Tacrolimus Capsules

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

Tacrolimus Capsules contain NLT 93.0% and NMT 105.0% of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂).

2 IDENTIFICATION

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

B. The UV absorption spectrum of the major peak of the Sample solution and that of the Standard solution exhibit maxima and minima at the

same wavelengths, as obtained in the Assay.

3 ASSAY

3.1 Procedure

Allow the Standard solution and Sample solution to stand for 3 h at ambient temperature before use. Protect solutions containing tacrolimus from light.

Solution A: 6 mM phosphoric acid

Solution B: 50 g/L of polyoxyethylene (23) lauryl ether. [Note—Polyoxyethylene (23) lauryl ether is also called Brij-35.]

Solution C: Acetonitrile and Solution B (7:3)

Mobile phase: Acetonitrile, tert-butyl methyl ether, and Solution A (335:55:600)

Standard solution: 50 μg/mL of USP Tacrolimus RS in Solution C

Sample solution: Equivalent to 50 μg/mL of tacrolimus from NLT 10 Capsules in Solution C. [Note—Sonicate, and stir with a magnetic stirrer.]

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 205 nm. When this procedure is used for Identification test B, use a diode array detector set at 200–400 nm.

Column: 4.0-mm × 5.5-cm; 3-μm packing L1

Column temperature: 60°

Flow rate: 1 mL/min

Injection volume: 5 μL

3.3 System suitability

Sample: Standard solution

[Note—The relative retention times for tacrolimus 19-epimer and tacrolimus are 0.67 and 1.0, respectively.]

3.4 Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 3.0% for the sum of the tacrolimus and tacrolimus 19-epimer peaks

3.5 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) in the portion of Capsules taken:

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

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

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

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

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

Acceptance criteria: 93.0%–105.0%

4 PERFORMANCE TESTS

Dissolution 〈711〉

Test 1

Medium: Hydroxypropylcellulose in water (1:2 × 104), adjusted with 6% phosphoric acid to a pH of 4.5; 900 mL

Apparatus 2: 50 rpm with sinker (see Dissolution 〈711〉, Figure 2a)

Time: 90 min

Mobile phase: Acetonitrile, methanol, water, and 6% phosphoric acid (46: 18: 36: 0.1)

Standard stock solution: (L/360) mg/mL in acetonitrile, where L is the Capsule label claim in mg

Standard solution: To 20.0 mL of the Standard stock solution add 50.0 mL of Medium, and mix to obtain solutions with known concentrations as indicated in Table 1. Allow the solution to stand for NLT 6 h at 25° before use.

Sample solution: Pass 10 mL of the solution under test through a G4 glass Filter. To 5.0 mL of the filtrate add 2.0 mL of acetonitrile, and mix. Allow the solution to stand for NLT 1 h at 25° before use.

Table 1

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 15-cm; 5-μm packing L7

Column temperature: 50°

Flow rate: Adjust the flow rate so that the retention time of tacrolimus is approximately 14 min.

Injection volume: See Table 2.

Table 2

[Note—For products with strengths other than those listed in Table 2, adjust the Injection volume to deliver an equivalent amount of tacrolimus into the column.]

System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 1.5 between tacrolimus 19-epimer and tacrolimus

Tailing factor: NMT 1.5

Relative standard deviation: NMT 1.5%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) dissolved:

Result = (r_U/r_S ) × C_S × D × V × (100/L)

r_U = peak response of tacrolimus from the Sample solution

r_S = peak response of tacrolimus from the Standard solution

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

D = dilution factor of the Sample solution

V = volume of Medium, 900 mL

L = label claim (mg/Capsule)

Tolerances: NLT 80% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 2: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 2.

[Note—Allow the Standard solution to stand for 3 h at ambient temperature before use. Protect solutions containing tacrolimus from light.]

Buffer: Dissolve 6 g of sodium dodecyl sulfate and 8.28 g of monobasic sodium phosphate in 6000 mL of water. Adjust with 2 N sodium

hydroxide to a pH of 7.0.

Medium: Buffer; 900 mL

Apparatus 2: 50 rpm, with sinkers

Time: 60 min

Standard stock solution: 0.2 mg/mL of USP Tacrolimus RS in alcohol and Medium (3:7). [Note—Dissolve USP Tacrolimus RS in alcohol using 30% of the final volume. Sonicate until dissolved, and dilute with Medium to volume.]

Standard solution: Dilute the Standard stock solution with Medium to obtain a final concentration of 5 μg/mL.

Sample solution: Pass a portion of the solution under test through a suitable fIlter.

