
Internal standard solution:  4.5 mg/mL of decanol in ethyl acetate
Standard stock solution:  9.0 mg/mL of USP Benzyl Alcohol RS in water
Standard solution:  0.45 mg/mL of benzyl alcohol from the Standard stock solution prepared as follows. Transfer 5.0 mL of Standard stock solution and 20.0 mL of ethyl acetate into a 50-mL centrifuge tube. Stopper, shake well for 30 s, and allow the phases to separate. Transfer 10.0 mL of the top (organic) layer and 5.0 mL of the Internal standard solution into a 50-mL volumetric flask. Dilute with ethyl acetate to volume.
Sample solution:  Transfer 5.0 mL of the Injection and 20.0 mL of ethyl acetate into a 50-mL centrifuge tube. Stopper, shake well for 30 s, and allow the phases to separate. Transfer 10.0 mL of the top (organic) layer and 5.0 mL of the Internal standard solution into a 50-mL volumetric flask. Dilute with ethyl acetate to volume.
Chromatographic system 
(See Chromatography 621, System Suitability.)
Mode:  GC
Detector:  Flame ionization
Column:  0.53-mm × 15-m fused-silica capillary column coated with a 1.0-µm layer of liquid phase G16
Temperatures 
Detector:  250
Injection port:  220
Column:  140
Carrier gas:  Helium
Flow rate:  5.0 mL/min
Injection volume:  1 µL
Injection type:  Split ratio, 2.5: 1
System suitability 
Sample:  Standard solution
[Note—The relative retention times for decanol and benzyl alcohol are 0.62 and 1.0, respectively. ]
Suitability requirements 
Resolution:  NLT 2.0 between decanol and benzyl alcohol
Relative standard deviation:  NMT 2.0% for peak area ratios of benzyl alcohol to decanol
Analysis 
Samples:  Standard solution and Sample solution
Calculate the percentage of the labeled amount of benzyl alcohol in the portion of Injection taken:
Result = (RU/RS) × (CS/L) × 100

RU== peak area ratio of benzyl alcohol to the internal standard from the Sample solution
RS== peak area ratio of benzyl alcohol to the internal standard from the Standard solution
CS== concentration of USP Benzyl Alcohol RS in the Standard stock solution (mg/mL)
L== label claim of benzyl alcohol (mg/mL)
Acceptance criteria:  90.0%–110.0% of the labeled amount of benzyl alcohol
IMPURITIES
•  Organic Impurities
Mobile phase, Diluent, System suitability solution, Standard solution, Sample solution, Chromatographic system, and System suitability:  Proceed as directed in the Assay.
Sensitivity solution:  0.4 µg/mL of USP Cisatracurium Besylate RS in Diluent
Analysis 
Samples:  Standard solution, Sample solution, and Sensitivity solution
Calculate the percentage of each impurity in the portion of Injection taken:
Result = (rU/rS) × (CS/CU) × (Mr1/Mr2) × 100

rU== peak response for each impurity from the Sample solution
rS== peak response of cisatracurium from the Standard solution
CS== concentration of USP Cisatracurium Besylate RS in the Standard solution (mg/mL)
CU== nominal concentration of cisatracurium in the Sample solution (mg/mL)
Mr1== molecular weight of cisatracurium, 929.14
Mr2== molecular weight of cisatracurium besylate, 1243.48
Acceptance criteria:  See Table 1. Disregard any peaks with responses less than the peak from the Sensitivity solution.
Table 1
Name Relative
Retention
Time 		Acceptance
Criteria,
NMT (%)
Besylatea 0.10 —
cis-Quaternary acidb 0.13 4.3
(R)-N-Methyllaudanosinec,d 0.15 —
(R)-Laudanosinee 0.19 4.0
cis-Quaternary methyl
esterf,d and benzyl alcoholg 		0.22 —
cis-Quaternary alcoholh 0.27 5.0
Benzaldehydei 0.40 —
R-trans-R¢-trans-Atracuriumj,d 0.72 —
R-cis-R¢-trans-Atracuriumk,d 0.88 —
Cisatracurium 1.0 —
trans-Monoquaternary
compoundl,d 		1.19 —
trans-Monoacrylatem,d 1.30 —
cis-Monoquaternary
compoundn,d 		1.42 —
cis-cis-Triester analogo,d 1.47 —
cis-Monoacrylatep 1.58 2.5
Any individual unspecified degradation product — 0.2
Total degradation products — 14.4
a   This peak is due to the counterion and is not to be reported or included in Total degradation products.
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   This is a process impurity that is included in the table for identification only. This impurity is controlled in the drug substance. It is not to be reported for the drug product and is not to be included in the total degradation products.
e   (R)-1,2,3,4-Tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinoline.
f   (1R,2R)-1,2,3,4-Tetrahydro-6,7-dimethoxy-2-[2-(methoxycarbonyl)ethyl]-2-methyl-1-veratrylisoquinolinium benzenesulfonate.
g   Benzyl alcohol co-elutes with cis-quaternary methyl ester. It is monitored using the test for Benzyl Alcohol Content.
h   (1R,2R)-1,2,3,4-Tetrahydro-2-(9-hydroxy-3-oxo-4-oxanonyl)-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium benzenesulfonate.
i   Benzaldehyde is a degradant of benzyl alcohol and is not included in the total impurities.
j   (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.
k   (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.
l   (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.
m   (1R,2S)-2-(3,11-Dioxo-4,10-dioxa-12-tridecenyl)-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) benzenesulfonate.
n   (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.
o   (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.
p   (1R,2R)-2-(3,11-Dioxo-4,10-dioxa-12-tridecenyl)-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl-1-veratrylisoquinolinium) benzenesulfonate.
SPECIFIC TESTS
•  pH 791: 3.0–3.8
•  Bacterial Endotoxins Test 85: NMT 8.17 USP Endotoxin Units/mg of cisatracurium besylate
•  Sterility Tests 71: Meets the requirements for the test for Sterility of the Product to Be Examined, Membrane Filtration
•  Other Requirements: Meets the requirements for Injections 1
ADDITIONAL REQUIREMENTS
•  Packaging and Storage: Preserve in single-dose or multiple-dose containers, preferably of Type I glass. Store in a cool place, protected from freezing and light.
•  USP Reference Standards 11
USP Benzyl Alcohol RS Click to View Structure
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.
USP Endotoxin RS
USP38
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Pharmacopeial Forum: Volume No. 39(5)