Lopinavir and Ritonavir Tablets

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 and Ritonavir Tablets contain NLT 90.0% and NMT 110.0% of the labeled amounts of lopinavir (C₃₇H₄₈N₄O₅) and ritonavir (C₃₇H₄₈N₆O₅S₂).

2 IDENTIFICATION

A. The retention times of the major peaks of the Sample solution correspond to those of the Standard solution, as obtained in the Assay.

3 ASSAY

3.1 LOPINAVIR AND RITONAVIR

Buffer 1: 4.1 g/L of monobasic potassium phosphate in water Solution A: Acetonitrile and Buffer 1 (50:50)

Buffer 2: 2.1 g/L of monobasic potassium phosphate in water Solution B: Acetonitrile and 1-butanol (13:3)

Solution C: Acetonitrile, 1-butanol, Buffer 1, and water (65:15:10:10)

Standard solution: 6.25 µg/mL of USP Ritonavir RS and 25 µg/mL of USP Lopinavir RS in Solution A

Sample solution: Place a number of Tablets equivalent to 1000 mg of lopinavir and 250 mg of ritonavir in a 250-mL volumetric flask, add 25 mL of Buffer 2, and agitate to dissolve the Tablet coating, if necessary. Add 100 mL of Solution B, and shake mechanically until the Tablets are dissolved. Dilute with Solution C to volume. Centrifuge a portion of this solution, and then further dilute with Solution A to a nominal concentration of 6.25 µg/mL of ritonavir and 25 µg/mL of lopinavir.

Mobile phase: Acetonitrile, methanol, tetrahydrofuran, and Buffer 1 (175:100:100:625)

3.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 215 nm

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

Column temperature: 40°

Flow rate: 1.5 mL/min

Injection volume: 50 µL

3.1.2 System suitability

Sample: Standard solution

[NOTE—The elution order is ritonavir, then lopinavir.]

Suitability requirements

Capacity factor: 15–24 for the ritonavir peak Tailing factor: 0.8–1.2 for the ritonavir peak

Theoretical plates: More than 5000 for the ritonavir peak

Relative standard deviation: NMT 2.0% for the ritonavir and lopinavir peaks

3.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of the labeled amount of lopinavir (C₃₇H₄₈N₄O₅) and ritonavir (C₃₇H₄₈N₆O₅S₂) in the portion of Tablets taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of lopinavir or ritonavir from the Sample solution

r_S = peak response of lopinavir or ritonavir from the Standard solution

C_S = concentration of lopinavir or ritonavir in the Standard solution (µg/mL)

C_U = nominal concentration of lopinavir or ritonavir in the Sample solution (µg/mL)

Acceptance criteria: 90.0%–110.0% of the labeled amounts of lopinavir (C₃₇H₄₈N₄O₅) and ritonavir (C₃₇H₄₈N₆O₅S₂)

4 PERFORMANCE TESTS

4.1 DISSOLUTION 〈711〉

Medium: 60 mM polyoxyethylene 10 lauryl ether (37.56 g/L) in water; 900 mL

Apparatus 2: 75 rpm

Time: 90 min

Mobile phase: Acetonitrile and 4.1 g/L potassium phosphate monobasic (55:45). Adjust with phosphoric acid to an apparent pH of 4.0 ± 0.05.

Standard solution: Dissolve USP Lopinavir RS in methanol to obtain a solution containing 2.6 mg/mL. Dissolve USP Ritonavir RS in methanol to obtain a solution containing 1.3 mg/mL. Combine portions of these solutions to make a solution containing approximately 0.104 mg/mL of lopinavir and 0.026 mg/mL of ritonavir in Medium.

Sample solutions: Pass a portion of the solution under test through a suitable filter. If necessary, dilute the solution with Medium to obtain a final sample solution containing approximately 0.104 mg/mL of lopinavir and 0.026 mg/mL of ritonavir.