Solution A: 6 mM phosphoric acid

Mobile phase: Acetonitrile, tert-butyl methyl ether, and Solution A (335:50:600)

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 205 nm

Column: 4.0-mm × 5.5-cm; 3-μm packing L1

Column temperature: 60°

Flow rate: 1.2 mL/min

Injection volume: 100 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for tacrolimus 19-epimer and tacrolimus are 0.67 and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 5.0% for the sum of the areas of tacrolimus and tacrolimus 19-epimer

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus ( C₄₄H₆₉NO₁₂ ) dissolved:

Result = (r_U/r_S ) × (C_S /L) × V × 100

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

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

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

L = label claim (mg/Capsule)

V = volume of Medium, 900 mL

Tolerances: NLT 80% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 3: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 3.

Medium: 50 mg/L of hydroxypropylcellulose in water. Adjust with phosphoric acid to a pH of 4.5; 900 mL.

Apparatus 2 (without sinker) and Time: Proceed as directed in Test 1.

Buffer: 3.6 g/L of monobasic potassium phosphate in water. Adjust with diluted phosphoric acid to a pH of 2.5.

Mobile phase: Buffer and acetonitrile (1:1)

Standard stock solution: 0.1 mg/mL of USP Tacrolimus RS in acetonitrile

Standard solution: Dilute the Standard stock solution with Medium to obtain a final concentration of (L/900) mg/mL, where L is the Capsule label claim in mg.

Sample solution: Pass a portion of the solution under test through a suitable fIlter.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 10-cm; 5-μm packing L1

Column temperature: 60°

Flow rate: 1.3 mL/min

Injection volume: 100 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for tacrolimus 19-epimer, tacrolimus open ring, and tacrolimus are 0.67, 0.79, and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus ( C₄₄H₆₉NO₁₂) dissolved:

Result = (r_U/r_S ) × (C_S /L) × V × 100

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

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

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

L = label claim (mg/Capsule)

V = volume of Medium, 900 mL

Tolerances: NLT 75% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 4: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 4.

Medium: Hydroxypropylcellulose in water (1 in 20,000), adjusted with phosphoric acid to a pH of 4.5. See Table 3 for the volume.

Table 3

Apparatus 2: 50 rpm, with sinkers

Time: 120 min

Diluent: 1 mg/mL of hydroxypropylcellulose in water. Sonicate as needed to dissolve.

Buffer: To a solution of 1 g/L of sodium 1-hexanesulfonate in water add 0.1 mL/L of trifluoroacetic acid.

Mobile phase: Acetonitrile, methanol, and Buffer (550:50:400)

Standard stock solution: Dissolve USP Tacrolimus RS in acetonitrile. See Table 4 for the concentrations (L is the Capsule label claim in mg).

Table 4

Standard solution: Dilute the Standard stock solution with Diluent. See Table 5 for the concentrations (L is the Capsule label claim in mg).

Table 5

Sample solution: Pass a portion of the solution under test through a suitable fIlter.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 15-cm; 5-μm packing L1

Column temperature: 60°

Flow rate: 1 mL/min

Injection volume: 100 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) dissolved:

Result = (r_U /r_S ) × (C_S /L) × V × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

L = label claim (mg/Capsule)

V = volume of Medium (mL) (see Table 3)

Tolerances: NLT 75% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 5: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 5.

Medium: 0.05 g/L hydroxypropylcellulose in water. Adjust with phosphoric acid to a pH of 4.5; 900 mL.

Apparatus 2: 50 rpm, with sinkers

Time: 90 min

Solution A: 0.1 mL/L of trifluoroacetic acid in water

Mobile phase: Acetonitrile and Solution A (50:50)

Standard stock solution: 0.22 mg/mL of USP Tacrolimus RS in acetonitrile

Standard solution: (L/900) mg/mL of USP Tacrolimus RS from the Standard stock solution in Medium, where L is the label claim in mg/Capsule

Sample solution: Centrifuge a portion of the solution under test. Use the supernatant.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 205 nm

Column: 2.1-mm × 15-cm; 3.5-μm packing L7

Column temperature: 60°

Flow rate: 0.8 mL/min

Injection volume: 750 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for tacrolimus 19-epimer (tautomer 1), tacrolimus open-ring (tautomer 2), and tacrolimus are 0.55, 0.79, and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 1.5

Relative standard deviation: NMT 4.0% for the peaks due to tautomer 1, tautomer 2, and tacrolimus.

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) dissolved:

Result = (r_U /r_S) × (C_S /L) × V × 100

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

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

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

L = label claim (mg/Capsule)

V = volume of Medium, 900 mL

Tolerances: NLT 75% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 6: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 6.

