Zidovudine

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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C₁₀H₁₃N₅O₄    267.24

Thymidine, 3′-azido-3′-deoxy-;

3′-Azido-3′-deoxythymidine CAS RN®: 30516-87-1.

1 DEFINITION

Zidovudine contains NLT 97.0% and NMT 102.0% of zidovudine (C₁₀H₁₃N₅O₄), calculated on the anhydrous basis. 

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 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.

3 ASSAY

Change to read:

Procedure

Buffer: 2 g/L of ammonium acetate in water. Adjust with dilute acetic acid or dilute ammonium hydroxide (1 in 10) to a pH of 6.8.

Solution A: Acetonitrile

Mobile phase: See Table 1.

Table 1

Diluent: Acetonitrile, methanol, and Buffer (4:20:76)

Standard solution: 0.2 mg/mL of USP Zidovudine RS in Diluent

Sample solution: 0.2 mg/mL of Zidovudine in Diluent

3.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 265 nm

Column: 4.6-mm × 25-cm; 5-µm packing L1

Flow rate: 1.5 mL/min

Injection volume: 20 µL

3.2 System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 2.0% (USP 1-May-2022)

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of zidovudine (C₁₀H₁₃N₅O₄) in the portion of Zidovudine taken:

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

r_U = peak response of zidovudine from the Sample solution

r_S = peak response of zidovudine from the Standard solution

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

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

Acceptance criteria: 97.0%–102.0% on the anhydrous basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.25%

Delete the following:

4.1 Limit of Triphenylmethanol and Other Impurities

Standard solution: 0.1 mg/mL each of USP Zidovudine RS and triphenylmethanol in methanol

Sample solution: 20 mg/mL of Zidovudine in methanol

Chromatographic system

(See Chromatography 〈621〉, Thin-Layer Chromatography.)

Mode: TLC

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture containing a fluorescent indicator having an optimal intensity at 254 nm

Application volume: 10 µL

Developing solvent system: Chloroform and methanol (9:1)

Spray reagent: 5 mg/mL of carbazole in alcohol and sulfuric acid (95:5)

Analysis: Develop the chromatogram until the solvent front has moved three-fourths of the length of the plate. Remove the plate from the chamber, mark the solvent front, and allow the solvent to evaporate. Examine the plate under short-wavelength UV light, and compare the intensities of any secondary spots observed in the chromatogram of the Sample solution with those of the principal spot in the chromatogram of the Standard solution.

Acceptance criteria 1: No secondary spot from the chromatogram of the Sample solution is larger or more intense than the principal spot from the Standard solution, and the sum of the intensities of the secondary spots obtained from the Sample solution corresponds to NMT 3.0%.

Spray the plate with Spray reagent, heat for 10 min at 120°, and compare the intensities of any secondary spots observed in the chromatogram of the Sample solution with those of the principal spots in the chromatogram of the Standard solution.

Acceptance criteria 2: No spot corresponding to triphenylmethanol (R_F about 2.3 relative to the R_F of zidovudine) is more intense than the corresponding spot from the Standard solution. No secondary spot from the chromatogram of the Sample solution is larger or more intense than the principal spot obtained from the Standard solution. The sum of the intensities of the secondary spots from the Sample solution corresponds to NMT 3.0%. (USP 1-May-2022)

Delete the following:

4.2 Limit of Zidovudine Related Compounds B and C

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

Analysis

Sample: Sample solution

Calculate the percentage of zidovudine related compound B and zidovudine related compound C in the portion of Zidovudine taken:

Result = (r_U /r_T ) × 100

r_U = peak response for zidovudine related compound B or zidovudine related compound C

r_T = sum of the responses of all of the peaks

Acceptance criteria

Individual impurities: NMT 1.0% for zidovudine related compound B and NMT 2.0% for zidovudine related compound C Total impurities: NMT 3.0% for all impurities from the tests for Limit of Triphenylmethanol and Other Impurities and Limit of Zidovudine Related Compounds B and C. (USP 1-May-2022)

Add the following:

4.3 Organic Impurities

Buffer, Solution A, Mobile phase, Diluent, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 1 mg/mL of USP Zidovudine RS and 2 µg/mL of USP Zidovudine Related Compound B RS in Diluent Standard solution: 1 µg/mL of USP Zidovudine RS in Diluent

Sample solution: 1 mg/mL of Zidovudine in Diluent

4.3.1 System suitability

Samples: System suitability solution and Standard solution

[Note—See Table 2 for the relative retention times.]

