Tacrolimus

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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C₄₄H₆₉NO₁₂· H₂ O 822.03

15,19-Epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro- 5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-, monohydrate, [3S-[3R*,E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-;

(−)-(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone monohydrate CAS RN®: 109581-93-3; UNII: WM0HAQ4WNM.

1 DEFINITION

Tacrolimus contains NLT 98.0% and NMT 102.0% of Tacrolimus (C₄₄H₆₉NO₁₂), calculated on the anhydrous and solvent-free basis.

2 IDENTIFICATION

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

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 solutions containing tacrolimus from light.

Solution A: 6 mM phosphoric acid

Solution B: Acetonitrile and tert-butyl methyl ether (81:19)

Solution C: Solution A and Solution B (4:1)

Solution D: Solution A and Solution B (1:4)

Mobile phase: See Table 1.

Table 1

Diluent: Acetonitrile and water (7:3)

System suitability solution: 3 mg/mL of USP Tacrolimus System Suitability Mixture RS in Diluent. Allow the solution to stand for 3 h at ambient temperature before use.

Standard solution: 3 mg/mL of USP Tacrolimus RS in Diluent. Allow the solution to stand for 3 h at ambient temperature before use.

Sample solution: 3 mg/mL of Tacrolimus in Diluent. Allow the solution to stand for 3 h at ambient temperature before use.

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

Column: 4.6-mm × 15-cm; 3-μm packing L1

Temperatures

Column: 60°

Autosampler: 4°

Flow rate: 1.5 mL/min

Injection volume: 20 μL

3.3 System suitability

Samples: System suitability solution and Standard solution

[Note—See Table 3 for relative retention times.]

3.4 Suitability requirements

Resolution: NLT 3.0 between ascomycin and tacrolimus, System suitability solution

Relative standard deviation: NMT 1.0% for the sum of the responses of tacrolimus, tacrolimus open ring, and tacrolimus 19-epimer,

Standard solution

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of tacrolimus (C₄₄H₆₉NO₁₂) in the portion of Tacrolimus taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = sum of the peak responses of tacrolimus open ring, tacrolimus 19-epimer, and tacrolimus from the Sample solution

r_S = sum of the peak responses of tacrolimus open ring, tacrolimus 19-epimer, and tacrolimus from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the anhydrous and solvent-free basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.1%

Organic Impurities, Procedure 1

Use Organic Impurities, Procedure 1 when the impurity prodfie includes tacrolimus methylacrylaldehyde and tacrolimus diene. It is suggested that new columns be conditioned with about 500 mL of alcohol before use to meet the resolution criterion.

Mobile phase: Hexane, n-butyl chloride, and acetonitrile (7:2:1). Add n-butyl chloride to hexane, and mix well before adding acetonitrile. After adding acetonitrile, mix the Mobile phase for 2 h to get a clear solution. Any deviations from the ratio of components in the Mobile phase and the order of mixing will result in a two-phase solution.

System suitability solution: 0.1 mg/mL each of USP Tacrolimus RS and USP Tacrolimus Related Compound A RS in Mobile phase

Sample solution: 2.0 mg/mL of Tacrolimus in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 225 nm

Column: Two 4.6-mm × 25-cm columns; 5-μm packing L20

Column temperature: 28 ± 2°

Flow rate: 1.5 mL/min. Adjust the Flow rate so that the retention time of tacrolimus is approximately 15 min.

Injection volume: 20 μL

System suitability

Sample: System suitability solution

Suitability requirements

Resolution: NLT 1.1 between tacrolimus and tacrolimus related compound A

Tailing factor: NMT 1.5

Relative standard deviation: NMT 2.0%

Analysis

Sample: Sample solution

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

Result = (r_U /F_i ) × {1/[r_T + Σ(r_U /F_i )]} × 100

r_U = peak response of each impurity from the Sample solution

F_i = relative response factor for each corresponding impurity (see Table 2)

r_T = peak response of tacrolimus from the Sample solution

Acceptance criteria: See Table 2.

Table 2

^a (E)-3-[[(1R,3R,4R)-4-Hydroxy-3-methoxycyclohexyl]-2-methylacrylaldehyde.

^b (14E,18E)-17-Allyl-1-hydroxy-12-[(E)-2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28- dioxa-4-azatricyclo[22.3.1.04,9] octacosa-14,18-diene-2,3,10,16-tetrone.

^c Specified unidentified impurity.

^d For informational purposes only; not to be reported.

^e (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19S,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a,hexadecahydro-5,19-dihydroxy-3- {(E)-2-[1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16,dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

^f (3S,4R,5S,8R,12S,14S,15R,16S,18R,26aS,E)-8-Allyl-5,6,11,12,13,14,15,16,17,18,24,25,26,26a-tetradecahydro-5,15,20,20-tetrahydroxy-3- {(E)-2-[1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,19,21(4H,8H,20H,23H)-tetrone.

