Allopurinol

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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C₅H₄N₄O 136.11 

4H-Pyrazolo[3,4-d]pyrimidin-4-one, 1,5-dihydro-; 

1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one; 

1H-Pyrazolo[3,4-d]pyrimidin-4-ol CAS RN®: 315-30-0; UNII: 63CZ7GJN5I. 

1 DEFINITION 

Allopurinol contains NLT 98.0% and NMT 102.0% of allopurinol (C₅H₄N₄O), calculated on the dried basis. 

2 IDENTIFICATION

Change to read: 

Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K (CN 1-May-2020)

3 ASSAY

3.1 Procedure

[Note—Store and inject the System suitability solution, Standard solution, and Sample solution at 8°, using a cooled autosampler.]

Mobile phase: 1.25-g/L solution of monobasic potassium phosphate in water, ltered and degassed

System suitability solution: 0.5 µg/mL each of USP Allopurinol RS, USP Allopurinol Related Compound B RS, and USP Allopurinol Related Compound C RS, prepared as follows. Transfer weighed quantities of USP Allopurinol RS, USP Allopurinol Related Compound B RS, and USP Allopurinol Related Compound C RS to three separate suitable volumetric asks, dissolve in a small volume of 0.1 N sodium hydroxide, and immediately dilute with Mobile phase to volume to obtain solutions containing 0.05 mg/mL each. Transfer 1.0 mL of each of these three solutions to a 100-mL volumetric ask and dilute with Mobile phase to volume.

Standard stock solution: 0.5 mg/mL of USP Allopurinol RS, prepared as follows. Transfer a weighed quantity of USP Allopurinol RS to a suitable volumetric ask, dissolve in a small volume of 0.1 N sodium hydroxide, and immediately dilute with Mobile phase to volume. Standard solution: 0.08 mg/mL of USP Allopurinol RS in Mobile phase from the Standard stock solution

Sample stock solution: 0.5 mg/mL of Allopurinol, prepared as follows. Transfer 50 mg of Allopurinol to a 100-mL volumetric ask, dissolve in 5.0 mL of 0.1 N sodium hydroxide, and immediately dilute with Mobile phase to volume.

Sample solution: 0.08 mg/mL of Allopurinol in Mobile phase from the Sample stock solution

3.2 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 230 nm

Column: 4.6-mm × 25-cm; packing L1

Flow rate: 1.8 mL/min

Injection volume: 20 µL

System suitability

Samples: System suitability solution and Standard solution

[Note—The relative retention times for allopurinol related compound B, allopurinol related compound C, and allopurinol are about 0.7, 0.8, and 1.0, respectively.]

Suitability requirements

Resolution: NLT 1.1 between allopurinol related compound B and allopurinol related compound C; NLT 6.0 between allopurinol related compound C and allopurinol, System suitability solution

Relative standard deviation: NMT 2.0% for replicate injections, Standard solution

3.3 Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of allopurinol (C H N O) in the portion of Allopurinol taken:

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

U S S U 

r_U = peak response from the Sample solution 

r_S = peak response from the Standard solution 

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

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

Acceptance criteria: 98.0%–102.0% on the dried basis 

4 IMPURITIES 

4.1 Organic Impurities 

[Note—Store and inject the Standard solution and the Sample solution at 8°, using a cooled autosampler.] 

Solution A: 1.25-g/L solution of monobasic potassium phosphate in water, ltered and degassed 

Solution B: Methanol 

Mobile phase: See Table 1. 

Table 1 

Diluent: Solution A and Solution B (90:10)

Standard stock solution: 0.05 mg/mL each of USP Allopurinol RS, USP Allopurinol Related Compound A RS, USP Allopurinol Related Compound B RS, USP Allopurinol Related Compound C RS, USP Allopurinol Related Compound D RS, and USP Allopurinol Related Compound E RS, prepared as follows. Transfer 5 mg each of USP Allopurinol RS, USP Allopurinol Related Compound A RS, USP Allopurinol Related Compound B RS, USP Allopurinol Related Compound C RS, USP Allopurinol Related Compound D RS, and USP Allopurinol Related Compound E RS to a 100-mL volumetric ask. Add 2.0 mL of 0.1 N sodium hydroxide, and promptly sonicate with swirling for NMT 1 min to dissolve. Add 80 mL of Diluent, and sonicate for an additional 5 min. Dilute with Diluent to volume. [Note—This solution is stable for 48 h when stored at 8°.]

Standard solution: 0.5 µg/mL each of USP Allopurinol RS, USP Allopurinol Related Compound A RS, USP Allopurinol Related Compound B RS, USP Allopurinol Related Compound C RS, USP Allopurinol Related Compound D RS, and USP Allopurinol Related Compound E RS in Diluent from the Standard stock solution

Sample solution: 0.25 mg/mL of Allopurinol, prepared as follows. Transfer 25 mg of Allopurinol to a 100-mL volumetric ask. Add 5.0 mL of 0.1 N sodium hydroxide to dissolve, promptly sonicate with swirling for NMT 1 min, add 80 mL of Diluent, and sonicate for an additional 5 min. Dilute with Diluent to volume.

