Ritonavir 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 

Ritonavir Capsules contain NLT 90.0% and NMT 110.0% of the labeled amount of ritonavir (C₃₇H₄₈N₆O₅S₂). 

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. 

3 ASSAY 

Procedure 

Buffer: 4.1 g/L of monobasic potassium phosphate 

Diluent: Acetonitrile and Buffer (50:50) 

Mobile phase: Acetonitrile, methanol, tetrahydrofuran (stabilizer-free), and Buffer (7:4:4:25). Separately filter the Buffer and the pre-mixed solvents before combining to make the Mobile phase. 

Standard solution: 25 µg/mL of USP Ritonavir RS in Diluent 

Sample stock solution: Nominally 1 mg/mL of ritonavir prepared as follows. Transfer Capsules (NLT 5) equivalent to 500 mg of ritonavir into a 500-mL volumetric flask, add about 250 mL of Diluent, and shake for at least 30 min or until the Capsules have visually disintegrated. Add 150 mL of acetonitrile, allow to cool to room temperature, and dilute to volume with Diluent. 

Sample solution: Nominally 25 µg/mL of ritonavir in Diluent from the Sample stock solution 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 240 nm 

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

Column temperature: 40° 

Flow rate: 1.5 mL/min 

Injection volume: 50 µL 

System suitability 

Sample: Standard solution 

Suitability requirements 

Capacity factor: NLT 15 

Tailing factor: 0.8–1.2 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of the labeled amount of ritonavir (C₃₇H₄₈N₆O₅S₂) in the portion of Capsules 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 ritonavir RS in the Standard solution (mg/mL)  

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

Acceptance criteria: 90.0%–110.0% 

4 PERFORMANCE TESTS 

Change to read: 

Dissolution 〈711〉 

Test 1 identification 

Medium: 0.1 N hydrochloric acid with 25 mM polyoxyethylene 10 lauryl ether, 900 mL Apparatus 2: 50 rpm, with sinkers 

Time: 30 min 

Buffer: 4.1 g/L of monobasic potassium phosphate 

Mobile phase: Acetonitrile and Buffer (55:45). Adjust with phosphoric acid to a pH of 4.0 ± 0.1. Standard stock solution: 5.2 mg/mL of USP Ritonavir RS in methanol 

Standard working solution: 104 µg/mL of USP Ritonavir RS in Medium 

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 240 nm 

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

Flow rate: 1.5 mL/min 

Injection volume: 25 µL 

System suitability 

Sample: Standard solution 

Suitability requirements 

Tailing factor: NMT 1.5 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of the labeled amount of ritonavir (C₃₇H₄₈N₆O₅S₂) 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 Ritonavir RS in the Standard solution (mg/mL) 

L = ritonavir label claim (mg/Capsule) 

V = volume of Medium, 900 mL 

Tolerances: NLT 80% (Q) of the labeled amount of ritonavir (C₃₇H₄₈N₆O₅S₂) is dissolved. 

Test 2: If the product complies with this test, the labeling indicates that it meets USP Dissolution Test 2. Medium: 0.1 N hydrochloric acid with 25 mM polyoxyethylene 10 lauryl ether; 900 mL, degassed 

Apparatus 2: 50 rpm, with sinker 

Time: 20 and 120 min 

Solution A: Water and phosphoric acid (98:2) 

Buffer: Water adjusted with Solution A to a pH of 3.5 

Mobile phase: Acetonitrile, methanol, and Buffer (500:100:400). 

Standard stock solution: 0.56 mg/mL of USP Ritonavir RS in methanol 

Standard solution: 0.11 mg/mL of USP Ritonavir RS in Medium from Standard stock solution 

Sample solution: Pass a portion of the solution under test through a suitable filter of 0.45-μm pore size. Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 240 nm 

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

Column temperature: 30° 

Flow rate: 1.8 mL/min 

Injection volume: 20 µL 

System suitability 

Sample: Standard solution 

Suitability requirements 

Tailing factor: NMT 2.0 

Relative standard deviation: NMT 2.0% 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the concentration (C ) of ritonavir (C₃₇H₄₈N₆O₅S₂) in the sample withdrawn from the vessel at each time point (i): i  

Result = (r_U/r_S) × C_S 

r_U = peak response of ritonavir 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) 

Calculate the percentage of the labeled amount of ritonavir (C₃₇H₄₈N₆O₅S₂) dissolved at each time point (i): 

Result = C₁ × V × (1/L) × 100 

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

C_i = concentration of ritonavir in the portion of sample withdrawn at the specified time point i (mg/mL) 

V_S = volume of Medium, 900 mL 

L = label claim of ritonavir (mg/Capsule) 

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

Tolerances: See Table 1. 

Table 1 

identification 

Uniformity of Dosage Units 〈905〉: Meet the requirements 

5 IMPURITIES 

Change to read: 

Organic Impurities 

[Note—Ritonavir is alkali sensitive. All glassware should be pre-rinsed with distilled water before use to remove residual detergent contamination.] 

Buffer A: 4.1 g/L of monobasic potassium phosphate 

Buffer B: 3.8 g/L of monobasic potassium phosphate and 0.25 g/L of dibasic potassium phosphate 

Solution A: Acetonitrile and Buffer A (50:50) 

Solution B: Acetonitrile and Buffer A (65:35) 

Solution C: Butyl alcohol and Buffer A (8:92) 

Mobile phase: Acetonitrile, butyl alcohol, tetrahydrofuran (stabilizer-free), and Buffer B (18:5:8:69). Adjust apparent pH to 6.3 ± 0.1 with 1 M phosphoric acid or 1 M potassium hydroxide if necessary. 

