Everolimus

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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C₅₃H₈₃NO₁₄ 958.24 (CN 1-Aug-2024)

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-Dihydroxy-12-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone;

(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone CAS RN®: 159351-69-6.

1 DEFINITION

Everolimus contains NLT 95.0% and NMT 102.0% of everolimus (C₅₃H₈₃NO₁₄), calculated on the anhydrous and solvent-free basis. It may contain a suitable antioxidant.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

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

Protect everolimus from exposure to oxygen. Protect solutions containing everolimus from light.

Solution A: 0.27 g/L of monobasic potassium phosphate

Solution B: Acetonitrile

Mobile phase: See Table 1.

Table 1

System suitability solution: 0.5 mg/mL of USP Everolimus System Suitability Mixture RS in acetonitrile

Standard solution: 0.5 mg/mL of USP Everolimus RS in acetonitrile

Sample solution: 0.5 mg/mL of Everolimus in acetonitrile

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 275 nm

Column: 3-mm × 25-cm; 5-μm packing L1

Column temperature: 50°

Flow rate: 1.1 mL/min

Injection volume: 10 μL

3.3 System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times for 9-O-hydroxyethyl sirolimus, everolimus, and everolimus oxepane isomer are about 0.92, 1.0, and 1.1,respectively.]

3.4 Suitability requirements

Resolution: NLT 1.2 between 9-O-hydroxyethyl sirolimus and everolimus and NLT 1.5 between everolimus and everolimus oxepane isomer,

3.5 System suitability solution

Relative standard deviation: NMT 2.0% for the sum of the everolimus and everolimus oxepane isomer peaks, Standard solution

3.6 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of everolimus (C₅₃H₈₃NO₁₄) in the portion of Everolimus taken:

Result = [(r_U /r_S ) × (C_S /C_U ) × 100] − F

r_U = sum of the peak responses of everolimus and everolimus oxepane isomer from the Sample solution

r_S = sum of the peak responses of everolimus and everolimus oxepane isomer from the Standard solution

C_S = concentration of USP Everolimus RS in the Standard solution (mg/mL)

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

F = percentage of sirolimus from the test for Limit of Sirolimus

Acceptance criteria: 95.0%–102.0% on the anhydrous and solvent-free basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.1%

4.1 Limit of Alcohol

Analysis: See Residual Solvents 〈467〉.

Acceptance criteria: NMT 1.5%

4.1.1 Organic Impurities

Protect everolimus from exposure to oxygen. Protect solutions containing everolimus from light and inject them within 2 h of preparation to minimize the effects of isomerization between everolimus and everolimus oxepane isomer.

Solution A, Solution B, and Mobile phase: Prepare as directed in the Assay.

System suitability solution: 5 mg/mL of USP Everolimus System Suitability Mixture RS in acetonitrile. Use the solution within 24 h.

Sensitivity solution: 0.0025 mg/mL of USP Everolimus RS in acetonitrile

Standard solution: 0.05 mg/mL of USP Everolimus RS in acetonitrile

Sample solution: 5 mg/mL of Everolimus in acetonitrile

4.1.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 3-mm × 25-cm; 5-μm packing L1

Column temperature: 50°

Flow rate: 1.1 mL/min

Injection volume: 10 μL

4.1.3 System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

[Note—See Table 2 for relative retention times.]

4.1.4 Suitability requirements

Resolution: NLT 1.2 between 9-O-hydroxyethyl sirolimus and everolimus; NLT 1.5 between everolimus and everolimus oxepane isomer,

4.1.5 System suitability solution

Relative standard deviation: NMT 5% for everolimus, Standard solution

Signal-to-noise ratio: NLT 10 for everolimus, Sensitivity solution

4.1.6 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Everolimus taken:

Result = (r_U /r_S ) × (C_S /C_U ) × (1/F) × 100

r_U = peak response of each impurity from the Sample solution

r_S = peak response of everolimus from the Standard solution

C_S = concentration of USP Everolimus RS in the Standard solution (mg/mL)

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

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. The reporting threshold is 0.05%.

Table 2

^a(S)-1-{2-[(2R,3R,6S)-2-Hydroxy-6-{(2S,3E,5E,7E,9S,11R,13R,14R,15E,17R,19E,21R)-14-hydroxy-22-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-2,13-dimethoxy-3,9,11,15,17,21-hexamethyl-12,18-dioxodocosa-3,5,7,15,19-pentaen-1-yl}-3-methyltetrahydro-2H-pyran-2-yl]-2-oxoacetyl}piperidine-2-carboxylic acid.