^g Total impurities limit does not include tacrolimus open ring and tacrolimus 19-epimer.

Organic Impurities, Procedure 2

Use Organic Impurities, Procedure 2 when the impurity profile includes ascomycin, desmethyl tacrolimus, tacrolimus 8-epimer, and tacrolimus 8- propyl analog. Protect solutions containing tacrolimus from light.

Solution A, Solution B, Solution C, Solution D, Mobile phase, Diluent, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Standard solution: 30 μg/mL of USP Tacrolimus RS in Diluent. Allow the solution to stand for 3 h at ambient temperature before use.

Reporting threshold solution: 1.5 μg/mL of USP Tacrolimus RS in Diluent

Peak identification solution 1: 10 μg/mL of USP Tacrolimus 8-epimer RS in acetonitrile

Peak identification solution 2: 10 μg/mL of USP Tacrolimus 8-propyl Analog RS in acetonitrile

System suitability

[Note—Identify the related compounds by the relative retention times provided in Table 3.]

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 3.0 between tacrolimus and ascomycin, System suitability solution

Relative standard deviation: NMT 10.0% for the sum of the responses of tacrolimus and tacrolimus 19-epimer, Standard solution

Analysis

Samples: Sample solution, Standard solution, Reporting threshold solution, Peak identification solution 1, and Peak identification solution 2

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

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of each impurity from the Sample solution

r_S = sum of the peak responses of tacrolimus 19-epimer and tacrolimus from the Standard solution

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

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

Acceptance criteria: See Table 3. Identify tacrolimus 8-epimer and tacrolimus 8-propyl analog using Peak identification solution 1 and Peak identification solution 2. Report impurity peaks with responses NLT that of the peak in the Reporting threshold solution (0.05%). Disregard peaks with retention times less than 3 min.

Table 3

^a (3S,4R,5S,8R,12S,14S,15R,16S,18R,26aS,E)-8-Allyl-5,6,11,12,13,14,15,16,17,18,24,25,26,26a-tetradecahydro-5,15,20,20-tetrahydroxy-3- {(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,19,21(4H,8H,20H,23H)-tetrone.

^b Tacrolimus open ring and tacrolimus 19-epimer are isomers of tacrolimus, which are present in equilibrium with the active ingredient. They are not to be reported as degradation products.

^c (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19S,26aS)-8-Ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3- [(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

^d If possible from the manufacturing process.

^e (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19S,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3- {(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

^f (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3- [(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

^g (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3- [(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-14,16-dimethoxy-4,12,18-trimethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

^h (3S,4R,5S,8S,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3- {(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

ⁱ (3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)- 5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-Hexadecahydro-5,19-dihydroxy-3-{(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-8-propyl-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

^j Total impurities limit does not include tacrolimus open ring and tacrolimus 19-epimer.

5 SPECIFIC TESTS

Optical Rotation, Specific Rotation 〈781S〉

Sample solution: 10 mg/mL in N,N-dimethylformamide

Acceptance criteria: −110° to −115° on the “as-is” basis

Water Determination, Method I 〈921〉: NMT 4.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers. Store at controlled room temperature.

Labeling: If a test for Organic Impurities other than Procedure 1 is used, then the labeling states with which test for Organic Impurities the article complies.

Change to read:

USP Reference Standards 〈11〉

USP Tacrolimus RS

15,19-Epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone-5,6,8,11,12,13,14,15, 16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-, monohydrate, [3S-[3R*,E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*, 18S*,19S*,26aR*]]-.

C₄₄H₆₉NO₁₂ · H₂O 822.03

USP Tacrolimus Related Compound A RS

(E)-8-Ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24, 25,26,26a-Hexadecahydro-5,19-dihydroxy-3-[(E)-2-(4-hydroxy-3-methoxycyclohexyl)-1- methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

C₄₃H₆₉NO₁₂ 792.02 (ERR 1-Sep-2021)

USP Tacrolimus 8-epimer RS

(3S,4R,5S,8S,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Allyl-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-{(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

C₄₄H₆₉NO₁₂ 804.03 (ERR 1-Sep-2021)

USP Tacrolimus 8-propyl Analog RS

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-Hexadecahydro-5,19-dihydroxy-3-{(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-8-propyl-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

C₄₄H₇₁NO₁₂ 806.03

USP Tacrolimus System Suitability Mixture RS

This is a mixture of tacrolimus, ascomycin

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-8-Ethyl-5,6,8,11,12,13,14,15,16,17,18,19,24, 25,26,26a-hexadecahydro-5,19-dihydroxy-3-[(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

C₄₃H₆₉NO₁₂ 792.01

and tacrolimus 8-propyl analog

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R, 19R,26aS)-5,6,8,11,12,13,14,15,16,17,18,19,24, 25,26,26a-Hexadecahydro-5,19-dihydroxy-3-{(E)-2- [(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-8-propyl-3H-pyrido[2,1-c] [1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone.

C₄₄H₇₁NO₁₂ 806.03