Cleaning solution: Acetonitrile, butyl alcohol, tetrahydrofuran (stabilizer-free), and Buffer A (30:8:13:49) 

Standard stock solution: 0.1 mg/mL of USP Ritonavir RS in Solution A 

Standard solution: 10 µg/mL of USP Ritonavir RS in Solution C from Standard stock solution 

Peak identification solution: Transfer 5–10 g from contents of Capsules into a suitable sealed container. Add an amount of citric acid equivalent to 1% of the Capsule weight taken, and mix until dissolved. Seal the container, and heat at 60° for about 24 h. Transfer about 2 g to a 100-mL volumetric flask, and dilute with Solution B to volume. Transfer 5.0 mL of the solution to a 50-mL centrifuge tube that has been previously rinsed with methanol and dried. Add 20.0 mL of heptane, and seal the tube with a stopper. Shake vigorously until a uniform emulsion is obtained, making sure to vent periodically. The emulsion formed yields distinct layers when centrifuged. The top layer (clear heptane) and the bottom layer (clear sample solution) are separated by a viscous white cloudy layer. The middle layer is part of the heptane layer. Carefully remove the clear heptane layer and the middle layer. Pass the bottom layer through a solid phase extraction cartridge containing strong anion-exchange packing in acetate form as described below. 

Sample stock solution: Nominally 2 mg/mL of ritonavir prepared as follows. Empty the contents of Capsules (NLT 6) into a suitable container, and accurately weigh and transfer an equivalent to 200 mg of ritonavir to a 100-mL volumetric flask. Dissolve and dilute with Solution B to volume. 

Sample solution: Nominally 1 mg/mL of ritonavir prepared as follows. Transfer 25.0 mL of Sample stock solution into a 50-mL volumetric flask, and dilute with Solution C to volume. Add 15.0 mL of this solution into a 50-mL centrifuge tube that has been previously rinsed with methanol and dried. Add 20.0 mL of heptane, and seal the tube with a stopper. Shake vigorously until a uniform emulsion is obtained, making sure to vent periodically. The emulsion formed yields distinct layers when centrifuged. The top layer (clear heptane) and the bottom layer (clear sample solution) are separated by a viscous white cloudy layer. The middle layer is part of the heptane layer. Carefully remove the clear heptane layer and the middle layer. Pass the bottom layer through a solid phase extraction cartridge containing strong anion exchange packing in acetate form as described below. 

Condition a solid phase extraction cartridge with methanol and Solution B two separate times, and dry for 10 min under low vacuum. Add 5.0 mL of the clear sample solution into the reservoir. Collect the sample solution at a slow rate into a 5-mL volumetric flask using low vacuum. Dilute with Solution B to volume. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 240 nm 

Column: 4.6-mm × 15-cm; 3-µm packing L26. Wash the column after each injection of the Peak identification solution and each injection of the Sample solution with Cleaning solution for about 26 min, and equilibrate with Mobile phase for about 30 min. Store in Cleaning solution after the analysis is completed. 

Column temperature: 60° 

Flow rate: 1 mL/min 

Injection volume: 50 µL 

Run time: 1.8 times the retention time of ritonavir 

System suitability 

Sample: Standard solution 

Suitability requirements 

Capacity factor: NLT 13 

Tailing factor: 0.8–1.2 

Relative standard deviation: NMT 3.0% 

Analysis 

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

Calculate the percentage of each impurity in the portion of Capsules 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 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 (see Table 2) identification 

Acceptance criteria: See Table 2. Reporting threshold is identification 0.05%. 

Table 2 identification 

a [N-Methyl[(2-isopropyl-4-thiazolyl)methyl]amino]carbonyl-l-valine (not quantied by this method due to solvent front and placebointerferences). 

b Process impurity. 

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

d Degradation impurity. 

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

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

g 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. 

h Thiazol-5-ylmethyl (2S,3S,5S)-3-hydroxy-5-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-1,6-diphenylhexan-2-ylcarbamate. i 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 (report as ethanol adduct due to possible co-elution). 

j Thiazol-5-ylmethyl (2S,3S,5S)-5-[(S)-2-ethoxycarbonylamino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate. k(4S,5S)-Thiazol-5-ylmethyl 4-benzyl-5-{(S)-2-[(S)-4-isopropyl-2,5-dioxoimidazolidin-1-yl]-3-phenylpropyl}-2-oxooxazolidine-3-carboxylate. l 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. 

m (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. 

n Thiazol-5-ylmethyl (2S,3S,5S)-(5-t-butoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate (may co-elute with isobutoxycarbonyl aminoalcohol; report as isobutoxycarbonyl aminoalcohol). 

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

p (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. 

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

r 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. 

s 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. 

t 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. u 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. 

v 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. 

w (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 SPECIFIC TESTS 

Microbial Enumeration Tests 〈61〉 and Tests for Specified Microorganisms 〈62〉: The total aerobic microbial count does not exceed 102 cfu/g. It meets the requirements of the test for absence of Escherichia coli and Salmonella. 

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight, light-resistant containers. Store between 2° and 8°. 

Add the following: 

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

USP Reference Standards 〈11〉 

USP Ritonavir RS