^b(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,10,27-trihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-21-methoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.

^c(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-27-hydroxy-9-(2-hydroxyethoxy)-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.

^d3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,28R,34aS)-9,10,12,13,14,21,22,23,24,25,26,28,32,33,34,34a-Hexadecahydro-9,28-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,28-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,27,29(4H,6H,31H)-pentone.

^e Everolimus oxepane isomer is formed in polar protic solvents. It is listed here for information only; it is not to be reported.

^f Butylated hydroxytoluene is an antioxidant that may be used to stabilize everolimus. It is listed here for information only; it is not to be reported.

^g(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-formyloxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.

^h The system may resolve formyl sirolimus and specified impurity 4. The limit is for the sum of the two peaks.

ⁱ(3S,6R,7E,9R,10R,11R,12R,14S,15S,16E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,15,18,21,22,23,24,25,26,27,32,33,34,34a-Octadecahydro-9,11,27-trihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-18-ethoxy-6,8,12,14,20,26-hexamethyl-23,27:11,15-diepoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,28,29(4H,6H,31H)-tetrone.

4.2 Limit of Sirolimus

Protect everolimus from exposure to oxygen. Protect solutions containing everolimus from light.

Solution A: 0.32 g of ammonium formate dissolved in 450 mL of water, 100 mL of methanol, 450 mL of acetonitrile, and 0.05 mL of formic acid

Solution B: 0.32 g of ammonium formate dissolved in 330 mL of water, 100 mL of methanol, 570 mL of acetonitrile, and 0.05 mL of formic acid

Mobile phase: See Table 3. [Note—The procedure is sensitive to the percentage of methanol in the Mobile phase.]

Table 3

System suitability solution: 2 mg/mL of USP Everolimus System Suitability Mixture RS and 0.016 mg/mL of USP Sirolimus RS in acetonitrile

Sensitivity solution: 0.001 mg/mL of USP Everolimus RS in acetonitrile

Standard solution: 0.016 mg/mL of USP Everolimus RS in acetonitrile

Sample solution: 2 mg/mL of Everolimus in acetonitrile

4.2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 278 nm

Column: 2.1-mm × 15-cm; 2.7-μm packing L1

4.2.2 Temperatures

[Note—A detector temperature of 40° was shown to improve the chromatography.]

Autosampler: 6°

Column: 60°

Flow rate: 0.9 mL/min

Injection volume: 3.5 μL

4.2.3 System suitability

Samples: System suitability solution, Sensitivity solution, and Standard solution

[Note—See Table 4 for relative retention times.]

4.2.4 Suitability requirements

Resolution: NLT 1.2 between sirolimus and everolimus, System suitability solution

Relative standard deviation: NMT 5% for the sum of the peak responses for everolimus and everolimus oxepane isomer, Standard solution

Signal-to-noise ratio: NLT 10 for the everolimus peak, Sensitivity solution

4.2.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of sirolimus in the portion of Everolimus taken:

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

r_U = peak response of sirolimus from the Sample solution

r_S = sum of the peak responses of everolimus and everolimus oxepane isomer from the Standard solution

C_S = concentration of USP Everolimus RS in the Standard solution (mg/mL)

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

Acceptance criteria: See Table 4. The reporting threshold is 0.05%.

Table 4

^a These impurities are controlled in the test for Organic Impurities. They are listed here for information only.

^b(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-Hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone.

^c Everolimus oxepane isomer is formed in polar protic solvents. It is listed here for information only; it is not to be reported.

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 1.5%

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 10 mg/mL in methanol

Acceptance criteria: −141.0° to −153.0° on the anhydrous basis

Microbial Enumeration Tests 〈61〉 and Tests for Specified Organisms 〈62〉: The total aerobic microbial count does not exceed 103cfu/g. The total yeasts and molds count does not exceed 102 cfu/g.

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers in an atmosphere of protective gas. Store at refrigerated temperatures.

Labeling: Label it to indicate the nature and the content of the antioxidant, if present.

USP Reference Standards 〈11〉

USP Everolimus RS

USP Everolimus System Suitability Mixture RS

This is a mixture of everolimus, everolimus oxepane isomer, and 9-O-hydroxyethyl sirolimus. Other minor components may be present.

USP Sirolimus RS