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Mode: LC

Detector: UV 215 nm

Column: 4.6-mm × 15-cm; 5-µm packing L1 Flow rate: 1.5 mL/min

Injection volume: 25 µL

4.1.2 System suitability

Sample: Standard solution

Suitability requirements

Resolution: NLT 2.0 between lopinavir and ritonavir Tailing factor: 0.9–1.5 for the lopinavir and ritonavir peaks

Relative standard deviation: NMT 2.0% for the lopinavir and ritonavir peaks

4.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of lopinavir lopinavir (C₃₇H₄₈N₄O₅) and ritonavir (C₃₇H₄₈N₆O₅S₂) dissolved:

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

r_U = peak response of lopinavir or ritonavir from the Sample solution

r_S = peak response of lopinavir or ritonavir from the Standard solution

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

L = Tablet label claim for lopinavir or ritonavir (mg)

D = dilution factor of the Sample solution

V = volume of Medium, 900 mL

Tolerances: NLT 80% (Q ) of the labeled amounts of lopinavir (C₃₇H₄₈N₄O₅) and ritonavir (C₃₇H₄₈N₆O₅S₂) are dissolved.

4.2 UNIFORMITY OF DOSAGE UNITS 〈905〉

Meet the requirements

5 IMPURITIES

5.1 ORGANIC IMPURITIES

Buffer 1: 4.1 g/L of monobasic potassium phosphate in water Solution A: Buffer 1 and acetonitrile (50:50)

Buffer 2: 2.1 g/L of monobasic potassium phosphate in water Solution B: Acetonitrile, 1-butanol, and Buffer 1 (15:5:80)

Solution C: Acetonitrile, 1-butanol, Buffer 1, and water (65:15:10:10) Solution D: Acetonitrile and 1-butanol (13:3)

Buffer solution: 3.8 g/L of monobasic potassium phosphate and 0.25 g/L of dibasic potassium phosphate in water

Mobile phase: Acetonitrile, tetrahydrofuran,1-butanol, and Buffer solution (18:8:5:69). Adjust with 1 M phosphoric acid or 1 M potassium hydroxide, if necessary, to a pH of 6.3 ± 0.1.

Standard stock solution: 0.025 mg/mL of USP Ritonavir RS in Solution A

Standard solution: 2.5 µg/mL of USP Ritonavir RS in Solution B from Standard stock solution

Ritonavir degradant identification solution: Transfer two 5.0 mL portions of a 1 mg/mL solution of USP Ritonavir RS in Solution A to separate 50-mL volumetric flasks. Add 1 g of citric acid to one flask, and shake until dissolved. Heat both flasks at 80° for approximately 24 h. Cool the flasks, and add 13 mL of 1 N sodium hydroxide to the flask containing the citric acid. Dilute both flasks with Solution B to volume.

Combine equal volumes of both solutions. This solution contains ritonavir and the ritonavir degradation products (N-deacylvaline ritonavir, hydantoin aminoalcohol, O -acyl isomer, and oxazolidinone derivative).

Ritonavir related compounds identification solution: 1 mg/mL of USP Ritonavir Related Compounds Mixture RS dissolved in Solution C and further diluted with Solution B to 0.5 mg/mL.

Sample solution: Place a number of Tablets equivalent to 1000 mg of lopinavir and 250 mg of ritonavir into a 250-mL volumetric flask. Add 25 mL of Buffer 2, and agitate to dissolve the Tablet coating, if necessary. Add 100 mL of Solution D, and shake mechanically until the Tablets are dissolved. Dilute with Solution C to volume. Centrifuge a portion of this solution, and further dilute with Solution B to a concentration of 2 mg/mL of lopinavir and 0.5 mg/mL of ritonavir.

5.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.) Column: 4.6-mm × 15-cm; 3-µm packing L26 Column temperature: 60°

Detector: UV 240 nm Injection volume: 50 µL Flow rate: 1.0 mL/min

5.1.2 System suitability

Samples: Ritonavir degradant identification solution, Ritonavir related compounds identification solution, and Standard solution Suitability requirements

Resolution: NLT 1.0 between the peaks for O -acyl isomer and oxazolidinone derivative, Ritonavir degradant identification solution. NLT 0.7 between the peaks for hydroxyritonavir and hydantoin aminoalcohol, Ritonavir related compounds identification solution

Capacity factor: NLT 10.8, Standard solution Tailing factor: 0.8–1.2, Standard solution Column efficiency: NLT 5000, Standard solution

Relative standard deviation: NMT 5.0%, Standard solution

5.1.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each ritonavir degradation product in the Sample solution:

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

r_U = peak area of individual degradation product from the Sample solution

r_S = peak response of ritonavir from the Standard solution

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

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

F = relative response factor

Acceptance criteria: See Table 1. [NOTE—Disregard all peaks eluting before the retention time of the N-deacylvaline ritonavir peak from the Ritonavir degradant identification solution.]