Suitability requirements

Resolution: NLT 2.0 between zidovudine and zidovudine related compound B, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

4.3.2 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of any individual impurity in the portion of Zidovudine 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 zidovudine from the Standard solution

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

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

F = relative response factor (see Table 2)

Acceptance criteria: See Table 2. Disregard the triphenylmethanol peak, any peak eluting after triphenylmethanol impurity, and any peak less than 0.05%.

Table 2

a Thymine; also known as 5-Methylpyrimidine-2,4(1H,3H)-dione.

b 1-[(2R,5S)-5-(Hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione.

c 3'-Chloro-3'-deoxythymidine.

d 1-{3-[3-(3-Azido-2,3-dideoxy-β-d-pentofuranosyl)-5-methyl-2,6-dioxo-3,6-dihydropyrimidin-1-yl]-2,3-dideoxy-β-d-pentofuranosyl]-5- methylpyrimidine-2,4-dione.

e See the test for Limit of Triphenylmethanol.

f Includes impurities from the tests for Organic Impurities and Limit of Triphenylmethanol.

(USP 1-May-2022)

Add the following:

4.4 Limit of Triphenylmethanol

Mobile phase: Acetonitrile and water (70:30)

System suitability solution: 0.01 mg/mL of USP Zidovudine RS and 5 µg/mL of USP Triphenylmethanol RS prepared as follows. Transfer a suitable amount of USP Zidovudine RS and USP Triphenylmethanol RS to a suitable volumetric ask and add 10% of the nal ask volume of acetonitrile. Dilute with Mobile phase to volume.

Standard stock solution: 0.5 mg/mL of USP Triphenylmethanol RS in acetonitrile

Standard solution: 5 µg/mL of USP Triphenylmethanol RS in Mobile phase from Standard stock solution

Sample solution: 5 mg/mL of Zidovudine prepared as follows. Transfer a suitable amount of Zidovudine to a suitable volumetric ask and add 10% of the nal ask volume of acetonitrile. Dilute with Mobile phase to volume.

4.4.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 210 nm

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

Flow rate: 1 mL/min

Injection volume: 20 µL

Run time: NLT 10 times the retention time of zidovudine

4.4.2 System suitability

Samples: System suitability solution and Standard solution

Suitability requirements

Resolution: NLT 5.0 between zidovudine and triphenylmethanol, System suitability solution

Relative standard deviation: NMT 5.0%, Standard solution

4.4.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of triphenylmethanol in the portion of Zidovudine taken:

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

r_U = peak response of triphenylmethanol from the Sample solution

r_S = peak response of triphenylmethanol from the Standard solution

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

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

Acceptance criteria: NMT 0.25% (USP 1-May-2022)

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 1.0%

Optical Rotation 〈781S〉, Procedures, Specific Rotation

Sample solution: 10 mg/mL, in alcohol

Acceptance criteria: +60.5° to +63°

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers. Store at 25°, excursions permitted between 15° and 30°. Change to read:

USP Reference Standards 〈11〉

USP Triphenylmethanol RS

Triphenylmethanol.

C₁₉H₁₆O    260.33

(USP 1-May-2022)

USP Zidovudine RS

USP Zidovudine Related Compound B RS

3′-Chloro-3′-deoxythymidine.

C₁₀H₁₃ClN₂O₄ 260.68

10 13 2 4

(USP 1-May-2022)