Ritonavir Oral Solution

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 Oral Solution contains 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 

Mobile phase: Acetonitrile, methanol, tetrahydrofuran (stabilizer-free), and Buffer (17.5:10:10:62.5). Filter the required solutions individually prior to use. 

Diluent: Acetonitrile and Buffer (50:50) 

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

Sample solution: Nominally 25 µg/mL of ritonavir in Diluent from a measured volume of Oral 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 

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 Oral Solution 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 

Deliverable Volume 〈698〉 

For multiple-unit containers 

Acceptance criteria: Meets the requirements 

5 IMPURITIES 

Organic Impurities 

[Note—Ritonavir is alkali sensitive. All glassware should be prerinsed 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 with 1 M phosphoric acid or 1 M potassium hydroxide to a pH of 6.3 ± 0.1, if necessary. 

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

Peak identification solution: Transfer 5–10 g of Oral Solution to a suitable sealed container. Add an amount of citric acid equivalent to 1% by weight of Oral Solution taken, and mix until dissolved. Seal the container, and heat at 70° for 24 h. Transfer 5.0 mL of the thermally degraded sample to a 200-mL volumetric flask. Dissolve and dilute with Solution B to volume. 

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 the Standard stock solution 

Sample stock solution: Nominally 2 mg/mL of ritonavir in Solution B prepared as follows. Transfer a measured volume of Oral Solution equivalent to 400 mg of ritonavir to a 200-mL volumetric flask, and dilute with Solution B to volume. 

Sample solution: Nominally 1 mg/mL of ritonavir prepared as follows. Transfer 25.0 mL of the Sample stock solution to a 50-mL volumetric flask, and dilute with Solution C to volume. Transfer 15.0 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, then stopper the tube. Shake the tube vigorously until a uniform emulsion is obtained, making sure to vent periodically. Centrifuge the resulting emulsion for about 5 min. Carefully aspirate off the top layer (heptane), leaving the clear bottom layer (Sample solution) in the tube. The centrifuged emulsion will have three distinct layers. The top layer (clear heptane) and the bottom layer (Sample solution) are separated by a viscous white cloudy layer. The middle layer should be considered part of the top layer for removal by aspiration. Repeat the extraction steps, and analyze the Sample solution. 

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 with the Cleaning solution after each injection of the Peak identification solution and each injection of the Sample solution for about 26 min, and equilibrate with Mobile phase for about 30 min. Store in the 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 

Tailing factor: 0.8–1.2 

Relative standard deviation: NMT 3.0% 

Analysis 

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

[Note—Determine the relative retention value (r) for the components listed in Table 1 as directed in Chromatography 〈621〉, using the time measured at the baseline deflection of the Standard solution chromatogram as the void volume (t_M).] 

Calculate the percentage of each impurity in the portion of Oral Solution 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 1) 

Acceptance criteria: See Table 1. Disregard peaks less than 0.05%. 

Table 1 

a{N-Methyl[(2-isopropyl-4-thiazolyl)methyl]amino}carbonyl-l-valine (not quantied by this method due to solvent front and placebointerferences; controlled as a synthetic impurity in the drug substance). 

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 product. 

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. (If two peaks appear with a relative retention of 0.24, the second peak is identified as the 2,5-thiazolylmethyl dicarbamate impurity. If a single peak with a relative retention of 0.24 appears, report as Hydroxypropyl carbamate analog.) 

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 (unspecied degradant; report as Ethyl carbamate analog due to possible coelution). j Thiazol-5-ylmethyl (2S,3S,5S)-5-[(S)-2-ethoxycarbonylamino-3-methylbutanamido]-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate (possible coelution with ritonavir hydroperoxide). 

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 (unspecified degradant). 

n Thiazol-5-ylmethyl (2S,3S,5S)-(5-t-butoxycarbonylamino)-3-hydroxy-1,6-diphenylhexan-2-ylcarbamate (may coelute with isobutoxycarbonyl aminoalcohol; report as isobutoxycarbonyl aminoalcohol using a relative response factor of 0.74). 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 (unspecied degradant). 

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 

Alcohol Content 

Internal standard solution: Transfer 10.0 mL of butyl alcohol to a 200-mL volumetric flask and dilute with methanol to volume. Internal standard identity solution: Dilute 5.0 mL of the Internal standard solution with methanol to 100 mL. 

Standard stock solution: 4.0% (v/v) of dehydrated alcohol in methanol 

Standard solution: 0.4% (v/v) of dehydrated alcohol prepared as follows. Transfer 10.0 mL of the Standard stock solution and 5 mL of the Internal standard solution to a 100-mL volumetric flask, and dilute with methanol to volume. 

Sample stock solution: Transfer 5.0 mL of Oral Solution to a 50-mL volumetric flask with the aid of several portions of methanol, and dilute with methanol to volume. 

Sample solution: Transfer 10.0 mL of the Sample stock solution and 5.0 mL of the Internal standard solution to a 100-mL volumetric flask, and dilute with methanol to volume. 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: GC 

Detector: Flame ionization 

Column: 0.53-mm × 30-m fused silica capillary; 1-µm layer of phase G16 

Temperatures 

Injection port: 185° 

Detector: 220° 

Column: See Table 2. 

Table 2 

Carrier gas: Helium 

Flow rate: 4.5 mL/min 

Makeup gas ow: 30 mL/min 

Injection volume: 1 µL 

Injection type: Split injection with a split ratio, 4:1 

System suitability 

Sample: Standard solution 

Suitability requirements 

Tailing factor: 0.8–1.2 for the alcohol peak 

Relative standard deviation: NMT 3.0% for the peak area ratio of alcohol to butyl alcohol 

Analysis 

Samples: Internal standard identity solution, Standard solution, and Sample solution 

Calculate the percentage of alcohol in the portion of Oral Solution taken: 

Result = (R_U/R_S) × C × D 

R_U = peak area ratio of alcohol to butyl alcohol from the Sample solution 

R_S = average peak area ratio of alcohol to butyl alcohol from the Standard solution  

C = concentration of dehydrated alcohol in the Standard solution (% v/v) 

D = dilution factor used to prepare the Sample solution, 100 

Acceptance criteria: 40%–47% (v/v) of alcohol (C₂H₆O)

Microbial Enumeration Tests 〈61〉andTests for Specified Microorganisms 〈62〉: The total aerobic microbial count does not exceed 102 cfu/mL. 

7 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in tight, light-resistant containers. Store at room temperature. 

USP Reference Standards 〈11〉 

USP Ritonavir RS