Lopinavir

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

Lopinavir contains NLT 98.0% and NMT 102.0% of lopinavir (C₃₇H₄₈N₄O₅), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197A (CN 1-May-2020)

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

3 ASSAY

Procedure

Buffer: 2.7 g/L of monobasic potassium phosphate and 0.9 g/L of dibasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 6.0. Pass the solution through a suitable filter of 0.45-μm pore size.

Diluent: Acetonitrile and water (1:1)

Solution A: Acetonitrile and Buffer (9:11)

Mobile phase: Solution A

Standard solution: 0.025 mg/mL of USP Lopinavir RS in Diluent

Sample solution: 0.025 mg/mL of Lopinavir in Diluent

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 25-cm; 4-μm packing L1

Column temperature: 50°

Flow rate: 1 mL/min

Injection volume: 20 μL

Run time: 60 min

System suitability

Sample: Standard solution

Suitability requirements

Column efficiency: NLT 8000 theoretical plates

Capacity factor: NLT 15

Tailing factor: 0.8–1.5

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of lopinavir (C₃₇H₄₈N₄O₅) in the portion of Lopinavir taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response from the Sample solution

r_S = peak response from the Standard solution

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

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

Acceptance criteria: 98.0%–102.0% on the anhydrous basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.2%

Organic Impurities: Procedure 1

[Note—For early-eluting impurities.]

Buffer, Diluent, and Solution A: Prepare as directed in the Assay.

Solution B: Acetonitrile and Buffer (3:1)

Mobile phase: See Table 1.

Table 1

System suitability solution: 0.5 mg/mL of USP Lopinavir System Suitability Mixture RS in Diluent

Standard solution: 0.005 mg/mL of USP Lopinavir RS in Diluent

Sample solution: 0.5 mg/mL of Lopinavir in Diluent

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 25-cm; 4-μm packing L1

Column temperature: 50°

Flow rate: 1 mL/min

Injection volume: 20 μL

Run time: 100 min

[Note—Data collection is only for the first 60 min. The remaining gradient steps wash out the late-eluting impurities and re-equilibrate the column.]

System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times are listed in Table 2.]

Suitability requirements

Resolution: NLT 1.2 between lopinavir N-formylphenoxyacetamide and lopinavir N-acetylphenoxyacetamide, System suitability solution

Capacity factor: NLT 15, Standard solution

Column efficiency: NLT 8000, Standard solution

Tailing factor: 0.8–1.5, Standard solution

Relative standard deviation: NMT 3.0%, Standard solution

Analysis

Samples: Diluent, System suitability solution, Standard solution, and Sample solution

Calculate the percentage of each lopinavir related impurity and unidentified impurity in the portion of Lopinavir 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 lopinavir from the Standard solution

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

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

F = relative response factor (see Table 2)

Table 2

^a (S)-N-[(2S,4S,5S)-5-Amino-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^b (S)-N-[(2S,4S,5S)-5-Formamido-4-hydroxy-1,6-diphenylhexan-2-yl]-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^c (2S,2′S)-N,N′-[(2S,3S,5S)-3-Hydroxy-1,6-diphenylhexane-2,5-diyl]bis{3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide}.

^d (2S,3S,5S)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-{(S)-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamido}-1,6-diphenylhexan-3-yl hydrogen sulfate.

^e N-[(2S,3S,5S)-5-Amino-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-dimethylphenoxy)acetamide.

^f 2-(2,6-Dimethylphenoxy)-N-[(2S,3S,5S)-5-formamido-3-hydroxy-1,6-diphenylhexan-2-yl]acetamide.

^g N-[(2S,3S,5S)-5-Acetamido-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-dimethylphenoxy)acetamide.

^h N-{(S)-1-[(4S,6S)-4-Benzyl-2-oxo-1,3-oxazinan-6-yl]-2-phenylethyl}-2-(2,6-dimethylphenoxy)acetamide.

ⁱ (S)-N-{(2S,3S,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-3-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^j (S)-N-{(2S,4S,5S)-5-[2-(2,4-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^k (R)-N-{(2S,4S,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^l (Z)-N,N′-(Ethene-1,2-diyl)bis[2-(2,6-dimethylphenoxy)acetamide].