Dilute phosphoric acid: Transfer 7.1 mL of phosphoric acid to a 100 mL volumetric flask, and dilute with water to volume.

Medium: 50 mg/L of hydroxypropyl cellulose in water. Adjust with Dilute phosphoric acid to a pH of 4.5; 900 mL.

Apparatus 2: 50 rpm

Time: 60 min

Buffer: 3.6 g/L of monobasic potassium phosphate in water. Adjust with Dilute phosphoric acid to a pH of 2.5.

Mobile phase: Acetonitrile and Buffer (1:1)

Standard stock solution: 0.11 mg/mL of USP Tacrolimus RS in acetonitrile

Standard solution: Dilute the Standard stock solution with Medium to obtain a final concentration of (L/900) mg/mL, where L is the label claim in mg/Capsule.

Sample solution: Centrifuge a portion of the solution under test. Use the supernatant.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 10-cm; 5-μm packing L1

Column temperature: 60°

Flow rate: 1.3 mL/min

Injection volume: 100 μL

System suitability

Sample: Standard solution

[Note—The relative retention times for tacrolimus 19-epimer, tacrolimus open ring, and tacrolimus are 0.77, 0.89, and 1.0, respectively.]

Suitability requirements

Tailing factor: NMT 2.0 for tacrolimus

Relative standard deviation: NMT 2.0% for the sum of tacrolimus 19-epimer, tacrolimus open ring, and tacrolimus

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) dissolved:

Result = (r_U /r_S ) × (C_S /L) × V × 100

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

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

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

L = label claim (mg/Capsule)

V = volume of Medium, 900 mL

Tolerances: NLT 80% (Q) of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) is dissolved.

Test 8: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 8.

Medium: 50 mg/L of hydroxypropylcellulose in water. Adjust with 6% phosphoric acid to a pH of 4.5; 500 mL.

Apparatus 2: 75 rpm with sinker

Times: 15, 30, and 90 min

Diluent: 1 mg/mL of hydroxypropylcellulose in water

Buffer: Dissolve 1 g of sodium 1-hexanesulfonate in 1 L of water and add 0.1 mL of tri

uoroacetic acid.

Mobile phase: Acetonitrile, methanol, and Buffer (55:5:40)

Standard stock solution: Dissolve USP Tacrolimus RS in acetonitrile. See Table 6 for the concentrations.

Table 6

Standard solution: Dilute the Standard stock solution with Diluent to obtain a final concentration of (L/500) mg/mL, where L is the label claim in mg/Capsule. Using the Standard stock solution to prepare the final Standard solution, acetonitrile will not exceed 10%.

Sample solution: Pass 10 mL of the solution under test through a 1-um glass fIlter. Replace the portion of solution withdrawn with an equal volume of Medium.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

Column: 4.6-mm × 15-cm; 5-μm packing L1

Column temperature: 60°

Flow rate: 1.0 mL/min

Injection volume: 100 uL

Run time: NLT 1.6 times the retention time of tacrolimus

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 3.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the concentration (C_i ) of tacrolimus (C₄₄H₆₉NO₁₂) in the sample withdrawn from the vessel at each time point (i):

Result_i = (r_U /r_S ) × C_S

r_U = peak response of tacrolimus from the Sample solution

r_S = peak response of tacrolimus from the Standard solution

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

Calculate the percentage of the labeled amount of tacrolimus (C₄₄H₆₉NO₁₂) dissolved at each time point (i):

Result₁ = C₁ × V_S × (1/L) × 100

Result₂ = [(C₂ × V_S) + (C₁ × V )] × (1/L) × 100

Result₃ = {(C₃ × V_S) + [(C₂ + C₁ ) × V ]} × (1/L) × 100

C_i = concentration of tacrolimus in the portion of sample withdrawn at each time point (mg/mL)

V_S = volume of Medium, 500 mL

L = label claim (mg/Capsule)

V = volume of the Sample solution withdrawn at each time point (mL)

Tolerances: See Table 7.

Table 7

Uniformity of Dosage Units 〈905〉: Meet the requirements

5 IMPURITIES

5.1 Organic Impurities, Procedure 1

Use Organic Impurities, Procedure 1 when the impurity profile includes tacrolimus diene and tacrolimus regioisomer. It is suggested that new columns be conditioned with about 500 mL of Ethanol 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: Transfer the contents of a suitable number of Capsules (equivalent to about 5 mg of tacrolimus for 0.5-mg Capsules or 10 mg of tacrolimus for 1-mg and 5-mg Capsules) into a centrifuge tube. Add 1.5 mL of a mixture of n-butyl chloride and acetonitrile (2:1), sonicate in an ultrasonic bath for 2 min, add 3.5 mL of n-hexane, and mix. Centrifuge this solution, and collect the supernatant or pass the solution through a 0.5-μm membrane fIlter. Use the solution within 30 min of preparation.

