Atazanavir Sulfate

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

DOWNLOAD PDF HERE

C₃₈H₅₂N₆O₇ · H₂SO₄                802.93

2,5,6,10,13-Pentaazatetradecanedioic acid, 3-12-bis(1,1-dimethylethyl)-8-hydroxy-4,11-dioxo-9-(phenylmethyl)-6-[]-4-(2-pyridinyl)phenyl]methyl]-. dimethyl ester, (3S,8S,95,12S)-, sulfate (1:1) (salt);

Dimethyl (3S,85,9S,12S)-9-benzyl-3,12,di-tert-butyl-8-hydroxy-4,11-dioxo-6-(p-2-pyridylbenzyl)-2,5,6,10,13-pentaazatetradecanedioate, sulfate (1:1) (salt) CAS RN: 229975-97-7.

1 DEFINITION

Atazanavir Sulfate contains NLT 98.0% and NMT 102.0% of atazanavir sulfate (C₃₈H₅₂N₆O₇ · H₂SO₄), calculated on the anhydrous and solvent-free basis.

2 IDENTIFICATION

A. SPECTROSCOPIC IDENTIFICATION TESTS (197), Infrared Spectroscopy: 197A or 197K

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

C. IDENTIFICATION TESTS GENERAL (191), Chemical Identification Tests. Sulfate: Meets the requirements

3 ASSAY

PROCEDURE

Buffer: 2.73 g/L of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 3.5.

Mobile phase: Acetonitrile and Buffer (50:50)

System suitability solution: 0.4 mg/mL of USP Atazanavir System Suitability Mixture RS in Mobile phase. Sonicate, if necessary, to dissolve prior to final dilution.

Standard solution: 0.4 mg/mL of USP Atazanavir Sulfate RS in Mobile phase. Sonicate, if necessary, to dissolve prior to final dilution.

Sample solution: 0.4 mg/mL of Atazanavir Sulfate in Mobile phase, Sonicate, if necessary, to dissolve prior to final dilution.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm x 15-cm; 3-µm packing 11

Flow rate: 1 mL/min

Injection volume: 10 µL

Run time: NLT 1.5 times the retention time of atazanavir

System suitability

Samples: System suitability solution and Standard solution

[NOTE-The relative retention times for atazanavir R,S,S,S-diastereomer and atazanavir are 0.9 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.5 between atazanavir R,S,S,S-diastereomer and atazanavir, System suitability solution

Tailing factor: 0.8-1.5 for the atazanavir peak, System suitability solution

Relative standard deviation: NMT 0.73%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of atazanavir sulfate (C₃₈H₅₂N₆O₇ · H₂SO₄) in the portion of Atazanavir Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u = peak response of atazanavir from the Sample solution

r_s = peak response of atazanavir from the Standard solution

C_s = concentration of USP Atazanavir Sulfate RS in the Standard solution (mg/mL)

C_u = concentration of Atazanavir Sulfate in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the anhydrous and solvent-free basis

Acceptance criteria: 98.0%-102.0% on the anhydrous and solvent-free basis

4 IMPURITIES

RESIDUE ON IGNITION (281): NMT 0.2%

ORGANIC IMPURITIES

[NOTE-Perform either Procedure 1 or Procedure 2, depending on the synthetic route. Procedure 2 is recommended if atazanavir formyl, ethyl, amine, or valine analogs are potential impurities.]

4.1 PROCEDURE 1

Buffer, System suitability solution, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

Solution A: Acetonitrile and Buffer (25:75)

Solution B: Acetonitrile and Buffer (75:25)

Mobile phase: See Table 1.

Table 1

Diluent: Solution A and Solution B (50:50)

Standard solution: 0.4mu*g / m * L of USP Atazanavir Sulfate RS in Diluent. Sonicate, if necessary, to dissolve prior to final dilution.

System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 1.5 between atazanavir R,S,S,S-diastereomer and atazanavir, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Atazanavir Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × (1/F) × 100

r_u = peak response of any individual impurity from the Sample solution

r_s = peak response of atazanavir from the Standard solution

C_s = concentration of USP Atazanavir Sulfate RS in the Standard solution (mg/mL)

C_u = concentration of Atazanavir Sulfate in the Sample solution (mg/mL)

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. The reporting threshold is 0.05%.

Table 2

^a For information only; disregard if not used in the process.

^b This impurity is determined by the Limit of Atazanavir Related Compound A test.

^c 4-(Pyridin-2-yl)benzoic acid.