4.1.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 220 nm

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

Column temperature: 30°

Flow rate: 1.0 mL/min

Injection volume: 40 µL

System suitability

Sample: Standard solution

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

Suitability requirements

Resolution: NLT 0.8 between allopurinol related compound C and allopurinol related compound B

Tailing factor: NMT 1.5 for the allopurinol peak

4.1.2 Analysis

Samples: Standard solution and Sample solution

Calculate the percentages of allopurinol related compounds A, B, C, D, and E in the portion of Allopurinol taken:

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

r_U = peak response of each individual impurity from the Sample solution

r_S = peak response of each individual impurity from the Standard solution

C_S = concentration of each individual impurity in the Standard solution (mg/mL)

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

Calculate the percentage of any other individual impurity in the portion of Allopurinol taken:

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

r_U = peak response of each impurity from the Sample solution

r_S = peak response of allopurinol from the Standard solution

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

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

Acceptance criteria: See Table 2.

Table 2

4.2 Limit of Hydrazine

[Note—Under the following conditions, any hydrazine present in the sample will react with benzaldehyde to form benzalazine.] Mobile phase: Hexane and isopropyl alcohol (95:5)

2 N sodium hydroxide solution: Dissolve 8.5 g of sodium hydroxide in water, and dilute with the same solvent to 100 mL. Alternatively, a commercially available 2 N sodium hydroxide solution can be used.

Diluent: Methanol and 2 N sodium hydroxide solution (1:1)

Benzaldehyde solution: 40 mg/mL of benzaldehyde in Diluent. [Note—Prepare immediately before use.]

Hydrazine solution: 2.0 µg/mL of hydrazine sulfate in Diluent. Use sonication if necessary.

Standard solution: Transfer 5.0 mL of Hydrazine solution to a suitable ask and add 4 mL of Benzaldehyde solution. Mix and allow to stand for 2.5 h at room temperature. Add 5.0 mL of hexane, and shake for 1 min. Allow the layers to separate, and use the upper (hexane) layer. Allopurinol solution: Dissolve 250 mg of Allopurinol in 5 mL of Diluent.

Sample solution: Transfer the Allopurinol solution to a suitable ask, and add 4 mL of Benzaldehyde solution. Mix, and allow to stand for 2.5 h at room temperature. Add 5.0 mL of hexane, and shake for 1 min. Allow the layers to separate, and use the upper (hexane) layer.

Blank solution: Mix 5.0 mL of Diluent and 4 mL of Benzaldehyde solution, and allow to stand for 2.5 h at room temperature. Add 5.0 mL of hexane, and shake for 1 min. Allow the layers to separate, and use the upper (hexane) layer.

4.2.1 Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 310 nm

Column: 4.0-mm × 25-cm; 5-µm packing L10

Column temperature: 30°

Flow rate: 1.5 mL/min

Injection volume: 20 µL

System suitability

Sample: Standard solution

[Note—The relative retention times for benzalazine and benzaldehyde are about 0.8 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.0 between benzalazine and benzaldehyde

Relative standard deviation: NMT 15.0% for the benzalazine peak

4.2.2 Analysis

Samples: Standard solution and Sample solution

Calculate the amount, in ppm, of hydrazine in the portion of Allopurinol taken:

Result = (r_U /r_S ) × (C_S /C_U ) × (M_r1 /M_r2 ) × F

r_U = peak response of benzalazine from the Sample solution

r_S = peak response of benzalazine from the Standard solution

C_S = concentration of hydrazine sulfate in the Hydrazine solution (µg/mL)

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

M_r1 = molecular weight of hydrazine, 32.05

M_r2 = molecular weight of hydrazine sulfate, 130.12

F = unit conversion factor (from µg/mg to ppm), 1000

Acceptance criteria: NMT 10 ppm of hydrazine

5 SPECIFIC TESTS

Loss on Drying 〈731〉

Analysis: Dry under vacuum at 105° for 5 h.

Acceptance criteria: NMT 0.5%

ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in well-closed containers. Store at room temperature.

Change to read:

USP Reference Standards 〈11〉

USP Allopurinol RS

USP Allopurinol Related Compound A RS

3-Amino-4-carboxamidopyrazole hemisulfate.

(C₄H₆N₄O)₂ · H₂SO₄ 350.31    (ERR 1-Dec-2018)

USP Allopurinol Related Compound B RS

5-(Formylamino)-1H-pyrazole-4-carboxamide.

C₅H₆N₄O₂ 154.13

USP Allopurinol Related Compound C RS

5-(4H-1,2,4-Triazol-4-yl)-1H-pyrazole-4-carboxamide.

C₆H₆N₆O 178.15

USP Allopurinol Related Compound D RS

Ethyl 5-amino-1H-pyrazole-4-carboxylate.

C₆H₉N₃O₂ 155.15

USP Allopurinol Related Compound E RS

Ethyl 5-(formylamino)-1H-pyrazole-4-carboxylate.

C₇H₉N₃O₃ 183.16