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 225 nm

Columns: 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

Run time: 3 times the retention time of tacrolimus

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 Capsules 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 8)

r_T = peak response of tacrolimus from the Sample solution

Acceptance criteria: See Table 8. Disregard peaks due to the solvent.

Table 8

^a (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.

^b (4E,11E)-10-Allyl-7,8,10,13,14,15,16,17,18,19,20,21,26,22,28,28a-hexadecahydro-7,21-dihydroxy-3-(4-hydroxy-3-methoxycyclohexyl)-16,18-dimethoxy-4,6,12,14,20-pentamethyl-17,21-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclopentacosine-1,9,22,23(6H,25H)-tetrone.

^c Tacrolimus impurity 1 is a specified, unidentified impurity.

^d Tacrolimus related compound A is listed here to indicate the relative retention time of this compound. It is used in the procedure to evaluate system suitability and is not to be reported. It is not to be included in total impurities.

^e 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 and are not included in total impurities.

^f (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.

^g (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.

5.2 Organic Impurities, Procedure 2

Use Organic Impurities, Procedure 2 when the impurity profile includes tacrolimus hydroxy acid and tacrolimus 8-epimer. It is suggested to equilibrate the column overnight with a mixture of Solution C and Solution D (17:3) before performing this procedure. Allow the System suitability solution, Standard solution, and Sample solution to stand for 3 h at ambient temperature before use. Protect solutions containing tacrolimus from light.

Solution A: 6 mM phosphoric acid

Solution B: Acetonitrile and tert-butyl methyl ether (81:19). [Note—The ratio of acetonitrile to tert-butyl methyl ether is critical.]

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

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

Mobile phase: See Table 9.

Table 9

Solution E: 50 g/L of polyoxyethylene (23) lauryl ether in Solution A. [Note—Polyoxyethylene (23) lauryl ether is also called Brij-35.]

Diluent: Acetonitrile and Solution E (7:3)

System suitability solution: 1.5 mg/mL of USP Tacrolimus System Suitability Mixture RS in Diluent

Standard solution: 7.5 μg/mL of USP Tacrolimus RS in Diluent

Sensitivity solution: 1.5 μg/mL of USP Tacrolimus RS in Diluent from Standard solution

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

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

Sample solution: Equivalent to 1.5 mg/mL of tacrolimus in Diluent. [Note—Shake the mixture on a mechanical shaker for 30 min, and pass through a suitable fIlter.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

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

Column temperature: 60°

Flow rate: 1.5 mL/min

Injection volume: 40 μL

System suitability

Samples: System suitability solution, Standard solution, and Sensitivity 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

Signal-to-noise ratio: NLT 10.0, Sensitivity solution

Analysis

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

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

Result = (r_U /r_S) × (C_S /C_U ) × P × (1/F) × 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 = nominal concentration of tacrolimus in the Sample solution (mg/mL)

P = potency of tacrolimus in USP Tacrolimus RS (mg/mg)

F = relative response factor (see Table 10)

Acceptance criteria: See Table 10. Identify tacrolimus 8-epimer and tacrolimus 8-propyl analog using Peak identification solution 1 and Peak identification solution 2. Disregard peaks that are smaller than the tacrolimus peak in the Sensitivity solution.

Table 10

^a (3S,4R,5S,8R,12S,14S,15R,16S,18R,25aS,E)-8-Allyl-5,15,19-trihydroxy-3-{(E)-1-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]prop-1-en-2-yl}-14,16-dimethoxy-4,10,12,18-tetramethyl-1,7,20-trioxo-1,3,4,5,6,7,8,11,12,13,14,15,16,17,18,19,20,22,23,24,25,25a-docosahydropyrido[2,1-c] [1]oxa[4]azacyclodocosine-19-carboxylic acid.

^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 and are not included in total impurities.

^c (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.

^d (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.

^e These are process impurities that are controlled in the drug substance. They are not to be reported in the drug product.

^f (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.

^g (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.

^h (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.

ⁱ (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.

^j (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.

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. When more than one Dissolution test is given, the labeling states the Dissolution test used only if Test 1 is not used.

Change to read:

USP Reference Standards 〈11〉

USP Tacrolimus RS

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 (CN 1-Aug-2023) 

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 804.02

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

It contains 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 792.02 (CN 1-Aug-2023)

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