Cyclosporine Injection

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Cyclosporine Injection is a sterile solution of Cyclosporine in a suitable vehicle. It contains NLT 90.0% and NMT 110.0% of the labeled amount of cyclosporine (C₆₂H₁₁₁N₁₁O₁₂).

2 IDENTIFICATION

2.1 A. THIN-LAYER CHROMATOGRAPHY

Solution A: 17 mg/mL of Bismuth subnitrate in 20% acetic acid

Solution B: 400 mg/mL of potassium iodide

Standard solution: 0.5 mg/mL of USP Cyclosporine RS in methanol

Sample solution: Nominally 0.5 mg/mL of cyclosporine from Injection in methanol

2.1.1 Chromatographic system

(See Chromatography (621), Thin-Layer Chromatography.)

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture

Application volume: 10 µL

Developing solvent system 1: Ethyl ether

Developing solvent system 2: Ethyl acetate, methyl ethyl ketone, water, and formic acid (60:40:2:1)

Spray reagent 1: Mix 5 mL of Solution A with 5 mL of Solution B and 20 mL of glacial acetic acid, and dilute with water to 100 mL. Prepare freshly.

Spray reagent 2: Hydrogen peroxide TS

2.1.2 Analysis

Samples: Standard solution and Sample solution

Apply the Standard solution and the Sample solution to the plate. Allow the spots to dry in a current of air, place the plate in a suitable chromatographic chamber, and develop the chromatogram, using Developing solvent system 1, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and allow it to dry. Place the plate in a second chromatographic chamber, and develop the chromatogram in Developing solvent system 2 until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and allow it to dry. Spray the plate with Spray reagent 1. Immediately again spray the plate with Spray reagent 2. Cyclosporine appears as a brown spot having an R, value of about 0.45.

Acceptance criteria: The R_F value of the principal spot of the Sample solution corresponds to that of the Standard solution. Disregard any spots at the origin.

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 PROCEDURE

Mobile phase: Acetonitrile, methanol, water, and phosphoric acid (550:50:400:0.5)

Standard solution: 0.5 mg/mL of USP Cyclosporine RS in methanol. Use this solution promptly after preparation.

Sample solution 1 (where it is represented as being in a single-dose container): Nominally 0.5 mg/mL of cyclosporine from Injection in methanol, prepared as follows. Using a suitable hypodermic needle and syringe, withdraw all of the withdrawable contents from 1 container of Injection, and dilute with methanol. Use this solution promptly after preparation.

Sample solution 2 (where the label states the quantity of cyclosporine in a given volume): Nominally 0.5 mg/mL of cyclosporine from Injection in methanol, prepared as follows. Dilute a suitable aliquot of Injection with methanol. Use this solution promptly after preparation.

3.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm x 25-cm; packing L16

Column temperature: 70"

Flow rate: 1 mL/min

Injection volume: 20 µL

3.1.2 System suitability

Sample: Standard solution

3.1.3 Suitability requirements

Capacity factor: NLT 3-NMT 10

Column efficiency: NLT 700 theoretical plates

Tailing factor: NMT 1.5

Relative standard deviation: NMT 1.5%

3.1.4 Analysis

Samples: Standard solution and Sample solution 1 or Sample solution 2

Calculate the percentage of the labeled amount of cyclosporine (C₆₂H₁₁₁N₁₁O₁₂) in the portion of Injection taken: 

                 Result = (r_U/r_S) x (C_S/C_U) × 100

r_U = peak response from Sample solution 1 or Sample solution 2

r_S = peak response from the Standard solution S

C_S = concentration of the Standard solution (mg/mL) C_{s} = 1

C_U = nominal concentration of Sample solution 1 or Sample solution 2 (mg/mL)

Acceptance criteria: 90.0%-110.0%

4 OTHER COMPONENTS

4.1 CONTENT OF ALCOHOL (where present)

Internal standard solution: n-Propyl alcohol and butyl alcohol (3:50)

Standard stock solution: 64 mg/mL of dehydrated alcohol in butyl alcohol

Standard solution: 12.8 mg/mL of alcohol, prepared as follows. Transfer a suitable aliquot of Standard stock solution to a suitable volumetric flask. Add Internal standard solution, using 24% of the final volume, and dilute with butyl alcohol to volume.

4.1.1 Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: GC

Detector: Flame ionization

Column: 2-mm x 2-m glass; packed with support S3

4.1.2 Temperatures

Injection port: 280°

Detector: 290°

Column: See Table 1.

Sample solution: Nominally 12.8 mg/mL of alcohol from Injection, prepared as follows. Transfer a suitable aliquot of Injection to a suitable volumetric flask. Add Internal standard solution, using 24% of the final volume, and dilute with butyl alcohol to volume.

Table 1

Carrier gas: Nitrogen

Flow rate: 35 mL/min

Injection volume: 1 μL. [Note—Make adjustments, if necessary, to obtain satisfactory chromatography.]

4.1.3 System suitability

Sample: Standard solution

Suitability requirements: [NOTE-The elution order is alcohol, n-propyl alcohol, and butyl alcohol.]

Relative standard deviation: NMT 2.0%

4.1.4 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of alcohol (C₆₂H₁₁₁N₁₁O₁₂) in the portion of Injection taken:

                 Result = (R_U/R_S) x (C_S/C_U) × 100

R_U = peak area ratio of alcohol to n-propyl alcohol from the Sample solution U

R_S = peak area ratio of alcohol to n-propyl alcohol from the Standard solution S

C_S = concentration of alcohol in the Standard solution (mg/mL)

C_U = concentration of the Sample solution (mg/mL)

Acceptance criteria: 80.0%-120.0% of the labeled amount

5 SPECIFIC TESTS

BACTERIAL ENDOTOXINS TEST (85)

Sample solution: Make a 1:10 dilution of the Injection with Water for Injection.

Analysis: Add 0.1 mL of Sample solution and 0.1 mL of appropriately constituted LAL reagent to a suitable pyrogen-free test tube. Mix on a vortex mixer for about 5 s.

Acceptance criteria: NMT 0.84 USP Endotoxin Unit/mg of cyclosporine

STERILITY TESTS (71): Meets the requirements

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in single-dose or multiple-dose containers.

LABELING: Label it to indicate that it is to be diluted with a suitable parenteral vehicle before intravenous infusion.

USP REFERENCE STANDARDS (11)

USP Cyclosporine RS