Mercaptopurine

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

Mercaptopurine contains NLT 97.0% and NMT 102.0% of mercaptopurine (C₅H₄N4_S), calculated on the anhydrous basis.

2 IDENTIFICATION

Change to read:

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197A or (IRA 1-Sep-2021) 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

Solution A: 0.77 g/L of ammonium acetate in water

Mobile phase: Methanol and Solution A (25:75)

Standard stock solution: 0.2 mg/mL of USP Mercaptopurine RS in a mixture of methanol and water (50:50). (IRA 1-Sep-2021) Transfer USP

Mercaptopurine RS into a suitable volumetric flask, and add methanol equivalent to 50% of the final volume. Shake mechanically to dissolve, and dilute with water to volume.

Standard solution: 0.02 mg/mL of USP Mercaptopurine RS in Mobile phase from the Standard stock solution

Sample stock solution: 0.4 mg/mL of Mercaptopurine prepared as follows. (IRA 1-Sep-2021) Transfer 25 mg of Mercaptopurine into a 50-mL volumetric flask. Add 25 mL of methanol, shake mechanically to dissolve, (IRA 1-Sep-2021) and dilute with water to volume. Transfer 20 mL of this solution into a 25-mL volumetric flask, and dilute with Mobile phase to volume.

Sample solution: 0.02 mg/mL of Mercaptopurine in Mobile phase from the Sample stock solution

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 325 nm

Column: 4.6-mm × 15-cm; 5-μm packing L68

Flow rate: 1.0 mL/min

Injection volume: 20 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: NMT 2.0

Relative standard deviation: NMT 0.73%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of mercaptopurine (C₅H₄N4_S) in the portion of Mercaptopurine taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of mercaptopurine (IRA 1-Sep-2021) from the Sample solution

r_S = peak response of mercaptopurine (IRA 1-Sep-2021) from the Standard solution

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

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

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

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.1%

Change to read:

Organic Impurities

Solution A: 0.1% (v/v) formic acid in water

Solution B: Methanol and Solution A (2:98)

Solution C: Methanol and Solution A (50:50) (IRA 1-Sep-2021)

Mobile phase: See Table 1.

Table 1

Standard stock solution: 0.06 mg/mL of USP Mercaptopurine RS in Solution A. [Note—Use methanol equivalent to 2.5% of the final volume to help dissolve.]

Standard solution: 1.2 μg/mL of USP Mercaptopurine RS in Solution B from the Standard stock solution

Sensitivity solution: 0.06 μg/mL of USP Mercaptopurine RS in Solution B from the Standard solution

Sample stock solution: 0.5 mg/mL of Mercaptopurine in a mixture of methanol and Solution A (50:50) (IRA 1-Sep-2021) prepared as follows.

Transfer a suitable quantity of Mercaptopurine to an appropriate volumetric flask, add methanol equivalent to 50% (IRA 1-Sep-2021) of the final volume, and shake to dissolve. Dilute with Solution A to volume.

Sample solution: 0.12 mg/mL of Mercaptopurine in Solution A from the Sample stock solution. [Note—Inject the Sample solution within 1 h of preparation.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 260 nm

Column: 4.6-mm × 10-cm; 3-μm packing L1

Temperatures

Column: 30°

Sample: 4°

Flow rate: 1.0 mL/min

Injection volume: 50 μL

System suitability

Samples: Standard solution and Sensitivity solution

Suitability requirements

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 2.0%, Standard solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of each impurity in the portion of Mercaptopurine 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 mercaptopurine from the Standard solution

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

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

F = relative response factor for each individual impurity (see Table 2)

Acceptance criteria: See Table 2. The reporting threshold is (IRA 1-Sep-2021) 0.05%.

Table 2

^a Hypoxanthine.

^b 1,2-Di(9H-purin-6-yl)disulfane.

5 SPECIFIC TESTS

Phosphorus

Standard phosphate solution: 43.96 μg/mL of dried monobasic potassium phosphate (equivalent to 10 μg of phosphorus)

Standard solution: Transfer 2 mL of Standard phosphate solution to a 25-mL volumetric flask. Add 1 mL of 15 N sulfuric acid, 0.5 mL of nitric acid, 0.75 mL of ammonium molybdate TS, and 1 mL of aminonaphtholsulfonic acid TS; then dilute with water to volume; and mix. Allow to stand for 5 min.

Sample solution: Digest 200 mg with 2 mL of 15 N sulfuric acid in a large test tube, periodically adding nitric acid, dropwise and with caution.

Continue heating until practically all of the liquid has evaporated and the residue is colorless. Transfer the residue, with the aid of small portions of water, to a 25-mL volumetric flask. Add 1 mL of 15 N sulfuric acid, 0.5 mL of nitric acid, 0.75 mL of ammonium molybdate TS, and 1 mL of aminonaphtholsulfonic acid TS, and then dilute with water to volume. Allow to stand for 5 min.

Blank: Transfer 2 mL of 15 N sulfuric acid to a large test tube, periodically adding nitric acid, dropwise and with caution. Continue heating until practically all of the liquid has evaporated and the residue is colorless. Transfer the residue, with the aid of small portions of water, to a 25-mL volumetric flask. Add 1 mL of 15 N sulfuric acid, 0.5 mL of nitric acid, 0.75 mL of ammonium molybdate TS, and 1 mL of aminonaphtholsulfonic acid TS, and then dilute with water to volume. Allow to stand for 5 min.

Instrumental conditions

(See Ultraviolet-Visible Spectroscopy 〈857〉.)

Mode: UV-Vis

Analytical wavelength: 750 nm

Analysis

Samples: Standard solution, Sample solution, and Blank

Acceptance criteria: The absorbance of the Sample solution is NMT that of the Standard solution (NMT 100 ppm).

Water Determination 〈921〉, Method I

Medium: 30 mL of methanol and 5 g of salicylic acid in the titration vessel

Sample: 0.3 g of Mercaptopurine

Acceptance criteria: NMT 12.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers, protected from light. Store at room temperature.

USP Reference Standards 〈11〉

USP Mercaptopurine RS