Table 1

^** Disregard any peak less than 0.05%.

^a  Thiazol-5-ylmethyl (2S,3S,5S)-5-[(S)-2-amino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.

^b  Thiazol-5-ylmethyl (2S,3S,5S)-5-acetamido-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.

^c  Bis(thiazol-5-ylmethyl) (2S,3S,5S)-3-hydroxy-1,6-diphenylhexane-2,5-diyldicarbamate.

^d Thiazol-5-ylmethyl (2S,3S,5S)-3-hydroxy-5-[(S)-2-(3-{[2-(2-hydroxypropan-2-yl)thiazol-4-yl]methyl}-3-methylureido)-3- methylbutanamido]-1,6-diphenylhexan-2-ylcarbamate.

^e Thiazol-5-ylmethyl (2S,3S,5S)-3-hydroxy-5-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-1,6-diphenylhexan-2-ylcarbamate.

^f Thiazol-5-ylmethyl (2S,3S,5S)-5-[(S)-2-(3-{[2-(2-hydroperoxypropan-2-yl)thiazol-4-yl]methyl}-3-methylureido)-3-methylbutanamido]-3- hydroxy-1,6-diphenylhexan-2-ylcarbamate.

^g (4S,5S)-Thiazol-5-ylmethyl 4-benzyl-5-{(S)-2-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-3-phenylpropyl}-2-oxooxazolidine-3-carboxylate.

^h Thiazol-5-ylmethyl (2S,3S,5S)-5-[(S)-2-{3-[(2-ethylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan- 2-ylcarbamate.

ⁱ (S)-{(2S,3S,5S)-5-Amino-1,6-diphenyl-2-[(thiazol-5-ylmethoxy)carbonylamino]hexan-3-yl} 2-{3-[(2-isopropylthiazol-4-yl)methyl]-3- methylureido}-3-methylbutanoate.

^j Thiazol-5-ylmethyl (2S,3S,5S)-(5-t-butoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.

^k  Thiazol-5-ylmethyl (2S,3S,5S)-(5-isobutoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate.

^l (S)-N-[(S)-1-[(4S,5S)-4-Benzyl-2-oxooxazolidin-5-yl]-3-phenylpropan-2-yl]-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3- methylbutanamide.

^m  (S)-Isobutyl 2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanoate.

ⁿ Thiazol-5-ylmethyl (2S,4S,5S)-4-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6- diphenylhexan-2-ylcarbamate.

^o Thiazol-5-ylmethyl (2S,3R,5S)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6- diphenylhexan-2-ylcarbamate.

^p  Bis(thiazol-5-ylmethyl) (2S,2'S,3S,3'S,5S,5'S)-5,5'-carbonylbis(azanediyl)bis(3-hydroxy-1,6-diphenylhexane-5,2-diyl)dicarbamate.

^q Thiazol-5-ylmethyl (2S,3R,5R)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6- diphenylhexan-2-ylcarbamate.

^r Thiazol-5-ylmethyl (2S,3S,5R)-3-hydroxy-5-[(S)-2-{3-[(2-isopropylthiazol-4-yl)methyl]-3-methylureido}-3-methylbutanamido]-1,6- diphenylhexan-2-ylcarbamate.

^s (3S,4S,6S,10S,13S,15S,16S)-Bis(thiazol-5-ylmethyl)-4,15-dihydroxy-10-isopropyl-8,11-dioxo-3,6,13,16-tetrabenzyl-2,7,9,12,17- pentaazaoctadecanedioate.

6 ADDITIONAL REQUIREMENTS

USP REFERENCE STANDARDS 〈11〉

USP Lopinavir RS USP Ritonavir RS

USP Ritonavir Related Compounds Mixture RS