Thioguanine

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

Thioguanine is anhydrous or contains one-half molecule of water of hydration. It contains NLT 96.0% and NMT 100.5% of thioguanine, calculated on the dried basis.

2 IDENTIFICATION

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

B. Spectroscopic Identification Tests 〈197〉, Ultraviolet-Visible Spectroscopy: 197U

Sample solution: Transfer 100 mg of Thioguanine, previously dried, to a 100-mL volumetric flask. Dissolve in a mixture of 15 mL of water and 1.5 mL of 1 N sodium hydroxide, dilute with water to volume, and mix. Transfer 10.0 mL of this solution to a second 100-mL volumetric flask, add 1% hydrochloric acid to volume, and mix. Finally, transfer 5.0 mL of the last solution to a third 100-mL volumetric flask, then add 1% hydrochloric acid to volume, and mix.

Acceptance criteria: The UV absorption spectrum of the Sample solution exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Thioguanine RS.

3 ASSAY

Procedure

Phosphoric acid solution: Add 1 mL of phosphoric acid to 99 mL of water.

Mobile phase: 0.05 M monobasic sodium phosphate. Adjust with phosphoric acid to a pH of 3.0.

Standard stock solution: 0.4 mg/mL of USP Thioguanine RS in 0.01 N sodium hydroxide

Standard solution: 0.04 mg/mL of USP Thioguanine RS in Phosphoric acid solution from Standard stock solution

Sample stock solution: 0.4 mg/mL of Thioguanine in 0.01 N sodium hydroxide

Sample solution: 0.04 mg/mL of Thioguanine in Phosphoric acid solution from Sample stock solution

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 248 nm

Column: 4.6-mm × 5-cm; packing L1

Flow rate: 2.0 mL/min

Injection volume: 10 μL

System suitability

Sample: Standard solution

Suitability requirements

Relative standard deviation: NMT 2.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of thioguanine in the portion of Thioguanine taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of thioguanine from the Sample solution

r_S = peak response of thioguanine from the Standard solution

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

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

Acceptance criteria: 96.0%–100.5% on the dried basis

4 OTHER COMPONENTS

Nitrogen Determination 〈461〉, Method II

Sample: 100 mg

Analysis: Each milliliter of 0.1 N sulfuric acid is equivalent to 1.401 mg of nitrogen (N).

Acceptance criteria: 40.2%–43.1% on the dried basis

5 IMPURITIES

Delete the following:

Selenium 〈291〉

Sample: 200 mg

Acceptance criteria: NMT 30 ppm (USP 1-Dec-2021)

Free Sulfur

Sample solution: Dissolve 50 mg of Thioguanine in 5 mL of 1 N sodium hydroxide.

Acceptance criteria: The Sample solution is clear.

Phosphorous-Containing Substances

Solution A: Dissolve 8.3 g of ammonium molybdate in 40 mL of water, add 33 mL of dilute sulfuric acid (2 in 7), and dilute with water to 100.0 mL. [Note—This solution is stable for about 2 weeks.]

Instrumental conditions

Mode: UV-Vis

Cell: 1 cm

Analytical wavelength: 620 nm

Analysis: Transfer 50.0 mg of Thioguanine to a large test tube, add 1 mL of dilute sulfuric acid (2 in 7), and heat in a boiling water bath for 5 min. Cautiously add nitric acid, dropwise, continue heating until the mixture becomes colorless, and then heat for 1 min longer. Cool, dilute with water to about 10 mL, and transfer the solution to a 25-mL volumetric flask with the aid of a few milliliters of water. To the flask add 0.75 mL of Solution A and 1.0 mL of aminonaphtholsulfonic acid TS, dilute with water to volume, and mix.

Acceptance criteria: NMT 0.03% as phosphate; the absorbance is not greater than that produced by 1.5 mL of a similar solution of monobasic potassium phosphate in water having a known concentration of 10 μg/mL of phosphate (PO -3), concomitantly measured.

Change to read:

Organic Impurities

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

Standard stock solution: 0.04 mg/mL of USP Guanine RS in 0.01 N sodium hydroxide

Standard solution: 0.4 μg/mL of USP Guanine RS in Mobile phase from Standard stock solution

Sample solution: 0.04 mg/mL of Thioguanine in Mobile phase from Sample stock solution

System suitability solution: Transfer 1.0 mL of Standard stock solution into a 100-mL volumetric flask, and dilute with the Sample solution to volume.

System suitability

Samples: Standard solution and System suitability solution

[Note—The relative retention times for guanine and thioguanine are about 0.60 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 3.0 between guanine and thioguanine, System suitability solution

Tailing factor: NMT 2.0, Standard solution

Relative standard deviation: NMT 5.0%, Standard solution

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of guanine in the portion of Thioguanine taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of guanine from the Sample solution

r_S = peak response of guanine from the Standard solution

C_S = concentration of USP Guanine RS (USP 1-Dec-2021) in the Standard solution (mg/mL)

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

Acceptance criteria: NMT 2.5%

6 SPECIFIC TESTS

Loss on Drying 〈731〉

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

Acceptance criteria: NMT 6.0%

7 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight containers.

Labeling: Label it to indicate its state of hydration.

USP Reference Standards 〈11〉

USP Guanine RS

USP Thioguanine RS