^d 4-(Pyridin-2-yl)benzaldehyde.

^e Methyl [(5S,10S,11S,14S)-11-benzyl-5-(tert-butyl)-10-hydroxy-15,15-dimethyl-3,6,13-trioxo-2-oxa-4,7,8,12-tetraazahexadecan-14-yl]carbamate.

^f (2S,3S)-3-Amino-4-phenyl-1-{1-[4-(pyridin-2-yl)benzyl]-2-[4-(pyridin-2-yl)benzylidene]hydrazinyl}butan-2-ol.

^g Methyl {(5S,10R,11S,14S)-11-benzyl-5-(tert-butyl)-10-hydroxy-15,15-dimethyl-3,6,13-trioxo-8-[4-(pyridin-2-yl)benzyl]-2-oxa-4,7,8,12- tetraazahexadecan-14-yl}carbamate.

^h Methyl {(5R,10S,11S,14S)-11-benzyl-5-(tert-butyl)-10-hydroxy-15,15-dimethyl-3,6,13-trioxo-8-[4-(pyridin-2-yl)benzyl]-2-oxa-4,7,8,12- tetraazahexadecan-14-yl}carbamate.

ⁱ Methyl {(5S,10S,11S,14R)-11-benzyl-5-(tert-butyl)-10-hydroxy-15,15-dimethyl-3,6,13-trioxo-8-[4-(pyridin-2-yl)benzyl]-2-oxa-4,7,8,12- tetraazahexadecan-14-yl}carbamate.

^j Methyl {(5S,10R,11R,14S)-11-benzyl-5-(tert-butyl)-10-hydroxy-15,15-dimethyl-3,6,13-trioxo-8-[4-(pyridin-2-yl)benzyl]-2-oxa-4,7,8,12- tetraazahexadecan-14-yl}carbamate.

^k Methyl [(S)-1-{[(2S,3S)-3-hydroxy-1-phenyl-4-{1-[4-(pyridin-2-yl)benzyl]-2-[4-(pyridin-2-yl)benzylidene]hydrazinyl}butan-2-yl]amino}-3,3- dimethyl-1-oxobutan-2-yl]carbamate.

^l tert-Butyl 2-{(2S,3S)-3-[(tert-butoxycarbonyl)amino]-2-hydroxy-4-phenylbutyl}-2-[4-(pyridin-2-yl)benzyl]hydrazinecarboxylate.

^m Total impurities includes the sum of all impurities found in the tests for Limit of Atazanavir Related Compound A and Organic Impurities, Procedure 1.

4.2 PROCEDURE 2

[NOTE-This procedure is recommended if atazanavir formyl, ethyl, amine, or valine analogs are potential impurities.]

Buffer: 1.6 g/L of ammonium formate in water. Adjust with formic acid to a pH of 4.0. Add 50 mL of methanol per liter of the solution.

Mobile phase: Acetonitrile and Buffer (40:60)

Standard stock solution: 0.23 mg/mL of USP Atazanavir Sulfate RS in Mobile phase. Sonicate, if necessary, to dissolve prior to final dilution.

Standard solution: 0.023 mg/mL of USP Atazanavir Sulfate RS in Mobile phase from Standard stock solution. Prepare this solution fresh.

Sample solution: 0.23 mg/mL of Atazanavir Sulfate in Mobile phase. Sonicate, if necessary, to dissolve prior to final dilution.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 254 nm

Column: 4.6-mm x 25-cm; 5-µm packing 17

Column temperature: 30°

Flow rate: 1.2 mL/min

Injection volume: 10 µL

Run time: NLT 4.7 times the retention time of atazanavir

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 5.0%

Analysis

Samples:

Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Atazanavir Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u= peak response of any individual impurity from the Sample solution

r_s = peak response of atazanavir from the Standard solution

C_s = concentration of USP Atazanavir Sulfate RS in the Standard solution (mg/mL)

C_u = concentration of Atazanavir Sulfate in the Sample solution (mg/mL)

Acceptance criteria: See Table 3. The reporting threshold is 0.05%.

Table 3

^a Methyl [(S)-1-{[(2S,3S)-4-{2-[(S)-2-amino-3,3-dimethylbutanoyl]-1-[4-(pyridin-2-yl)benzyl]hydrazineyl}-3-hydroxy-1-phenylbutan-2- yl]amino}-3,3-dimethyl-1-oxobutan-2-yl]carbamate.

