A: Infrared Absorption 197K.

B: The UV absorption spectrum of a 1 in 200,000 solution of it, prepared as directed in the Assay, exhibits maxima and minima at the same wavelengths as that of a similar solution of USP Thioguanine RS, concomitantly measured.

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

Procedure— Transfer 50.0 mg, accurately weighed, to a large test tube, add 1 mL of dilute sulfuric acid (2 in 7), and heat in a boiling water bath for 5 minutes. Cautiously add nitric acid, dropwise, continue heating until the mixture becomes colorless, and then heat for 1 minute 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 mL of water. To the flask add 0.75 mL of Ammonium molybdate solution and 1.0 mL of aminonaphtholsulfonic acid TS, dilute with water to volume, and mix. Determine the absorbance of this solution in a 1-cm cell, with a suitable spectrophotometer, at a wavelength of about 620 nm, using a reagent blank to set the instrument: 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 of phosphate (PO4) in each mL, concomitantly measured (0.03% as phosphate).

Mobile phase— Proceed as directed in the Assay.

Standard solution— Dissolve an accurately weighed quantity of guanine in 0.01 N sodium hydroxide, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.04 mg per mL. Pipet 1.0 mL of this solution into a 100-mL volumetric flask, and dilute with Mobile phase to volume to obtain a solution having a known concentration of 0.4 µg per mL.

Test solution— Transfer about 40 mg of Thioguanine, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with 0.01 N sodium hydroxide to volume, and mix. Transfer 10.0 mL of this solution into a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 248-nm detector and a 4.6-mm × 5-cm column that contains packing L1. The flow rate is about 2.0 mL per minute. Chromatograph the Standard solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.60 for guanine and 1.0 for thioguanine; the resolution, R, between guanine and thioguanine is not less than 3.0; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 5.0% for the guanine peak.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of guanine in the portion of Thioguanine taken by the formula: in which C is the concentration, in µg per mL, of guanine in the Standard solution; W is the weight, in mg, of Thioguanine taken to prepare the Test solution; and rU and rS are the peak responses of guanine obtained from the Test solution and the Standard solution, respectively: not more than 2.5% is found.

Phosphoric acid solution— Carefully add 1 mL of phosphoric acid to 99 mL of water, and mix.

Mobile phase— Prepare a filtered and degassed solution of 0.05 M monobasic sodium phosphate. Adjust with phosphoric acid to a pH of 3.0. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Thioguanine RS in 0.01 N sodium hydroxide, and dilute quantitatively, and stepwise if necessary, to obtain a solution having a known concentration of about 0.4 mg per mL. Pipet 10.0 mL of this solution into a 100-mL volumetric flask, and dilute with Phosphoric acid solution to volume to obtain a solution having a known concentration of 0.04 mg per mL.

Assay preparation— Transfer about 40 mg of Thioguanine, accurately weighed, to a 100-mL volumetric flask, dissolve in and dilute with 0.01 N sodium hydroxide to volume, and mix. Transfer 10.0 mL of this solution to a 100-mL volumetric flask, dilute with Phosphoric acid solution to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 248-nm detector and a 4.6-mm × 5-cm column that contains packing L1. The flow rate is about 2.0 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.60 for guanine and 1.0 for thioguanine, and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C5H5N5S in the portion of Thioguanine taken by the formula: in which C is the concentration, in mg per mL, of USP Thioguanine RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.