^m (S)-N-{(2R,4R,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

ⁿ (S)-N-{(2S,4R,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

Organic Impurities: Procedure 2

[Note—For late-eluting impurities.]

Buffer, Diluent, and Solution A: Prepare as directed in the Assay.

Solution B: Acetonitrile and Buffer (3:1)

Mobile phase: Solution A and Solution B (3:7)

System suitability solution: 0.5 mg/mL of USP Lopinavir System Suitability Mixture RS in Diluent

Standard solution: 0.005 mg/mL of USP Lopinavir RS in Diluent

Sample solution: 0.5 mg/mL in Diluent

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 25-cm; 4-μm packing L1

Column temperature: 50°

Flow rate: 1 mL/min

Injection volume: 20 μL

Run time: 50 min

System suitability

Sample: Standard solution

[Note—The relative retention times are listed in Table 3.]

Suitability requirements

Capacity factor: NLT 1.5

Column efficiency: NLT 3000

Tailing factor: 0.8–1.5

Relative standard deviation: NMT 3.0%

Analysis

Samples: Diluent, System suitability solution, Standard solution, and Sample solution

Calculate the percentage of each lopinavir related impurity and unidentified impurity in the portion of Lopinavir 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 lopinavir from the Standard solution

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

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

F = relative response factor (see Table 3)

Table 3

^a (S)-{(2S,3S,5S)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-[(S)-3-methyl-2-(2-oxotetrahydropyrimidin-1(2H)-yl)butanamido]-1,6-diphenylhexan-3-yl} 3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanoate.

^b (S)-N-{(2R,4S,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamide.

^c N,N′-[(2S,3S,5S)-3-Hydroxy-1,6-diphenylhexane-2,5-diyl]bis[2-(2,6-dimethylphenoxy)acetamide].

^d (S)-N-{(2S,4S,5S)-5-[2-(2,6-Dimethylphenoxy)acetamido]-4-hydroxy-1,6-diphenylhexan-2-yl}-2-{3-[2-(2,6-dimethylphenoxy)acetyl]-2-oxotetrahydropyrimidin-1(2H)-yl}-3-methylbutanamide.

^e (2S,3S,5S)-2-[2-(2,6-Dimethylphenoxy)acetamido]-5-{(S)-3-methyl-2-[2-oxotetrahydropyrimidin-1(2H)-yl]butanamido}-1,6-diphenylhexan-3-yl 2-(2,6-dimethylphenoxy)acetate.

^f N,N′-(2S,2′S,3S,3′S,5S,5′S)-5,5′-Carbonylbis(azanediyl)bis(3-hydroxy-1,6-diphenylhexane-5,2-diyl)bis[2-(2,6-dimethylphenoxy)acetamide].

^g Exclude from Organic Impurities, Procedure 2, lopinavir (4R) epimer and any other peak eluting prior to this peak because these are already monitored in Procedure 1.

Acceptance criteria: See Table 2 and Table 3.

5 SPECIFIC TESTS

Water Determination, Method I〈921〉: NMT 4.4%

6 ADDITIONAL REQUIREMENTS

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

USP Reference Standards 〈11〉

USP Lopinavir RS

USP Lopinavir System Suitability Mixture RS

Lopinavir System Suitability Mixture contains lopinavir N-formylphenoxyacetamide, lopinavir N-acetylphenoxyacetamide, and several other minor components.

Lopinavir N-formylphenoxyacetamide is (2-(2,6-dimethylphenoxy)-N-[(2S,3S,5S)-5-formamido-3-hydroxy-1,6-diphenylhexan-2-yl]acetamide.

C₂₉H₃₄N₂O₄ 474.59

Lopinavir N-acetylphenoxyacetamide is (N-[(2S,3S,5S)-5-acetamido-3-hydroxy-1,6-diphenylhexan-2-yl]-2-(2,6-dimethylphenoxy)acetamide.

C₃₀H₃₆N₂O₄ 488.62