^b Methyl [(S)-1-{[(2S,3S)-4-{2-[(S)-2-formamido-3,3-dimethylbutanoyl]-1-[4-(pyridin-2-yl)benzyl]hydrazineyl}-3-hydroxy-1-phenylbutan-2- yl]amino}-3,3-dimethyl-1-oxobutan-2-yl]carbamate.

^c Methyl {(5S,10S,11S,14S)-11-benzyl-5-(tert-butyl)-10-hydroxy-15-methyl-3,6,13-trioxo-8-[4-(pyridin-2-yl)benzyl]-2-oxa-4,7,8,12- tetraazahexadecan-14-yl}carbamate sulfate.

^d Methyl {(6S,11S,12S,15S)-12-benzyl-6-(tert-butyl)-11-hydroxy-16,16-dimethyl-4,7,14-trioxo-9-[4-(pyridin-2-yl)benzyl]-3-oxa-5,8,9,13- tetraazaheptadecan-15-yl}carbamate.

^e Methyl {(5S,8S,13S,14S,17S)-14-benzyl-5,8-di-tert-butyl-13-hydroxy-18,18-dimethyl-3,6,9,16-tetraoxo-11-[4-(pyridin-2-yl)benzyl]-2-oxa4,7,10,11,15-pentaazanonadecan-17-yl}carbamate.

4.3 Limit of Atazanavir Related Compound A

[Note—Perform this test when Organic Impurities, Procedure 1 is used.]

Buffer: 2.73 g/L of monobasic potassium phosphate in water. Adjust with phosphoric acid to a pH of 3.5.

Solution A: Acetonitrile and Buffer (25:75)

Solution B: Acetonitrile and Buffer (75:25)

Mobile phase: See Table 4.

Table 4

Diluent: Solution A and Solution B (50:50)

System suitability solution: 1.1 mg/mL of USP Atazanavir Sulfate RS and 1 µg/mL of USP Atazanavir Related Compound A RS in Diluent.

Sonicate, if necessary, to dissolve prior to final dilution.

Standard stock solution: 0.01 mg/mL of USP Atazanavir Related Compound A RS in Diluent

Standard solution: 1 µg/mL of USP Atazanavir Related Compound A RS in Diluent from Standard stock solution. Store this solution at 5°.

Sample solution: 1.14 mg/mL of Atazanavir Sulfate in Diluent. Sonicate, if necessary, to dissolve prior to final dilution.

Chromatographic system

(See Chromatography (621), System Suitability.)

Mode: LC

Detector: UV 215 nm

Column: 4.6-mm x 15-cm; 3-µm packing 11

Flow rate: 1 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

[NOTE-The relative retention times for atazanavir related compound A and atazanavir are 0.4 and 1.0, respectively.]

Suitability requirements

Tailing factor: 0.8-1.5 for the atazanavir peak, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of atazanavir related compound A in the portion of Atazanavir Sulfate taken:

Result = (r_u /r_s ) × (C_s /C_u ) × 100

r_u= peak response of atazanavir related compound A from the Sample solution

r_s = peak response of atazanavir related compound A from the Standard solution

C_s = concentration of USP Atazanavir Related Compound A RS in the Standard solution (mg/mL)

C_u = concentration of Atazanavir Sulfate in the Sample solution (mg/mL)

Acceptance criteria: NMT 0.1%. Disregard the atazanavir related compound A peak when it is less than 0.05%, and do not include it in the

total impurities calculation.

5 SPECIFIC TESTS

WATER DETERMINATION (921): NMT 2.5%

OPTICAL ROTATION (781S), Procedures, Specific Rotation

Sample solution: 10 mg/mL of Atazanavir Sulfate in methanol

Acceptance criteria: -40° to -44°

6 ADDITIONAL REQUIREMENTS

PACKAGING AND STORAGE: Preserve in tight, light-resistant containers. Store at controlled room temperature.

LABELING: If a test for Organic Impurities other than Organic Impurities, Procedure 1 is used, then the labeling states the test with which the article complies.

USP REFERENCE STANDARDS (11)

USP Atazanavir Related Compound A RS

(S)-2-[(Methoxycarbonyl)amino]-3,3-dimethylbutanoic acid.

C₈H₁₅NO₄                        189.21

USP Atazanavir Sulfate RS

USP Atazenavir System Suitability Mixture RS

This is a mixture of atazanavir sulfate, atazanavir R,S,S,S-diastereomer, and atazanavir S,R,S,S-diastereomer (other impurities may also be present) _{(USP 1-Aug-2022)}